Insulin Restores Gestational Diabetes Mellitus–Reduced Adenosine Transport Involving Differential Expression of Insulin Receptor Isoforms in Human Umbilical Vein Endothelium

Abstract

OBJECTIVETo 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 METHODSPrimary 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-NG-nitro-l-arginine methyl ester (l-NAME; NO synthase inhibitor) or ZM-241385 (an A2A-adenosine receptor antagonist).RESULTSUmbilical 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.CONCLUSIONSGDM 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 A2A-adenosine receptors and insulin receptor A, will play crucial roles in this phenomenon in GDM.

Insulin requires A1 adenosine receptors expression to reverse gestational diabetes-increased L-arginine transport in human umbilical vein endothelium

“…Reduced adenosine transport leads to increased extracellular levels of adenosine in HUVECs primary cultures from GDM pregnancies, a finding that agrees with the elevated adenosine plasma level detected in human umbilical vein blood from GDM pregnancies [8,13]. We reported that activation of A 2A adenosine receptors (A 2A AR) is required for the increase caused by insulin on L-arginine transport via the human cationic amino acid transporter 1 (hCAT-1), a Na + -and pH-independent membrane transporter (apparent K m 100 μM) [14,15], and eNOS activity in HUVECs from normal pregnancies [6,12]. Additionally, insulin restores GDM-reduced adenosine transport [15] in this cell type.…”

“…PBS and Krebs solutions were also supplemented with 2 U/mL adenosine deaminase 1. ATP, ADP, AMP, or adenosine was not detected in PBS or Krebs solutions as assayed by high-performance light chromatography (not shown) as described [12]. Cell monolayers were rinsed with ice-cold Krebs to terminate tracer uptake.…”

“…GDM also associates with increased uptake of the cationic amino acid L-arginine [4], the substrate for nitric oxide (NO) synthesis by the endothelial NO synthase (eNOS) [5], in human umbilical vein endothelial cells (HUVECs), changes referred as GDM-associated fetoplacental endothelial dysfunction [3,6]. The latter is reinforced with findings showing that uptake of the endogenous nucleoside adenosine, a potent vasodilator in the human fetoplacental vasculature [7,8] and other vascular beds [9][10][11], is also reduced in HUVECs from GDM pregnancies [8,12]. Reduced adenosine transport leads to increased extracellular levels of adenosine in HUVECs primary cultures from GDM pregnancies, a finding that agrees with the elevated adenosine plasma level detected in human umbilical vein blood from GDM pregnancies [8,13].…”

Gestational Diabetes Mellitus Impairs Fetal Endothelial Cell Functions Through a Mechanism Involving MicroRNA-101 and Histone Methyltransferase Enhancer of Zester Homolog-2

“…For instance, molecular abnormalities, especially in the nitric oxide production machinery and the adenosine transport system, have been described in HUVECs from GDM pregnancies. 38,39 However, to the best of our knowledge, our study has provided a functional characterization of the effect of GDM on HUVECs. In fact, here, we report that cultured GDM-HUVECs demonstrated decreased survival and functional capacity in comparison with HUVECs obtained from healthy nondiabetic mothers who gave birth at the same obstetric unit.…”

Gestational Diabetes Mellitus Is Associated With Changes in the Concentration and Bioactivity of Placenta-Derived Exosomes in Maternal Circulation Across Gestation

“…HUVECs were used to determine the bioactivity of exosomes isolated from maternal plasma. HUVEC primary cultures (37°C, 5% CO 2 ) were isolated by enzymatic digestion using Collagenase Type II (Gibco Life Technologies, Carlsbad, CA) as previously described (11,18). Cells were cultured in primary culture medium containing 2% exosome-depleted FBS (culture media was depleted of the contaminating exosomes using the same protocol for exosome isolation described previously, and exosome-free culture media medium was confirmed by electron microscopy) for 24 h before experiments.…”