Retinal MCs exposed to HG increase Beclin-1, which marks the induction of autophagosome formation, and LC3II (MAP1LC3B/LC3-microtubule-associated protein 1 light chain 3), which marks the existence of autophagosomes in the cell. Cells cultured under NG conditions (
Fig. 1A,
P = 0.005 and 0.003, respectively) do not increase these markers, indicating that HG conditions induce initiation of autophagic machinery. To evaluate whether autophagic flux was functioning properly, we examined the autophagic substrate p62/SQTSM1, a cargo protein degraded by an autophagy–lysosome system.
28 We observed increases in the p62/SQTSM1 levels of cells exposed to HG (
P < 0.0001), suggesting a reduction in autophagosome degradation by lysosome systems (
Fig. 1A). Similar findings were observed after 48 and 72 hours of exposure to HG conditions and were steady across exposure times (
Supplementary Figs. S1A, S1C). Stress markers of ER were also assessed under HG conditions. rMCs under HG conditions for 24 hours showed increased eif2α phosphorylation and CHOP expression, which are markers of ER stress (
Fig. 1A,
P = 0.01 and 0.02, respectively). Electron microscopy shows a massive increase in the autophagosome accumulation in rMCs exposed to HG conditions; in NG conditions, rMCs have normal autophagosome (AP) and autophagolysosome (APL) distribution throughout the cytosol (
Fig. 1B). In order to investigate whether autophagy and ER stress are involved in cell death, caspase 8 activity, and TUNEL assays were performed (
Figs. 1B,
1C). There was a clear increase in caspase 8 activity and TUNEL-positive cells in HG conditions (
P = 0.02 and 0.004, respectively), but caspase 9 activity did not change in cells exposed to HG conditions (data not shown). A similar increase in TUNEL-positive cells was observed after 48 or 72 hours of HG exposure and was steady throughout those periods (
Supplementary Fig. S1D). Collectively, these data show that rMCs in HG conditions show autophagic flux dysfunction and ER stress response, leading to increased risk of apoptosis.