Elevated glucose attenuates human insulin gene promoter activity in INS-1 pancreatic beta-cells via reduced nuclear factor binding to the A5/core and Z element.
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.
Pino
Maria F
MF
Department of Physiology, Michigan State University, 3176 Biomedical and Physical Sciences Building, East Lansing, MI 48824, USA.
Ye
Diana Z
DZ
Linning
Katrina D
KD
Green
Christopher D
CD
Wicksteed
Barton
B
Poitout
Vincent
V
Olson
L Karl
LK
D024506:5' Flanking Region; D000818:Animals; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation / Q000502:physiology*; D005947:Glucose / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism*; D051473:Maf Transcription Factors, Large; D011401:Promoter Regions, Genetic; D051381:Rats; D015534:Trans-Activators / Q000378:metabolism
D018398:Homeodomain Proteins; D007328:Insulin; C485159:MAFA protein, human; D051473:Maf Transcription Factors, Large; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D005947:Glucose