High glucose induces mitochondrial dysfunction in photoreceptor cells

Abstract

Purpose : Photoreceptors have high metabolic activity, yet limited reserve capacity for mitochondrial oxidative phosphorylation. While mitochondrial dysfunction has been implicated in the cell death of various retinal cell types, including retinal endothelial cells, pericytes, and Müller cells, under simulated hyperglycemic stress, its effect on photoreceptor cells remains unclear. Here we investigated the effects of high glucose on mitochondrial membrane potential (ΔΨM), morphology, bioenergetics, and cell apoptosis of 661w photoreceptor-like cells. Methods : The effect of high glucose (55mM) on ΔΨM of 661w cells over time was evaluated with tetramethylrhodamine ethyl ester (TRME) fluorescein intensity level relative to normal glucose condition (5.5mM). Mitochondrial network of 661w cells incubated in these two conditions for 48 hours was identified using MitoTracker Green staining. Mitochondrial images were captured in live cells with confocal microscopy and analyzed for mitochondrial morphology changes based on form factor (FF) and aspect ratio (AR) values. Mitochondrial bioenergetics was assessed by measuring oxygen consumption rate (OCR) using the Seahorse XFe24 Extracellular Flux Analyzer. Cell apoptosis was evaluated by annexin V assay. Results : 661w cells incubated in high glucose condition exhibited a multiphasic change in ΔΨM over time (Fig 1). Significant increase in mitochondrial fragmentation was also observed when compared to normal glucose condition (FF= 2.95±0.40 vs 4.82±0.42, p<0.01; AR= 1.96±0.10 vs 2.44±0.08, p<0.001). OCRs were significantly reduced in cells grown in high glucose condition compared to those in normal glucose condition (ATP production: 7.62±0.84 vs 9.86±0.40; Maximal respiration: 10.52±0.86 vs 14.33±0.89, p<0.05). Cell apoptosis was also increased by approximately 1.4-fold in high glucose condition compared to normal glucose condition (p<0.001). Conclusions : High glucose affects the mitochondria of 661w cells by disrupting the homeostasis of ΔΨM, changing mitochondrial morphology, and increasing mitochondrial fragmentation. Impaired mitochondrial function and increased cell apoptosis were also observed. The detrimental effects of glucose on mitochondrial function and cellular metabolism may lead to photoreceptor cells apoptosis, contributing to the pathogenesis of diabetic retinopathy.