Abstract
Abstract: :
Purpose:Diacylglycerol (DG) activates protein kinase C (PKC), which increases VEGF expression in retinal endothelial cells in diabetic eyes. Diacylglycerol kinase (DGK) regulates cell functions by regulating DG level, inositol–phospholipid turnover and PKC activity. Recent studies have identified that PKC–DGK–VEGF axis affects the pathological changes of retinal vessels by in diabetic eyes, however, the effects of this axis on retinal neuron are remaining to be resolved. The aim of this study is to investigate whether high glucose concentration and/or oxidative stress affects retinal neurons through PKC–DGK–VEGF axis using retinoblastoma (RB) cell lines as retinal neuron–derived cell lines. Methods:We compared the expression pattern of DGK isoforms in 2 RB cell lines (WERI–Rb–1 and Y–79) and in retina. Two cell lines were treated in 4 arms (normal glucose =5.5mM of glucose (NG); high glucose (22.5mM of glucose = HG); NG+H2O2(H2O2=50–500µM); HG+H2O2. The expression patterns at mRNA level of DGK isoforms and VEGF were observed by RT–PCR. The expression at the protein levels of DGKαwas observed by immunoblot analysis and the total enzymatic activity was measured. PKC expression was investigated by immunoblot analysis and the translocation was observed immunocytochemiccally. Results:All the isoforms of DGK were expressed in normal retina. DGKγwas not expressed in neither Y–79 nor WERI and DGKα&ßwere not expressed in Y–79. HG increased the DGK activity in both cell lines and induced DGKαexpression in Y–79. The high–glucose induced DGK activity was decreased by the addition of H2O2 in both cell lines. PKC translocation was detected by stimulation of HG in both cell lines. The expression of DGKαwas in parallel with that of VEGF. Conclusions:The DGK–PKC–VEGF axis exerts certain effects on retinal neurons in high glucose concentration and oxidative stress, which may be related to the pathogenesis of diabetic retinopathy.
Keywords: diabetic retinopathy • pathology: experimental • retinoblastoma