The Effect And Mechanism Of Tribbles Pseudokinase2(TRIB2) In Hepatic Insulin Resistance

Objective: To investigate the effect and regulatory mechanism of TRIB2 on insulin resistance in HepG2 model and diabetic mice.Methods:We did conduct cell experiments in cell model.Firstly,HepG2 cells were used to establish insulin resistance cell model induced by palmitate for 24 hours,cell models were used to detect the insulin signaling pathway and observed the change of TRIB2 in transcription and protein level.Then,the cell models,overexpressed or knock down the intracellular TRIB2 level by plasmid or siRNA,were used to detect glucose consumption in the culture medium,and the changes in protein expression of the insulin signaling pathway.Besides,in vivo experiment,we constructed high fat diet(HFD)induced obesity animal model in 8 weeks of C57BL/6J for 12 weeks.We detected the changes of TRIB2 transcription and protein levels in fresh liver tissue of animal model.At 8 weeks,HFD-induced-C57BL/6J mice were injected with over-expressed/low-expressed adeno-associated virus through the tail vein to specifically interfere with the expression of TRIB2 in the liver.Finally,we observed the effect of TRIB2 on insulin resistance by body weight,fasting blood glucose,fasting glucose tolerance,fasting insulin tolerance and glycogen PAS staining,and observed the effect on steatosis by liver H&E,oil red staining,content of TG.Results:TRIB2 was elevated in transcription and protein levels in insulin resistance cell model.The cell model overexpressing TRIB2 reduced the glucose consumption of the medium and increased the damage of insulin signaling pathway.Knocking down the level of TRIB2 in the cell increased the glucose consumption of the medium and reduced the damage of the insulin signaling pathway.Additionally,compared with control,the m RNA and protein level of TRIB2 in the liver tissue of obese mice were both increased.Finally,in obese mice model,the liver specific overexpression of TRIB2 mice had significantly worse insulin resistance and steatosis compared with the control group,the degree of steatosis and insulin resistance were improved in mouse liver lower expressing TRIB2 compared with control.Conclusions: The expression of TRIB2 increased in insulin resistance model whatever in vivo or vitro.And overexpression of TRIB2 aggravated insulin resistance,and knockdown of TRIB2 improved insulin resistance condition.The results suggested that TRIB2 is involved in the occurrence and development of insulin resistance.Objective:In the past,we have used overexpression or interference technology in insulin resistance model in vivo and in vitro and verified that TRIB2 is involved in the process of insulin resistance.This section is to describe the specific molecular mechanism of TRIB2 regulating insulin resistance.Methods:Firstly,we predicted and screened the proteins(AMPKβ1/2)that may be bind to TRIB2 through the online database.Then,we used protein immunoprecipitation(Co-IP)to verify the endogenous interaction of TRIB2 and AMPKβ1/2 subunits in the cell,and verify the exogenousness interaction between Flag-TRIB2 and HA-AMPKβ1 by cell transfection in HEK293 T.Cellular immunefluorescence(IF)technology detected the intracellular localization of TRIB2 and AMPKβ1/2 subunits.Then,Western blot was used to detect the expression of AMPK and its downstream genes with the changes of TRIB2.Finally,we detected glucose consumption in the culture medium and insulin signaling pathway changes in cells overexpressing or interfering with TRIB2,with the stimulation of AMPK activator AICAR or inhibitor Compound C.Results:Firstly,Co-IP found that TRIB2 can bind to AMPKβ1/2 subunits in endogenous interaction and bind to AMPKβ1 subunits in exogenousness,IF results suggested that there is intracellular co-localization.Besides,overexpression of TRIB2 decreased the phosphorylation level of AMPK complex and downstream lipid metabolism gene ACC in cell model.Inhibition of intracellular TRIB2 increased the phosphorylation level of AMPK complex and ACC.Changes in the intracellular of TRIB2 had no effect on content of AMPKβ1/2.Compared with control group overexpressing TRIB2,the glucose consumption of the medium increased,and the damage of the insulin signaling pathway was reduced after AICAR treatment of the cell model;compared with the control group,glucose consumption is reduced,and insulin signaling pathway damage is aggravated with low expression of TRIB2 in cell model while were treated with Compound C.Conclusion : TRIB2 can affect the phosphorylation activity of the complex by binding AMPKβ1/2 subunits,which in turn affects the insulin resistance status of downstream lipid metabolism genes.