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

摘要

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. A₁ adenosine receptors (A₁AR) and A_2AAR expression (Western blot, quantitative PCR) was determined. Experiments were done in cells expressing or siRNA-suppressed expression of A₁AR or A_2AAR. 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 GDM-associated 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 A₁AR, but not A_2AAR expression and activity in this cell type. In the absence of insulin, GDM-increased hCAT-1 expression and activity required A_2AAR expression and activity. HUVECs from GDM pregnancies exhibit a differential requirement of A₁AR or A_2AAR 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.