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Proinflammatory cytokines activate the intrinsic apoptotic pathway in beta-cells.

Grunnet LG, Aikin R, Tonnesen MF, Paraskevas S, Blaabjerg L, Størling J, Rosenberg L, Billestrup N, Maysinger D, Mandrup-Poulsen T - Diabetes (2009)

Bottom Line: We found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser136, mitochondrial stress, cytochrome c release, activation of caspase-9 and -3, and DNA fragmentation.Inhibition of Bad Ser136 dephosphorylation or Bax was found to inhibit cytokine-induced intrinsic proapoptotic signaling.Our findings demonstrate that the intrinsic mitochondrial apoptotic pathway contributes significantly to cytokine-induced beta-cell death and suggest a functional role of calcineurin-mediated Bad Ser136 dephosphorylation and Bax activity in cytokine-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Diabetology, Steno Diabetes Center, Gentofte, Denmark.

ABSTRACT

Objective: Proinflammatory cytokines are cytotoxic to beta-cells and have been implicated in the pathogenesis of type 1 diabetes and islet graft failure. The importance of the intrinsic mitochondrial apoptotic pathway in cytokine-induced beta-cell death is unclear. Here, cytokine activation of the intrinsic apoptotic pathway and the role of the two proapoptotic Bcl-2 proteins, Bad and Bax, were examined in beta-cells.

Research design and methods: Human and rat islets and INS-1 cells were exposed to a combination of proinflammatory cytokines (interleukin-1beta, interferon-gamma, and/or tumor necrosis factor-alpha). Activation of Bad was determined by Ser136 dephosphorylation, mitochondrial stress by changes in mitochondrial metabolic activity and cytochrome c release, downstream apoptotic signaling by activation of caspase-9 and -3, and DNA fragmentation. The inhibitors FK506 and V5 were used to investigate the role of Bad and Bax activation, respectively.

Results: We found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser136, mitochondrial stress, cytochrome c release, activation of caspase-9 and -3, and DNA fragmentation. Inhibition of Bad Ser136 dephosphorylation or Bax was found to inhibit cytokine-induced intrinsic proapoptotic signaling.

Conclusions: Our findings demonstrate that the intrinsic mitochondrial apoptotic pathway contributes significantly to cytokine-induced beta-cell death and suggest a functional role of calcineurin-mediated Bad Ser136 dephosphorylation and Bax activity in cytokine-induced apoptosis.

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Cell-permeable V5 inhibits cytokine-induced cytochrome c release and improves mitochondrial metabolic activity in human islets. Cells were pre-exposed to V5 (100 μmol/l) for 1 h before exposure to cytokines for 24 h. A and B: Immunoblotting was performed on fractionated cell lysates from human islets to assess the cellular localization of cytochrome c. COX4, a mitochondrial protein, was used as a control for the fractionation. Quantifications are shown as the ratio of cytosolic to mitochondrial cytochrome c (A). Representative gels from three independent experiments are shown (B). C: Changes in the mitochondrial activity in human islets were measured with the MTT assay as described in research design and methods. Bars represent means ± SE for five independent experiments. *P < 0.05, ** P < 0.01 vs. treatment without cytokines.
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Figure 1: Cell-permeable V5 inhibits cytokine-induced cytochrome c release and improves mitochondrial metabolic activity in human islets. Cells were pre-exposed to V5 (100 μmol/l) for 1 h before exposure to cytokines for 24 h. A and B: Immunoblotting was performed on fractionated cell lysates from human islets to assess the cellular localization of cytochrome c. COX4, a mitochondrial protein, was used as a control for the fractionation. Quantifications are shown as the ratio of cytosolic to mitochondrial cytochrome c (A). Representative gels from three independent experiments are shown (B). C: Changes in the mitochondrial activity in human islets were measured with the MTT assay as described in research design and methods. Bars represent means ± SE for five independent experiments. *P < 0.05, ** P < 0.01 vs. treatment without cytokines.

Mentions: To investigate if cytokines induce mitochondrial stress in human islets, we investigated the effects of cytokine exposure on mitochondrial cytochrome c release and mitochondrial metabolic activity. We used cell fractionation followed by immunoblotting to observe a movement of cytochrome c from the mitochondrial fraction into the cytosolic fraction after cytokine exposure (Fig. 1A and B). Immunoblotting for COX4 was used to confirm that the cytosolic fractions were not contaminated with mitochondrial proteins. Cytokine exposure also led to a significant decrease in mitochondria metabolic activity examined with the MTT assay (Fig. 1C).


Proinflammatory cytokines activate the intrinsic apoptotic pathway in beta-cells.

Grunnet LG, Aikin R, Tonnesen MF, Paraskevas S, Blaabjerg L, Størling J, Rosenberg L, Billestrup N, Maysinger D, Mandrup-Poulsen T - Diabetes (2009)

Cell-permeable V5 inhibits cytokine-induced cytochrome c release and improves mitochondrial metabolic activity in human islets. Cells were pre-exposed to V5 (100 μmol/l) for 1 h before exposure to cytokines for 24 h. A and B: Immunoblotting was performed on fractionated cell lysates from human islets to assess the cellular localization of cytochrome c. COX4, a mitochondrial protein, was used as a control for the fractionation. Quantifications are shown as the ratio of cytosolic to mitochondrial cytochrome c (A). Representative gels from three independent experiments are shown (B). C: Changes in the mitochondrial activity in human islets were measured with the MTT assay as described in research design and methods. Bars represent means ± SE for five independent experiments. *P < 0.05, ** P < 0.01 vs. treatment without cytokines.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2712790&req=5

Figure 1: Cell-permeable V5 inhibits cytokine-induced cytochrome c release and improves mitochondrial metabolic activity in human islets. Cells were pre-exposed to V5 (100 μmol/l) for 1 h before exposure to cytokines for 24 h. A and B: Immunoblotting was performed on fractionated cell lysates from human islets to assess the cellular localization of cytochrome c. COX4, a mitochondrial protein, was used as a control for the fractionation. Quantifications are shown as the ratio of cytosolic to mitochondrial cytochrome c (A). Representative gels from three independent experiments are shown (B). C: Changes in the mitochondrial activity in human islets were measured with the MTT assay as described in research design and methods. Bars represent means ± SE for five independent experiments. *P < 0.05, ** P < 0.01 vs. treatment without cytokines.
Mentions: To investigate if cytokines induce mitochondrial stress in human islets, we investigated the effects of cytokine exposure on mitochondrial cytochrome c release and mitochondrial metabolic activity. We used cell fractionation followed by immunoblotting to observe a movement of cytochrome c from the mitochondrial fraction into the cytosolic fraction after cytokine exposure (Fig. 1A and B). Immunoblotting for COX4 was used to confirm that the cytosolic fractions were not contaminated with mitochondrial proteins. Cytokine exposure also led to a significant decrease in mitochondria metabolic activity examined with the MTT assay (Fig. 1C).

Bottom Line: We found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser136, mitochondrial stress, cytochrome c release, activation of caspase-9 and -3, and DNA fragmentation.Inhibition of Bad Ser136 dephosphorylation or Bax was found to inhibit cytokine-induced intrinsic proapoptotic signaling.Our findings demonstrate that the intrinsic mitochondrial apoptotic pathway contributes significantly to cytokine-induced beta-cell death and suggest a functional role of calcineurin-mediated Bad Ser136 dephosphorylation and Bax activity in cytokine-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Diabetology, Steno Diabetes Center, Gentofte, Denmark.

ABSTRACT

Objective: Proinflammatory cytokines are cytotoxic to beta-cells and have been implicated in the pathogenesis of type 1 diabetes and islet graft failure. The importance of the intrinsic mitochondrial apoptotic pathway in cytokine-induced beta-cell death is unclear. Here, cytokine activation of the intrinsic apoptotic pathway and the role of the two proapoptotic Bcl-2 proteins, Bad and Bax, were examined in beta-cells.

Research design and methods: Human and rat islets and INS-1 cells were exposed to a combination of proinflammatory cytokines (interleukin-1beta, interferon-gamma, and/or tumor necrosis factor-alpha). Activation of Bad was determined by Ser136 dephosphorylation, mitochondrial stress by changes in mitochondrial metabolic activity and cytochrome c release, downstream apoptotic signaling by activation of caspase-9 and -3, and DNA fragmentation. The inhibitors FK506 and V5 were used to investigate the role of Bad and Bax activation, respectively.

Results: We found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser136, mitochondrial stress, cytochrome c release, activation of caspase-9 and -3, and DNA fragmentation. Inhibition of Bad Ser136 dephosphorylation or Bax was found to inhibit cytokine-induced intrinsic proapoptotic signaling.

Conclusions: Our findings demonstrate that the intrinsic mitochondrial apoptotic pathway contributes significantly to cytokine-induced beta-cell death and suggest a functional role of calcineurin-mediated Bad Ser136 dephosphorylation and Bax activity in cytokine-induced apoptosis.

Show MeSH
Related in: MedlinePlus