The Effect Of PA On Pancreatic Derived Factor Expression In βTC6Cells As Well As The Protective Role Of Exendin-4

Objective The mouse insulinoma cell line βTC6cells were treated withdifferent concentration of palmitic acid (PA) for different times, and the c-junNH2-terminal kinase (JNK)-specific inhibitor, SP600125was added to observe theexpression of PANDER as well as the level of phosphorylated JNK and its substrateprotein c-jun, to explore the effects of PA on PANDER expression in β cells and therole of JNK signaling pathway plays on this process. A small interfering (si)RNA wasconstructed to interfere with the PANDER expression, the PA induced β-cell apoptosisand the activation of a major apoptotic molecule, cysteine containing aspartatespecific protease(caspase)-3, were measured to investigate the relationship betweenPANDER and PA mediated β-cell apoptosis. βTC6cells were pretreated withExendin-4and then treated with combination of PA and Exendin-4to observe theeffects of Exendin-4on PA-induced apoptosis and PANDER expression, to determinethe possible mechanism of Exendin-4on protecting β cells from lipoapoptosis.Part One The effects of PA on the expression of PANDER in βTC6cellsMethods (1) βTC6cells were treated with different concentration of PA (0,0.125,0.25,0.5或1.0mmol/L) for different times (0,6,12,24或48h), the expressionof PANDER mRNA and protein were detected by quantitative Real Time RT-PCR andWestern blotting respectively.(2) βTC6cells were treated with0.5mmol/L of PA for0,0.1,0.25,0.5,1,6,12and24h, the expression of JNK, p-JNK, c-jun, p-c-junwas detected using Western blotting.(3) βTC6cells were divided into four groups:control (treated with fresh medium), PA (treated with0.5mmol/L of PA for24h),SP600125(treated with25umol/L of SP600125for24h) and PA+SP600125 (pretreated with25umol/L of SP600125for1h, and then treated with thecombination of PA and SP600125for24h). The expression of PANDER, JNK, p-JNK,c-jun and p-c-jun was detected using Western blotting.Results (1) PANDER expression could be enhanced when the cells werestimulated with higher concentration of PA for increasing lengths of time. The peakvalue of PA-induced PANDER mRNA expression appeared at0.5mmol/L with12hincubation period as well as0.5mmol/L with24h incubation period at the proteinlevels of PANDER expression.(2) JNK could be actived by PA, and the peak valueappeared at6min and12h.(3)24-h exposure to PA significantly increased thephosphorylation of JNK, pretreatment of βTc6cells with SP600125reducedPA-induced PANDER expression.Conclusions PA up-regulates PANDER expression in β-cells through theactivation of JNK signaling pathway.Part Two The contribution of PANDER to PA-induced apoptosis in βTC6cellsMethods (1) Six siRNAs were designed and synthesized in vitro according tothe sequence of mouse PANDER gene(GenBank Accession Number:52793). ThesiRNAs were transfected respectively into βTC6cells using Lipofectamine2000. Cellscultured in medium (Blank Control, BC) or transfected with Negative siRNA(Negative Control, NC) or exposured to Lipofectamine2000(mock) served as thecontrol. The expression of PANDER mRNA and protein were detected by quantitativeReal Time RT-PCR and Western blotting respectively.(2) βTC6cells were dividedinto four groups: NC, siRNA, NC+PA and siRNA+PA. The concentration and theintervention time of PA were chosen according to the results of the part-oneexperiment of this study (0.5mmol/L of PA for24h). The apoptosis of βTC6cellswere detected by the terminal dexynucleotidyl transferase (TdT)-mediated dUTP nickend labeling(TUNEL) and the flow cytometric analysis.The cleaved caspase-3wasdetected by Western blotting to assess the activated level of caspase-3.Results (1) βTC6cells transfected with PANDER siRNA(Fam3b-mus-208) caused about72%decrease in the expression of PANDER mRNA as well as about60%decrease at the protein level of PANDER when compared with NC.(2) The24-hexposure to PA significantly increased the level of cleaved caspase-3as well as thepercentage of apoptotic cells in NC, whereas after24-h exposure to PA, the level ofcleaved caspase-3and apoptosis rate in PANDER siRNA transfected cells was lessthan that in NC transfected cells.Conclusions PANDER may play a role in β-cell lipoapoptosis throughactivating caspase-3.Part three The protective role of Exendin-4against βTC6-cell lipoapoptosisMethods (1) βTC6cells were pretreated with increasing concentration ofExendin-4(0,12.5,25,50and100nmol/L) for24h, and then treated withcombination of PA and Exendin-4for24h, the expression of PANDER wasdetected by Western blotting.(2) βTC6cells were divided into four groups: control(treated with fresh medium), Exendin-4(treated with50nmol/L of Exendin-4for24h)PA(treated with0.5mmol/L of PA for24h), and Exendin-4+PA (pretreated with50nmol/L of Exendin-4for24h, and then treated with the combination of PA andExendin-4for24h). The concentration and the intervention time of Exendin-4werechosen according to the results of the previous experiment of this part. The apoptosisof βTC6cells were detected by TUNEL and the flow cytometric analysis. Theexpression of PANDER, JNK, p-JNK, c-jun, p-c-jun and cleaved caspase-3wasdetected using Western blotting.Results (1) Exendin-4decreased PA-induced PANDER expression in adose-dependent manner in βTC6cells.(2) The PA-induced toxicity could be partlyprevented in the presence of Exendin-4, and24-h exposure to PA significantlyincreased the expression of p-JNK, p-c-jun, PANDER and cleaved caspase-3, whereasthese enhancements could be partly weakened by the addition of Exendin-4in βTC6cells.Conclusions The reverse role of Exendin-4on lipapoptosis of β-cells may bepartly related with interfering JNK-PANDER-caspase-3pathway.