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. 2008 Feb;57(2):415-23.
doi: 10.2337/db07-0993. Epub 2007 Nov 14.

Inhibition of AMP-activated protein kinase protects pancreatic beta-cells from cytokine-mediated apoptosis and CD8+ T-cell-induced cytotoxicity

Audrey Riboulet-Chavey  1 , Frédérique Diraison, L Khai Siew, F Susan Wong, Guy A Rutter
Affiliations

Inhibition of AMP-activated protein kinase protects pancreatic beta-cells from cytokine-mediated apoptosis and CD8+ T-cell-induced cytotoxicity

Audrey Riboulet-Chavey et al. Diabetes. 2008 Feb.

Abstract

Objective: Apoptotic destruction of insulin-producing pancreatic beta-cells is involved in the etiology of both type 1 and type 2 diabetes. AMP-activated protein kinase (AMPK) is a sensor of cellular energy charge whose sustained activation has recently been implicated in pancreatic beta-cell apoptosis and in islet cell death posttransplantation. Here, we examine the importance of beta-cell AMPK in cytokine-induced apoptosis and in the cytotoxic action of CD8(+) T-cells.

Research design and methods: Clonal MIN6 beta-cells or CD1 mouse pancreatic islets were infected with recombinant adenoviruses encoding enhanced green fluorescent protein (eGFP/null), constitutively active AMPK (AMPK-CA), or dominant-negative AMPK (AMPK-DN) and exposed or not to tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma. Apoptosis was detected by monitoring the cleavage of caspase-3 and DNA fragmentation. The cytotoxic effect of CD8(+) purified T-cells was examined against pancreatic islets from NOD mice infected with either null or the AMPK-DN-expressing adenoviruses.

Results: Exposure to cytokines, or expression of AMPK-CA, induced apoptosis in clonal MIN6 beta-cells and CD1 mouse pancreatic islets. By contrast, overexpression of AMPK-DN protected against the proapoptotic effect of these agents, in part by preventing decreases in cellular ATP, and lowered the cytotoxic effect of CD8(+) T-cells toward NOD mouse islets.

Conclusions: Inhibition of AMPK activity enhances islet survival in the face of assault by either cytokines or T-cells. AMPK may therefore represent an interesting therapeutic target to suppress immune-mediated beta-cell destruction and may increase the efficacy of islet allografts in type 1 diabetes.

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Figures

Fig. 1
Fig. 1. Cytokines induce apoptosis in clonal MIN6 β-cells.
MIN6 cells were cultured with a cytokine mix containing IL-1β (50 U/ml), TNF-α (1000 U/ml) and IFN-γ (1000 U/ml), for the times indicated, in their culture medium (see Research Design and Methods). Induction of apoptosis was measured by western (immuno-) blotting for cleaved caspase-3 (Asp-175) (see Research Design and Methods) and total protein content was estimated using anti-ERK1/2. Data are given as means ± S.E.M. of three independent experiments: *p<0.05 for effect of cytokines compared with basal without cytokines.
Fig. 2
Fig. 2. Effect of AMPK CA adenovirus on caspase-3 activity in MIN6 β-cells.
(a) Cells were infected with the indicated adenoviruses at 100 MOI (Null/GFP, AMPK CA) for the times indicated. Induction of apoptosis was measured by immunocytochemistry with an antibody to cleaved-caspase-3. Primary antibody was revealed using TRITC-conjugated secondary antibody against rabbit IgG and a Hoechst coloration of the nucleus was performed. (b) The data from (a) are given as means ± SEM of triplicate analyses from three independent experiments. Results were analysed by densitometry and statistical differences were assessed by unpaired Students t-test (*p<0.05 for effect of AMPK CA virus compared with Null virus). Scale bar, 20 µm.
Fig. 3
Fig. 3. Effect of cytokines on AMPK activity in clonal MIN6 β-cells and mouse pancreatic islets.
(a) MIN6 cells or (b) mouse pancreatic islets were treated with cytokine mix for 48 h and incubated in KRB medium (see Research Design and Methods) at the indicated concentrations of glucose for 30 min. Phospho-Thr-172 AMPK phosphorylation was assessed using the appropriate phospho-specific antibody. Total protein was estimated using anti-ERK1/2 antibody. Results shown are from three independent experiments and statistical differences were assessed by unpaired Students t-test: *p<0.05 and **p<0.01 for the effect of cytokines compared without cytokines.
Fig. 4
Fig. 4. Dominant-negative AMPK blocks cytokine-mediated apoptosis in MIN6 β-cells.
(a) MIN6 cells were infected with the indicated adenoviruses at 100 MOI (Null/GFP, AMPK DN) for 48 h, before treatment with cytokines as in Fig. 1, for a further 48 h. Apoptosis was measured by TUNEL assay using the In Situ Cell Death Detection Kit, TMR Red, as described in Research Design and Methods, followed by nucleus coloration with Hoechst dye. Results were analysed by densitometry and statistical differences were assessed by unpaired Students t-test: **p<0.01 for the effects of AMPK DN virus in the presence of cytokines compared with Null virus. (b) The data from (a) are given as means ± SEM of triplicate analyses from three independent experiments involving 100 individual cells per condition. Scale bar, 20 µm.
Fig. 5
Fig. 5. Dominant-negative AMPK blocks cytokine-activation of caspase 3 in MIN6 β-cells.
MIN6 cells were treated as in Fig. 4, and active caspase 3 was measured by (a, b) immunocytochemistry or (c) by western blot with anti cleaved-caspase-3 antibody. Results were analysed as in Fig. 4: *p<0.05 and ***p<0.001 for the effect of AMPK DN virus in the presence of cytokines compared with Null virus. (b) Data are given as means ± S.E.M. of triplicate analyses from three independent experiments involving 50 individual cells per condition for the immunocytochemistry and (c) as means ± S.E.M. of three independent experiments for the western blot. Scale bar, 10 µm.
Fig. 6
Fig. 6. Effect of dominant-negative AMPK and AMPK CA on cytokine-mediated apoptosis in dispersed mouse pancreatic islets.
(a) Dispersed islets were infected or not with the indicated adenoviruses at 100 MOI (Null/GFP, AMPK DN, AMPK CA) for 48 h. Then, islets infected with Null/GFP or AMPK DN were treated or not with cytokines, as in Fig. 1, for a further 24 h or 48 h. Islets infected with AMPK CA were maintained for a further 24h. Apoptosis was measured by TUNEL assay as described in Fig. 4, followed by nucleus coloration with Hoechst dye (BF represents the bright field). (b) Data from (a) are given as means ± SEM of duplicate analyses from five experiments involving 50 individual cells per condition. Scale bar, 5 µm. *p<0.05 for the effect of AMPK DN with cytokines compared with Null virus in presence of cytokines. (c) Dispersed islets were treated with cytokines during 24 h and caspase-3 cleavage was measured by immunocytochemistry with the appropriate antibody, followed by nucleus coloration with Hoechst dye. (d) Data from (c) are given as means ± SEM of duplicate analyses from three experiments involving 50 individual cells per condition. Scale bar, 5 µm. **p<0.01 for the effect of the cytokines compared with the control without cytokines.
Fig. 7
Fig. 7. Effect of cytokines on total cellular ATP content in MIN6 cells.
MIN6 cells were infected with the indicated adenoviruses at 100 MOI (Null/GFP, AMPK DN) for 48 h, before treatment with cytokines as in Fig. 1, for the indicated times. Prior to ATP assay, cells were incubated with the indicated concentrations of glucose for 30 min (see Fig. 3). Cells were then extracted in perchloric acid as described under “Research Design and Methods”, before assay of total ATP. *p<0.05, **p<0.01 and ***p<0.001 for effects of glucose or cytokines compared to the corresponding controls. Data are the means ± SEM of triplicates from observations on three separate cell cultures. Results shown are representative of three independent experiments.
Fig. 8
Fig. 8. Dominant-negative AMPK decreases the cytotoxicity of insulin-reactive CD8+ T cells to NOD pancreatic islets.
(a) Scheme explaining the cytotoxic assay (see Research Design and Methods). (b) P185 control cells were labelled with 51chromium sulphate during 1 h, washed, and incubated for 16 h with insulin B15-23 reactive CD8+ T cells at a ratio of 1:10, in presence of increasing concentrations of insulin B15-23 peptide. The percentage of lysis was determined measuring the gamma radioactivity released in the supernatant by the lysed cells: % of lysis = ((sample release-min) / (max-min))*100. (c) The same procedure was used with islets from NOD mice which were infected with the AMPK DN adenovirus at 100 MOI for 48 h. 20 islets per condition were used in presence of 400 000 CD8+ T cells, estimating 1000 cells / islet, and insulin peptide was added or not at 1 μg/ml. *p<0.05 for the effect of AMPK DN virus in the presence of the peptide compared with Null virus. Data are given as means ± S.E.M. of triplicate analyses from three independent experiments.
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