Cytokine-induced β-cell apoptosis is NO-dependent, mitochondria-mediated and inhibited by BCL-X(L)

Pro-inflammatory cytokines are implicated as the main mediators of β-cell death during type 1 diabetes but the exact mechanisms remain unknown. This study examined the effects of interleukin-1β (IL-1β), interferon-γ (IFNγ) and tumour necrosis factor α (TNFα) on a rat insulinoma cell line (RIN-r) in order to identify the core mechanism of cytokine-induced β-cell death. Treatment of cells with a combination of IL-1β and IFNγ (IL-1β/IFNγ)induced apoptotic cell death. TNFα neither induced β-cell death nor did it potentiate the effects of IL-1β, IFNγ or IL-1β/IFNγ . The cytotoxic effect of IL-1β/IFNγ 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-1β/IFNγ -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-1β/IFNγ -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 β-cell death, and have identified Bcl-X(L) as a potential target to combatβ-cell apoptosis.