Autophagy is believed to play an important role in maintaining homeostasis in pancreatic β-cells during insulin resistance. This study investigated the role of autophagy in β-cell damage induced by cholesterol and its possible activation mechanism. Rat and mouse pancreatic β-cell lines INS-1 and βTC-6 were incubated with cholesterol alone or in combination with autophagy inhibitors E-64d/Pepstatin A or bafilomycin A1. DAPI staining, western blotting, transmission electron microscopy and immunofluorescence were conducted to assess the effects of autophagy inhibitors on cholesterol-induced apoptosis and autophagy activity. An increase in FITC-LC3 fluorescence dots, autophagic vacuoles and LC3-II protein indicated that autophagy was activated in cells treated with cholesterol. This was further confirmed by blocking the natural turnover processes in lysosomes and autolysosomes with autophagy inhibitors, suggesting enhanced autophagic activity rather than blockage of autophagy. Furthermore, inhibition of autophagy significantly augmented the activation of caspase 3 and the percentage of cholesterol-induced apoptotic nuclei. These results demonstrate that autophagy plays a protective role against cholesterol-induced apoptosis in pancreatic β-cells.
Keywords: Apoptosis; Autophagy; Cholesterol; LC3-II; β-cell.
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