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Elevated Glucose Attenuates Human Insulin Gene Promoter Activity in INS-1 Pancreatic ß-Cells via Reduced Nuclear Factor Binding to the A5/Core and Z Element

Departments of Physiology (M.F.P., K.D.L., L.K.O.), Pharmacology (D.Z.Y.), and Biochemistry and Molecular Biology (C.D.G.), Michigan State University, East Lansing, Michigan 48824; Pacific Northwest Research Institute (B.W., V.P.), Seattle, Washington 98122; and Department of Medicine (V.P.), University of Washington, Seattle, Washington 98195

Address all correspondence and requests for reprints to: L. Karl Olson, Ph.D., Michigan State University, Department of Physiology, 3176 Biomedical and Physical Sciences Building, East Lansing, Michigan 48824. E-mail: olsonla{at}msu.edu.

Chronic exposure of pancreatic ß-cells to elevated glucose reduces insulin gene promoter activity, and this is associated with diminished binding of two ß-cell-enriched transcription factors, Pdx-1 and MafA. In this study using INS-1 ß-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 minienhancer 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 minienhancer constructs containing the A5/core were suppressed by glucose and strongly activated by MafA. Glucose-mediated suppression of the Z minienhancer 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 ß-cell lines, also participate in glucose suppression of insulin promoter activity.

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