Limits...
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.

Show MeSH

Related in: MedlinePlus

Cytokines induce caspase-9 activation in INS-1 cells and rat islets. Cleavage of caspase-9 after cytokine exposure was assessed by immunoblotting in INS-1 cells (A) and rat islets (C). Representative gels of five independent experiments (A) or three independent experiments (C) are shown; results were adjusted for actin and tubulin levels. Caspase-9 activity in INS-1 cells (n = 5) (B) and rat islets (n = 2) (D) in the presence or absence of cytokines was measured as described in research design and methods. Bars represent means ± SE (A, B, and C) or means with indication of the data from the individual experiments (D). *P < 0.05, ** P < 0.01 vs. the control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2712790&req=5

Figure 3: Cytokines induce caspase-9 activation in INS-1 cells and rat islets. Cleavage of caspase-9 after cytokine exposure was assessed by immunoblotting in INS-1 cells (A) and rat islets (C). Representative gels of five independent experiments (A) or three independent experiments (C) are shown; results were adjusted for actin and tubulin levels. Caspase-9 activity in INS-1 cells (n = 5) (B) and rat islets (n = 2) (D) in the presence or absence of cytokines was measured as described in research design and methods. Bars represent means ± SE (A, B, and C) or means with indication of the data from the individual experiments (D). *P < 0.05, ** P < 0.01 vs. the control.

Mentions: Data are presented as means ± SE. Statistical significance was determined by Student's paired t test (on the planned comparisons reported) or by two-way ANOVA with Dunnett's post hoc test (time course experiments) (Figs. 3A and 5A), with P < 0.05 as the level of significance.


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)

Cytokines induce caspase-9 activation in INS-1 cells and rat islets. Cleavage of caspase-9 after cytokine exposure was assessed by immunoblotting in INS-1 cells (A) and rat islets (C). Representative gels of five independent experiments (A) or three independent experiments (C) are shown; results were adjusted for actin and tubulin levels. Caspase-9 activity in INS-1 cells (n = 5) (B) and rat islets (n = 2) (D) in the presence or absence of cytokines was measured as described in research design and methods. Bars represent means ± SE (A, B, and C) or means with indication of the data from the individual experiments (D). *P < 0.05, ** P < 0.01 vs. the control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Cytokines induce caspase-9 activation in INS-1 cells and rat islets. Cleavage of caspase-9 after cytokine exposure was assessed by immunoblotting in INS-1 cells (A) and rat islets (C). Representative gels of five independent experiments (A) or three independent experiments (C) are shown; results were adjusted for actin and tubulin levels. Caspase-9 activity in INS-1 cells (n = 5) (B) and rat islets (n = 2) (D) in the presence or absence of cytokines was measured as described in research design and methods. Bars represent means ± SE (A, B, and C) or means with indication of the data from the individual experiments (D). *P < 0.05, ** P < 0.01 vs. the control.
Mentions: Data are presented as means ± SE. Statistical significance was determined by Student's paired t test (on the planned comparisons reported) or by two-way ANOVA with Dunnett's post hoc test (time course experiments) (Figs. 3A and 5A), with P < 0.05 as the level of significance.

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