| Objective:G protein-coupled receptor (GPR40) is a specific FFA recptor expressed preferentially inβcells. It is activated by long-chain fatty acids and has been implicated in mediating physiological and pathological effects of long-chain fatty acids onβcells. This study aimed to observe the effect of difference glucose and palmitate(PA) on the gene expression of insulin and GPR40, on the cell viability,apoptosis induced by PA;Examine the role of GPR40 in chronic regulation of insulin secretion in vitro, as well as its implication in long-term lipotoxicity by free fatty acids.Methods:Apply small interference RNA(siRNA)technology and vector transfer methods to changge the expression of GPR40 in INS-1 cells; Culturing the INS-1 cells at different concentrations of glucose and PA for 24-48h, and examined viability with MTT assay, detected insulin levels by radioimmunoassay, and examined cell apoptosis by immuno-fluorescence and flow-cytometry analysis. Reactive oxygen species levels were detected by enzyme linked immunosorbent assay;phosphorylation of IRS,PKB inβcell were tested by Western-blot methods .Results:1. INS-1 cells incubated in 5.6 mM glucose for 24–48h demonstrated no change in basal and glucose simulated insulin secretion. After incubated in 16.7mG or 33.3mG for 48 h had an approximately 30% and 34% decrease in GSIS compared with control INS-1 cells incubated in 5.6 mM glucose (P<0.01). After plus of 0.5 mM PA, GSIS in high glucose and high PA group showed a significant decrease in GSIS as compared with control group. SiRNA transfected cells in the presence of PA for 48h resulted in augment of GSIS compared to untransfected cells. In contrast, insulin secretion from cells transfected with GPR40-pIRESpuro decreased significantly as compared to control group by 48h exposure to PA. The results suggesting that GPR40 is implicated in mediating the long-term negative effect of FFA on GSIS.2.The expression of insulin mRNA in INS-1 cells was decreased in dose and time dependent manner when they were cultured in different concentration of glucose. INS-1 cells incubated in 5.6 mM glucose for 24–48h demonstrated no change in insulin mRNA levels. In contrast, cells incubated in 16.7 mM glucose showed a small decrease in insulin mRNA levels that became apparent after 24 h. INS-1 cells incubated in 33.3 mM glucose showed even further decreases in insulin mRNA levels that were also apparent after 24 h. INS-1 cells incubated in 16.7 or 33.3 mM glucose for 48 h had an approximately 36% or 50% decrease in insulin mRNA levels compared with control INS-1 cells incubated in 5.6 mM glucose (P<0.01). After PA plus, insulin mRNA decreased significantly in high glucose, but has small change in 5.6mG group.3. Difference glucose cultured for 24–48h demonstrated no change in GPR40 mRNA levels. Incubation INS-1 cells with 0.5 mM PA for 48h decrease GPR40 mRNA under 33.3mG conditions as compared with control group.4. Exposure of INS-1cells to 33.3mG for 48h significantly decreased cell viability as compared with 5.6mG group. PA at the dose of 0.5mmol/L for 48 h significantly decreased cell viability at high glucose, suggesting that both high concentrations of glucose and PA may produce cytotoxic effects on INS-1 cells. Viability rates of INS-1 treated with high glucose plus PA decreased more markedly than those treated with high concentrations of glucose or PA alone. After siRNA or GPR40-pIRESpuro transfection to change the expression of GPR40, the viability difference between the high glucose and high PA group was not significant.After culture in different glucose for 48h, the apoptosis rate was increased in high glucose as compared with normal control group. After treating with 0.5mol/l PA for 48 h, the percentage of apoptotic cells in the PA treated group was greater than that in control (P<0.01). To further evaluate whether GPR40 gene in INS-1 cells might affect lipo- apoptosis of INS-1 cells, siRNA and GPR40- pIRES puro transfected cells were supplemented with 0.5mM PA for 48 h. No significant differences of the percentages of apoptotic cells were indicated among the three groups treated with PA.5. High glucose can significantly increase the ROS production after culture for 48h. After treating with 0.5mM PA for 48 h, the production of ROS increase significantly as compared with control group without PA. SiRNA and GPR40-pIRESpuro transfection to change the expression of GPR40 have no obviously effect on the production of ROS as compared with untransfected control group.6. PA treatment can increase the phosphorylation of IRS and decrease the phosphorylation of PKB, which may be partly imprvoved by the inhibion of GPR40 expression. Increase the GPR40 can increase the phosphorylation of IRS and decrease the phosphorylation of PKB. GPR40 may be involed in the impairement of insulin signaling.Conclusion:High glucose and high palmitate can impair the GSIS and insulin signal inβcells. Inhibiting the expression of GPR40 can partly improve the GSIS and insulin signal impaired by PA。...
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