Protease CLPP Changes Mitochondrial Dynamics And Affects Mitochondrial Function

Part One Effects of high glucose and high fat on Clpp and mitochondrial dynamicsObjective To investigate the effects of high glucose and high fat on Clpp and mitochondrial dynamics.Methods Mouse islet Min6 cells were treated with different concentrations of glucose(5.6 mmol/l,16.7 mmol/l and 33.3 mmol/l)and different concentrations of palmitic acid(0.2 mmol/l,0.5 mmol/l and 1.0 mmol/l)at different times(24 hours,36 hours and 48 hours),respectively.The changes of CLPP expression levels were observed.The optimum concentration and time of glucose and palmitic acid treatment were screened out.After treating Min6 cells with 33.3 mmol/l glucose and 0.5 mmol/l palmitic acid for 48 hours,the expression of CLPP was analyzed by qPCR,and the expression of mitochondrial dynamics-related genes(MFN1,MFN2,OPA1,DRP1 and FIS1)was analyzed by qPCR and Western blotting.Results Compared with the Normal group,the effects of glucose and palmitic acid on CLPP expression were time and dose dependent(except for 1.0 mmol/l palmitic acid).After treating Min6 cells with 33.3 mmol/l glucose and 0.5 mmol/l palmitic acid for 48 hours,the expression level of CLPP was the highest(p<0.01),which was independent of osmotic pressure.Compared with Normal group,after treating Min6 cells with 33.3 mmol/l glucose and 0.5 mmol/l palmitic acid for 48 hours,the expression of MFN1,a mitochondrial fusion-related protein,decreased(p<0.05),while the expression of DRP1,a mitochondrial fission-related protein,increased(p<0.01).Conclusion High glucose and high fat can promote mitochondrial fission and inhibit mitochondrial fusion.Part Two Effects of high glucose and high fat on mitochondrial dynamics and mitochondrial function in the absence of CLPPObjective To investigate the effects of high glucose and high fat on mitochondrial dynamics and mitochondrial function in the absence of CLPP.Methods The expression of CLPP in Min6 cells was inhibited by siRNA transfection.The expression of mitochondrial dynamics-related genes(MFN1,MFN2,OPA1,DRP1 and FIS1)in Min6 cells treated with different media was analyzed by qPCR and Western blotting.The expression of mitochondrial autophagy genes(PINK1 and PARKIN)was analyzed by qPCR and Western blotting.Flow cytometry was used to analyze the difference of apoptotic rate of Min6 cells in each group.To evaluate the difference of mitochondrial function of cells treated with different media by the following indicators and methods:ELISA method to detect insulin,kit to detect ATP,flow cytometry to analyze ROS,JC-1 method to detect mitochondrial membrane potential.Finally,the morphological changes of mitochondria of Min6 cells treated with different media were analyzed by transmission electron microscopy.Results CLPP siRNA reduced the expression of CLPP protein(p<0.05).Compared with HG+NC group and HF+NC group,CLPP siRNA inhibited the expression of MFN1 and increased the expression of DRP1 at the level of expression of protein and mRNA(p<0.05).Compared with HG+NC group and HF+NC group,CLPP siRNA increased the expression of PINK1 and PARKIN at the level of mRNA and protein expression(p<0.05).By flow cytometry analysis,compared with HG+NC group and HF+NC group,CLPP siRNA increased early and late apoptosis of Min6 cells(p<0.05).By flow cytometry analysis,compared with HG+NC group and HF+NC group,CLPP siRNA decreased insulin secretion(p<0.05).Compared with HG+NC group and HF+NC group,CLPP siRNA decreased ATP secretion of Min6 cells(p<0.05).CLPP siRNA increased ROS production in Min6 cells(p<0.05).CLPP siRNA decreased mitochondrial membrane potential(p<0.05).Transmission electron microscopy showed that in the Min6 cells of siCLPP group,the number of mitochondria decreased significantly,the area of mitochondria increased significantly,the density of mitochondria decreased significantly,the cristae of mitochondria was not obvious,the density of cytoplasm decreased significantly and vacuoles increased significantly.Conclusion In the absence of CLPP,high glucose and high fat aggravate mitochondrial fission,inhibit mitochondrial fusion,and deteriorate the multiple functions of mitochondria.