REDD1 Activates a ROS-Generating Feedback Loop in the Retina of Diabetic Mice.

William P Miller,Michael D Dennis,Alistair J Barber,Allyson L Toro

doi:10.1167/iovs.19-26606

Abstract

PurposeThe present study was designed to evaluate the role of the stress response protein REDD1 in diabetes-induced oxidative stress and retinal pathology.MethodsWild-type and REDD1-deficient mice were administered streptozotocin to induce diabetes. Some mice received the antioxidant N-acetyl-l-cysteine (NAC). Visual function was assessed by virtual optometry. Retinas were analyzed by Western blotting. Reactive oxygen species (ROS) were assessed by 2,7-dichlorofluoroscein. Similar analyses were performed on R28 retinal cells in culture exposed to hyperglycemic conditions, NAC, and/or the exogenous ROS source hydrogen peroxide.ResultsIn the retina of diabetic mice, REDD1 expression and ROS were increased. In cells in culture, hyperglycemic conditions enhanced REDD1 expression, ROS levels, and the mitochondrial membrane potential. However, similar effects were not observed in the retina of diabetic mice or cells lacking REDD1. In the retina of diabetic mice and cells exposed to hyperglycemic conditions, NAC normalized ROS and prevented an increase in REDD1 expression. Diabetic mice receiving NAC also exhibited improved contrast sensitivity as compared to diabetic controls. Hydrogen peroxide addition to culture medium increased REDD1 expression and attenuated Akt/GSK3 phosphorylation in a REDD1-dependent manner. In REDD1-deficient cells exposed to hyperglycemic conditions, expression of a dominant negative Akt or constitutively active GSK3 increased the mitochondrial membrane potential and promoted ROS.ConclusionsThe findings provide new insight into the mechanism whereby diabetes-induced hyperglycemia causes oxidative stress and visual dysfunction. Specifically, hyperglycemia-induced REDD1 activates a ROS-generating feedback loop that includes Akt/GSK3. Thus, therapeutic approaches targeting REDD1 expression and ROS may be beneficial for preventing diabetes-induced visual dysfunction.

Highlights

MethodsWild-type and regulated in development and DNA damage 1 (REDD1)-deficient mice were administered streptozotocin to induce diabetes
The present study was designed to evaluate the role of the stress response protein regulated in development and DNA damage 1 (REDD1) in diabetes-induced oxidative stress and retinal pathology
The findings provide new insight into the mechanism whereby diabetes-induced hyperglycemia causes oxidative stress and visual dysfunction

Summary

Methods

Wild-type and REDD1-deficient mice were administered streptozotocin to induce diabetes. Similar analyses were performed on R28 retinal cells in culture exposed to hyperglycemic conditions, NAC, and/or the exogenous ROS source hydrogen peroxide. Male C57BL6/N mice (Charles River, Wilmington, MA, USA) and wild-type/REDD1 knockout B6;129 mice[23] were administered either 50 mg/kg streptozotocin (STZ) or sodium citrate buffer for 5 consecutive days to induce diabetes. Cells were cultured on plates (CellBIND; Corning, Corning, NY, USA) with Dulbecco’s modified Eagle’s medium (DMEM) containing 5 mM glucose and 10% fetal bovine serum (FBS). Culture medium was supplemented with 100 nM insulin (Lilly, Indianapolis, IN, USA), 10 mM NAC (Sigma-Aldrich Corp., St. Louis, MO, USA), or 1 mM hydrogen peroxide. To evaluate mitochondrial membrane potential, cells were transferred to Opti-MEM reduced-serum media lacking phenol red and stained with 1 lg/mL dye (JC-1; Life Technologies)

Results

Discussion

Conclusion