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

Abstract

Gestational diabetes mellitus (GDM) associates with increased L-arginine transport and extracellular concentration of adenosine in human umbilical vein endothelial cells (HUVECs). In this study we aim to determine whether insulin reverses GDM-increased L-arginine transport requiring adenosine receptors expression in HUVECs. Primary cultured HUVECs from full-term normal (n = 38) and diet-treated GDM (n = 38) pregnancies were used. Insulin effect was assayed on human cationic amino acid transporter 1 (hCAT1) expression (protein, mRNA, SLC7A1 promoter activity) and activity (initial rates of L-arginine transport) in the absence or presence of adenosine receptors agonists or antagonists. A 1 adenosine receptors (A 1 AR) and A 2A AR expression (Western blot, quantitative PCR) was determined. Experiments were done in cells expressing or siRNA-suppressed expression of A 1 AR or A 2A AR. HUVECs from GDM exhibit higher maximal transport capacity (maximal velocity (V max )/ apparent Michaelis Menten constant (K m ), V max /K m ), which is blocked by insulin by reducing the V max to values in cells from normal pregnancies. Insulin also reversed the GDMassociated increase in hCAT-1 protein abundance and mRNA expression, and SLC7A1 promoter activity for the fragment −606 bp from the transcription start point. Insulin effects required A 1 AR, but not A 2A AR expression and activity in this cell type. In the absence of insulin, GDM-increased hCAT-1 expression and activity required A 2A AR expression and activity. HUVECs from GDM pregnancies exhibit a differential requirement of A 1 AR or A 2A AR depending on the level of insulin, a phenomenon that represent a condition where adenosine or analogues of this nucleoside could be acting as helpers of insulin biological effects in GDM.

Intracellular acidification increases adenosine transport in human umbilical vein endothelial cells

“…Human umbilical vein endothelial cells (HUVECs) were isolated by digestion with 95 collagenase from umbilical cord veins and cultured (5% O 2 , 5% CO 2 , 37°C) in primary culture medium (PCM: medium 199 (M199, Gibco Life Technologies, Carlsbad, CA, USA), 5 mmol/L D-glucose, 10% new born calf serum, 10% foetal calf serum (Gibco), 3.2 mmol/L Lglutamine, 100 U/mL penicillin-streptomycin (Gibco)) as described [10,15]. Experiments were in primary cultured cells in passage 3 in the absence or presence of 1 or 10 µmol/L S-(4M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 5 nitrobenzyl)-6-thio-inosine (NBTI) (Sigma, Atlanta, GA, USA), inhibitory concentrations for hENT1 or hENT1+hENT2 transport, respectively, 2 mmol/L hypoxanthine, and 2 mmol/L adenine (nucleobases taken up via ENT2) [11].…”

“…Some of these transport systems depend on the pHo, such as those mediating adenosine and 5-hydroxytryptamine (5HT) [6]. The endogenous nucleoside adenosine is reported to increase 60 the L-arginine transport and synthesis of nitric oxide (NO) in the human foetoplacental micro and macrovascular endothelium from normal or pathological pregnancies such as gestational diabetes mellitus (GDM) [7][8][9][10]. This phenomenon results from activation of adenosine receptors due to the extracellular accumulation of this nucleoside arising from a reduced uptake by the foetoplacental endothelium [8,10,11].…”

“…The endogenous nucleoside adenosine is reported to increase 60 the L-arginine transport and synthesis of nitric oxide (NO) in the human foetoplacental micro and macrovascular endothelium from normal or pathological pregnancies such as gestational diabetes mellitus (GDM) [7][8][9][10]. This phenomenon results from activation of adenosine receptors due to the extracellular accumulation of this nucleoside arising from a reduced uptake by the foetoplacental endothelium [8,10,11]. Thus, a proper function of membrane 65 transport mechanisms modulating the extracellular concentration of adenosine is critical to mantaining the physiological cell metabolism in this vascular bed [4,[8][9][10] as in other tissues [5,6].…”

“…This phenomenon results from activation of adenosine receptors due to the extracellular accumulation of this nucleoside arising from a reduced uptake by the foetoplacental endothelium [8,10,11]. Thus, a proper function of membrane 65 transport mechanisms modulating the extracellular concentration of adenosine is critical to mantaining the physiological cell metabolism in this vascular bed [4,[8][9][10] as in other tissues [5,6].…”

Pharmacological targeting of adenosine receptor signaling

“…Additionally, A 2A AR receptors seem essential in developing or maintaining endothelial dysfunction in the fetoplacental vasculature in diseases of pregnancy such as gestational diabetes mellitus or preeclampsia Sobrevia et al, 2016). However, the A 1 AR is required for the effect of insulin correcting the gestational diabetes mellitus-enhanced L-arginine transport in this vascular bed (Guzman-Gutierrez et al, 2016).…”