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


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.
Grunnet
Lars G
LG
Department of Translational Diabetology, Steno Diabetes Center, Gentofte, Denmark.
Aikin
Reid
R
Tonnesen
Morten F
MF
Paraskevas
Steven
S
Blaabjerg
Lykke
L
Størling
Joachim
J
Rosenberg
Lawrence
L
Billestrup
Nils
N
Maysinger
Dusica
D
Mandrup-Poulsen
Thomas
T
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002102:Cadaver; D053453:Caspase 9 / Q000378:metabolism; D016923:Cell Death; D016207:Cytokines / Q000494:pharmacology*; D006801:Humans; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D007371:Interferon-gamma / Q000494:pharmacology*; D053583:Interleukin-1beta / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D014019:Tissue Donors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016207:Cytokines; D053583:Interleukin-1beta; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D053453:Caspase 9