The Role Of GPC4 In The Browning Of White Fat Cells And Its Mechanism

PART I EXPRESSION OF GPC4 IN MOUSE FAT AND THE CONSTRUCTION OF GPC4 OVEREXPRESSION AND SHRNA PLASMIDObjective: To detect the expression of GPC4 in mouse fat;to construct the overexpression plasmid and shRNA of mouse-based GPC4 and to provide conditions for further study of the role of GPC4 in fat browning.Methods: The expression of GPC4 protein in iWAT,eWAT and BAT in C57BL/6J was detected by Western blot.A primer for mouse GPC4 was designed,and a GPC4 overexpression plasmid was constructed using the vector pCDNA3.1(+);a mouse GPC4 inhibitor plasmid primer was designed to construct a shRNA of GPC4.Successfully induced differentiation of 3T3-L1 preadipocytes,transfected GPC4 overexpression plasmid and shRNA,and detected the mRNA and protein expression of GPC4 by RT-QPCR and Western blot.Results: GPC4 was significantly higher in iWAT than eWAT and BAT(P<0.001);GPC4 overexpression plasmid sequencing showed consistent with GPC4;GPC4 mRNA and protein expression were significantly increased after overexpression of 3T3-L1 cells(P<0.001).GPC4 mRNA and protein expression levels of GPC4 were significantly decreased after 3T3-L1 cell inhibition(P < 0.001).Conclusion: GPC4 may be associated with browning of white fat,the GPC4 overexpression plasmid and the shRNA were successfully constructed.PART II GPC4 PROMOTES WHITE FAT BROWNING IN FAT CELLSObjective: To investigate the role of GPC4 in the browning of 3T3-L1 preadipocytes.Methods: After successful differentiation of 3T3-L1 preadipocytes,GPC4 overexpression and inhibition plasmids were transfected respectively,and cold exposure or rosiglitazone stimulated browning.RT-QPCR was used to detect the expression changes of m RNAs such as UCP1,PGC1?,PPAR?,TMEM26,P2RX5,DIO2,CIDEA,ELOVL3,PRDM16,Hoxc8,FASN and TCF21.The oil red staining was used to observe the browning of fat cells.Western blot was used to detect the protein expression levels of GPC4,UCP1,p-mTOR/t-mtor,rictor,raptor,p-AMPK/t-AMPK,p-ERK/t-ERK,and internal reference GAPDH.Results: In the absence of cold exposure and rosiglitazone stimulation,the expression of PPAR? m RNA was significantly increased in GPC4 overexpressing adipocytes(P<0.05),and other genes did not change significantly;under cold exposure and rosiglitazone treatment Overexpression of GPC4 enhanced browning of 3T3-L1,and the expression of brown fat-related gene m RNA was significantly increased compared with the control group(P<0.05),while the expression of brown fat-related gene m RNA was significantly reduced after transfected GPC4 sh RNA(P<0.05).Oil red staining showed that there were more lipid droplets in the adipocytes of the GPC4 overexpression group,but the number of cells in the GPC4 inhibition group was decreased;the protein expression of UCP1 was significantly increased in the GPC4 overexpression group(P< 0.01),at the same time,the protein expression levels of p-mTOR and raptor also increased significantly,and the expression levels of UCP1,p-mTOR and raptor protein in GPC4 inhibition group were significantly decreased(P<0.01),while there was no significant difference in the expression of protein p-AMPK/t-AMPK,p-ERK/t-ERK and rictor.Conclusion: GPC4 is involved in browning under cold exposure and rosiglitazone treatment and promotes browning and protein UCP1 production,and GPC4 may participate in browning by interacting with mTOR/raptor.PART III GPC4 PROMOTES BROWNING BY INTERACTING WITH RAPTORObjective: To explore whether raptor is involved in the role of GPC4 in browning and its possible mechanism.Methods: After cultured 3T3-L1 preadipocytes and successfully induced differentiation,the GPC4 overexpression plasmid and raptor overexpression plasmid were co-transfected,and the cell protein was collected by IP lysate,and some were used for subsequent Western blot detection,and the remaining parts were immunoprecipitated with the Ig G antibody and GPC4 antibody coated with magnetic beads respectively.Then the protein expression of raptor and GPC4 in Ig G group and GPC4 group and Input group were detected by Western blot.Results: Co-IP results showed that there was raptor proteins in the GPC4 group,but not in the Ig G group.The expression of GPC4 and raptor protein were appeared in Input group,and there was no significant difference.Raptor can be precipitated by GPC4 antibodies,indicating a direct or indirect interaction between the protein raptor and GPC4.Conclusion: GPC4 promotes browning of 3T3-L1 cells by interacting with raptor.