title: Different effects of low- and high-dose insulin on ROS production and VEGF expression in bovine retinal microvascular endothelial cells in the presence of high glucose.
abstract: BACKGROUND
  Clinical trials have demonstrated that acute intensive insulin therapy may cause transient worsening of retinopathy in type 1 and type 2 diabetes patients. However, the related mechanism still remains controversial. The purpose of the present study was to investigate the effect of insulin on the mitochondrial membrane potential (△Ψm), reactive oxygen species (ROS) production, UCP-2 and VEGF expression in bovine retinal microvascular endothelial cells (BRECs) in the presence of normal or high glucose and the related mechanisms.
  
  
  METHODS
  BRECs were isolated as primary cultures and identified by immunostaining. Passage BRECs were initially exposed to normal (5 mM) or high glucose (30 mM) for 3 days, with equimolar L: -glucose supplemented for osmotic equation. Then the cells were treated with 1 nM, 10 nM, or 100 nM insulin for 24 h: △Ψm and ROS production were determined by JC-1 and CM-H2DCFDA, respectively. Expression of UCP-2 and VEGF mRNA was determined by real-time RT-PCR; expression UCP-2 and VEGF protein was determined by Western-blotting analysis. A general ROS scavenger N-acetylcysteine (NAC, 10 mM) and an NADPH oxidase inhibitor apocynin (1 mmol/l) were added 1 h before treatment with 100 nM insulin.
  
  
  RESULTS
  Insulin increased △Ψm, ROS production, and expression of UCP-2 and VEGF in BRECs at normal glucose (5 mM) in a dose-dependent manner. Low-dose insulin (1 nM) decreased △Ψm, ROS production, and UCP-2, VEGF expression in BRECs at high glucose (30 mM); and high-dose insulin (10 nM, 100nM) recovered △Ψm, ROS production, and UCP-2, VEGF expression. Pretreatment of cells with NADPH oxidase inhibitor apocynin significantly suppressed 100 nM insulin-induced ROS production (p < 0.01, one-way ANOVA). Pretreatment of cells with ROS scavenger N-acetylcysteine completely blocked insulin-induced UCP-2 expression (p < 0.01, one-way ANOVA) and significantly suppressed VEGF expression (p < 0.01, one-way ANOVA).
  
  
  CONCLUSIONS
  High-dose insulin-induced ROS production and VEGF expression in BRECs in the presence of high glucose might be one of the reasons for the transient worsening of diabetic retinopathy during intensive insulin treatment.
authors.affiliation: Department of Ophthalmology, Eye Ear Nose and Throat Hospital of Fudan University, #83 Fenyang Road, Shanghai 200031, China.
authors.forename: Haixiang
authors.initials: H
authors.lastname: Wu
authors.forename: Chunhui
authors.initials: C
authors.lastname: Jiang
authors.forename: Dekang
authors.initials: D
authors.lastname: Gan
authors.forename: Yujie
authors.initials: Y
authors.lastname: Liao
authors.forename: Hui
authors.initials: H
authors.lastname: Ren
authors.forename: Zhongcui
authors.initials: Z
authors.lastname: Sun
authors.forename: Meng
authors.initials: M
authors.lastname: Zhang
authors.forename: Gezhi
authors.initials: G
authors.lastname: Xu
mesh_terms: D000818:Animals; D002417:Cattle; D003930:Diabetic Retinopathy / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012171:Retinal Vessels / Q000166:cytology*; D000071246:Uncoupling Protein 2; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
chemical_list: D007004:Hypoglycemic Agents; D007328:Insulin; D007473:Ion Channels; D024101:Mitochondrial Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D000071246:Uncoupling Protein 2; D042461:Vascular Endothelial Growth Factor A; D005947:Glucose