Functional Studies On The TBC1D15 Gene In The Regulation Of Glycometabolism

Objective TBC1D15 was knocked out by CRISPR/Cas9 in L02 and L6 cells to study the relationship between TBC1D15 and glycometabolism at cellular level,which may serve as a potential target in the development of anti-diabetic strategies.Methods First of all,five recombinant plasmids containing sgRNA targeting sequences were designed and constructed.Optimum purinomycin concentration was determined by MTT test in L02 and L6 cells.Subsequently,L02 cells were transfected with the pX462 recombinant plasmids,and L6 cells were transfected with pX459 recombinant plasmids.T7E1 nucleic acid endonuclease was used to detect the knockout efficiency,and the monoclonal cell lines were screened by the finite dilution method.Then the clonal cells losing expression of TBC1D15 were screened by Western Blotting and genomic PCR.In functional studies,the number of cells containing 2-NBDG and the respective fluorescence intensity were determined by flow cytometry.To further address the effects of TBC1D15 on glucose uptake and potentially on glucose metabolism,we checked the expression of GLUT4,Rab7 and LC3B in TBC1D15knockout cells,with or without insulin stimulation.We screened the lowest G-418 concentration that could kill all L02 and L6 cells in 10¹4 d.The HA-GLUT4-EGFP plasmid was constructed to transfect WT and TBC1D15^-/-L02 cells,and the stable cell lines were screened.Immunofluorescence technique was used to observe the distribution of Rab7,GLUT4 and Lamp1 in WT and TBC1D15^-/-L02 cells.Results The optimal purinomycin concentration was 1.5 and 5.0?g/mL in L02 and L6 cells respectively.The knockout efficiency of pX459-1,pX459-2,pX459-3 and pX459-4 was 56%,28%,45%and 28%,respectively.The expression of TBC1D15 was not detected by Western Blotting in L02 and L6 monoclonal cell lines,indicating that the cell lines were successfully knocked out TBC1D15.The fluorescence absorption peaks in TBC1D15^-/-L02 and L6 cells shifted to the left compared with those in WT cells,showing that the 2-NBDG uptake in TBC1D15^-/-cells was reduced.The expression of GLUT4 and LC3 II were significantly decreased in TBC1D15^-/-L02 and L6 cells,compared to those in WT cells,but the total expression of Rab7 was not changed.The distribution of Rab7 was more concentrated in certain areas in cytoplasm in TBC1D15^-/-L02 cells,compared to that in WT cells.We next tested whether there were changes in Rab7 and GLUT4 co-localization.Indeed,we detected an increase in co-localization between endogenous Rab7 and GLUT4 in TBC1D15^-/-cells.Further study showed that the localization of Rab7 relative to Lamp1 was strikingly increased,which is expressed in late endosomes/lysosomes,in TBC1D15^-/-L02 cells than that in WT cells.Conclusions The same as the results of HeLa cells,knocking out TBC1D15 in non cancerous cells also caused a decrease in glucose uptake.TBC1D15 could regulate the translocation of GLUT4 by affecting the activity of Rab7.In the absence of TBC1D15,a large number of GLUT4 was sorted into the late endosomal pathway,possibly resulting in the final degradation of GLUT4 and impairment of glucose uptake.Moreover,the decrease of LC3 II after knockout of TBC1D15 meant that TBC1D15 was associated with autophagy.