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K. Trudeau, A. Molina, G. Las, O. Shirihai, S. Roy; Increased Autophagy is Associated With Transient Recovery of High Glucose-Induced Mitochondrial Fragmentation in Retinal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5635.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate whether recovery from high glucose (HG)-induced mitochondrial fragmentation is associated with altered autophagy in rat retinal endothelial cells (RRECs), we assessed mitochondria shape change and autophagy induction after acute (24hours) exposure to HG.
To assess mitochondrial shape, RRECs were grown in normal (5mM) and HG (30mM) medium for 0, 15, 30, 60, 120 minutes and 6 days. Cells were stained with (8nM) tetramethylrhodamine-ethyl-ester-perchlorate and imaged live using confocal microscopy. Average mitochondria shape within a cell was determined using Form Factor (FF) and Aspect Ratio (AR) values of the mitochondria, to assess branching and length. To analyze HG-induced formation and turnover of autophagosomes in RRECs, expression of LC3-II, a marker for autophagosomes, and p62, a marker for autophagic protein degradation, were examined by Western Blot analysis at 1, 2, 6, 48 hours and 6 days of HG.
Acute exposure of RRECs to HG media resulted in transient fragmentation of mitochondria within 30 minutes (0 min: FF=2.38, AR=2.11; 30min: FF=1.519, p=0.001; AR=1.89, p=0.009), followed by partial morphology recovery after 120 minutes (FF=2.01, p<0.001, AR=2.06, p=0.004). LC3-II expression was increased after 2 hours of HG (180±58% of normal, p=0.004) and p62 expression was reduced after 6 hours of HG (64±31% of normal, p=0.025). After 24 hours of HG mitochondrial fragmentation was observed again and remained for the rest of the 6 day experiment (FF=1.377, p<0.001, AR=1.793, p=0.006). LC3-II and p62 expression showed no change after 6 days of HG exposure.
Findings from this study show that transient recovery from HG-induced mitochondrial fragmentation occurs with changes in autophagy-related proteins LC3-II and p62. Permanent mitochondrial fragmentation persists after 24 hours of HG with no change in LC3-II and p62. Together these observations suggest that autophagy may play a role in the recovery from HG-induced mitochondrial fragmentation. This could shed light on how HG contributes to mitochondrial dysfunction associated with diabetic retinopathy.
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