IDENTIFICATION OF AN INSULIN-RESPONSIVE ELEMENT IN THE RAT INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-3 GENE

The hepatic expression and serum levels of insulinlike growth factor-b inding protein-3 (IGFBP-3) are decreased in insulin-dependent and insu lin-resistant diabetes. Insulin increases hepatic IGFBP-3 expression b y enhancing gene transcription. This report identifies sequences withi n the IGFBP-3 promoter that are necessary and sufficient for the respo nse to insulin in hepatic nonparenchymal cells. By transient transfect ion, we mapped the insulin response element to the -1150 to -1124 base pair (bp) region of the rat IGFBP-3 promoter. Three tandem repeats of the -1150 to -1117 bp region conferred insulin responses in a heterol ogous promoter. Gel shift analyses revealed a 3-fold increase in DNA-p rotein complex formation with nuclear extracts obtained from insulin-s timulated nonparenchymal cells compared with cells incubated without i nsulin and revealed 3-4-fold decrease in complex formation with nuclea r extracts obtained from the livers of streptozotocin-diabetic rats co mpared with control rats. Mutational analysis of this 34-bp region sho wed a core sequence of 10 bp (-1148 to -1139) that is critical for int eraction with insulin-induced trans-acting factors. Southwestern blott ing revealed a similar to 90-kDa protein that was increased 23-fold by the addition of insulin. Thus, we have identified cis-acting DNA sequ ences that are responsible for regulation of IGFBP-3 transcription by insulin and essential for binding of insulin-responsive nuclear factor s.