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Sayon Roy, Rupa Bhattacharya, Dana Graves; Insulin Reduces High Glucose-Induced FOXO1 Nuclear Translocation and Apoptosis in Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3585.
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Retinal vascular cell loss, a prominent characteristic lesion of early stage diabetic retinopathy, involves activation of a nuclear transcription factor, forkhead box 1 (FOXO1). This study determined if insulin inhibits FOXO1 nuclear translocation, a key event in FOXO1 activation, in retinal endothelial cells grown in high glucose (HG) medium.
Rat retinal endothelial cells (RRECs) were transfected with plasmid carrying FLAG tagged FOXO1 (pcDNA3 Flag FKHR). FOXO1 transfected and untransfected cells were maintained in either normal glucose (5 mM) or HG (30 mM) medium for two days and immunostained with anti FLAG antibody and counterstained with DAPI. In parallel experiments untransfected RRECs were maintained in HG for 5 days with or without insulin treatment for the last 24 hours. Cells were immunostained with anti-flag and anti-FOXO1 antibodies and images photographed under fluorescence microscopy were analyzed for transfection efficiency and FOXO1 nuclear localization. The effect of insulin treatment and FOXO1 knockdown by siRNA was determined by TUNEL assay.
Data shows that ~60% of FOXO1 transfected RRECs maintained in HG have FOXO1 nuclear translocation compared to less than 10% cells grown in N medium. Longer exposure of untransfected RRECs to high glucose resulted in a 2.5 fold increase in FOXO1 nuclear translocation compared to those of cells grown in N medium, which was blocked by insulin treatment. Furthermore, both insulin treatment and silencing FOXO1 by siRNA significantly blocked high glucose induced apoptosis by 70% and 60%, respectively (P<0.05).
Findings from this study provide further support that HG promotes FOXO1 activation, and that insulin reduces this activation by inhibiting FOXO1 nuclear transport. Insulin’s beneficial effect in inhibiting apoptosis occurs at least in part from reduced FOXO1 nuclear localization. Thus, targeting FOXO1 could have therapeutic potential for inhibiting apoptosis of retinal vascular cells, a characteristic lesion of diabetic retinopathy.
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