title: High glucose activates ChREBP-mediated HIF-1α and VEGF expression in human RPE cells under normoxia.
abstract: OBJECTIVE
  Because retina-damaging angiogenesis is controlled by vascular endothelial growth factor (VEGF) and people with higher glucose intakes are more susceptible to retinal complications that may be due to increased VEGF, it is crucial to elucidate relations between glucose exposure and VEGF expression. We aimed to determine if a carbohydrate response element binding protein (ChREBP) plays a role in the transcriptional up-regulation of hypoxia-inducible factor-1α (HIF-1α) and the downstream VEGF expression in retinal pigment epithelial (RPE) cells exposed to high glucose under normoxic conditions.
  
  
  METHODS
  ARPE19 cells were exposed to 5.6, 11, 17, 25 and 30 mM glucose for 48 h in serum-free culture media under normoxic (21 % O2) conditions. Protein and mRNA expression of indicated genes were determined by immunoblot analyses and real-time RT-PCR, respectively. An enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of VEGF in the media. Immunofluorescence (IF) and chromatin immunoprecipitation (ChIP) for ChREBP were used to demonstrate nuclear translocation and HIF-1α gene promoter association, respectively.
  
  
  RESULTS
  Immunoblot analyses showed that HIF-1α levels were positively related to levels of glucose exposure between 5.6-25 mM in the RPE cells, indicating the induction and stabilization of HIF-1α by elevated glucose under normoxic conditions. Human lens epithelial cells and HeLa cells did not respond to high glucose, implying that this phenomenon is cell type-specific. Real-time RT-PCR for HIF-1α and VEGF and ELISA for VEGF indicated that high glucose is associated with elevated production of HIF-1α-induced VEGF, an established inducer of neovascularization, in the RPE cells. IF analyses showed that, although ChREBP was expressed under both low (5.6 mM) and high (25 mM) glucose conditions, it appeared more in the nuclear region than in the cytosol of the RPE cells after the high glucose treatment. ChIP analyses suggested a HIF-1α gene promoter association with ChREBP under the high glucose condition. These results imply that RPE cells use cytosolic ChREBP as a glucose sensor to up-regulate HIF-1α expression.
  
  
  CONCLUSIONS
  These results suggest a high glucose-induced, ChREBP-mediated, and normoxic HIF-1α activation that may be partially responsible for neovascularization in both diabetic and age-related retinopathy.
authors.affiliation: Laboratory for Nutrition and Vision Research, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, 02111, Boston, MA, USA, Min-Lee.Chang@tufts.edu.
authors.forename: Min-Lee
authors.initials: ML
authors.lastname: Chang
authors.forename: Chung-Jung
authors.initials: CJ
authors.lastname: Chiu
authors.forename: Fu
authors.initials: F
authors.lastname: Shang
authors.forename: Allen
authors.initials: A
authors.lastname: Taylor
mesh_terms: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / Q000378:metabolism*; D015687:Cell Hypoxia / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D008268:Macular Degeneration / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000494:pharmacology; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C116385:MLXIPL protein, human; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D005947:Glucose; D010100:Oxygen