Hypoxia alters phosphorylation status of insulin-like growth factor (IGF)-binding protein-1 and attenuates biological activities of IGF-I in HepG2 cell cultures

Abstract

Aim: Insulin-like growth factor (IGF)-I is known to stimulate fetal growth. One of the IGF-binding proteins, IGFBP-1, suppresses IGF-I activity, and thereby inhibits fetal growth. Because hypoxic stress in the uterus is known to cause fetal growth restriction, we examined the effects of hypoxia on IGFBP-1 production and phosphorylation status.

Methods: Because liver is a main IGFBP-1 production site in the fetus, we used a hepatoma cell line, HepG2 cells, that secrete a large amount of IGFBP-1, express IGF-I receptors and model fetal liver metabolism in vitro. IGFBP-1 was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (PAGE) following immunoblotting, and IGFBP-1 phosphorylation status was analyzed by native PAGE following immunoblotting.

Results: Total concentrations of IGFBP-1 in media were higher and the highly phosphorylated isoforms were dominant in low oxygen conditions. Phosphorylation of IGF-I receptor by IGF-I was attenuated in low oxygen conditions. IGF-I-induced phosphorylation of insulin receptor substrate-1 (IRS-1) was attenuated in low oxygen conditions as well. However, attenuated phosphorylation of IGF-I receptor and IRS-1 were not observed in low oxygen conditions if the cells were stimulated with LR³IGF-I that has a similar binding affinity to IGF-I receptor but much less binding affinity to IGFBP-1 compared to those of native IGF-I. While IGF-I-induced cell proliferation was also inhibited in low oxygen conditions, LR³IGF-I-stimulated cell proliferation was not inhibited. These findings indicate that low oxygen conditions inhibit IGF-I action by increasing IGFBP-1, especially phosphorylated IGFBP-1, which inhibits IGF-I action.

Conclusion: This study has indicated that hypoxia-induced IGFBP-1 production in the fetus may be a conserved physiological mechanism for restricting IGF-I-stimulated fetal growth.

Keywords: fetal growth; hypoxia; insulin-like growth factor-binding protein-1.