Mitochondrial transport of glutathione in diabetic retinopathy

Diabetes increases free radical production and impairs the antioxidant defense system; and increased oxidative stress-mitochondrial damage plays a central role in the development of diabetic retinopathy. Glutathione (GSH), a negatively charged molecule at physiological pH, is biosynthesized in the cytosol, and cannot pass through mitochondrial inner membranes. Interestingly, mitochondria contain ∼15 % of the total GSH and depend on their inner membrane solute carriers for its import from cytosol; in diabetes, retinal cellular and mitochondrial GSH (mtGSH) levels are downregulated. Our aim was to investigate the role of solute carriers in the subnormal levels of mtGSH in diabetic retinopathy. Human retinal endothelial cells, regulated for solute carriers dicarboxylate (DIC), or 2-oxyglutarate (OGC) by their respective siRNAs or by overexpressing plasmids, and incubated in 20 mM glucose, were analyzed for cytosolic and mitochondrial GSH levels, mitochondrial respiration, membrane potential and cell apoptosis. Key results were confirmed in the retina from streptozotocin-induced C57BL/6J diabetic mice. High glucose decreased DIC and OGC expression, and downregulated cytosolic and mtGSH. While mtGSH was further decreased by inhibition of DIC or OGC and protected by their overexpression, cytosolic GSH was not affected by DIC or OGC regulation. Overexpression of these solute carriers also prevented glucose-induced decrease in mitochondrial structural damage, impaired membrane potential and respiration and increased cell death. Consistent with in vitro results, retinal DIC, OGC and mtGSH levels were significantly downregulated in diabetic mouse, and GSH co-staining with DIC, or with OGC, was also significantly decreased. Thus, DIC and OGC downregulation pays a major role in the impaired GSH import inside the mitochondria, making them more susceptible to the damage. Damaged mitochondria accelerate cell death, culminating in the development of diabetic retinopathy. Restoring mtGSH levels via upregulating GSH transporters could provide a novel approach to inhibit diabetic retinopathy.