The Intervening Effects Of Exendin-4 On The Resistin-induced Impairment And PDX-1 Gene Expression In βTc6 Cells

Objective:1.To investigate the changes of pancreatic-duodenal homeobox-1(PDX-1) mRNA expression, insulin secretion, morphological change, proliferation,and apoptosis induced by resistin inβcells.2. To evaluate the intervening effects of Exendin-4 on the resistin induced impairment and PDX-1 gene expression inβTc6 cells.Methods:1.Insulinoma cell lineβTc6 cells were cultured in vitro, treating with resistin (20 ng/ml, 40 ng/ml, 60 ng/ml, 100 ng/ml, 200 ng/ml) for 6 or12 or 24 hours, then the expression of PDX-1 mRNA, glucose stimulated insulin secretion(GSIS),theβTc6 cells'proliferation and cell apoptosis were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), radioimmunoassay,CCK-8 viability assay and DNA laddering assay respectively. In addition the morphological change ofβTc6 cells was observed.2.βTc6 cells were treated with Exendin-4 (5 nmol/L, 10 nmol/L, 50 nmol/L) for different duration(6h, 12h, 24h) prior to 24 hours resistin intervention, then the expression of PDX-1 mRNA, glucose stimulated insulin secretion (GSIS) ,theβTc6 cells'proliferation and cell apoptosis were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), radioimmunoassay,CCK-8 viability assay and DNA laddering assay respectively. In addition the morphological change ofβTc6 cells was observed.Results:1. After treating with resistin 60～200 ng/ml for 6～24 hours, theβTc6 cells tended to be apoptosis in morphology, and the corresponding proliferation and GSIS also decreased gradually. But theβTc6 cells treating with resisntin 20～40 ng/ml didn't appear aforesaid similar changes.2. The expression of PDX-1 mRNA didn't change after intervening with 60～100 ng/ml resistin for 6 hours, but it decreased when resistin was 200 ng/ml.When the intervening duration prolonged to 24 hours, the expression of PDX-1 mRNA was decreased, espectially with 60ng/ml resistin for 24 hours.But theβTc6 cells treating with resisntin 20～40 ng/ml didn't appear aforesaid similar changes.3. After treating with resistin 20~200 ng/ml for 6 hours, resistin didn't induce the formation of the DNA ladder. When treating with resistin 200 ng/ml for 12 hours, it could induce the formation of the DNA ladder. When the intervening duration prolonged to 24 hours, both 100 ng/ml and 200 ng/ml resistin treatment could induce the formation of the DNA ladder.4. The intervening effects of Exendin-4 on insulin secretion and proliferation ofβTc6 cells in resistin state as below.(1)Treating with 5 nmol/L Exendin-4 for 6～12 hours beforehand, the insulin secretion and proliferation ofβTc6 cells in resistin state would not improve.When Exendin-4 intervening duration prolonging to 24 hours, the insulin secretion and proliferation ofβTc6 cells would become better.(2)When Exendin-4 concentration setted in the range from 5 nmol/L to 50 nmol/L, the insulin secretion and proliferation ofβTc6 cells in resistin state would become better along with the Exendin-4 intervening duration prolonging to 24 hours.(3)When Exendin-4 concentration raised from 10 nmol/L to 50 nmol/L, the insulin secretion and proliferation ofβTc6 cells in resistin state could not improve furthermore.5. The intervening effects of Exendin-4 on the PDX-1 mRNA expression ofβTc6 cells in resisntin state as below.(1)βTc6 cells treating with 5 nmol/L～50 nmol/L Exendin-4 for 6 hours in advance would not up-regulate the expression n of PDX-1 mRNA in resistin state. (2)βTc6 cells treating with 5 nmol/L or 10 nmol/L Exendin-4 for 12 hours in advance would up-regulate the expression of PDX-1 mRNA in resiatin state gradually, but it did not keep on up-regulating when Exendin-4 concentration raised to 50 nmol/L furthermore.(3)Treating with 5 nmol/L～50 nmol/L Exendin-4 for 24 hours beforehand, theβTc6 cells in resistin state would up-regulate the expression of PDX-1 mRNA markedly, and there was no statistical difference among different Exendin-4 concentration groups.6. The protection effects of Exendin-4 on the cell apoptosis ofβTc6 cells in resisntin state as below.(1) Resistin inducedβTc6 cells apoptosis would not be prevented by treating with 5 nmol/L～50 nmol/L Exendin-4 for 6 hours in advance.(2) When Exendin-4 intervening duration prolonging to 12 hours, although 5 nmol/L Exendin-4 would not preventβTc6 cells from apoptosis, but 10~50 nmol/L Exendin-4 could preventβTc6 cells from apoptosis markly.(3) Treating with 5 nmol/L～50 nmol/L Exendin-4 for 24 hours beforehand, theβTc6 cells apoptosis induced by resistin would be prevented.Conclusions:1. Resistin with lower concentration did not affect the expression of PDX-1, and can not affectβcells'proliferation, insulin secretion or cell apoptosis in a short-term.2.Along with the increase of resistin intervening concentration and duration, the PDX-1 mRNA expression ofβcells is down-regulated, cell apoptosis is increased, and its proliferation and insulin secretion are impaired.3.Treating with Exendin-4 in optimal concentration and duration beforehand, the PDX-1 mRNA expression ofβcells is up-regulated,βcells apoptosis is prevented, and its proliferation and insulin secretion are improved obviously.