RESUMO
Metformin is used by women during pregnancy to manage diabetes and crosses the placenta, yet its effects on the fetus are unclear. We show that the liver is a site of metformin action in fetal sheep and macaques, given relatively abundant OCT1 transporter expression and hepatic uptake following metformin infusion into fetal sheep. To determine the effects of metformin action, we performed studies in primary hepatocytes from fetal sheep, fetal macaques, and juvenile macaques. Metformin increases AMP-activated protein kinase (AMPK) signaling, decreases mammalian target of rapamycin (mTOR) signaling, and decreases glucose production in fetal and juvenile hepatocytes. Metformin also decreases oxygen consumption in fetal hepatocytes. Unique to fetal hepatocytes, metformin activates stress pathways (e.g., increased PGC1A gene expression, NRF-2 protein abundance, and phosphorylation of eIF2α and CREB proteins) alongside perturbations in hepatokine expression (e.g., increased growth/differentiation factor 15 [GDF15] and fibroblast growth factor 21 [FGF21] expression and decreased insulin-like growth factor 2 [IGF2] expression). Similarly, in liver tissue from sheep fetuses infused with metformin in vivo, AMPK phosphorylation, NRF-2 protein, and PGC1A expression are increased. These results demonstrate disruption of signaling and metabolism, induction of stress, and alterations in hepatokine expression in association with metformin exposure in fetal hepatocytes. ARTICLE HIGHLIGHTS: The major metformin uptake transporter OCT1 is expressed in the fetal liver, and fetal hepatic uptake of metformin is observed in vivo. Metformin activates AMPK, reduces glucose production, and decreases oxygen consumption in fetal hepatocytes, demonstrating similar effects as in juvenile hepatocytes. Unique to fetal hepatocytes, metformin activates metabolic stress pathways and alters the expression of secreted growth factors and hepatokines. Disruption of signaling and metabolism with increased stress pathways and reduced anabolic pathways by metformin in the fetal liver may underlie reduced growth in fetuses exposed to metformin.
Assuntos
Metformina , Gravidez , Feminino , Animais , Ovinos , Metformina/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Hepatócitos/metabolismo , Glucose/metabolismo , Feto/metabolismo , Mamíferos/metabolismoRESUMO
von Willebrand factor (VWF) plays a key role in normal hemostasis, and deficiencies of VWF lead to clinically significant bleeding. We sought to identify novel modifiers of VWF levels in endothelial colony-forming cells (ECFCs) using single-cell RNA sequencing (scRNA-seq). ECFCs were isolated from patients with low VWF levels (plasma VWF antigen levels between 30 and 50 IU/dL) and from healthy controls. Human umbilical vein endothelial cells were used as an additional control cell line. Cells were characterized for their Weibel Palade body (WPB) content and VWF release. scRNA-seq of all cell lines was performed to evaluate for gene expression heterogeneity and for candidate modifiers of VWF regulation. Candidate modifiers identified by scRNA-seq were further characterized with small-interfering RNA (siRNA) experiments to evaluate for effects on VWF. We observed that ECFCs derived from patients with low VWF demonstrated alterations in baseline WPB metrics and exhibit impaired VWF release. scRNA-seq analyses of these endothelial cells revealed overall decreased VWF transcription, mosaicism of VWF expression, and genes that are differentially expressed in low VWF ECFCs and control endothelial cells (control ECs). An siRNA screen of potential VWF modifiers provided further evidence of regulatory candidates, and 1 such candidate, FLI1, alters the transcriptional activity of VWF. In conclusion, ECFCs from individuals with low VWF demonstrate alterations in their baseline VWF packaging and release compared with control ECs. scRNA-seq revealed alterations in VWF transcription, and siRNA screening identified multiple candidate regulators of VWF.