Chronic activation of neutral ceramidase protects beta-cells against cytokine-induced apoptosis.
OBJECTIVE
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-1beta, 10 ng/mL TNF-alpha, and 50 ng/mL interferon-gamma). 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 NCDase. 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.


CONCLUSIONS
The N-CDase pathway is active in INS-1 cells, and the chronic activation of N-CDase is involved in the pathological response of beta-cells to cytokines, potentially providing protection against cytokine toxicity.
Zhu
Qun
Q
Department of Endocrinology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
Jin
Jun-Fei
JF
Shan
Xiao-Hong
XH
Liu
Cui-Ping
CP
Mao
Xiao-Dong
XD
Xu
Kuan-Feng
KF
Liu
Chao
C
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D004396:Coloring Agents / Q000378:metabolism; D016895:Culture Media, Serum-Free; D016207:Cytokines / Q000235:genetics / Q000633:toxicity*; D004789:Enzyme Activation / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D009336:Necrosis / Q000378:metabolism; D055576:Neutral Ceramidase / Q000378:metabolism*; D011419:Propidium / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000633:toxicity; D013997:Time Factors
D017304:Annexin A5; D004396:Coloring Agents; D016895:Culture Media, Serum-Free; D016207:Cytokines; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011419:Propidium; D055576:Neutral Ceramidase; D016650:Fluorescein-5-isothiocyanate