Understanding placental function in pregnancies complicated by Diabetes mellitus: a systems biology approach

Pregnancies complicated with diabetes mellitus (DM) are associated with poor maternal and fetal outcomes, such as birth trauma, fetal overgrowth (macrosomia) and programming of the fetus to develop metabolic syndrome in adult life. Maternal hyperglycemia is thought to contribute to fetal macrosomia, however the role of the placenta in these pregnancies is incompletely understood, therefore we aimed to investigate the specific consequences of high glucose on placental metabolism. To achieve this aim an in vitro model of placental exposure to high glucose was developed. This model was used with the aim of analysing how high glucose alters the transcriptome and metabolome of these cells, using a systems biology approach to identify candidate functional pathways which may be altered in placenta as a result of hyperglycemia. These candidate functional pathways were validated in an ex vivo model of placenta exposed to high glucose and in placental tissue from pregnancies complicated by DM. A trophoblast cell line (BeWo) was cultured in low (5 mM) and high (12 mM or 25 mM) D-glucose conditions for 48 hours. Transcriptomic and metabolomic analysis of these cells was performed using microarrays, and gas- and liquid-chromatography-mass spectrometry, respectively. Transcript and metabolite changes were independently analysed and integrated, using network analysis. From the integrated analysis of the ‘omic datasets, β-fatty acid oxidation (β-FAO), purine metabolism, phosphatidylinositol/PI3K phosphate pathway and lipid metabolism, were identified as candidates for further study. Changes within the PI3K pathway and lipid metabolism/β-fatty acid oxidation were validated in an ex vivo placental explant model of high glucose and in placental tissue from women with DM, compared to uncomplicated pregnancies. mRNA, protein expression and protein activation of key molecules within the PI3K pathway were not significantly altered in placenta as a response of high glucose ex vivo or DM in vivo. The second candidate functional pathway, lipid metabolism, has previously been implicated in association with placental dysfunction in pregnancies complicated by DM. Placental fatty acid transporter and lipase protein expression, as well as, relative abundance of different fatty acids were unaltered in response to high glucose or DM. High glucose levels increased triglyceride levels within the placenta, indicating reduced rates of β-FAO. The effect of high glucose could be ameliorated using a PPARα agonist. This may provide a novel therapeutic intervention to prevent excess esterification of fatty acids to triglycerides in maternal diabetes, which may in turn influence fetal growth.This study illustrates how a systems biology approach can be used to identify novel candidate functional pathways that are altered within the trophoblast in response to high glucose. Thus, improving understanding of placental dysfunction in these pregnancies and providing novel candidate pathways for future study, which may represent potential therapeutic targets for intervention of fetal macrosomia in pregnancies complicated by DM. CD of Excel Worksheets (Appendix b)Containing:1.Gene changes from the microarray1a. All microarray data with no cut-offs1b. Gene changes for 5 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following overnight culture (Time 0); fold change cut-off ±1.3.1c. Gene changes for 12 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following overnight culture (Time 0); fold change cut-off ±1.3.1d. Gene changes for 25 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following overnight culture (Time 0); fold change cut-off ±1.3.1e. Gene changes for 12 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following 24 hours of culture; fold change cut-off ±1.3.1f. Gene changes for 25 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following 24 hours of culture; fold change cut-off ±1.3.1g. Summary of gene changes for 12 mM and 25 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following 24 hours of culture; fold change cut-off ±1.3.2. Transcriptome analysis datasets2a. Transcriptome analysis of gene changes for 12 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following 24 hours of culture. 2a.1. Inferred network analysis 2a.2 Non-inferred network analysis2b. Transcriptome analysis of gene changes for 12 mM D-glucose following 24 hours of culture compared to 5 mM D-glucose following 24 hours of culture 2b.1. Inferred network analysis 2b.2 Non-inferred network analysis2c. Transcriptome analysis of gene changes unique to different levels of high glucose 2c.1. Analysis of genes unique to 12 mM compared to 5 mM D-glucose2c.2. Analysis of genes unique to 25 mM compared to 5 mM D-glucose3.Metabolome analysis datasets3a. Footprint metabolites identified using GC-MS3b. Fingerprint metabolites identified using GC-MS3c. Footprint metabolites identified using LC-MS3d. Fingerprint metabolites identified using LC-MS4.Summary of network analysis from the transcriptome and metabolome datasets4a. Master spreadsheet of all data PDF copy of publication arising from this thesis.Hulme C. H., Westwood M., Myers J.E., Heazell A.E. (2012). A high-throughput colorimetric-assay for monitoring glucose consumption by cultured trophoblast cells and placental tissue. Placenta, 33 (11), 949-51.