Insulin requires normal expression and signaling of insulin receptor A to reverse gestational diabetes‐reduced adenosine transport in human umbilical vein endothelium

Abstract

Reduced adenosine uptake via human equilibrative nucleoside transporter 1 (hENT1) in human umbilical vein endothelial cells (HUVECs) from gestational diabetes mellitus (GDM) is reversed by insulin by restoring hENT1 expression. Insulin receptors A (IR-A) and B (IR-B) are expressed in HUVECs, and GDM results in higher IR-A mRNA expression vs. cells from normal pregnancies. We studied whether the reversal of GDM effects on transport by insulin depends on restoration of IR-A expression. We specifically measured hENT1 expression [mRNA, protein abundance, SLC29A1 (for hENT1) promoter activity] and activity (adenosine transport kinetics) and the role of IR-A/IR-B expression and signaling [total and phosphorylated 42 and 44 kDa mitogen-activated protein kinases (p44/42(mapk)) and Akt] in IR-A, IR-B, and IR-A/B knockdown HUVECs from normal (n = 33) or GDM (n = 33) pregnancies. GDM increases IR-A/IR-B mRNA expression (1.8-fold) and p44/42(mapk):Akt activity (2.7-fold) ratios. Insulin reversed GDM-reduced hENT1 expression and maximal transport capacity (V(max)/K(m)), and GDM-increased IR-A/IR-B mRNA expression and p44/42(mapk):Akt activity ratios to values in normal pregnancies. Insulin's effect was abolished in IR-A or IR-A/B knockdown cells. Thus, insulin requires normal IR-A expression and p44/42(mapk)/Akt signaling to restore GDM-reduced hENT1 expression and activity in HUVECs. This could be a protective mechanism for the placental macrovascular endothelial dysfunction seen in GDM.

Intracellular acidification increases adenosine transport in human umbilical vein endothelial cells

“…HUVECs [11] and hPMECs [12], we hypothesize that adenosine transport mediated via hENT1 and hENT2 is under regulation by an acidic pHi in HUVECs.…”

“…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].…”

“…Adenosine nucleoside is taken up via the human equilibrative nucleoside transporters (hENTs) in the human umbilical vein (HUVECs) [11] and placental microvascular (hPMECs) 70 [12] endothelial cells. hENTs corresponds to a family of at least four proteins, i.e., hENT1, hENT2, hENT3, and hENT4.…”

“…hENTs corresponds to a family of at least four proteins, i.e., hENT1, hENT2, hENT3, and hENT4. hENT1 and hENT2 mediate adenosine uptake in HUVECs from normal or pathological pregnancies, including GDM [11]. Despite the proposed activation of hENT4-mediated adenosine and 5HT transport in response to an acidic pHo in HUVECs [6], the role of a change in pHi or pHo regulating hENT1 and hENT2 transport activity in this or 75 other cell type is unknown [3,5].…”

Pharmacological targeting of adenosine receptor signaling

“…However, adenosine's concentration varies a lot over time and current methodological approaches destroy some cells locally, potentially leading to higher false positive measurements (Chen et al, 2013). Interestingly, the adenosine plasma concentration in the human umbilical vein at birth is higher in gestational diabetes mellitus compared with normal pregnancies (Westermeier et al, 2015), and maternal plasma concentration of this nucleoside is elevated at early stages of pregnancy in women that later develop preeclampsia (Escudero and Sobrevia, 2008;Espinoza et al, …”