Background: Small extracellular vesicles (sEVs) play key roles in regulating maternal metabolism by transfer of molecular signals (proteins and miRNAs) mediating cell communication. The aim of the present study is to determine the association between longitudinal changes in insulin sensitivity during pregnancy with changes in the protein and miRNA profile of circulating sEVs in healthy and gestational diabetes mellitus (GDM) pregnancies.
Method: Samples were obtained from a multicentre randomized controlled trial conducted from 2012 to 2014, the DALI (vitamin D and lifestyle intervention for GDM prevention) lifestyle study. sEVs were isolated from plasma samples from early (<20 weeks), mid (24-28 weeks) and late (35-37 weeks) gestation by differential centrifugation followed by size exclusion chromatography (NGT, n=67; GDM, n=63). sEVs were characterised by nanoparticle tracking analysis, western blots and electron microscopy. sEV-associated miRNAs and proteins were isolated and characterized by liquid chromatography mass spectrometry (LC-MS/MS) and next generation sequencing respectively. Insulin sensitivity was accessed using by Homeostatic model assessment (HOMA-IR) index. Using a longitudinal study design sEV-associated proteins and miRNAs were analysed in conjunction with HOMA-IR across gestation using linear mixed modelling and hierarchical clustering analysis.
Results: The sEV concentration increased across gestation in healthy and GDM pregnancies and sEV concentration correlated to HOMA-IR (p=0.0007) in GDM subjects. A total of 1155 proteins were identified with 50 proteins differentially expressed between healthy and GDM conditions. A total of 390 proteins were significantly correlated with HOMA-IR. This include proteins FABP4, SAMP and PAPP-A which are associated with fatty acid metabolism, inflammation and placental dysfunction. A specific set of miRNAs (miR-30c-5p, miR-574-5p, miR-378i, miR-503-5p, miR-548l and miR-218-5p) were differentially expressed in healthy and GDM pregnancies. When correlated to HOMA-IR, 24 miRNA were found to be significantly associated (22 positively correlated and 2 negatively correlated). Finally, bioinformatic analysis revealed the downstream pathways and targets of these miRNA and proteins associated with glucose metabolism and insulin signalling suggesting the role of sEV associated proteins and miRNAs in target cells.
Conclusion: The findings from this study elucidate the role of sEVs as mediators of insulin resistance in GDM and provide insights into the potential of sEVS as targeted therapeutics and early detection biomarkers in GDM.
Funding: National Health and Medical Research Council (NHMRC, 1114013), and EU FP7 (242187).