E3 Ubiquitin Ligase-Itch Regulates The Expression Of Key Proteins In Insulin Signaling Pathway And Molecular Mechanism Research

The biological function of insulin depends on the cascade activation of signal transduction molecules at all levels in the insulin signaling pathway.The inhibition or degradation of any signal molecule will cause signal transduction to be interrupted and cause metabolic disorders and other insulin resistance phenomena in the body.The degradation of 80% of proteins in cells depends on the ubiquitin-proteasome system(UPS),and the specificity of the substrate ubiquitination process depends on the E3 ubiquitin ligases.Itch is a highly conserved E3 ubiquitin ligase of the HECT family.Under physiological conditions,Itch specifically binds to substrate proteins,catalyzes the ubiquitination of substrate proteins and their degradation by the proteasome,and participates in substrate-mediated immune regulation,cell cycle and other biological processes.Studies have confirmed that insulin resistance is associated with UPS,but it is still unclear how Itch is involved in the development of insulin resistance.Our previous research results showed that the expression of Itch gene is up-regulated at the translation level in the state of insulin resistance in human liver cancer Hep G2 cells.In order to further study how Itch affects insulin sensitivity in human liver cancer Hep G2 cells,two models of Itch gene suppression and overexpression were constructed in this experiment.Small interfering RNA(si RNA)and recombinant plasmid p En CMV-Itch-3×flag were transfected into Hep G2 cells by Lipofectamine.The optimal transfection conditions and Itch si RNA with the best inhibitory effect were determined by real-time fluorescence quantitative PCR(q RT-PCR).After successfully constructing the Itch gene suppression and overexpression Hep G2 cell model,we first tested the effect of Itch gene suppression and overexpression on the glucose utilization rate of Hep G2 cells.The results showed that when the expression of Itch gene was inhibited by si RNA,the glucose uptake of Hep G2 cells increased,which was about10% higher than that of the normal control group(P<0.05);while when the recombinant plasmid-mediated Itch gene expression increased,the glucose uptake by the cells decreased,which was about 20% lower than that of the normal control group(P<0.05).This suggests that the expression of E3 ubiquitin ligase Itch negatively regulates the glucose utilization of Hep G2 cells,and inhibition of Itch gene expression can improve the insulin sensitivity of Hep G2 cells.In insulin signaling pathway,the cascade of signaling molecules determines the function of insulin,and the blockade of signal transduction will lead to the occurrence of insulin resistance.Therefore,we further explored the effect of Itch on the expression of insulin receptor-β subunit(IR-β),insulin receptor substrate 1(IRS1)and protein kinase B(PKB/Akt)in insulin signaling pathway.Based on the successful establishment of Itch gene suppression and overexpression models,Western blot technology was used to detect the total IR-β,IRS1 and Akt protein levels and their phosphorylated protein levels.The results showed that compared with the normal control group,when the expression of Itch protein was inhibited,the expression of total IR-β and IRS1 protein increased(P<0.05),and insulinstimulated tyrosine phosphorylation of IR-β was significantly greater,serine307 phosphorylation of IRS1 was markedly reduced.There was no significant difference in the total of Akt protein in cells(P>0.05),but the serine 473 phosphorylation of Akt protein was increased(P<0.05).In the state of Itch gene inhibition,the greater tyrosine phosphorylation of IR-β and serine phosphorylation of Akt,reduced serine phosphorylation of IRS1,indicating that insulin signal transduction was enhanced,which suggested that inhibition of Itch gene may prevent or improve the occurrence of insulin resistance in Hep G2 cells.On the contrary,when the expression of Itch protein increased,the expression of total IR-β and IRS1 protein were decreased(P<0.05),and insulin-stimulated tyrosine phosphorylation of IR-β was markedly reduced,and the serine 307 phosphorylation of IRS1 was increased(P<0.05).The Serine 473 phosphorylation of Akt was decreased(P<0.05).In the state of Itch gene overexpression,the reduced tyrosine phosphorylation of IR-β and serine phosphorylation of Akt,increased serine phosphorylation of IRS1,indicating that the insulin signal transduction was inhibited,which may promote the occurrence of insulin resistance in hepatocytes.This suggested that the expression of Itch protein negatively affects the signal transduction of insulin pathway.Since Itch belongs to family members of E3 ubiquitin ligation enzymes,their functionality depends on the binding of the WW domain to the substrate for ubiquitination degradation.Next,we discussed whether Itch targets the key protein IR-β and IRS1 in the insulin pathway to degrade it,thereby affecting the normal transduction of insulin signals.We applied immunoprecipitation techniques to detect if Itch protein interacts with IR-βand IRS1.The results confirmed that Itch protein interacts with IR-β and IRS1 at the endogenous level of cells.As previously mentioned,the expression of Itch protein is negatively correlated with the expression of IR-β and IRS1,which suggests that Itch interacts with IR-β and IRS1 to promote their ubiquitination degradation.As mentioned above,our research further confirmed that Itch was closely related to insulin resistance,and the expression level of Itch genes affected Hep G2 cells to insulin sensitivity.Itch regulated the expression of key protein IR-β and IRS1 in insulin signal pathway,and the phosphorylation of IR-β,IRS1 and Akt.When overexpression of itch,it’s tended to promote insulin resistance.At the endogenous level of cells,Itch was detected to interact with IR-β and IRS1,and it was speculated that Itch may participate in the insulin signaling pathway to regulate cells by targeting an IR-β protein and IRS1 protein.