El papel del sustrato del receptor de la insulina tipo 2 (IRS2) en el desarrollo de la enfermedad colestásica

Abstract

ABSTRACT Hepatocellular injury is the major triggering event of the wound healing response that leads to liver fibrosis. Insulin receptor substrate 2 (IRS2) is a key downstream mediator of insulin signaling pathway with a major role in liver metabolism. However, its effects on liver fibrosis have been poorly studied. In this Thesis we have investigated whether IRS2 deficiency influences the hepatocellular stress responses of the liver triggered by cholestatic injury. For that goal, bile duct ligation (BDL) was performed in wild-type (WT) and IRS2-deficient (IRS2KO) female mice. Histological and biochemical analysis, inflammatory and fibrogenic responses were evaluated in livers from mice at 3, 7 and 28 days following BDL. Although no differences between genotypes were found at 28 days post-BDL, IRS2KO mice displayed less BDL-induced histological alterations in the liver, including hepatocyte damage and excess deposition of extracellular matrix components compared to WT mice at 3 and 7 days post-BDL. Surprisingly, no differences in total bile acid content accumulated after BDL were found, although the percentage of the different species of bile acid gathered in the gallbladder was different between genotypes. Moreover, hepatic expression of fibrogenic and proinflammatory markes was reduced in IRS2KO mice compared to WT animals after 3 and 7 days of the BDL. Interestingly, hemeoxygenase 1 expression, used as a maker of oxidative stress, was also down-regulated in livers lacking IRS2. Next, we explored whether activation of human hepatic stellate cells (HSC) LX2 induced by IGF1 is modulated by IRS2. Stable silencing of IRS2 significantly reduced IGF1 activation of LX2 since cells it preserved almost the same quiescent phenotype than untreated cells and showed a reduced expression of α-SMA and collagen 1. Indeed, IRS2 knockdown impaired the phosphorylation of ERK1/2 as well as the induction of MMP9 upon IGF1 challenge. Lastly, we delineate the role of IGF1R-ERK1/2-MMP9 axis in HSC activation. To achieve this, we pre-treated LX2 with IGF1 combined with PD98059, a well-known inhibitor of ERK1/2 activation. Under these conditions IGF1-mediated activation of LX2 cells was abolished. Similar results were obtained upon IRS2 silencing. Lastly, the analysis of of the components of the IGF1/IGF1R system in the two genotypes of mice mice revealed that hepatic levels of IGF1R mRNA significantly increased while IRS mRNA levels decreased after BDL challenge. Moreover, the expression of MMP9 after BDL was higher in WT mice than in those animals lacking IRS2. Taken together, our results indicate that IRS2 contributes to the progression of cholestatic liver injury since its deficiency reduced both inflammatory and fibrogenic responses triggered by BDL, at least in part by reducing HSC activation induced by IGF1.