title: Insulin reverses D-glucose-increased nitric oxide and reactive oxygen species generation in human umbilical vein endothelial cells.
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.
authors: 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.
authors: Marcelo
authors: M
authors: González
authors: Vascular Physiology Laboratory, Department of Physiology, Faculty of Biological Sciences, Universidad de Concepción, P.O. Box 160-C, Concepción 4070386, Chile.
authors: Susana
authors: S
authors: Rojas
authors: Vascular Physiology Laboratory, Department of Physiology, Faculty of Biological Sciences, Universidad de Concepción, P.O. Box 160-C, Concepción 4070386, Chile.
authors: Pía
authors: P
authors: Avila
authors: Vascular Physiology Laboratory, Department of Physiology, Faculty of Biological Sciences, Universidad de Concepción, P.O. Box 160-C, Concepción 4070386, Chile; Department of Morphophysiology, Faculty of Medicine, Universidad Diego Portales, Santiago 8370076, Chile.
authors: Lissette
authors: L
authors: Cabrera
authors: Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynaecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago 8330024, Chile.
authors: Roberto
authors: R
authors: Villalobos
authors: Vascular Physiology Laboratory, Department of Physiology, Faculty of Biological Sciences, Universidad de Concepción, P.O. Box 160-C, Concepción 4070386, Chile.
authors: Carlos
authors: C
authors: Palma
authors: Department of Clinical Biochemistry and Immunology, Faculty of Pharmacy, 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.
authors: Claudio
authors: C
authors: Aguayo
authors: Department of Physiopathology, Faculty of Biological Sciences, Universidad de Concepción, P.O. Box 160-C, Concepción 4070386, Chile.
authors: Eduardo
authors: E
authors: Peña
authors: Department of Physiopathology, Faculty of Biological Sciences, Universidad de Concepción, P.O. Box 160-C, Concepción 4070386, Chile.
authors: Victoria
authors: V
authors: Gallardo
authors: Group of Research and Innovation in Vascular Health (GRIVAS-Health), PO-Box 114-D, Chillán 3800708, Chile; Faculty of Health Sciences, Universidad San Sebastián, Concepción 4080871, Chile.
authors: Enrique
authors: E
authors: Guzmán-Gutiérrez
authors: Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynaecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago 8330024, Chile.
authors: Tamara
authors: T
authors: Sáez
authors: Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynaecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago 8330024, Chile.
authors: Rocío
authors: R
authors: Salsoso
authors: Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynaecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago 8330024, Chile.
authors: Carlos
authors: C
authors: Sanhueza
authors: Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynaecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago 8330024, Chile.
authors: Fabián
authors: F
authors: Pardo
authors: Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynaecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago 8330024, Chile.
authors: Andrea
authors: A
authors: Leiva
authors: University of Queensland Centre for Clinical Research (UQCCR), Faculty of Medicine and Biomedical Sciences, University of Queensland, Herston, QLD 4029, Queensland, Australia; Department of Physiology, Faculty of Pharmacy, Universidad de Sevilla, Seville E-41012, Spain; Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynaecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago 8330024, Chile.
authors: Luis
authors: L
authors: Sobrevia
mesh_terms: D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000098:Acetophenones / Q000494:pharmacology; D001120:Arginine / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology; D027201:Cationic Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism; D003497:Cyclic N-Oxides / Q000494:pharmacology; D005786:Gene Expression Regulation; D005947:Glucose / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D009569:Nitric Oxide / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D013113:Spin Labels; D046509:Tissue Culture Techniques; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014471:Umbilical Veins / Q000187:drug effects* / Q000378:metabolism; D014662:Vasoconstrictor Agents / Q000494:pharmacology
chemical_list: D000098:Acetophenones; D027201:Cationic Amino Acid Transporter 1; D003497:Cyclic N-Oxides; D007328:Insulin; D017382:Reactive Oxygen Species; C420825:SLC7A1 protein, human; D013113:Spin Labels; D014157:Transcription Factors; D014662:Vasoconstrictor Agents; C478359:WDR77 protein, human; D001708:Biopterins; D009569:Nitric Oxide; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D001120:Arginine; C056165:acetovanillone; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; C003402:sapropterin; D005947:Glucose; D015740:Calcitonin Gene-Related Peptide; C001803:tempol