Sphingosine-1-phosphate And Neutral Ceramidase Protect Islet β-cells Against Cytokine-induced Apoptosis

Objectives:(1)To observe whether exogenous sphingosine- 1-phosphate(S1P)alleviates the cytokine-induced apoptosis in isletβ-cells.(2)To investigate the probable mechanism by which S1P exerts its protective effects on isletβ-cells.(3)To observe whether the activity and the expression of neutral ceramidase (N-CDase)are present in isletβ-cells.(4)To observe the activity and expression of N-CDase in isletβ-cells in response to cytokines.(5)To observe whether the inhibition of the activity of N-CDase in isletβ-cells affects the cytokine-induced apoptosis.Methods:(1)Rat isletβ-cells line INS-1 cells were grown in RPMI-1640 medium supplemented with 10 mM Hepes,10%fetal calf serum(FCS),2 mM L-glutamine,1 mM sodium pyruvate,50μM 2-mercaptoethanol.Then, INS-1 cells were stimulated by a cytokine mixture(IL-1β5 ng/mL;TNF-α 10 ng/mL;IFN-γ50 ng/mL),and the apoptosis in INS-1 cells was detected by flow cytometry.(2)INS-1 cells were pretreated by 1μM SIP for 1 h,then the cells were co-incubated by SIP and cytokines for 24 h.The apoptosis in INS-1 cells was detected by flow cytometry.(3)Western-blot method was applied to detect the protein expression of cleaved-caspase-3 in INS-1 cells.(4)Organic extract and HPLC methods were applied to quantificationally assay the activity of N-CDase in INS-1 cells treated or untreated by cytokines.(5)Semiquantitative PCR and quantitative real-time PCR were performed to detect the gene expression of N-CDase in INS-1 cells treated or untreated by cytokines.Western-blot analysis was applied to detect the protein expression of N-CDase in INS-1 cells.(6)D-MAPP,an inhibitor of N-CDase,was applied to inhibit the activity of N-CDase,and HPLC assay was performed to confirm the decreased activity of N-CDase by D-MAPP.(7)INS-1 cells were treated by cytokines and D-MAPP,and the apoptosis in INS-1 cells was detected by flow cytometry.Results: (1)INS-1 cells growed well by adhering to the wall.There was no obvious apoptosis in normal cultured INS-1 cells,whereas treatment with a cytokine mixture(IL-1β,TNF-αand IFN-γ)for 24 h induced significant apoptosis in INS-1 cells.(2)Pretreatment and co-incubation with 1μM S1P significantly alleviated the apoptosis in INS-1 cells induced by cytokines.(3)The expression of cleaved-caspase-3 in INS-1 cells treated with cytokines was significantly inhibited by pretreatment and co-incubation with 1μM S1P.(4)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.(5)Cytokines upregulated the protein expression of N-CDase in the INS-1 cells,and this upregulation was consistent with the time-course effects of cytokines on the activation of N-CDase in INS-1 cells.(6)A marked increase in N-CDase mRNA was observed as early as 4 h after treatment by cytokines,and the mRNA levels peaked at 12 h.(7)Pretreatment and co-incubation with 10μM D-MAPP significantly inhibited the activity of N-CDase in INS-1 cells treated or untreated with cytokines.(8)Pretreatment and co-incubation with 10μM D-MAPP markedly enhanced the apoptosis in INS-1 cells induced by cytokines.Conclusions:(1)Treatment with a cytokine mixture induces significant apoptosis in INS-1 cells,whereas treatment by the low concentration of S1P alleviates the apoptosis in INS-1 cells induced by cytokines.(2)Treatment with S1P reduces the expression of proapoptotic protein, which mediates the protective effects on cytokine-induced apoptosis in INS-1 cells.(3)N-CDase is active in INS-1 cells,and the activation of N-CDase can protect pancreaticβ-cells against cytokine-induced apoptosis.