Abstract
Purpose :
Pars plana vitrectomy (PPV) is associated with higher rates of cataract development, however, retrospective studies have found that individuals with diabetes had a decreased risk of requiring cataract extraction after PPV. We investigated the effects of high-glucose conditions on human lens epithelial cells (hLECs). We focused on the changes in autophagic flux and effects on autophagic or cell survivability markers, such as LC3A/B-I and -II, p63, Beclin-1, ZO-1, Bcl-2, and Bax, in response to high-glucose conditions. Lastly, we compared autophagic inhibitory effects between high-glucose conditions and chloroquine, a known inhibitor of autophagy.
Methods :
Immortalized hLECs (SRA01/04, Riken Research Institute, Japan) were cultured in DMEM with 1% FBS, to mimic starvation status found in vivo, and subjected to low (5.5 mM), medium (30 mM), and high (60 mM) glucose (HG) treatments. Chloroquine (CQ) 0.1 μM was added to high glucose conditions. Cells in control conditions were cultured in DMEM with 20% FBS and low glucose. Western blot analysis, immunofluorescence for LC3A/B and p62, and CCK-8 cell viability assays were performed.
Results :
CCK-8 assay showed that an HG condition with CQ significantly increased viability compared to cells treated with CQ only, but both showed lower viability compared to the control condition. Western blot showed increased Bcl-2 and decreased Bax in HG conditions compared to low and medium at 72 hours of culture, indicating reduced apoptotic activity. LC3A/B-II, an autophagosome marker, markedly increased in the HG with CQ condition over 72 hours of exposure, indicating an accumulation of autophagic intermediates due to an inhibition of terminal autophagic processes. A similar effect was found with p62, a marker for autophagic flux, after 24 hours of exposure. Beclin-1, a mediator of autophagy, was decreased in the HG with CQ condition over 24 hours. ZO-1, an indicator of structural integrity, was found to decrease from 4 to 8 hours but increase by 24 hours. This indicated successful inhibition of autophagy initially by CQ, which dwindled by 8 hours, then by HG by 24 hours.
Conclusions :
High glucose culture conditions inhibit autophagic processes in hLECs, which may decrease apoptosis and contribute to increased cell viability and structural integrity. High glucose inhibits autophagy at a later time of onset compared to chloroquine.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.