Lipotoxic Stress Induces Pancreatic β-Cell Apoptosis through Modulation of Bcl-2 Proteins by the Ubiquitin-Proteasome System

Figure 2

Palmitate and MG132 treatment induces a similar increase in levels of ubiquitinated proteins in pancreatic islets and β-cells. ((a)–(c)) Expression of ubiquitinated proteins measured by Western blot in human islets (a), mouse islets (b), or MIN6 cells (c) under control conditions or following 0.5 mM palmitate treatment as indicated. Band intensities were quantified, with values of the specific time points standardised to β-actin loading control levels. Results represent fold induction levels, relative to control. ((d)-(e)) MIN6 cells were treated with 0.5 mM palmitate (d) and 10 μM MG132 (e) for 0 (untreated), 2, 4, and 8 h. Proteasome activity was measured using the Proteasome 20S Activity Assay kit. Results represent fold induction levels relative to the control. (f) MIN6 cells were treated with 10 μM MG132 as indicated and levels of ubiquitinated proteins were measured by Western blot analysis. ((g)-(h)) MIN6 cells were treated with 0.5 mM palmitate (g) and 10 μM MG132 (h) for 24 h and cell death was visualised by HO/PI staining. White arrows indicate cell death. (i) Western blot for cleaved caspase-3 in MIN6 cells after treatment with MG132 as indicated. (j) Human islets isolated from organ donors were treated for 24 h with MG132 and % of β-cells detected by Newport green staining and flow cytometry. Results are the means ± SEM of 3-4 independent experiments, except for Figures 2(a), 2(b), 2(i), and 2(j) (data representative of 2 independent experiments). ; .

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