Identification and Characterization of a Glucose-Responsiveness Region Upstream of Human Insulin Gene in Transfected HIT-T 15 Cells

Abstract

To determine possible regulation of full-length human insulin gene promoter activity by glucose, we examined a 2-kilobase pair (kbp) 5′-flanking region of the human insulin gene and characterized the DNA elements in transfected HIT-T 15 cells. The expression of the 2-kilobase pair 5′-flanking region human insulin gene fused to the luciferase reporter gene occurred by transfection. In 0.8 mM glucose of the F-12 K medium, the element mediating the negative regulatory region was localized from −1782 to −1295 base pairs (bp) and stimulatory element from −1295 to −1138 bp. The elements from −1138 to −880 bp and from −356 to +252 bp possessed the elements dose-dependently responsive to 0.8 mM, 7.0 and 22.2 mM glucose. In fragment D, cotransfection of oligonucleotide that confers RIPE3b1 activator decreased the glucose-stimulated promoter activity, but the other oligonucleotide that confers STF-1 did not. The present data indicated that 2 kbp possesses glucose-responsive region in the element from −1138 to −880 bp, in addition to the previously reported element from −356 to initiation site. There may exist a RIPE3b1 activator binding site in the glucose-responsive element from −1138 to −880 bp. In addition, negatively regulatory region may exist from −1782 to −1295 bp.