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

Palin, Victoria Jayne

[Thesis]. Manchester, UK: The University of Manchester; 2017.

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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.

Additional content not available electronically

CD-ROM containing supplementary csv file of proteomic data, submitted in the pocket of the inside back cover of the print version of thesis

Bibliographic metadata

Type of resource:
Content type:
Administered thesis
Form of thesis:
Traditional
Type of submission:
Doctoral level ETD - final
Thesis title:
Glucose sensing by the placenta - the role of the hexosamine biosynthetic pathway in pregnancies complicated by maternal diabetes
Degree type:
Doctor of Philosophy
Degree programme:
PhD Medicine 4yr (DBM)
Publication date:
2017-08-15T13:16:25
Institution:
The University of Manchester
Location:
Manchester, UK
Total pages:
241
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.
Additional digital content not deposited electronically:
CD-ROM containing supplementary csv file of proteomic data, submitted in the pocket of the inside back cover of the print version of thesis
Keyword(s):
Thesis main supervisor(s):
Thesis co-supervisor(s):
Degree grantor:
Language:
en

Institutional metadata

University researcher(s):
Academic department(s):

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:310337
Created by:
Palin, Victoria
Created:
15th August, 2017, 12:16:25
Last modified by:
Palin, Victoria
Last modified:
4th January, 2021, 11:36:21

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