Maternal Fetal Medicine
Poster Only Presentation
Concurrent Session 2C - DOHaD
Introduction: Our previous studies strongly support a critical role for IGFBP-1 hyperphosphorylation, which results in reduced IGF-I availability, in the development of fetal growth restriction (FGR). Leucine deprivation, which is common in FGR fetuses, has been shown to increase IGFBP-1 phosphorylation by activation of amino acid response. Nutrient deprivation nonetheless is also potent inducer of endoplasmic reticulum (ER) stress and unfolded protein response (UPR). We tested the hypothesis that IGFBP-1-hyperphosphorylation in leucine deprivation is mediated by UPR pathway.
Methods: Using HepG2 cells, we inhibited UPR signaling using tunicamycin (Tu, 6uM) either with 450 uM leucine (L450) or without leucine (L0). To identify the UPR arms involved, cells were treated (24 hrs) with Tu in L450/L0 in presence or absence of Inositol-requiring enzyme 1( IRE-1) pathway inhibitor 4u8c (50uM), or PKR-like ER kinase (PERK )inhibitor GSK2656157 (1uM). Further, using Tu we activated UPR signaling in combination with/without mTOR activator MHY1485 (1uM) in L450 and L0. The subsequent effects on UPR activity were confirmed using Western blot. IGFBP-1 phosphorylation was assessed in cell media using phospho-site (pS101/ 119/169) specific IGFBP-1 antibodies.
Results: Immunoblot analysis demonstrated that activation of UPR markedly induced IGFBP-1 phosphorylation at Ser 101 (~70 fold, p=0.0015), Ser169 ((~73 fold, p=0.0207) compared to control. Leucine deprivation combined with Tu did not show significant additive effects on IGFBP-1-phosphorylation. Inhibition of IRE pathway reduced the Tu induced phosphorylation of IGFBP-1Ser101 (down-regulation ~100 fold, p<0.0001) while inhibition of PERK arm didn’t have any significant effect indicating IRE pathway is involved. Importantly, MHY1485 failed to influence Tu-induced-IGFBP-1 phosphorylation indicating UPR mediates effects independent of mTOR.
Conclusion: Induction of UPR via IRE-pathway constitutes a mechanistic link between leucine deprivation and increased IGFBP-1 phosphorylation. These data provide novel insights into the mechanisms modulating IGF-I bioavailability and potentially fetal growth under nutritional deprivation.