Abstract
Background
To investigate whether insulin can increase the production of reactive oxygen species (ROS) in bovine retinal microvascular endothelial cells (BRECs), the role of antioxidants in the insulin-induced exacerbation of diabetic retinopathy and the related mechanisms.
Methods
BRECs were cultured in either 5 or 30 mM glucose for 3 days before stimulation with 100 nM insulin for 24 h or incubated with 1 mM apocynin, 100 μM LY294002, 50 μM U0126, 2 μM GF109203X, 250 U/ml catalase, 100 μg/ml ascorbic acid, 100 μM α-lipoic acid and 50 μM α-tocopherol before stimulation with 100 nM insulin. H2O2 (200 μM) was added to cells to measure the VEGF protein expression. Intracellular ROS was measured by immunofluorescence and flow cytometry, superoxide anion measurement was done by cytochrome c reduction, and VEGF protein was measured by ELISA analysis.
Results
Insulin or (and) high glucose significantly increased intracellular ROS production in BRECs, and pretreatment of the cells with apocynin and LY294002 decreased insulin-induced superoxide anion production. Neither pretreatment with GF109203X nor U0126 showed an effect on the superoxide anion production. Ascorbic acid, α-lipoic acid, and α-tocopherol also decreased superoxide anion production. Furthermore, H2O2 increased VEGF protein expression in BRECs and catalase suppressed insulin-induced VEGF protein expression.
Conclusions
Insulin can increase ROS production through an NAD(P)H, phosphatidylinositol 3´-kinase-dependent mechanism in bovine retinal microvascular endothelial cells ex vivo. ROS can regulate insulin-induced VEGF expression. Supplementation with antioxidants may help to attenuate the transient worsening of retinopathy in diabetes caused by acute intensive insulin therapy.
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Acknowledgments
This article was supported by grants from the National Basic Research Program of China (973 program), (2007 CB512205), National Basic Research grants of China (81170857, 2011; 30872825, 2008), Plan of the Best Disciplines Leaders in Shanghai (09XD1400900).
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This article is supported by grants from the National Basic Research Program of China (973 program), (2007 CB512205), National Basic Research grants of China (81170857, 2011; 30872825, 2008), Plan of the Best Disciplines Leaders in Shanghai (09XD1400900).
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Wu, H., Xu, G., Liao, Y. et al. Supplementation with antioxidants attenuates transient worsening of retinopathy in diabetes caused by acute intensive insulin therapy. Graefes Arch Clin Exp Ophthalmol 250, 1453–1458 (2012). https://doi.org/10.1007/s00417-012-2079-4
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DOI: https://doi.org/10.1007/s00417-012-2079-4