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

  • Haixiang Wu
  • Chunhui Jiang
  • Dekang Gan
  • Yujie Liao
  • Hui Ren
  • Zhongcui Sun
  • Meng Zhang
  • Gezhi Xu
Basic Science

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.

Keywords

Diabetic retinopathy Oxidative damage Insulin 

Supplementary material

417_2011_1677_MOESM1_ESM.doc (25 kb)
(DOC 31 kb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Haixiang Wu
    • 1
  • Chunhui Jiang
    • 1
  • Dekang Gan
    • 1
  • Yujie Liao
    • 2
  • Hui Ren
    • 1
  • Zhongcui Sun
    • 1
  • Meng Zhang
    • 1
  • Gezhi Xu
    • 1
    • 3
  1. 1.Department of OphthalmologyEye Ear Nose and Throat Hospital of Fudan UniversityShanghaiChina
  2. 2.Department of OphthalmologyThe Fifth People’s Hospital of Shanghai, Fudan UniversityShanghaiChina
  3. 3.Institute of Brain Science, Fudan University, ShanghaiShanghaiChina

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