title: ChREBP rather than USF2 regulates glucose stimulation of endogenous L-pyruvate kinase expression in insulin-secreting cells.
abstract: 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 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.
authors.affiliation: Division of Clinical Biochemistry, Department of Internal Medicine, University Medical Center, CH-1211 Geneva 4, Switzerland. Haiyan.Wang@medicine.unige.ch
authors.forename: Haiyan
authors.initials: H
authors.lastname: Wang
authors.forename: Claes B
authors.initials: CB
authors.lastname: Wollheim
mesh_terms: D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D015152:Blotting, Northern; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005799:Genes, Dominant; D005947:Glucose / Q000378:metabolism* / Q000494:pharmacology; D022781:Hepatocytes / Q000378:metabolism; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011770:Pyruvate Kinase / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013347:Subcellular Fractions; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D015854:Up-Regulation; D051720:Upstream Stimulatory Factors
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D007328:Insulin; C442825:Mlxipl protein, rat; D012333:RNA, Messenger; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; C497186:Usf2 protein, mouse; D008156:Luciferases; D011770:Pyruvate Kinase; D005947:Glucose