Modulation of Apoptosis Pathways by Oxidative Stress and Autophagy in β Cells
Figure 3
Induction of autophagy in human islets. (a) Human islets were incubated for 8 h in Miami medium (control) or in Hank’s balanced salt solution for amino acid (AA) starvation. Treated islets were fixed in paraformaldehyde and embedded in OCT. Frozen sections (7 μm thickness) were immunostained for insulin with FITC (Green) and for LC3-II with cy3 (red). Formation of autophagosomes was visualized by the punctated staining of LC3-II in β cells (arrows). (b) Human islets were cultured under normoxic or hypoxic conditions in the absence and presence of inducers of autophagy, trehalose, rapamycin (Rapa) or salt solution (Salt) for 8 h. Cell lysates were processed for Western blot analysis of the active form of caspase-3. Induction of autophagy during hypoxia exacerbated apoptosis in islets. (c) Human islets were preincubated with inducers of autophagy for 4 h. Following change of medium, the cells were cultured under normoxic or hypoxic conditions for 12 h, lysed and processed for the Western blot analysis of active caspase-3. Autophagic preconditioning protected islets from hypoxia-induced apoptosis. Representative images from experiments with three independent batches of human islets are presented.
