Modulation of Apoptosis Pathways by Oxidative Stress and Autophagy in β Cells
Figure 5
Ceramide is a mediator of cytokine-mediated CREB downregulation. (a) Human islets were exposed to C2 (10 μM), a synthetic ceramide, for 24 h. The islets were fixed and immunostained for phospho Akt (Thr 308). The images were analyzed by digital deconvolution microscopy. Decrease in the active form of Akt was observed in ceramide-treated islets. (b) Islets were incubated in the absence and presence of C2 and a combination of cytokines for 24 h and processed for the Western blot analysis of phosphorylated form of CREB, total CREB and β actin. Band intensities were quantitated by scanning. The results are M ± SE of 3 independent experiments. *; **; # compared to untreated control. Active form of CREB was decreased by both C2 and cytokines. (c) MIN6 cells were transfected with a CRE site-containing bcl-2 promoter linked to a firefly luciferase reporter and a constitutively active renilla luciferase (for transfection efficiency). The transfected cells were exposed to increasing concentrations of C2 and 50 μM of an inactive analogue (dh) for 24 h. The cells were processed for luciferase activities using a dual luciferase assay kit. The ratio of firefly and renilla luciferase activities was taken as a measure of bcl-2 promoter activity. Bcl-2 promoter activity was inhibited by the ceramide. The results are M ± SE of 4 independent experiments. *; ** compared to untreated control.
