In cultured fetal liver cells, IGFBP-1 hyperphosphorylation in response to hypoxia and amino acid deprivation is mediated by inhibition of mechanistic target of rapamycin (mTOR) and activation of amino acid response (AAR) signalling and casein kinase CK2. We hypothesized that fetal liver mTOR inhibition, activation of AAR and CK2, and IGFBP-1 hyperphosphorylation occur prior to development of intrauterine growth restriction (IUGR). Pregnant baboons were fed a Control (C) or a Maternal Nutrient Restriction (MNR; 70% calories of Control) diet starting at gestational day (GD) 30 (Term GD185). Umbilical blood and fetal liver tissue were obtained at GD120 (C, n=7; MNR, n=10) and GD165 (C, n=7; MNR, n=8). Fetal weights were unchanged at GD120 but decreased at GD165 in the MNR group (-13%, p=0.03). IGFBP-1 phosphorylation, as determined by PRM-MS, immunohistochemistry and/or western blot, was enhanced in MNR fetal liver and umbilical plasma at GD120 and GD165. IGF-1 receptor autophosphorylationTyr1135 (-64%, p=0.05) was reduced in MNR fetal liver at GD120. Furthermore, fetal liver CK2 (α/α'/β) expression CK2β colocalization, proximity with IGFBP-1 and CK2 autophosphorylationTyr182 were greater at GD120 and GD165 in the MNR vs. Control. Additionally, mTORC1 (p-P70S6KThr389, -52%, p=0.05) and mTORC2 (p-AktSer473, -56%, p<0.001) activity was decreased, and AAR was activated ((p-GCN2Thr898, +117%, p=0.02), (p-eIF2αSer51, +294%, p=0.002), (p-ERKThr202, +111%, p=0.03)) in MNR liver at GD120. Our data suggest that fetal liver IGFBP-1 hyperphosphorylation, mediated by mTOR inhibition and both AAR and CK2 activation, is a key link between restricted nutrient and oxygen availability and the development of IUGR.

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Hypoxia increases fetal hepatic insulin‐like growth factor binding protein‐1 (IGFBP‐1) phosphorylation mediated by mechanistic target of rapamycin (mTOR) inhibition. Whether maternal nutrient restriction (MNR) causes fetal hypoxia remains unclear. We used fetal liver from a baboon (Papio sp.) model of intrauterine growth restriction due to MNR (70% global diet of Control) and liver hepatocellular carcinoma (HepG2) cells as a model for human fetal hepatocytes and tested the hypothesis that mTOR‐mediated IGFBP‐1 hyperphosphorylation in response to hypoxia requires hypoxia‐inducible factor‐1α (HIF‐1α) and regulated in development and DNA‐damage responses‐1 (REDD‐1) signaling. Western blotting (n = 6) and immunohistochemistry (n = 3) using fetal liver indicated greater expression of HIF‐1α, REDD‐1 as well as erythropoietin and its receptor, and vascular endothelial growth factor at GD120 (GD185 term) in MNR versus Control. Moreover, treatment of HepG2 cells with hypoxia (1% pO2) (n = 3) induced REDD‐1, inhibited mTOR complex‐1 (mTORC1) activity and increased IGFBP‐1 secretion/phosphorylation (Ser101/Ser119/Ser169). HIF‐1α inhibition by echinomycin or small interfering RNA silencing prevented the hypoxia‐mediated inhibition of mTORC1 and induction of IGFBP‐1 secretion/phosphorylation. dimethyloxaloylglycine (DMOG) induced HIF‐1α and also REDD‐1 expression, inhibited mTORC1 and increased IGFBP‐1 secretion/phosphorylation. Induction of HIF‐1α (DMOG) and REDD‐1 by Compound 3 inhibited mTORC1, increased IGFBP‐1 secretion/ phosphorylation and protein kinase PKCα expression. Together, our data demonstrate that HIF‐1α induction, increased REDD‐1 expression and mTORC1 inhibition represent the mechanistic link between hypoxia and increased IGFBP‐1 secretion/phosphorylation. We propose that maternal undernutrition limits fetal oxygen delivery, as demonstrated by increased fetal liver expression of hypoxia‐responsive proteins in baboon MNR. These findings have important implications for our understanding of the pathophysiology of restricted fetal growth.