Insulin-increased L-arginine transport requires A(2A) adenosine receptors activation in human umbilical vein endothelium

Abstract

Adenosine causes vasodilation of human placenta vasculature by increasing the transport of arginine via cationic amino acid transporters 1 (hCAT-1). This process involves the activation of A(2A) adenosine receptors (A(2A)AR) in human umbilical vein endothelial cells (HUVECs). Insulin increases hCAT-1 activity and expression in HUVECs, and A(2A)AR stimulation increases insulin sensitivity in subjects with insulin resistance. However, whether A(2A)AR plays a role in insulin-mediated increase in L-arginine transport in HUVECs is unknown. To determine this, we first assayed the kinetics of saturable L-arginine transport (1 minute, 37°C) in the absence or presence of nitrobenzylthioinosine (NBTI, 10 µmol/L, adenosine transport inhibitor) and/or adenosine receptors agonist/antagonists. We also determined hCAT-1 protein and mRNA expression levels (Western blots and quantitative PCR), and SLC7A1 (for hCAT-1) reporter promoter activity. Insulin and NBTI increased the extracellular adenosine concentration, the maximal velocity for L-arginine transport without altering the apparent K(m) for L-arginine transport, hCAT-1 protein and mRNA expression levels, and SLC7A1 transcriptional activity. An A2AAR antagonist ZM-241385 blocked these effects. ZM241385 inhibited SLC7A1 reporter transcriptional activity to the same extent in cells transfected with pGL3-hCAT-1(-1606) or pGL3-hCAT-1(-650) constructs in the presence of NBTI + insulin. However, SLC7A1 reporter activity was increased by NBTI only in cells transfected with pGL3-hCAT-1(-1606), and the ZM-241385 sensitive fraction of the NBTI response was similar in the absence or in the presence of insulin. Thus, insulin modulation of hCAT-1 expression and activity requires functional A(2A)AR in HUVECs, a mechanism that may be applicable to diseases associated with fetal insulin resistance, such as gestational diabetes.

Figure 1. L-Arginine transport and extracellular adenosine concentration.

(a) Overall (300 µmol/L) L-arginine transport in HUVECs cultured (8 hours) in absence (white bars) or presence (black bars) of 1 nmol/L insulin, without (–NBTI) or with (+NBTI) 10 µmol/L nitrobenzylthioinosine (NBTI). Cells were co-incubated in culture medium without (–) or with (+) CGS-21680 (30 nmol/L) or ZM-21385 (10 nmol/L). (b) L-Arginine transport in the absence or presence of NECA (10 nmol/L) or DPCPX (10 nmol/L) in cells as in (a). (c) Adenosine concentration in the culture medium in cells as in (a). *P<0.05 versus cells in the absence of NBTI, CGS-21680 and ZM-241385 (control). Values are mean ± SEM (n = 6–18).

Figure 2. L-Arginine transport kinetics.

L-Arginine transport kinetics (3 μCi/mL L-[³H] arginine, 1 minute, 37°C) in HUVECs cultured (8 hours) without (–NBTI; a, c) or with (+NBTI; b, d) 10 μmol/L nitrobenzylthioinosine (NBTI). (a, b) Saturable L-arginine transport in absence (control) or presence of 1 nmol/L insulin and/or ZM-241385 (10 nmol/L) or CGS-21680 (30 nmol/L) as indicated. (c, d) Eadie-Hofstee plot of the L-arginine transport data in (a) and (b), respectively. Values are mean ± SEM (n = 6–18).

Figure 3. hCAT-1 protein abundance in response to insulin and adenosine receptor agonists and antagonists.

(a) Western blots (representative of other 16 experiments) for hCAT-1 in HUVECs incubated (8 hours) without (–NBTI) or with (+NBTI) 10 μmol/L nitrobenzylthioinosine (NBTI), in the absence (–) or presence (+) of 1 nmol/L insulin and/or ZM-241385 (10 nmol/L) (ß-actin is internal control). Lower panel: hCAT-1/ß-actin ratio densitometries from data in absence (white bars) or presence (black bars) of NBTI, normalized to 1 in cells in the absence of NBTI, insulin and ZM-241385 (control). (b) Western blots for hCAT-1 in the absence or presence of insulin, CGS-21680 and/or ZM-241385 as in (a). Lower panel: hCAT-1/ß-actin ratio densitometries from data in absence (white bars) or presence (black bars) of NBTI normalized to 1 in cells in absence of NBTI, insulin, CGS-21680 and ZM-241385 (control). (c) hCAT-1 mRNA expression relative to 28S rRNA (internal reference) as in (a) and (b). *P<0.05 versus values in control. Values are mean ± SEM (n = 16).

Figure 4. hCAT-2A/B protein abundance in response to insulin and adenosine receptor agonists and antagonists.

(a) Western blots (representative of other 6 experiments) for hCAT-2A/B in HUVECs incubated (8 hours) without (–NBTI) or with (+NBTI) 10 μmol/L nitrobenzylthioinosine (NBTI), in the absence (–) or presence (+) of 1 nmol/L insulin, CGS-21680 (30 nmol/L) and/or ZM-241385 (10 nmol/L) (ß-actin is internal control). Lower panel: hCAT-2A/B/ß-actin ratio densitometries from data in absence (white bars) or presence (black bars) of NBTI normalized to 1 in cells in the absence of NBTI, insulin, CGS-21680 or ZM-241385 (control). Values are mean ± SEM (n = 6).

Figure 5. SLCA71 promoter activity in response to insulin.

(a) Luciferase (Luc) reporter constructs containing serial truncations of SLC7A1 promoter (−1606 bp (pGL3-hCAT-1⁻¹⁶⁰⁶) or −650 bp (pGL3-hCAT-1⁻¹⁶⁰⁶) from the transcriptional start point) were transfected in primary cultures of HUVECs incubated (8 hours) without (plain bars) or with (dashed bars) 10 μmol/L nitrobenzylthioinosine (NBTI). Cell transfection was done in the absence or presence of 1 nmol/L insulin and/or ZM-241385 (10 nmol/L), along with Renilla reporter plasmid, and assayed for Firefly and Renilla luciferase activity, respectively. Results depict ratio of Firefly/Renilla luciferase activity. Cells were also transfected with the empty pGL3-basic vector or pGL3-control vector (SV40 pGL3) as negative or positive controls, respectively. (b) Fraction of SLC7A1 reporter constructs activity inhibited (sensitive) by ZM-241385 in absence (Basal) or presence of insulin or NBTI as in (a). (c) SLC7A1 reporter constructs fraction activity non-inhibited (insensitive) by ZM-241385 as in (a). In (a), *P<0.05 versus Control, †P<0.05 versus corresponding values in the presence of ZM-241385. In (b), *P<0.05 versus corresponding Basal, †P<0.05 versus values in pGL3-hCAT-1⁻¹⁶⁰⁶ in the presence of insulin, ‡P<0.05 versus values in pGL3-hCAT-1⁻⁶⁵⁰ in the presence of insulin or insulin + NBTI. Values are mean ± SEM (n = 6).

Figure 6. Involvement of adenosine receptors on insulin effect in human umbilical vein rings reactivity.

(a) Endothelium-intact (circles) or endothelium-denuded (squares) human umbilical vein rings were exposed (8 hours) to increasing concentrations of insulin in absence (○,□) or presence (•,▪) of ZM-241385 (10 nmol/L). (b) Endothelium-intact human umbilical vein rings incubated in absence (–, control) or presence (+) of 10 μmol/L nitrobenzylthioinosine (NBTI) and/or ZM-241384 as in (a). Reactivity was measured in vessel rings co-incubated without (white bars) or with (black bars) 1 nmol/L insulin. Relative responses are given as a percentage fraction of the initial vessel response to KCl (see Methods). (c) Umbilical vein rings with (+ Endothelium) or without (– Endothelium) were exposed for 2 minutes or 8 hours 30 nmol/L CGS-21680 (white bars) in the absence or presence of 10 nmol/L ZM-241385 (black bars) or 10 nmol/L DPCPX (grey bars). Relative responses are as in (a). In (a), *P<0.05 versus corresponding values in endothelium-intact vessels. In (b), *P<0.05 versus control. In (c), *P<0.05 versus all other values except in DPCPX for 2 minutes in endothelium intact vessels. Values are mean ± SEM (n = 7–9).

Figure 7. Proposed model for insulin action requiring adenosine receptors on L-arginine transport in human fetal endothelial cells.

Human umbilical vein endothelial cells respond to insulin via activation of insulin receptors (IR) leading to a reduced (–) adenosine uptake via the human equilibrative nucleoside transporters (hENTs). Insulin reduced adenosine uptake leading to extracellular accumulation of this nucleoside, which turns into activation of A_2A adenosine receptors (A_2A) at the plasma membrane. Activation of these membrane receptors leads to a mechanism mediated by transcription factors acting as activators (+) between −1606 and −650 bp from the transcription start point of SLC7A1 (for hCAT-1) gene promoter. Alternatively, insulin activates transcription factors acting as activators (+) between −650 bp and the transcription start point of SLC7A1. This phenomenon increases (+) by adenosine receptor-activated transcription factors increasing hCAT-1 mRNA expression and protein abundance resulting in stimulation of L-arginine transport by HUVECs in response to insulin.