Aim:

To investigate the activity and expression of neutral ceramidase (N-CDase) in the insulin-secreting cell line INS-1 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-1 cells treated with a cytokine mixture (5 ng/mL interleukin-1, 10 ng/mL TNF-, and 50 ng/mL interferon-). 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-1 cells.

Results:

The INS-1 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 -cells to cytokines, potentially providing protection against cytokine toxicity.

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