Abstract
Mitochondrial dysfunction has been implicated in diabetic complications; however, it is unknown whether hyperglycemia affects mitochondrial morphology and metabolic capacity during development of diabetic retinopathy. We investigated high glucose (HG) effects on mitochondrial morphology, membrane potential heterogeneity, cellular oxygen consumption, extracellular acidification, cytochrome c release, and apoptosis in retinal endothelial cells. Rat retinal endothelial cells grown in normal (5 mmol/L) or HG (30 mmol/L) medium and double-stained with MitoTracker Green and tetramethylrhodamine-ethyl-ester-perchlorate were examined live with confocal microscopy. Images were analyzed for mitochondrial shape change using Form Factor and Aspect Ratio values, and membrane potential heterogeneity, using deviation of fluorescence intensity values. Rat retinal endothelial cells grown in normal or HG medium were analyzed for transient changes in oxygen consumption and extracellular acidification using an XF-24 flux analyzer, cytochrome c release by Western blot, and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Rat retinal endothelial cells grown in HG medium exhibited increased mitochondrial fragmentation concurrent with membrane potential heterogeneity. Metabolic analysis showed increased extracellular acidification in HG with reduced steady state/maximal oxygen consumption. Cytochrome c and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were also increased in HG. Thus, HG-induced mitochondrial fragmentation with concomitant increase in membrane potential heterogeneity, reduced oxygen consumption, and cytochrome c release may underlie apoptosis of retinal endothelial cells as seen in diabetic retinopathy.