González Marcelo, Rojas Susana, Avila Pía, Cabrera Lissette, Villalobos Roberto, Palma Carlos, Aguayo Claudio, Peña Eduardo, Gallardo Victoria, Guzmán-Gutiérrez Enrique, Sáez Tamara, Salsoso Rocío, Sanhueza Carlos, Pardo Fabián, Leiva Andrea, Sobrevia Luis
Vascular Physiology Laboratory, Department of Physiology, Faculty of Biological Sciences, Universidad de Concepción, P.O. Box 160-C, Concepción 4070386, Chile; Group of Research and Innovation in Vascular Health (GRIVAS-Health), PO-Box 114-D, Chillán 3800708, Chile.
Vascular Physiology Laboratory, Department of Physiology, Faculty of Biological Sciences, Universidad de Concepción, P.O. Box 160-C, Concepción 4070386, Chile.
PLoS One. 2015 Apr 14;10(4):e0122398. doi: 10.1371/journal.pone.0122398. eCollection 2015.
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.
血管张力由L-精氨酸/一氧化氮（NO）途径控制，而高血糖诱导的氧化应激会强烈影响NO的生物利用度。胰岛素可导致人阳离子氨基酸转运体1（hCAT-1）的高表达和活性、NO合成及血管舒张；因此，胰岛素可能对高D-葡萄糖引起的内皮功能改变具有保护作用。在正常（5 mmol/L）或高（25 mmol/L）D-葡萄糖中孵育的氯化钾预收缩人脐静脉环（线肌张力测定法）中，测量血管对U46619（血栓素A2类似物）和降钙素基因相关肽（CGRP）的反应性。通过蛋白质印迹法和qRT-PCR测定hCAT-1、内皮型一氧化氮合酶（eNOS）、42和44 kDa丝裂原活化蛋白激酶（p42/44mapk）、蛋白激酶B/Akt（Akt）的表达和活性，通过高效液相色谱法测定四氢生物蝶呤（BH4）水平，并在第2代人脐静脉内皮细胞（HUVECs）中测量响应5-25 mmol/L D-葡萄糖（0-36小时）的L-精氨酸转运（0-1000 μmol/L）。检测在有无胰岛素和/或阿朴吗啡（烟酰胺腺嘌呤二核苷酸磷酸氧化酶[NADPH氧化酶]抑制剂）、tempol或Mn(III)TMPyP（超氧化物歧化酶模拟物）的情况下进行。高D-葡萄糖增加hCAT-1的表达和活性，呈双相性（峰值：孵育6小时和24小时）。胰岛素可阻断高D-葡萄糖增加的最大转运速度，且与较低的hCAT-1表达和SLC7A1基因启动子活性相关。高D-葡萄糖增加的转运与更高的活性氧（ROS）和超氧阴离子（O2•-）生成平行，并增加了U46619引起的静脉环收缩以及降低了CGRP引起的静脉环舒张。胰岛素和阿朴吗啡可减轻ROS和O2•-的生成，并恢复血管对U46619和CGRP的反应性。胰岛素可逆转高D-葡萄糖增加的NO合成，但阿朴吗啡或tempol则不能；然而，tempol和Mn(III)TMPyP可逆转高D-葡萄糖降低的BH4水平。胰岛素和tempol可阻断高D-葡萄糖增加的p42/44mapk磷酸化。高D-葡萄糖引起的血管功能障碍可能通过L-精氨酸/NO和O2•-/NADPH氧化酶途径被胰岛素减轻。这些发现对于更好地理解胎儿胰岛素抵抗和高血糖状态下的血管功能障碍具有重要意义。
