Glucose sensing by the placenta - the role of the hexosamine biosynthetic pathway in pregnancies complicated by maternal diabetes

UoM administered thesis: Phd

Abstract

The hexosamine biosynthetic pathway (HBP) utilises glucose to produce uridine diphosphate N-acetylglucosamine for post-translational addition to side chain hydroxyl (O-GlcNAc) on serine (S) or threonine (T), altering protein function. Pregnancies complicated by maternal diabetes are associated with poor maternal and fetal outcomes, such as overgrowth (macrosomia), trauma at birth and an increased likelihood of developing metabolic syndrome in adult life. Maternal hyperglycaemia is thought to exert its effects in part by altering placental function, but the mechanisms are not well understood. This project investigated the hypothesis that hyperglycaemia changes placental hexosamine metabolism, thereby altering the functions controlled by this nutrient-sensitive molecular switch. Such changes may contribute to aberrant placental action and impaired fetal health. The first aim was to compare O-GlcNAc-modified proteins from normal pregnancies and those complicated by maternal diabetes. O-GlcNAc-proteomes were obtained from placentas of mothers with type 1 diabetes (n=6; T1DM), type 2 diabetes (n=6; T2DM), obesity (maternal body mass index (BMI) 30-35kg/m2, n=6; obese control) or no complications (BMI 20-25kg/m2, n=5; normal control). Ingenuity pathway analysis identified canonical pathways affected by alterations to the O-GlcNAc-omes, including clathrin-mediated endocytosis (CME). The functional relevance of this finding was investigated by assessing uptake of the classical CME ligand transferrin (TF) using flow cytometry. Stimulating protein O-GlcNAcylation using glucosamine (2.5mM) increased the rate of TF endocytosis in the trophoblast cell line BeWo (p=0.02). Furthermore, accumulation of iron, transferrin's in vivo cargo, was significantly increased in term placenta from mothers with T2DM (p=0.01). The effect of glucosamine-induced O-GlcNAcylation on signalling pathways activated by three growth factors (GF) was investigated. Glucosamine treatment diminished GF-stimulated receptor tyrosine kinase (RTK) autophosphorylation, reduced phosphorylation of the cytoplasmic tyrosine phosphatase SHP2 at Y580 and reduced the cells' rate of transition into S phase. The O-GlcNAcylation status of downstream signalling proteins AKT and SHP2 was confirmed in trophoblast and two serine residues in the Src homology (SH2-1 & SH2-2) regions of SHP2 that are important for binding of phosphorylated RTKs were identified as O-GlcNAc targets. This study, the first analysis of the placental GlcNAc-ome, adds to our understanding of the molecular mechanisms governing placental function. Components of the CME pathway are differentially GlcNAcylated in T2DM, leading to altered membrane dynamics in endocytosis and increased accumulation of iron. O-GlcNAcylation of proteins involved in GF-signalling changes signal transduction across the plasma membrane, altering control of the cell cycle. O-GlcNAcylation may be attractive as a target pathway for therapies to aid pregnancies complicated by maternal diabetes.

Details

Original languageEnglish
Awarding Institution
Supervisors/Advisors
Award date31 Dec 2017