Abstract
Purpose:
The present study was designed to evaluate the role of the stress response protein REDD1 in diabetes-induced oxidative stress and retinal pathology.
Methods:
Wild-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.
Results:
In 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.
Conclusions:
The 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.
Hyperglycemia is a major causative factor in the development of complications associated with diabetes.
1 The Diabetes Control and Complications Trial demonstrated that intensive glycemic control is associated with a reduction in both the onset and progression of diabetic retinopathy (DR),
2 yet the molecular events that contribute to early diabetes-induced retinal dysfunction remain incompletely understood. A unifying theory for the pathophysiology of diabetic complications suggests that the principle pathways responsible for hyperglycemia-induced tissue damage are all linked to the overproduction of reactive oxygen species (ROS).
3 Oxidative stress results from an imbalance between the production of ROS, such as superoxide anion (O
2−), hydroxyl radical (OH), hydrogen peroxide (H
2O
2), and singlet oxygen (
1O
2), and the antioxidant defense system. In diabetes, an increase in the production of ROS and an impaired capacity to reduce free radicals contributes to retinal pathogenesis.
4 Experimental evidence demonstrates that hyperglycemia-induced mitochondrial superoxide production drives the development of oxidative stress in the retina.
5,6 In fact, overexpression of the mitochondrial superoxide dismutase is sufficient to prevent diabetes-induced oxidative stress and the development of acellular capillaries in the retina.
6,7
Hyperglycemic conditions activate a multicomponent feedback loop that promotes mitochondrial ROS production.
8 Hyperglycemia provides an abundance of substrate for glucose oxidation, leading to increased generation of NADH and pyruvate, enhanced electron transport chain flux, and a high inner mitochondrial trans-membrane potential (ΔΨ
m).
9,10 Mitochondrial ROS generation is dependent on a high proton gradient, as O
2− and H
2O
2 formation dramatically increase when ΔΨ
m exceeds a threshold level.
11 Mitochondrial hexokinase (HK) activity is an important regulator of ΔΨ
m. Specifically, the enzyme provides local ADP recycling through the VDAC (voltage-dependent anion channel)/ANT (adenine nucleotide translocator) complex to the F
1F
0 ATP synthase complex, which phosphorylates the ADP at the expense of ΔΨ
m.12 The association of HK with VDAC is reduced by glycogen synthase kinase 3β (GSK3β)-dependent phosphorylation of VDAC.
13 In turn, GSK3β activity is inhibited by Akt-dependent phosphorylation of the GSK3 N-terminus, which reduces kinase activity by obstructing substrate binding.
14,15 In response to hyperglycemic conditions, protein phosphatase 2A (PP2A) inhibits Akt kinase activity by dephosphorylation of the kinase.
8 This reduction in Akt activity leads to increased GSK3β-dependent phosphorylation of VDAC, reduced mitochondrial association with HK, and increased ROS production.
The stress response protein regulated in development and DNA damage 1 (REDD1) promotes association of PP2A with Akt, leading to site-specific dephosphorylation of the kinase and subsequent reduction in Akt-mediated phosphorylation of GSK3.
16 Thus, we speculated that REDD1 expression may be a critical element in the feedback loop that increases ROS production in response to hyperglycemic conditions. Since its discovery, REDD1 has been linked to the regulation of ROS.
17–20 Our laboratory recently demonstrated that REDD1 expression is increased in the retina of diabetic mice by hyperglycemia,
21 coincident with attenuated Akt kinase activity and increased retinal cell death.
22 In the retina of diabetic mice lacking REDD1, Akt activity remains elevated and cell death is similar to that observed in nondiabetic controls.
22 In fact, genetic ablation of REDD1 prevents electroretinogram defects or visual threshold deficits in diabetic mice.
22 Similarly, REDD1 has also been linked to the development of ischemic proliferative retinopathy.
23 Remarkably, the DEGAS study demonstrates that intravitreal injection of a siRNA targeting the REDD1 mRNA is more effective than laser photocoagulation in improving visual acuity in patients with diabetic macular edema.
24 Improved vision in patients treated with a siRNA targeting the REDD1 mRNA occurred in the absence of altered anatomical features or fluorescein leakage, suggesting a mechanism independent of changes in vascular permeability. In the present study, we investigated the mechanism responsible for the protective effects of REDD1 deletion on diabetes-induced visual dysfunction.
NAC Supplementation Attenuates Retinal ROS and REDD1 Expression in a Model of Type 1 Diabetes
Hyperglycemic Conditions Increase ROS, REDD1 Expression, and Cell Death in R28 Cells in Culture
NAC Prevents Glucose-Induced ROS, REDD1 Expression, and Cell Death in R28 Cells in Culture
ROS Promotes REDD1 Expression to Attenuate Akt/GSK3 Phosphorylation in R28 Cells in Culture
The authors thank Elena Feinstein (Quark Pharmaceuticals) for permission to use the REDD1 knockout mice and Leif Ellisen (Harvard Medical School) for providing REDD1 wild-type and REDD1-knockout MEF. We also gratefully acknowledge Leonard Jefferson and Scot Kimball (Penn State College of Medicine) for critically evaluating the manuscript. Parts of this study were presented in abstract form at the 78th Scientific Session of the American Diabetes Association, Orlando, Florida, United States, June 2018.
Supported by the American Diabetes Association Pathway to Stop Diabetes Grant 1-14-INI-04 and National Eye Institute Grant EY023612.
Disclosure: W.P. Miller, None; A.L. Toro, None; A.J. Barber, None; M.D. Dennis, None