Acta Pharmacologica Sinica 2008 May; 29 (5): 593-599; doi: 10.1111/j.1745-7254.2008.00781.x

 
ORIGINAL ARTICLES
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Chronic activation of neutral ceramidase protects β-cells against cytokine-induced apoptosis1
 

Qun ZHU2,6,7, Jun-fei JIN3,4,6, Xiao-hong SHAN5, Cui-ping LIU2, Xiao-dong MAO2, Kuan-feng XU2, Chao LIU2,8

2Department of Endocrinology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China; 3Research Center of Life Science, University of South China, Hengyang 421001, China; 4Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA; 5The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China

 

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-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-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 β-cells to cytokines, potentially providing protection against cytokine toxicity.

 

Keywords: neutral ceramidase; cytokines; β-cells; apoptosis; diabetes

 
1 This work was supported by Medical Key Subject grants from Jiangsu Province of China (No SK200214).
6These authors contributed equally to this work.
7Present address: Department of Endocrinology, Second Affiliated Hospital of Nanjing Medical University, Nanjing 210011, China.

8 Correspondence to Prof Chao LIU.
Phn 86-25-8371-8836, ext 6466.
Fax 86-25-8367-4006.
E-mail drchliu@hotmail.com
Received 2007-10-14     Accepted 2008-01-24

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