Regulation of Hepatic Insulin-Like Growth Factor-Binding Protein-1 Gene Expression by Insulin: Central Role for Mammalian Target of Rapamycin Independent of Forkhead Box O Proteins

Abstract

The expression of IGF-binding protein-1 (IGFBP-1) is induced in rat liver by dexamethasone and glucagon and is completely inhibited by 100 nm insulin. Various studies have implicated phosphatidylinositol 3-kinase, protein kinase B (Akt), phosphorylation of the transcription factors forkhead in rhabdomyosarcoma 1 (Foxo1)/Foxo3, and the mammalian target of rapamycin (mTOR) in insulin’s effect. In this study we examined insulin regulation of IGFBP-1 in both subconfluent and confluent hepatocytes. In subconfluent hepatocytes, insulin inhibition of IGFBP-1 mRNA levels was blocked by inhibiting PI3 kinase activation, and there was a corresponding inhibition of Foxo1/Foxo3 phosphorylation. In these same cells, inhibition of the insulin effect by rapamycin occurred in the presence of insulin-induced Foxo1/Foxo3 phosphorylation. In confluent hepatocytes, insulin could not activate the phosphatidylinositol 3-kinase (PI3 kinase)-Akt-Foxo1/Foxo3 pathway, but still inhibited IGFBP-1 gene expression in an mTOR-dependent manner. In subconfluent hepatocytes, the serine/threonine phosphatase inhibitor okadaic acid (100 nm) partially inhibited IGFBP-1 gene expression by 40%, but did not produce phosphorylation of either Akt or Foxo proteins. In contrast, 1 nm insulin inhibited the IGFBP-1 mRNA level by 40% and correspondingly activated Akt and Foxo1/Foxo3 phosphorylation to a level comparable to that observed with 100 nm insulin. These results suggest a potential role for a serine/threonine phosphatase(s) in the regulation of IGFBP-1 gene transcription, which is not downstream of mTOR and is independent of Akt. In conclusion, we have found that in rat liver, insulin inhibition of IGFBP-1 mRNA levels can occur in the absence of the phosphorylation of Foxo1/Foxo3, whereas activation of the mTOR pathway is both necessary and sufficient.