Chronic activation of neutral ceramidase protects β-cells against cytokine- induced apoptosis

Aim： To investigate the activity and expression of neutral ceramidase （N-CDase） in the insulin-secreting cell line INS-I and its role in the cellular response to cytokines. Methods： HPLC, Western blotting, and quantitative real-time PCR were performed to detect the activity and expression of N-CDase in INS-I cells treated with a cytokine mixture （5 ng/mL interleukin- 1β, 10 ng/mL TNF-α, and 50 ng/mL interferon-γ）. The expression and activity of N-CDase in the INS- 1 cells were specifically inhibited using N-CDase-siRNA transfection. Annexin V-fluorescein-isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis in the INS-I cells. Results： The INS-I cells exhibited some basal N-CDase activity, and cytokines induced a time-dependent delay in the activation of N- CDase. As a result, the activation of N-CDase was first detectable at 8 h after stimulation. It peaked at 16 h and remained elevated at 24 h. Cytokines also upregulated the mRNA and protein expression of N-CDase in the INS-1 cells. Furthermore, when N-CDase activity was inhibited by RNA interference, cytokine- induced apoptosis in the INS- 1 cells was markedly increased. Conclusion： The N-CDase pathway is active in INS-1 cells, and the chronic activation of N-CDase is involved in the pathological response of l-cells to cytokines, potentially providing protection against cytokine toxicity.