Suppression of the proapoptotic proteins Bid, Bim, or Bad does not protect pancreatic beta cells against pro‐inflammatory cytokines

Abstract

Pro-inflammatory cytokines such as interleukin-1β (IL-1β) and interferon-γ (IFN-γ) mediate the autoimmune destruction of pancreatic beta cells in type 1 diabetes. It has been suggested that apoptosis plays a major role in cytokine-mediated beta cell death. Therefore, we hypothesized that disrupting the apoptotic program would prevent cytokine-mediated cytotoxicity in 832/13 INS-1-derived cells. Thus, we transfected small interfering RNAs (siRNAs) specific for the BH3-only proapoptotic proteins Bid, Bim, and Bad into cytokine-sensitive 832/13 cells, yielding respective reductions in protein content of 75, 75, and 85 % relative to a scrambled control siRNA. After 48 h, cells were exposed to media alone or that containing 10 ng/mL IL-1β and 100 U/mL IFN-γ for 24 h. Compared to control siRNA-treated cells, reductions in Bid, Bim, or Bad did not improve cell viability (73±2, 75±2, 59±3, and 58±5 %) or decrease nitrite production (12±2, 14±4, 13±2, and 15±1 μ M) following exposure to cytokines. Importantly, pro-inflammatory cytokines did not increase caspase 3 activity (a hallmark of apoptosis), but the apoptotic inducers campothecin and etoposide robustly increased caspase 3 activity (1.2±0.1, 7.8±1.1, and 5.0±1.2 fold relative to a media-treated control). Interestingly, suppression of Bid, Bim, or Bad alone was not able to block cell death induced by camptothecin or etoposide, presumably due to the redundancy of the role of BH3-only proteins in apoptotic signaling cascades. In summary, selective suppression of BH3-only proapoptotic proteins does not afford any protection against pro-inflammatory cytotoxicity. Therefore, we conclude that apoptosis may not be a major mechanism of cytokine-mediated cytotoxicity in rat insulinoma cells. Supported by NIH grant DK55188-04 (to CBN)