The Effect Of FFAR1on Pioglitazone-attenuating Palmitic Acid-induced Lipoapoptosis And Oxidative Stress In β Cells

Background:It is the key pathway to block β cells lipotoxicity-induced damage for preventingand treating diabetes, but it still lacks of specific intervention to lipotoxicity. In ourprevious study, we have found that down-regulating the expression of free fatty acidreceptor-1(FFAR1) could weaken the anti-lipotoxicity effect of PPARγ agonistPioglitazone(PIO) on insulin impaired secretion of β cells. Our results indicated thatFFAR1might mediate the anti-lipotoxicity role of PPARγ agonist. Therefore, thisstudy was conducted to investigate whether FFAR1could mediate the effect of PIO inprotecting β cells against lipotoxicity, and discuss the potential underlying mechanismto understand the value of FFAR1in lipotoxicity protection and its potential role indiabetes.Objective:(1) To observe whether FFAR1can mediate the effect of PIO in protecting β cellsagainst lipotoxicity.(2) To explore the relationship between the mediating action of FFAR1onPIO-attenuating lipoapoptosis and oxidative stress.(3) To investigate whether the influence of FFAR1on the anti-lipotoxic effect of PIOwas mediated through the PLCγ-ERK1/2-PPARγ pathway.Method:(1) The glucose-sensitive mouse beta pancreatic cell line βTC6was set as an object ofresearch. βTC6cells were infected with the RNAi lentiviral vector （siRNA）orFFAR1-expressing lentiviral vector（FFAR1+）to regulate FFAR1gene expression;βTC6cells were exposed to1.0mmol/L of PA for24hours to mimic conditions ofthe lipotoxic β-cell model, and then cultured with different concentrations of PIO fordifferent time periods. The effect of FFAR1on PIO in protecting β cells against lipotoxicity was observed.(2) βTC6cells infected without lentiviral vector or FFAR1siRNA or FFAR1+wereexposed with1.0mmol/L PA for24hours, and then were treated to PIO; H2O2inducing oxidative stress was used as the positive control to examine whether FFAR1is involved in the anti-oxidative stress effect of PIO.(3) Regulating the gene expression of FFAR1or using the signaling inhibitor toinhibit the activity of signaling proteins; TAK-875, a specific agonist of FFAR1, wasused as the positive control to examine whether FFAR1activation affected the PLCγ-ERK1/2-PPARγ.(4) Techniques: β-cell apoptosis was detected by flow cytometric analysis andTUNEL assay; expression of FFAR1gene was detected by RT-PCR; the protein levelsof FFAR1, oxidative stress-related proteins (NAPDH oxidase subunit p47phox, bcl-2,Bax, cleaved caspase3) and the signal proteins (PLCγ, ERK1/2, PPARγ) wereexamined by Western blot; the active oxygen content was detected by DCFH-DAprobe; the intracellular concentration of IP3was detected by ELISA assay.Results:(1) PIO reduced PA-induced lipoapoptosis in β cells and upregulated the expression ofFFAR1at the mRNA and protein levels in a dose-and time-dependent manner.Silencing of FFAR1expression was shown to weaken the protective effect of PIO onPA-induced lipoapoptosis in βTC6cells; while lentiviral-mediated overexpression ofFFAR1was shown to enhance the protective effect of PIO against lipoapoptosis in βcells. The levels of FFAR1expression were negatively correlated with β-cellapoptosis.(2) Downregulation of FFAR1expression reduced the attenuating effect of PIO on theexpression of NAPDH oxidase subunit p47phox, Bax, cleaved caspase3, and theproduction of reactive oxygen specific (ROS) induced by lipotoxicity, therebypreventing the upregulation of the expression of bcl-2. Inducing the overexpression ofFFAR1enhanced the anti-oxidative stress effect of PIO. Similarly, these effects of FFAR1on PIO were reproduced under conditions of oxidative stress and apoptosis inβTC6cells that were induced by H2O2.(3) PIO and TAK-875were found to increase the expression of PLCγ, ERK1/2,PPARγ and the intracellular concentration of IP3in lipotoxic β cells. SilencingFFAR1expression reduced the PIO-mediated increases in the expression of aboveproteins; while inducing FFAR1overexpression showed the opposite effect. Use of aninhibitor of PLCγ, ERK1/2, PPARγ was shown restrict the protective effect of PIO onoxidative stress and lipoapoptosis of β cells.Conclusions:FFAR1can mediate PIO suppression of β-cell lipoapoptosis through anti-oxidative stress, which may be related to the activation of the PLCγ-ERK1/2-PPARγpathway.