The Role Of Activated Foxo1 At Transcription Level In Liver Fibrosis Induced By Insulin Resistance

Objectives To investigate the role of activated Foxo1 at transcriptional level in liver fibrosis induced by insulin resistance.Methods Twenty 2-month-old Foxo1-S253 site mutation alanine(A/A)mice and twenty normal control(CNTR)mice were randomly divided into four groups: CNTR,A/A,CNTR+HFD and A/A+HFD.CNR+HFD and A/A+HFD were given 60% high fat diet(HFD)for 4M to induce insulin resistance(IR),and CNTR and A/A groups were given maintain normal diet for the same time.Then,after measuring body weight,fasting blood glucose and random feed blood glucose,all the mice were sacrificed under anesthesia.The liver tissues were weighed and the liver samples were taken and kept.At the same time,serums were taken and ALT and AST in the serums were detected,respectively.The hepatic pathological changes were observed by HE staining,and the distribution of liver fibrosis evaluated using Masson staining,respectively.The protein and/or gene expressions of ECMs,fibrosis-related genes,changes of both TGF-β1→Smad2/3 signaling pathway and IRS1/2→Akt→Foxo1 insulin signaling pathway in the livers were determined through Western-blot and/or RT-PCR,respectively.Results 1 Under without HFD stimulating condition,compared with CNTR mice,we found that in A/A mice:(1)There were no obvious changes in body weight and liver weight;(2)Both the concentrations of ALT and AST in the serums were significantly increased;(3)The blood glucose concentration under fasting condition had no significant changes,while blood glucose concentration under random fed condition obviously increased;(4)Hepatocytes were arranged disorderly,with smaller nuclei and irregular hepatic cord irregularly arrangement detected by HE staining.Collagen fibers were accumulated around blood vessels as revealed by Masson staining;(5)According to the results of Western-blot and R-T PCR,first,the expressions of extracellular matrix proteins,such as Collagen1 and Collagen3 increased significantly.Both the expressions of MMP2 and MMP9,which promote ECM degradation,were increased significantly,while Timp1,an inhibitor for ECM degradation,significantly increased too in the liver.Second,insulin pathway in liver: the expression of IRS1 and IRS2 and p-Akt/Akt was up-regulated,and without change of total Foxo1.However,the downstream target genes of Foxo1,such as Pepck and G6 pase were up-regulated in the liver.Third,the TGF-β1→Smad2/3 signaling pathway was activated too,proved by the expression of TGF-β1 and its downstream signaling molecules p-Smad2/3 and Smad2/3 in the liver.2 After 4 month HFD stimulation,compared with CNTR+HFD mice,we found that in A/A+HFD mice:(1)Both body weight and liver weight significantly decreased;(2)Both AST and ALT concentration in serums had no significant change;(3)The blood glucose concentrations under fasting condition reduced obviously,and was unchanged under random fed condition;(4)The number of hepatocytes were increased,vacuole-like and edema-like transformations were observed in CNTR+HFD mice,and in A/A+HFD mice live are fewer fat vacuoles in liver detected by HE staining,while the accumulation of perivascular fibers did not further increased determined by Masson staining;(5)The results of Western-blot or RT-PCR showed,first,the Collagen3 protein expression was further increased in the liver.All the expressions of MMP2 and MMP9,which promote ECM degradation,and the expressions of Timp1,which inhibits ECM degradation were further increased.Second,the Foxo1 upstream signal of insulin pathway was not further activated,exhibited by unchanged expression of p-Akt,while Foxo1 and it’s the downstream target genes were further slightly activated,demonstrated by increases in the expressions of Foxo1,Pepck and G6 pase.Third,the TGF-β1→Smad2/3 signaling pathway in the liver was weakly further activated because the expressions of TGF-β1 and p-Smad2/3 increased slightly.Conclusions After Foxo1 is activated at the transcriptional level,the liver developed fibrosis and impaired liver function,the mechanism underlying which is related to the activation of the TGF-β1→Smad2/3 signaling pathway in the liver.Figure29;Table6;Reference 151...