Cytokine-induced beta-cell apoptosis is NO-dependent, mitochondria-mediated and inhibited by BCL-XL.
Pro-inflammatory cytokines are implicated as the main mediators of beta-cell death during type 1 diabetes but the exact mechanisms remain unknown. This study examined the effects of interleukin-1beta (IL-1beta), interferon-gamma (IFNgamma) and tumour necrosis factor alpha (TNFalpha) on a rat insulinoma cell line (RIN-r) in order to identify the core mechanism of cytokine-induced beta-cell death. Treatment of cells with a combination of IL-1beta and IFNgamma (IL-1beta/IFNgamma)induced apoptotic cell death. TNFalpha neither induced beta-cell death nor did it potentiate the effects of IL-1beta, IFNgamma or IL-1beta/IFNgamma . The cytotoxic effect of IL-1beta/IFNgamma was associated with the expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide. Adenoviral-mediated expression of iNOS (AdiNOS) alone was sufficient to induce caspase activity and apoptosis. The broad range caspase inhibitor, Boc-D-fmk, blocked IL-1beta/IFNgamma -induced caspase activity, but not nitric oxide production nor cell death. However, pre-treatment with L-NIO, a NOS inhibitor, prevented nitric oxide production, caspase activity and reduced apoptosis. IL-1beta/IFNgamma -induced apoptosis was accompanied by loss of mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9, -7 and -3. Transduction of cells with Ad-Bcl-X(L) blocked both iNOS and cytokine-mediated mitochondrial changes and subsequent apoptosis, downstream of nitric oxide. We conclude that cytokine-induced nitric oxide production is both essential and sufficient for caspase activation and beta-cell death, and have identified Bcl-X(L) as a potential target to combat beta-cell apoptosis.
Holohan
C
C
Department of Biochemistry, National University of Ireland Galway, Galway, Ireland.
Szegezdi
E
E
Ritter
T
T
O'Brien
T
T
Samali
A
A
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D016207:Cytokines / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D007340:Insulinoma / Q000473:pathology; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D008954:Models, Biological; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D051381:Rats; D051020:bcl-X Protein / Q000378:metabolism*
D016207:Cytokines; D051020:bcl-X Protein; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D020169:Caspases