Extracellular vesicle-associated miR-515-5p from adipose tissue regulates placental metabolism and fetal growth in gestational diabetes mellitus

Abstract

Background: Gestational diabetes mellitus (GDM) affects 2-20% of pregnant women worldwide and is linked to fetal overgrowth, increased perinatal morbidity, and mortality, as well as a higher risk of developing cardiovascular disease later in life for mother and child. MicroRNAs (miRNAs), which regulate gene expression, can be transported within extracellular vesicles (EVs). Adipose tissue-derived EVs have been associated with changes in placental metabolism in GDM, potentially influencing cardiovascular health outcomes. This study aimed to evaluate the miRNA profile in EVs from omental adipose tissue in GDM and their effect on placental nutrient uptake and fetal growth.

Methods: This case-control study included patients with normal glucose tolerance (NGT) and GDM. We conducted a miRNA expression profiling on omental adipose tissue and its derived EVs from women with NGT (n = 20) and GDM (n = 36). Trophoblast cells were utilized to assess the effect of EVs on glucose and fatty acid uptake, pro-inflammatory cytokine, and chemokine release. Double-stranded miRNA mimics were used to investigate the effect of selected miRNAs on trophoblast cells. Subsequently, the impact of EVs from NGT and GDM, as well as miR-515-5p, on in vivo glucose tolerance and fetal growth was assessed in pregnant mice.

Results: Fifty-four miRNAs showed significant differences between EVs from the adipose tissue of NGT and GDM groups. EVs from GDM increased glucose uptake in trophoblast cells, whereas EVs from NGT increased the secretion of CXCL8, IL-6, CXCL1, CXCL4, and CXCL5 from trophoblasts compared to the effect without EVs. Specifically, miR-515-5p increased glucose uptake and abolished TNF-α-dependent increase in pro-inflammatory cytokines and chemokines from trophoblast cells. Injection of pregnant mice with EVs from NGT adipose tissue loaded with miR-515-5p resulted in increased fetal weight and glucose levels.

Conclusion: miR-515-5p, specifically encapsulated within EVs from omental adipose tissue in GDM, regulates placental nutrient uptake, glucose homeostasis, and fetal growth.

Keywords: Adipose tissue; Extracellular vesicles; Fetal growth; Gestational diabetes; Glucose uptake; Placenta.

Fig. 1

Characterization of miRNA enrichment adipose tissue and EVs in normal and gestational diabetes pregnancy. Volcano plot of miRNA expression in (A) NGT adipose compared to GDM adipose, (B) NGT EVs compared to GDM EVs, (C) NGT adipose compared to NGT EVs, and (D) GDM adipose compared to GDM EVs. (E) Comparative analysis of miR-146a-5p, miR-515-5p and miR-516-5p in (a-c) adipose tissue, (d-f) EVs and (g-i) placenta from NGT and GDM pregnancy (n = 8–12/ sample/condition). The fold change was calculated relative to NGT tissues. Non-parametric Mann–Whitney U test was performed. Data presented as mean ± SEM. *p< 0.05

Fig. 2

Effects of adipose tissue-derived EVs on placental cells. Effect of adipose tissue-derived EVs on placental (A) glucose uptake and (B) fatty acid uptake by 2-NBDG and BODIPY staining (n = 5–12 patients/condition). Glucose and fatty acid accumulation measured as relative fluorescence unit (rfu). The release of (C) CXCL8, (D) IL-6, (E) CXCL1, (F) CCL2, (G) CCL4, and (H) CCL5 measured using ELISA (n = 13 patients/condition). All assays were performed after 24 h of treatment with adipose tissue-derived EV. Repeated measure one-way ANOVA was performed with Bonferroni test. Data presented as mean ± SEM. *p < 0.05 **p < 0.005, ***p < 0.0005, ****p < 0.001

Fig. 3

Effects of adipose tissue-derived EVs associated miRNA on placental cells. (A) Schematic diagram of miRNA transfection in primary trophoblast experimental design. Effect of adipose tissue-derived EVs associated miRNA, (B) glucose uptake, and (C) fatty acid uptake by 2-NBDG and BODIPY staining (n = 6 patients). Glucose and fatty acid accumulation measured as relative fluorescence unit (rfu). The TNF- induced release of (D) CXCL8, (E) IL-6, (F) CXCL1, (G) CCL2, and (H) CCL5 measured using ELISA (n = 6 patients). All assays were performed after 48 h of transfection with miRControl and miRNA mimics. Repeated measure one-way ANOVA was performed with Bonferroni test. Data presented as mean ± SEM. *p < 0.05 **p < 0.005

Fig. 4

Enrichment of placental pathways associated with metabolism and inflammation. (A) Differentially enriched pathways in placental cells transfected with miRNAs. Panel A: miRCont, Panel B: miR-146a-5p, Panel C: miR-515-5p and Panel D: miR-516-5p. Volcano plot gene expression in (B) NGT, (C) GDM placental tissues, (D) miR-146a-5p, (E) miR-515-5p, and (F) miR-516-5p transfected placental cells (n = 5–6 patients)

Fig. 5

Adipose derived EVs and miRNA regulates glucose metabolism and fetal growth. (Right) Overview of experimental design. Pregnant mice (n = 4 per group) received daily doses of 8 µg/kg of EVs from women with normal pregnancy or GDM, EVs-transfected with miR-515-5p or PBS as Control. (A) Maternal blood glucose and (B) the area under the blood glucose response curve were measured via tail bleeding on GD17.5 after 4 days of EVs or PBS injection. (C) Mice weight, (D) litter size (number of fetuses per pregnant mouse), (E) Fetal weight and (F) placental weight was measured on post-operative after 5 days of EVs injection. (G) Fetal glucose was measured using fetal blood on PO post during fetal and tissue collection. P values in the figures represent the results of a one-way ANOVA, followed by a Bonferroni post hoc test. Data displayed as mean ± SEM. *p < 0.05 **p < 0.005, ***p < 0.0005,****p < 0.001. Fetal weight, placental weight and fetal glucose are presented as the average fetal weight per dam, adjusted for litter size, and compared these averages across treatment groups