31 Pino, M. F. Ye, D. Z. Linning, K. D. Green, C. D. Wicksteed, B. Poitout, V. Olson, L. K. 2005 Mol Endocrinol 19 5 1343-60 May 0888-8809 (Print) 15650027 Animals 5' Flanking Region Trans-Activators/metabolism Rats *Promoter Regions, Genetic Maf Transcription Factors, Large Islets of Langerhans/*metabolism Insulin/genetics/*metabolism Humans Homeodomain Proteins/metabolism Glucose/*metabolism Gene Expression Regulation/*physiology Enhancer Elements, Genetic *Promoter Regions Genetic Maf Transcription Factors Large Enhancer Elements Chronic exposure of pancreatic beta-cells to elevated glucose reduces insulin gene promoter activity, and this is associated with diminished binding of two beta-cell-enriched transcription factors, Pdx-1 and MafA. In this study using INS-1 beta-cells, overexpression of MafA, but not Pdx-1, was able to restore expression of a human insulin reporter gene (-327 to +30 bp) suppressed by elevated glucose. At issue, however, was that MafA also markedly stimulated an insulin reporter gene (-230 to +30 bp) that was only marginally suppressed by glucose, suggesting that glucose-mediated suppression of the insulin promoter involved elements upstream of -230. Using serial truncations and mini-enhancer constructs of the human insulin promoter, the majority of glucose suppression was localized to regulatory elements between -327 and -261. Nuclear extracts from INS-1 cells exposed to elevated glucose had reduced binding activities to the A5/core (-319 to -307), and to a palindrome (-284 to -267) and an E box (-273 to -257, E3) contained within the Z element. The A5/core binding complex was determined to contain MafA, Pdx-1, and an A2-like binding factor. Two mini-enhancer constructs containing the A5/core were suppressed by glucose and strongly activated by MafA. Glucose-mediated suppression of the Z mini-enhancer was not attenuated by overexpression of MafA or Pdx-1. Site-directed mutation of the A5/core, palindrome, and E3 elements attenuated glucose-mediated suppression. These data indicate that glucose suppression of human insulin promoter activity in INS-1 cells involves reduced binding of MafA to the A5/core. Changes in nuclear factor binding to the Z element, which functions as a strong activator element in primary islets and a negative regulatory element in simian virus 40 or T antigen transformed beta-cell lines, also participate in glucose suppression of insulin promoter activity. R01 DK 58096/DK/NIDDK NIH HHS/United StatesJournal ArticleResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.United States http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15650027