| Aims:To determine the role and molecular mechanism of Interferon regulatory factor9(IRF9) in the pathophysiological process of nonalcoholic fatty liver, hepatic insulin resistance and inflammation.Methods:Part Ⅰ:To investigate whether IRF9is involved in type2diabetes mellitus (T2DM) relates metabolic disorders, we used diet induced obesity (DIO) model (8week old, male, C57BL/6mice(WT) with26weeks’high-fat diet feeding) and genetic obesity model (9week old, female, ob/ob mice). We used immunefluorescence staining, real-time PCR and Western Blot to determine the expression of IRF9in the mice liver tissues.Part Ⅱ:To determine the critical role in the pathogenesis of T2DM, we established DIO model in WT and IRF9knockout (KO) mice.8week old, male, WT and IRF9KO mice were random divided into two groups, feeding with normal chow(NC) and high-fat diet(HFD) for26weeks. In the begain(0weeks) and final(26weeks) of experiment, the mice were anesthetizeed with ether inhalation, blood collected from orbital venous, separated the serum and detected serum lipid contents (TG, TC, HDL-C, LDL-C, NEFA and β-hydroxybutyrate) and inflammatory cytokines (IL-1β, IL-6, IL-4, TNF-a, MCP-1, leptin, resistin and Adiponectin). The food-intake in mice recorded weekly, body weight and fasting glucose detected once every four weeks, serum insulin level was determined every8weeks, liver function (ALT, AST, ALP) changes detected in26weeks. During the experiment, at the24th and25th week, we explored the intraperitoneal injection glucose test (IPGTT) and insulin experiments (IPITT) to evaluate the glucose tolerance and insulin sensitivity, respectively. After26weeks of diet treatment, the mice were anesthetized with3%pentobarbital sodium, and macroscopic pictures of their physical appearance were taken. Next, the mice were sacrificed, and their livers were rapidly harvested and weighed. Each liver was cut into two parts:one part was immediately frozen in liquid nitrogen and stored at-80℃for molecular biological analysis, and the other part was either fixed in10%formalin or frozen with Tissue-Tek(?) OCTTM Compound in dry ice and then embedded. By H&E staining (5μm per section) and Oil Red0staining (frozen liver tissue slices,10μm), two kinds of histological detection methods to evaluate the severity of fatty liver and the lipid accumulation in the liver. Furtherly, detected the liver tissue TG, TC, and NEFA content. The mRNA expression levels of lipid metabolic-related genes and M1/M2inflammatory genes in the mouse liver were detected by real-time PCR. In order to assess the degree of insulin resistance, western blot was used to detect the changes of IRS1/AKT signaling pathway.Part Ⅲ:To interpret IRF9overexpression whether reverse WT mice (DIO model) and ob/ob mice (genetic obesity model) obese phenotype. WT mice (DIO model,20weeks) and ob/ob mice transjugular injected with IRF9-expressing adenovirus, then continue with HFD feeding. Four weeks after adenovirus injection, fasting blood glucose, serum insulin levels and liver function changes were analysized. Furtherly, IPGTT and IPITT were used to evaluate insulin resistance alleviation in mice. Then sacrified mice and taken the liver, one part of liver tissue specimens as pathologic analysis (H&E staining and Oil Red O staining), the other part for molecular biological analysis.The IRF9protein expression in liver, white fat tissue and skeletal muscle were determined by western blot and immunofluorescence staining confirmed that overexpressed IRF9colocalized-expression with liver cell marker HNF4, these results indicate adenovirus mediate IRF9major overexpressed in the liver. Finally, real-time PCR determined the changes of M1/M2inflammatory genes mRNA levels and western blot detected protein expression of IRS1/AKT signaling pathway to assess the degree of improvement in insulin sensitivity.Part Ⅳ:To investigate whether IRF9interact with PPARa and activate PPARa target genes. The yeast acid two-hybrid screening system was employed to identify possible genes interact with IRF9. By co-immunoprecipitation and GST-Pulldown assay analysis to determine whether there is a interaction between IRF9and PPARα. IRF9and the PPARa whether co-localization by confocal microscopy imaging.Truncated of IRF9and PPARα was employed to mapping the domains of interaction between IRF9and PPARα. To confirmed the interaction and regulation relationship between IRF9and PPARα, both primary hepatocytes and mice (WT mice and ob/ob mice) were transfected with Ad-GFP and Ad-IRF9adenovirus and determined the mRNA levels of PPARα target genes.Furtherly, primary hepatocytes transfected with Ad-GFP and Ad-IRF9mutant adenovirus detected by real-time PCR to confirm the role and relations between IRF9and PPARa. Part Ⅴ:PPARα overexpressing adenovirus was used to reverse the obese phenotype of WT mice and IRF9KO mice (DIO model) and explicit the role of PPARα in metabolism disorders.WT mice and IRF9KO mice (DIO model,20weeks) transjugular injected PPARα overexpressing adenovirus, continue to feed HFD four weeks’, fasting blood glucose, serum insulin levels and liver functional changes were test in mice, the IPGTT and IPITT to evaluated the alleviation of insulin resistance. Terminally, one part of the liver as pathologic analysis (H&E staining and Oil Red O staining), the other part for molecular biological analysis and liver tissue lipid content analysis. Using real-time PCR and western blot detected the PPARα and its target gene mRNA and PPARa protein expression level in primary mouse hepatocytes and mouse liver tissue,respectively. Also detected the M1/M2inflammatory genes by real-time PCR and the change of IRS1/AKT signaling pathway by western blot in mice liver tissue.Results:Part Ⅰ:In mice of DIO model and of genetic obesity model, we observed percents of IRF9positive hepatocytes, IRF9mRNA and protein expression in the livers of mice were reduced.Part Ⅱ:In WT and IRF9KO mice DIO model,①both HFD or NC feeding conditions, IRF9KO mice showed obesity, and its weight was significantly higher than WT mice despite of the same kind of diet feeding. The comparing of food-intake energy in each group was no difference.②HFD group fasting glucose, fasting serum insulin level and HOMA-IR (homeostasis model assessment-insulin resistance) of IRF9KO mice were higher than WT mice. Key gluconeogenesis gene (PEPCK and G6Pase) mRNA expression levels in liver detected by real-time PCR in HFD fed IRF9KO mice were significantly higher than WT mice. The IPGTT and IPITT examination indicates HFD feeding IRF9KO mice decreased glucose tolerance and insulin sensitivity. IRS1/AKT signaling pathway also impaired.③Macroscopic pictures of liver, liver weight and liver/body weight ratio, histological analysis, lipid comtents analysis, all indicates that HFD feeding IRF9KO mice had a more severe fatty liver and lipid accumulation in liver increased significantly. Lipid metabolism genes analysis, the mRNA of genes participates in cholesterol synthesis, fatty acid biosynthesis, transport and lipogenesis were significantly increased,while the mRNA of genes participates in cholesterol output,fat mobilization and fatty acid β-oxidation were significantly reduced.The liver function examination in IRF9KO mice was worse than WT mice. Analysis of the AMPK signaling pathway, also confirmed the energy consumption in IRF9KO mice significantly inhibited,whereas enhanced lipid synthesis capability. Systemic and local inflammation in the liver of IRF9KO mice was significantly higher than the WT mice.Part Ⅲ:Adenovirus mediates IRF9gene overexpression in WT mice (DIO model) and ob/ob mice (genetic obesity model), we observed that:①IRF9protein expression and immunofluorescent staining confirmed that IRF9play a critical role in liver tissue.②IRF9gene can be overexpressed effectively to reverse the fatty liver caused by the high fat diet and congenital lacking of leptin, liver function was significantly improved.③Livers of mice overexpressed IRF9, glucose metabolism and insulin sensitivity were significantly improved, meanwhile, the level of inflammation of the liver tissue significantly reduced.Part Ⅳ:①Co-IP and GST pulldown detection shown co-interaction between IRF9and PPARa, and immunofluorescencent staining also confirmed thatIRF9and PPARa co-localized in the nucleus.②To map the PPARα-interacting region of IRF9, a series of IRF9deletion mutants were generated. only the less conserved IRF9intermediate region interact with PPARa. We also generated a series of PPARa deletion mutants. The mapping demonstrated that the DNA-binding domain (DBD, C domain), the hinge region (D domain) and the ligand-binding domain (LBD, E/F domain) of PPARa were all able to interact with IRF9.③Real-time PCR further confirmed PPARa target genes that in IRF9over-expressed primary hepatocytes, in the liver tissue of WT mice (DIO model), ob/ob mice (genetic obesity model) were significant upregulated consistently; while overex-pressed the mutant IRF9gene in mouse primary hepatocytes, PPARa downstream target genes did not change.Part Ⅴ:PPARa overexpressed in hepatocytes, the downstream target genes were significantly upregulated. PPARα gene overexpressed in WT mice and IRF9KO mice (DIO model), we observed:①PPARα protein expression level in mice liver, PPARa and target gene mRNA expression was significantly upregulated.②PPARa over-expression can be partial effectively reversed the high-fat diet caused fatty liver and also obviously improved liver function.③PPARa overexpressed livers signifycantly enhance the ability of glucose metabolism and improve insulin sensitivity, and significantly reduce the level of inflammation. Conclusion:IRF9interacts with PPARa, involved in the regulation of glucose and lipid metabolic disorders, hepatic insulin resistance and inflammation in the development and progression of T2DM. |
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