The Intervening Effects Of Exendin-4 On The Free Fatty Acids- Induced Impairment On GK And GLUT2 Expression In βTc6 Cells

Objective:1. To investigate the changes of glucokinase (GK) and glucose transporter 2 (GLUT2) gene expression induced by free fatty acid (FFA) inβTc6 cells.2.Exendin-4 was used to intervene the FFA-induced impairment onβcells, observing the changes of aforesaid research indexes, exploring the protective action of Exendin-4 onβcells in lipotoxic state.Methods:1.βTc6 cells were cultured in vitro, treating with FFA (0.25mmol/L, 0.5mmol/L and 1.0mmol/L) for 24 hours, then examined GK and GLUT2 expression by real-time PCR and western blot analysis. In addition the morphological change ofβTc6 cells was observed.2.βTc6 cells were incubated with Exendin-4 (5nmol/L, 10nmol/L and 50nmol/L) for different duration(6h, 12h and 24h) prior to 24 hours FFA intervention, then examined GK and GLUT2 expression by real-time PCR and western blot analysis. In addition the morphological change ofβTc6 cells was observed.Results:1. After intervening with 0.25 mmol/L FFA, apoptotic bodies increase gruadually between cells mass, butβTc6 cells did not change markedly in morphology. After intervening with 0.50～1.00 mmol/L FFA,βTc6 cells showed apoptosis in the trend. We can see the accumulation of lipid droplets in cells, connect fusion between cells decreased obviously and cells mass show the trend of disintegration. As the concentration increases, the above phenomenon is more visible.2. After intervening with 0.25 mmol/L FFA for 24 hours, the expression of GK and GLUT2 mRNA and protein did not change markedly.But after treating with 0.50～1.00 mmol/L FFA, the expression of GK and GLUT2 decreased gradually.3. The intervening effects of Exendin-4 on the morphological change ofβTc6 cells in lipotoxic state.(1) After treating with Exendin-4 for 6 hour, each concentration group ofβTc6 cells in lipotoxic state would not improve in morphology.(2) After treating with Exendin-4 for 12 hour, we could not see the improvement on the morphology of cells in the 5nmol/L Exendin-4 intervention group; In the 10nmol/L Exendin-4 intervention group although the cells continued to show shrinkage, rounded and nuclear condensed, but the accumulation of intracellular lipid droplets decreased, the disintegration of cells mass reduced and the activity of adherent in cells restored partially. When Exendin-4 concentration raised to 50nmol/L, the morphology ofβTc6 cells could not improve furthermore.(3) After treating with Exendin-4 for 24 hour, we could not see the improvement on the morphology of cells obviously in the 5nmol/L Exendin-4 intervention group, but the accumulation of intracellular lipid droplets decreased; When Exendin-4 concentration raised to 10nmol/L, theβTc6 cells showed the characteristics of holding together and the trend of connect fusion; the accumulation of intracellular lipid droplets reduced furtherly, and granular secretory vesicles can be seen in part of the cytoplasm.4. The intervening effects of Exendin-4 on the GK and GLUT2 mRNA and protein expression ofβTc6 cells in lipotoxic state.(1) After treating with Exendin-4 for 6 hour, each concentration group ofβTc6 cells would not up-regulate the expression of GK and GLUT2 mRNA and protein in lipotoxic state.(2) After treating with Exendin-4 for 12 hour, the GK and GLUT2 mRNA and protein expression ofβTc6 cells in lipotoxic state increased gradually along with the increasing concentration of Exendin-4. (3) After treating with Exendin-4 for 24 hour, the GK and GLUT2 mRNA and protein expression ofβTc6 cells in lipotoxic state up-regulated furtherly along with the increasing concentration of Exendin-4 in the 5nmol/L and 10nmol/L intervention group. But it did not keep on increasing when Exendin-4 concentration raised from 10nmol/L to 50nmol/L furthermore.Conclusions:1. FFA intervention with lower concentration would affect the GK and GLUT2 expression ofβTc6 cells in a short time. But along with the increase of FFA concentration, the GK and GLUT2 expression ofβcells down-regulated gradually.2.FFA intervention with lower concentration would affect the survival ofβTc6 cells in a short time. However, as the FFA concentration increases, the more detrimental toβcells survival.3. Treating with Exendin-4 in optimal concentration and duration, the GK and GLUT2 expression ofβcells up-regulated, and the morphology ofβcells in lipotoxic state improved obviously.