Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biochemistry

Supervisor

Gupta, Madhulika B.

Abstract

Impairment of fetal oxygen levels and nutrient delivery contributes to fetal growth restriction (FGR), which affects 20% of pregnancies. Such cellular stress induces hepatic Insulin-like Growth Factor Binding Protein 1 (IGFBP1) phosphorylation, which sequesters Insulin-like Growth Factor 1 (IGF-I) and markedly reduces fetal growth signaling. IGFBP1 hyperphosphoryaltion in hypoxia is mediated through the mTOR signaling pathway and through the Amino Acid Response (AAR) pathway during amino acid deprivation. Hypoxia stimulates upstream mTORC1 regulators, AMPK and REDD1 which are well-established upstream regulators of one of the two mTOR complexes, mTORC1. The molecular mechanisms by which upstream mTORC1-driven processes regulate IGFBP1 phosphorylation in hypoxia are unknown. We hypothesized that AMPK impacts IGFBP1 phosphorylation by modulating mTORC1 signaling due to hypoxia – a key factor in the development of reduced fetal growth in utero. Our results indicated that upregulation of AMPK phosphorylation at Thr172 via chemical activators leads to greater IGFBP1 phosphorylation. Additionally, we investigated the effects of combined hypoxia and amino acid deprivation (specifically leucine) on pIGFBP1 levels. We hypothesized that combined hypoxia and leucine deprivation results in greater IGFBP1 phosphorylation than either treatment alone, however we found that the combined conditions lead to IGFBP1 phosphorylation similar to leucine deprivation alone. The investigations in this study of nutrient sensing proteins (AMPK-MTORC1) and multiple cellular stressors (nutrient deprivation and hypoxia) mediating IGFBP1 hyperphosphorylation help provide greater insight of the underlying mechanisms regulating FGR.

Summary for Lay Audience

An estimated 20% of all pregnancies are affected by fetal growth restriction (FGR), a condition characterized by improper nutrient flow to the developing fetus, usually resulting in infants with low birthweight. The onset of FGR during pregnancy can also negatively impact physical and neurological health throughout childhood and even into adulthood. These children are disproportionately affected by learning disorders such as ADHD and are at a higher risk for the development of diabetes, obesity and cardiac diseases in later life. Thus, the effects of FGR have life-long health implications making better diagnosis and subsequent therapeutic intervention during pregnancy imperative.

Current diagnostic methods aim to identify physical manifestations of disease through evaluation of physical markers of proper fetal growth such as fetal height and maternal weight, as well as using radiological techniques to visualize the developing baby. However, these methods often identify FGR symptoms late into pregnancy, when symptoms have fully manifested and are irreversible. Our study aims to help identify the onset of FGR earlier on, through the detection of a network of proteins and their signaling activity at the cellular level. The hepatic protein Insulin-like Growth Factor Binding Protein 1 has been highly associated with FGR and is known to mitigate specific protein pathways crucial to fetal growth, especially when phosphorylated.

In this study we hypothesized that IGFBP1 protein and its’ phosphorylation is regulated through a network of proteins reliant on AMPK protein in low oxygen conditions, we also hypothesized that conditions of nutrient deprivation combined with low oxygen would amplify IGFBP phosphorylation and increase the levels of IGFBP1. We found that AMPK is among a network of several oxygen sensing proteins involved with increasing IGFBP1 protein and phosphorylation levels. We also found that conditions of low oxygen and nutrient restriction did not amplify the levels of IGFBP1 protein and phosphorylation levels greater than that observed when only one of the conditions is present. These findings help to further shed light about the network of proteins and conditions involved in manifesting FGR at cellular level and helps us to better understand the nature of the disease.

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