Abstract
Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose–alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A2 mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH4) level was determined by HPLC, and L-arginine transport (0–1000 μmol/L) was measured in response to 5–25 mmol/L D-glucose (0–36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose–increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose–increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O2 •–) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O2 •– generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose–increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose–reduced BH4 level. Insulin and tempol blocked the high D-glucose–increased p42/44mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O2 •–/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.
Highlights
Hyperglycaemia and diabetes mellitus are pathological conditions associated with foetal endothelial dysfunction [1,2,3,4] and type 2 diabetes mellitus (T2DM) [5] or cardiovascular disease (CVD) [6, 7] in adulthood
This study shows that high extracellular D-glucose increases L-arginine transport, nitric oxide (NO) synthesis and O2– generation through endothelial nitric oxide (NO) synthase (eNOS) and NADPH oxidase activation
Insulin reversed these effects of high D-glucose, leading to normal human cationic amino acid transporter 1 (hCAT-1) expression, NO synthesis, O2– generation and vasorelaxation
Summary
Hyperglycaemia and diabetes mellitus are pathological conditions associated with foetal endothelial dysfunction [1,2,3,4] and type 2 diabetes mellitus (T2DM) [5] or cardiovascular disease (CVD) [6, 7] in adulthood. Elevated extracellular D-glucose increases hCAT-1-mediated L-arginine transport and NO synthesis (the ‘L-arginine/NO pathway’) [14, 15] as well as intracellular ROS generation [16, 17], leading to endothelial dysfunction [2]. Insulin reverses the gestational diabetes mellitus (GDM) or high D-glucose-associated stimulation of the L-arginine/NO pathway in HUVECs [11]. We hypothesise that insulin has a beneficial antioxidant capacity that reverses the high D-glucose-associated increase in ROS generation. We studied the effect of high extracellular D-glucose on insulin modulation of the L-arginine/NO and NADPH oxidase/O2– pathways in fetoplacental vascular reactivity. Insulin likely acts as an antioxidant under conditions of hyperglycaemia, leading to protection of the fetoplacental endothelium in diseases associated with endothelial dysfunction such as GDM
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