title: Insulin restores gestational diabetes mellitus-reduced adenosine transport involving differential expression of insulin receptor isoforms in human umbilical vein endothelium.
abstract: OBJECTIVE
  To determine whether insulin reverses gestational diabetes mellitus (GDM)-reduced expression and activity of human equilibrative nucleoside transporters 1 (hENT1) in human umbilical vein endothelium cells (HUVECs).
  
  
  METHODS
  Primary cultured HUVECs from full-term normal (n = 44) and diet-treated GDM (n = 44) pregnancies were used. Insulin effect was assayed on hENT1 expression (protein, mRNA, SLC29A1 promoter activity) and activity (initial rates of adenosine transport) as well as endothelial nitric oxide (NO) synthase activity (serine(1177) phosphorylation, l-citrulline formation). Adenosine concentration in culture medium and umbilical vein blood (high-performance liquid chromatography) as well as insulin receptor A and B expression (quantitative PCR) were determined. Reactivity of umbilical vein rings to adenosine and insulin was assayed by wire myography. Experiments were in the absence or presence of l-N(G)-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) or ZM-241385 (an A(2A)-adenosine receptor antagonist).
  
  
  RESULTS
  Umbilical vein blood adenosine concentration was higher, and the adenosine- and insulin-induced NO/endothelium-dependent umbilical vein relaxation was lower in GDM. Cells from GDM exhibited increased insulin receptor A isoform expression in addition to the reported NO-dependent inhibition of hENT1-adenosine transport and SLC29A1 reporter repression, and increased extracellular concentration of adenosine and NO synthase activity. Insulin reversed all these parameters to values in normal pregnancies, an effect blocked by ZM-241385 and l-NAME.
  
  
  CONCLUSIONS
  GDM and normal pregnancy HUVEC phenotypes are differentially responsive to insulin, a phenomenon where insulin acts as protecting factor for endothelial dysfunction characteristic of this syndrome. Abnormal adenosine plasma levels, and potentially A(2A)-adenosine receptors and insulin receptor A, will play crucial roles in this phenomenon in GDM.
authors.affiliation: Cellular and Molecular Physiology Laboratory and Perinatology Research Laboratory, Medical Research Centre, Department of Obstetricsand Gynaecology, Faculty of Medicine, School of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile.
authors.forename: Francisco
authors.initials: F
authors.lastname: Westermeier
authors.forename: Carlos
authors.initials: C
authors.lastname: Salomón
authors.forename: Marcelo
authors.initials: M
authors.lastname: González
authors.forename: Carlos
authors.initials: C
authors.lastname: Puebla
authors.forename: Enrique
authors.initials: E
authors.lastname: Guzmán-Gutiérrez
authors.forename: Fredi
authors.initials: F
authors.lastname: Cifuentes
authors.forename: Andrea
authors.initials: A
authors.lastname: Leiva
authors.forename: Paola
authors.initials: P
authors.lastname: Casanello
authors.forename: Luis
authors.initials: L
authors.lastname: Sobrevia
mesh_terms: D000241:Adenosine / Q000378:metabolism* / Q000494:pharmacology; D000328:Adult; D001692:Biological Transport / Q000187:drug effects*; D015153:Blotting, Western; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D016640:Diabetes, Gestational / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D033721:Equilibrative Nucleoside Transporter 1 / Q000235:genetics; D005260:Female; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D011247:Pregnancy; D011401:Promoter Regions, Genetic / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014471:Umbilical Veins / Q000378:metabolism*
chemical_list: D033721:Equilibrative Nucleoside Transporter 1; D007328:Insulin; D020033:Protein Isoforms; C438649:SLC29A1 protein, human; D052250:Nitric Oxide Synthase Type III; D011972:Receptor, Insulin; D000241:Adenosine