title: NADPH Oxidase-Mediated ROS Production Determines Insulin's Action on the Retinal Microvasculature.
abstract: OBJECTIVE
  To determine whether insulin induces nitric oxide (NO) formation in retinal microvessels and to examine the effects of high glucose on the formation of NO.
  
  
  METHODS
  Freshly isolated rat retinal microvessels were incubated in normal (5.5 mM) or high (20 mM) glucose with or without insulin (100 nM). The levels of insulin-induced NO and reactive oxygen species (ROS) in the retinal microvessels were determined semiquantitatively using fluorescent probes, 4,5-diaminofluorescein diacetate, and hydroethidine, respectively, and a laser scanning confocal microscope. The insulin-induced changes of NO in rat retinal endothelial cells and pericytes cultured at different glucose concentrations (5.5 and 25 mM) were determined using flow cytometry. Nitric oxide synthase (NOS) protein levels were determined by Western blot analysis; intracellular levels of ROS were determined using fluorescence-activated cell sorting (FACS) analysis of ethidium fluorescence; and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase RNA expression was quantified using real-time PCR.
  
  
  RESULTS
  Exposure of microvessels to insulin under normal glucose conditions led to a significant increase in NO levels; however, this increase was significantly suppressed when the microvessels were incubated under high glucose conditions. Intracellular levels of ROS were significantly increased in both retinal microvessels and cultured microvascular cells under high glucose conditions. The expression of NOS and NADPH oxidase were significantly increased in endothelial cells and pericytes under high glucose conditions.
  
  
  CONCLUSIONS
  The increased formation of NO by insulin and its suppression by high glucose conditions suggests that ROS production mediated by NADPH oxidase is important by insulin's effect on the retinal microvasculature.
authors.forename: Teruyo
authors.initials: T
authors.lastname: Kida
authors.forename: Hidehiro
authors.initials: H
authors.lastname: Oku
authors.forename: Taeko
authors.initials: T
authors.lastname: Horie
authors.forename: Junko
authors.initials: J
authors.lastname: Matsuo
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Masanori
authors.initials: M
authors.lastname: Fukumoto
authors.forename: Tsunehiko
authors.initials: T
authors.lastname: Ikeda
mesh_terms: D000818:Animals; D015153:Blotting, Western; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D019793:Fluorescein; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D018613:Microscopy, Confocal; D055806:Microvessels / Q000187:drug effects* / Q000378:metabolism; D023421:Models, Animal; D009249:NADP / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D020286:Pericytes / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012171:Retinal Vessels / Q000187:drug effects* / Q000378:metabolism
chemical_list: C414689:4,5-diaminofluorescein diacetate; D007328:Insulin; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D009249:NADP; D019001:Nitric Oxide Synthase; D005947:Glucose; D019793:Fluorescein