The Study On The Role Of Autophagy In The Treatment Of Simple Hepatic Steatosis By Glucagon-Like Peptide-1

BackgroundNonalcoholic fatty liver disease (NAFLD) is a spectrum of disorders ranging from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH), and NASH can progress to fibrosis and cirrhosis, even hepatocellular carcinoma (HCC). NAFLD is characterized by the deposition of triglyceride (TG) as lipid droplets in the cytoplasm of hepatocytes. Excess alcoholic consumption, virus, drugs, total parenteral nutrition (TPN), congenital and autoimmune diseases should be excluded before making a diagnosis. With the popularization of western lifestyle and the improvement of living standard, the morbidity of NAFLD keeps rising year by year. Therefore, NAFLD has become a major health problem endangering the people’s health.Simple hepatic steatosis was concerned as a benign stage and often neglected by researchers. However, almost20percents of patients with simple hepatic steatosis can aggravate to NASH. Compared with normal liver, it is more susceptible to drugs, alcohol, industrial toxicants, ischemia and virus infection. Additionally, it can make insulin resistance and metabolic disorders worse. Lastly, simple hepatic steatosis is also the independent risk factor of the progress to diabetes, atherosclerosis and vascular dysfunction. Therefore, it is of great significance to actively intervene in simple hepatic steatosis for the early prevention and treatment of NAFLD.However, the pathogenesis of NAFLD has not been fully clarified. Diet therapy and appropriate exercise are thought as valid measures. At present, effective targeted drugs remains absent. Glucagon-like peptide-1(GLP-1) is secreted by the L cell in the small intestine. GLP-1chiefly promotes glucose-dependent insulin secretion. Moreover, it can increase satiety, slow gastric emptying, reduce body weight and enhance the proliferation of pancreatic β cells. Previous studies have proved that GLP-1receptor existed on the membrane of human hepatocyte, and further studies showed that GLP-1can reduce lipid accumulation in the hepatocytes. But whether GLP-1takes part in the stage of simple hepatic steatosis and which mechanism is involved in the process, still require further investigation.Autophagy is one pathway of material degradation widely existing in eukaryotic cells. It removes the superfluous proteins and damaged organelles, which can promote the recirculation and maintain internal homeostasis. Recent studies have shown that autophagy is involved in lipid degradation and in turn inhibition of autophagy can lead to excessive lipid inflow in the liver. Moreover, some researchers discovered that GLP-1analogs can reduce fatty acid-induced lipid deposition in hepatocytes. Meanwhile, they observed that the expression of autophagy-related molecular markers such as Beclin-1and LC3B were upregulated and the number of autophagosome was increased. Conclusively, it is extremely possible that autophagy participates in the process that GLP-1mitigates the lipid accumulation in the liver. However, whether autophagy plays a role in the occurrence and development of simple fatty liver demands further studies.Part one Construction and identification of cell model for investigating simple hepatic steatosisObjective:To construct cell model for investigating simple hepatic steatosis, and then to identify whether it can mimic the pathological features of human simple fatty liver.Methods:Cell model for investigating simple hepatic steatosis was induced by incubating L-02cells with a free fatty acids (FFA) mixture (oleate and palmitate at the ratio of2:1) for24h. Cell viability was evaluated by cell counting kit-8assay. The degree of lipid accumulation was evaluated by Oil Red O staining and triglyceride quantification. MDA was tested for comparing lipid peroxidation. Finally, Hoechst33342staining, flow cytometer after staining cells with Annexin V-FITC and propidium iodide (PI), and detection of caspase-3protein expression by western blot were conducted for evaluating cell apoptosis.Results:After incubation with FFA mixture for24h, a large quantity of lipid droplets was observed in the cell model. Triglyceride content of the model group was significantly higher than normal (p<0.01). Cell viability, lipid peroxidation, and apoptosis showed no significant difference between the normal and model group.Summary:The cell model, built by incubating L-02cells with DMEM containing a FFA mixture (OA:PA=2:1) for24h, can replicate the pathological features of human simple fatty liver.Part two The role of autophagy in the treatment of simple hepatic steatosis by glucagon-like peptide-1Objective:To investigate the treatment of human long-acting GLP-1analogs Liraglutide on simple hepatic steatosis and the role of autophagy in the process of the treatment.Methods:As described in the first part, we firstly construct the cell model of simple hepatic steatosis and then divide them into five groups. Change the medium, and treat them with saline (model group), lOnM liraglutide,100nM liraglutide,100nM Hraglutide+5mM3-MA (autophagic inhibitor),1μM Rapamycin (autophagic enhancer) for another24h respectively. L-02cell was cultured with DMEM for24h and then treated with saline for another24h as normal group. The cells of six groups above were processed by Oil Red O staining, triglyceride quantification. Total RNA was extracted, and the mRNA levels of Beclin-1, p62, LC3B were evaluated by Real-Time PCR. Total protein was analyzed by Western blot to detect the protein level of Beclin-1, p62, LC3B. At last, electron microscopy was applied to observe the autophagosome.Results:1. According to the results of intracellular triglyceride quantification, the levels of TG in the cells of the10nM and100nM liraglutide group were significantly reduced compared with the model group (p<0.05). And the level of TG in the100nM liraglutide group was significantly reduced compared with the10nM liraglutide group (p<0.05). When both100nM liraglutide and5mM3-MA were used, the level of TG was apparently increased compared with the100nM liraglutide group (p<0.05), but still lower than the level of TG in the model group. Meanwhile the level of TG in the Rapamycin group was reduced compared with the model group (p<0.05). The results of Oil Red O staining in six groups kept in consistence with the change of the levels of intracellular triglyceride quantification.2. The results of Real-time PCR showed that the mRNA level of Beclin-1among the six groups had no significant difference. The mRNA level of p62among the six groups (except the normal group) also had no significant difference. However, the mRNA level of LC3B in the model group was reduced compared with the normal group, with no apparent difference. The mRNA levels of LC3B in both lOnM and100nM liraglutide groups were increased compared with the model group, with significant difference in100nM liraglutide group (p<0.05). The mRNA level of LC3B in the100nM liraglutide plus5mM3-MA group was significantly lower than the100nM liraglutide group (p<0.05). The mRNA level of LC3B in the Rapamycin group was significantly increased compared with the model group (p<0.05). The protein levels of Beclin-1, p62, LC3B detected by Western blot analysis were basically consistent with the results of RT-PCR. In order to appraise the autophagic flux, the difference of LC3BII protein level before and after the treatment of Bafilomycin was calculated. The autophagic flux of the model group was smaller than the normal group. The autophagic flux of the100nM liraglutide group was distinctly larger than the model group. The autophagic flux of the100nM liraglutide plus5mM3-MA group was smaller than the100nM liraglutide group. The. autophagic flux of Rapamycin group was larger than the model group.3. Electron microscopy was conducted for observing the autophagosome formation. The results showed that there were plenty of mitochondria and no lipid droplets in the cytoplasm of the normal cell. Large quantity of lipid droplets and no autophagosome were seen in the model group. In the cells of the100nM liraglutide group, many autophagosome and several lipid droplets coexisted. The number of autophagosome in the cells of the100nM liraglutide plus5mM3-MA group was less than the100nM liraglutide group. There were many autophagosome and several lipid droplets in the cells of the Rapamycin group.Summary:Liraglutide can reduce the lipid accumulation in the cell model of smiple hepatic steatosis, which is dose-dependent. Liraglutide enhanced autophagy in the treating process of simple hepatic steatosis. When liraglutide and autophagic inhibitor were used together, autophagy was down-regulated and lipid deposition increased. Lipid accumulation was also reduced by only upregulating autophagy with Rapamycin. So autophagy might be involved in the treatment of liraglutide on simple hepatic steatosis.Conclusion1. The cell model, built by culturing L-02cells in DMEM containing a FFA mixture (OA:PA=2:1) for24h, can replicate the pathological features of human simple fatty liver.2. Liraglutide can reduce the lipid accumulation in the cell model of smiple hepatic steatosis, which is dose-dependent.3. Autophagy might be a new mechanism, by which liraglutide reduces lipid deposition in simple hepatic steatosis.