The Effects And Mechanism Of Exenatide On The Expression Of PGC-1? In Nonalcoholic Fatty Liver Cells Model

BackgroundsNonalcoholic fatty liver disease(NAFLD) is a kind of clinical syndrome that caused by the accumulation of excess lipid in hepatocyte, which should exclude the Alcoholic Hepatitis, viral hepatitis, autoimmune liver disease and other specific factors that lead to liver steatosis. NAFLD is the most common cause of chronic liver disease in both developed and developing countries. Although a lot of basic and clinical research have been done to reveal the pathogenesis of NAFLD, the mechanism of excessive lipid accumulation in hepatocyte and the abnormal metabolism in the progression of NAFLD remains to be further elucidated.Peroxisome proliferators-activated receptor-?coactivator-1?(PGC-1?) was fist found as the co-activator of peroxisome proliferators-activated receptor-? to adjust the adaptive thermogenesis. As research continues, a lot of experts have found that PGC-1? is abundant in the tissues of high requirements for energy, such as brown adipose tissue, heart, skeletal muscle, liver and kidney. The expression of PGC-1? in the hepatocyte would reach its peak in the early stages of puerperium, and it would coordinate the complex metabolic changes in the liver, such as gluconeogenesis,fatty acid ?-oxidation, ketogenesis, the synthesis of hemoglobin and the bile acidmetabolism. An increasing number of studies have found that PGC-1? play an important role in insulin resistance, lipid metabolism disturbance and mitochondrial dysfunction, which are well known as the pathogenesis of NAFLD. The expression of PGC-1? is regulated by multiple signaling pathways, for example, the AMP-activated protein kinase pathway, the mitogen-activeted protein kinase p38 pathway. Exenatide, a receptor agonist of glucagon-like peptide-1(GLP-1), has the similar ability of incretin to reduce insulin resistance, and it has been widely used in the treatment of type 2 diabetes. Recent studies have identified that GLP-1 could not only regulate blood sugar and lose weight, but also improve hepatocyte steatosis.AMPK is an important factor of the energy metabolism within cells. Shani BS and his team have found that GLP-1 could reduce the lipid accumulation in hepatocyte through the AMPK pathway. Now, there are few researches about the effects and mechanism of Exenatide on the expression of PGC-1?in LO2 cells. Therefore, doing research about whether GLP-1 could regulate the expression of PGC-1? through AMPK pathway in hepatocyte, would provide further evidence for the use of GLP-1in the treatment of NAFLD.Objective(1)To construct the cell model for NAFLD with free fatty acids.(2)To explore the effects of GLP-1R agonist Exenatide on the expression of PGC-1? and the lipid accumulation in nonalcoholic fatty liver cells model, and the relationship between the expression of PGC-1? and the lipid accumulation in hepatocyte.MethodsLO2 cells were cultured in vitro with ROMI1640 medium containing 10% fetal bovine serum. The experiment consists of two parts. The first part is divided into five groups:(1)normal group: there was no FFA in the medium;(2)FFA 0.5m M group;(3)FFA 0.75 m M group;(4)FFA 1m M group;(5)FFA 1.25 m M group;(6)FFA 1.5m M group. Each group was cultured for 24 h in the same environment. CCK-8 was used to reflect the proliferation of the cells and then determine the optimal concentration of FFA. Choose 1m M FFA as the model group. Oil Red O staining and triglyceride quantification was used to determine the degree of lipid accumulation. The second part is also divided into five groups:(1)control group: there was no FFA in the medium;(2)high-fat group: the cells were cultured in the medium containing1mmol/L FFA;(3)high-fat+ Exenatide 10nmol/L group: the cells were cultured in the medium containing 1mmol/L FFA and 10nmol/L Exenatide;(4)high-fat+ Exenatide100nmol/L group: the cells were cultured in the medium containing 1mmol/L FFA and 100nmol/L Exenatide;(5)high-fat+ Exenatide 100nmol/L +AMPK inhibitor group: the cells were cultured in the medium containing 1mmol/L FFA, 100nmol/L Exenatide and 15?mol/L Compound C. Each group was cultured for 24 h in the same environment. Oil Red O staining and triglyceride quantification was used to determine the degree of lipid accumulation. Real-time quantitative reverse transcription PCR was used to detect the level of PGC-1?m RNA and ACC m RNA.Western blotting was used to detect the level of PGC-1? protein.Results(1)(1) CCK-8 results: compared with the normal group, there were no significant effect on the proliferation of the cells when incubated with 0.5mmol/L,0.75mmol/L and 1mmol/L FFA for 24h(P>0.05), but the cells proliferation was decreased when incubated with 1.25mmol/L and 1.5mmol/L FFA for 24h(P<0.05).(2)Oil Red O staining results: there were a large number of lipid droplets in the model group, while the was no obvious lipid droplets in the normal group.(3)triglyceride quantification results: the level of intracellular triglyceride was higher than the normal group(P<0.05).(2)Compared with the normal group, the levels of the lipid droplets, the intracellular triglyceride and the ACC m RNA in high-fat group increased(P<0.05),the levels of PGC-1? m RNA and protein decreased(P<0.05); Compared with the high-fat group, when given Exenatide 10nmol/L or 100nmol/L, the levels of the lipid droplets, the intracellular triglyceride and the ACC m RNA decreased, and the decrease in Exenatide 100nmol/L group was more obvious than the Exenatide10nmol/L group, with statistical significance(P<0.05), the levels of PGC-1? m RNA and protein increased, and the increase in Exenatide 100nmol/L group was more obvious than the Exenatide 10nmol/L group, with statistical significance(P<0.05);Compared with the high-fat+ Exenatide 100nmol/L group, the levels of the lipid droplets, the intracellular triglyceride and the ACC m RNA in high-fat+ Exenatide100nmol/L +AMPK inhibitor group were increased, and the levels of PGC-1?m RNA and protein decreased, with statistical significance(P<0.05); The relevance of the results showed that, there was a negative correlation between the levels of PGC-1? expression and the level of intracellular triglyceride( r=-0.799,r=-0.826,P<0.01).Conclusions GLP-1R agonist Exenatide could regulate the expression of PGC-1? via the AMPK pathway and then reduce the lipid accumulation in the the nonalcoholic fatty liver cells model, which is dose-dependent.