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). RESEARCH DESIGN AND 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 (serine¹¹⁷⁷ 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.

Gestational diabetes mellitus (GDM) courses with increased fetal plasma adenosine concentration and reduced adenosine transport in placental macrovascular endothelium. Since insulin modulates human equilibrative nucleoside transporters (hENTs) expression/activity, we hypothesize that GDM will alter hENT2-mediated transport in human placental microvascular endothelium (hPMEC), and that insulin will restore GDM to a normal phenotype involving insulin receptors A (IR-A) and B (IR-B). GDM effect on hENTs expression and transport activity, and IR-A/IR-B expression and associated cell signalling cascades (p42/44 mitogen-activated protein kinases (p42/44^mapk) and Akt) role in hPMEC primary cultures was assayed. GDM associates with elevated umbilical whole and vein, but not arteries blood adenosine, and reduced hENTs adenosine transport and expression. IR-A/IR-B mRNA expression and p42/44^mapk/Akt ratios (‘metabolic phenotype’) were lower in GDM. Insulin reversed GDM-reduced hENT2 expression/activity, IR-A/IR-B mRNA expression and p42/44^mapk/Akt ratios to normal pregnancies (‘mitogenic phenotype’). It is suggested that insulin effects required IR-A and IR-B expression leading to differential modulation of signalling pathways restoring GDM-metabolic to a normal-mitogenic like phenotype. Insulin could be acting as protecting factor for placental microvascular endothelial dysfunction in GDM.

The global epidemics of obesity during pregnancy and excessive gestational weight gain (GWG) are major public health problems worldwide. Obesity and excessive GWG are related to several maternal and fetal complications, including diabetes (pregestational and gestational diabetes) and intrauterine programming of insulin resistance (IR). Maternal obesity (MO) and neonatal IR are associated with long-term development of obesity, diabetes mellitus, and increased global cardiovascular risk in the offspring. Multiple mechanisms of insulin signaling pathway impairment have been described in obese individuals, involving complex interactions of chronically elevated inflammatory mediators, adipokines, and the critical role of the endoplasmic reticulum (ER) stress-dependent unfolded protein response (UPR). However, the underlying cellular processes linking MO and IR in the offspring have not been fully elucidated. Here, we summarize the state-of-the-art evidence supporting the possibility that adverse metabolic postnatal outcomes such as IR in the offspring of pregnancies with MO and/or excessive GWG may be related to intrauterine activation of ER stress response.

Reduced expression of human equilibrative nucleoside transporter 1 (hENT1) results from nitric oxide (NO)-dependent reduced SLC29A1 transcriptional activity in human umbilical vein endothelial cells (HUVECs) from gestational diabetes. As expression of the transcription factor C/EBP homologous protein 10 (hCHOP, which forms heterodimers with C/EBPα transcription factor) is activated by NO and induced in diabetes mellitus, we hypothesize that hCHOP plays a role in the gestational diabetes-reduced hENT1 expression in HUVECs. HUVEC primary cultures from 42 normal and 42 gestational diabetic pregnancies were used for adenosine uptake assays. Real-time PCR (mRNA quantification), western blotting (protein abundance), and luciferase activity (SLC29A1 promoter activity) were used. hCHOP–C/EBPα activity was assayed by chromatin immunoprecipitation. Overlap extension mutagenesis was used to generate a mutated hCHOP–C/EBPα consensus site at the SLC29A1 promoter, and endothelial NO synthase (eNOS) siRNA recombinant adenovirus was used to knock down eNOS. hCHOP nuclear protein abundance and binding to DNA were higher in gestational diabetes, paralleled by reduced SLC29A1 promoter activity, hENT1 expression, and transport activity. These changes were blocked by hCHOP consensus sequence mutation (−1845G > T and −1844C > A), eNOS-siRNA-induced knockdown, and N^G-nitro-l-arginine methyl ester (NOS inhibitor), and were mimicked by S-nitroso-N-acetyl-l, d-penicillamine (NO donor) in cells from normal pregnancies. hCHOP and C/EBPα overexpression mimicked gestational diabetes effects in cells from normal pregnancies, but did not alter SLC29A1 promoter activity or hENT1-adenosine transport in cells from gestational diabetes. The hCHOP–C/EBPα complex down-regulates SLC29A1 expression in an NO-dependent manner in HUVECs from gestational diabetes.