Fig. 7.

Cu²⁺-inhibits amylin-evoked apoptosis in pancreatic cells. Human amylin (20 μM) was incubated in the absence or presence of 10 μM CuCl₂ and corresponding changes on cells’ viability were determined using MTT reduction (A and B) and apoptotic western-blot (C) and microscopy (D) assays. (A) Amylin induced a marked drop in the MTT signal, indicative of metabolic stress in cells. (B) Cu²⁺ impaired mitochondrial enzymatic activity in a dose-dependent manner. (C) Western blot analysis reveals an inhibitory effect of Cu²⁺ on caspase-3 and stress kinases activity in cells treated with human amylin. (D) Confocal microscopy analysis of apoptosis in RIN-m5F cells treated with human amylin, in the absence and in the presence of Cu²⁺. Arrows depict non-apoptotic nuclei (blue) in viable cells, whereas arrowheads depict caspase-3 (green) and annexin-V (red) positive apoptotic cells. Bar, 10 μm. Quantitative analysis of amylin-induced apoptosis in human islets is summarized in a bar graph. Significance established at **P < 0.01, n = 6, ANOVA followed by Newman–Keul post hoc test.