Insulin Response Sequence-Dependent and -Independent Mechanisms Mediate Effects of Insulin on Glucocorticoid-Stimulated Insulin-Like Growth Factor Binding Protein-1 Promoter Activity

Abstract

IGF binding protein-1 (IGFBP-1) gene expression is stimulated by glucocorticoids and suppressed by insulin in the liver. Insulin response sequences (IRSs) mediate effects of insulin on basal promoter function, whereas glucocorticoids stimulate promoter activity through a contiguous glucocorticoid response element. Here we examined the role of IRS-dependent and -independent mechanisms in mediating insulin and glucocorticoids effects on IGFBP-1 promoter activity. Dexamethasone (Dex) stimulates IGFBP-1 promoter activity in HepG2 cells, and mutation of IRSs reduces this effect, indicating that IRS-associated factors enhance glucocorticoid effects on promoter function. Conversely, insulin inhibits basal promoter activity by 40% and Dex-stimulated promoter activity by 65%, indicating that glucocorticoids enhance the ability of insulin to suppress promoter activity. Mutation of IRSs completely disrupts the insulin effect on basal promoter activity and reduces but does not abolish inhibition of Dex-stimulated promoter activity, indicating that insulin suppresses glucocorticoid-stimulated promoter activity through both IRS-dependent and -independent mechanisms. IRS-independent effects of insulin are context dependent because insulin does not suppress glucocorticoid-stimulated activity of a promoter containing multiple glucocorticoid response elements. Cotransfection studies indicate that suppression of peroxisomal proliferator-activated receptor-gamma coactivator-1alpha, an insulin-regulated coactivator of the glucocorticoid receptor, is not required for this effect of insulin. Studies with pharmacological inhibitors indicate that both phosphatidylinositol-3' kinase and mitogen-activated kinase kinase pathways contribute to IRS-independent effects. These studies indicate that glucocorticoids and IRS-associated factors function together to mediate effects of insulin and glucocorticoids on promoter activity and that glucocorticoid treatment creates a complex environment in which insulin regulates IGFBP-1 expression through both IRS-dependent and IRS-independent mechanisms.