The Intervening Effects Of Exendin-4 On The Free Fatty Acid Sinduced Impairment On PDX-1 Gene Expression And Insulin Secretion In βTc6 Cells

Objective:1.To investigate the changes of pancreatic-duodenal homeobox-1(PDX-1) mRNA transcription, related insulin secretion, morphological change and proliferation induced by free fatty acid (FFA) inβcells.2.Exendin-4 was used to intervene the free fatty acids-induced impairment onβcells, observing the changes of aforesaid research indexes, exploring the protective action of Exendin-4 onβcells in lipotoxic state, analyzing the underlying molecular biological mechanism.Methods:1.Insulinoma cell lineβTc6 cells were cultured in vitro, treating with FFA (0.25mmol/L, 0.5mmol/L, 1.0mmol/L ) for 24 or 48 hours, then the transcription of PDX-1 mRNA, glucose stimulated insulin secretion(GSIS) and theβTc6 cells'proliferation were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), radioimmunoassay and MTT viability assay respectively. In addition the morphological change ofβTc6 cells was observed.2.βTc6 cells were treated with Exendin-4 (5nmol/L, 10nmol/L, 50nmol/L) for different duration(6h, 12h, 24h) prior to 48 hours FFA intervention, then the transcription of PDX-1 mRNA, glucose stimulated insulin secretion (GSIS) and theβTc6 cells'proliferation were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), radioimmunoassay and MTT viability assay respectively. In addition the morphological change ofβTc6 cells was observed.Results:1.The expression of PDX-1 mRNA increased gradually after intervening with 0.25～1.00 mmol/L FFA for 24 hours, but it decreased when the intervening duration prolonged to 48 hours, espectially with 1.00 mmol/L FFA.2.After treating with 0.50～1.00 mmol/L FFA for 24～48 hours, theβTc6 cells tended to be apoptosis in morphology, and the corresponding proliferation and GSIS also decreased gradually. But theβTc6 cells treating with 0.25mmol/L FFA for 24 hours didn't appear aforesaid similar changes.3. The intervening effects of Exendin-4 on the PDX-1 mRNA transcription ofβTc6 cells in lipotoxic state.(1)βTc6 cells treating with 5nmol/L～50nmol/L Exendin-4 for 6 hours in advance would not up-regulate the transcription of PDX-1 mRNA in lipotoxic state.(2)βTc6 cells treating with 5nmol/L or 10nmol/L Exendin-4 for 12 hours in advance would up-regulate the transcription of PDX-1 mRNA in lipotoxic state gradually, but it did not keep on up-regulating when Exendin-4 concentration raised to 50nmol/L furthermore.(3)Treating with 5nmol/L～50nmol/L Exendin-4 for 24 hours beforehand, theβTc6 cells in lipotoxic state would up-regulate the transcription of PDX-1 mRNA markedly, and there was no statistical difference among different Exendin-4 concentration groups.4. The intervening effects of Exendin-4 on insulin secretion and proliferation ofβTc6 cells in lipotoxic state.(1)Treating with 5nmol/L Exendin-4 for 6～12 hours beforehand, the insulin secretion and proliferation ofβTc6 cells in lipotoxic state would not improve.(2)When Exendin-4 concentration setted in the range from 5nmol/L to 50nmol/L, the insulin secretion and proliferation ofβTc6 cells in lipotoxic state would become better along with the Exendin-4 intervening duration prolonging to 24 hours.(3)When Exendin-4 concentration raised from 10nmol/L to 50nmol/L, the insulin secretion and proliferation ofβTc6 cells in lipotoxic state could not improve furthermore.Conclusions:1.Abnormal FFA intervention was toxic for isletβcells. FFA with lower concentration would up-regulate the expression of PDX-1, and would not affectβcells'proliferation and insulin secretion in a short time.2.Along with the increase of FFA intervening concentration and duration, the PDX-1 mRNA transcription ofβcells down-regulated, and its proliferation and insulin secretion were impaired.3.Treating with Exendin-4 in optimal concentration and duration beforehand, the PDX-1 mRNA transcription ofβcells up-regulated, and its proliferation and insulin secretion improved obviously.