TNFα and SOCS3 regulate IRS-1 to increase retinal endothelial cell apoptosis

Rates of diabetes are reaching epidemic levels. The key problem in both type 1 and type 2 diabetes is dysfunctional insulin signaling, either due to lack of production or due to impaired insulin sensitivity. A key feature of diabetic retinopathy in animal models is degenerate capillary formation. The goal of this present study was to investigate a potential mechanism for retinal endothelial cell apoptosis in response to hyperglycemia. The hypothesis was that hyperglycemia-induced TNF alpha leads to retinal endothelial cell apoptosis through inhibition of insulin signaling. To test the hypothesis, primary human retinal endothelial cells were grown in normal glucose (5 mM) or high glucose (25 mM) and treated with exogenous TNF alpha. TNF alpha siRNA or suppressor of cytokine signaling 3 (SOCS3) siRNA. Cell lysates were processed for Western blotting and ELISA analyses to verify TNF alpha and SOCS3 knockdown, as well as key pro- and anti-apoptotic factors, IRS-1, and Akt. Data indicate that high glucose culturing conditions significantly increase TNF alpha and SOCS3 protein levels. Knockdown of TNF alpha and SOCS3 significantly increases anti-apoptotic proteins, while decreasing pro-apoptotic proteins. Knockdown of TNF alpha leads to decreased phosphorylation of IRS-1(ser307), which would promote normal insulin signaling. Knockdown of SOCS3 increased total IRS-1 levels, as well as decreased IRTyr960, both of which would inhibit retinal endothelial cell apoptosis through increased insulin signaling. Taken together, our findings suggest that increased TNF alpha inhibits insulin signaling in 2 ways: 1) increased phosphorylation of IRS-1(ser307), 2) increased SOCS3 levels to decrease total IRS-1 and increase IRTyr960, both of which block normal insulin signal transduction. Resolution of the hyperglycemia-induced TNF alpha levels in retinal endothelial cells may prevent apoptosis through disinhibition of insulin receptor signaling. (C) 2012 Elsevier Inc. All rights reserved.