| Nonalcoholic fatty liver disease(NAFLD) is a kind of liver injury caused by metabolic stress, which is closely associated with insulin resistance and genetic susceptibility. NAFLD represents a spectrum of liver impairment including nonalcoholic fatty liver, nonalcoholic steatohepatitis and related cirrhosis, and hepatocellular carcinoma, whose pathological change is similar to that of alcoholic liver disease. In contrast with alcoholic liver disease, patients with NAFLD do not have history of excess drinking. Epidemiological data have confirmed that NAFLD is one of important public health problems in 21 st century. However, there is no specific treatment against NAFLD, and new drugs shall be exploited on its development mechanisms.In recent years, fructose intake has increased in western countries. In our country, the consumption of coke and fruit juice also increase obviously due to influence of western diet. The development of NAFLD is associated with unhealthy diet, especially the excess intake of fructose. There is practical significance in using NAFLD animal models induced by high fructose diet to observe the influence of drug intervention. In previous studies of our group, high fructose diet led to fat deposition in liver of rodents, and induced NAFLD mice and rat models. We also confirmed that endogenous triglyceride synthesis in liver was the main mechanism of liver fat deposition caused by high fructose diet. Therefore, the NAFLD model induced by high fructose diet was chosen in this study.Gastric inhibitory polypeptide(GIP) and glucagon-like peptide-1(GLP-1) are two major active polypeptides of incretin. GLP-1 receptor agonists and dipeptidyl peptidase 4 inhibitors, which are exploited based on GLP-1, are focused as new antidiabetic drugs. GLP-1 receptors are widely distributed in the body, and its biological effects in the human body are extensive. In 2010, Gupta et al confirmed the existence of GLP-1 receptor in human primary liver cells for the first time, which made the foundation of biological research between GLP-1 and liver. Soon after, clinical, animal and cell studies found that GLP-1 improved liver lipid deposition; however, the exact mechanism was not clear.The main function of β-catenin is mediating inter-cellular adhesion and participating in gene expression. As the core molecule of Wnt signaling pathway, β-catenin was found to be associated with the development of obesity, diabetes, NAFLD and metabolic syndrome. Animal experiments showed that hepatic β-catenin gene knockout in mice led to fat deposition in liver. Accompanied with advanced researches, β-catenin was found to play an important role in the synthesis and function of GLP-1. However, there is no research on interaction between GLP-1 and β-catenin in the development of NAFLD. Whether GLP-1 can improve liver fat deposition through β-catenin remains to be investigated, and few related researches have been reported. Therefore, in the present study, we first established liver fat deposition in Wistar rats induced by high fructose diet, and observed the changes of de novo lipogenesis and β-catenin level in liver with the GLP-1 intervention. In addition,cell transfection was used in Hep G2 cells to intervene β-catenin pathway and explore whether GLP-1 improve liver fat deposition through β-catenin. By using two different experimental methods, we investigated the impact of GLP-1 and β-catenin in the development of NAFLD, explored the biological effects GLP-1 in liver and lipid metabolism, and then provided theoretical basis to exploit new drugs of NAFLD. Part One Influence of GLP-1 receptor agonist on liver fat deposition inrats with high fructose dietObjective: To investigate physiological, biological and histopathological changes in liver of Wistar rats fed with high fructose diet and GLP-1 intervention, and to explore the effect of GLP-1 on liver fat deposition in rats fed with high fructose diet.Method: Thirty six Wistar rats aged 6 weeks were used in this study. After a week of acclimation feeding, all rats were divided into 2 groups: normal diet group(ND, n=14) and high fructose diet group(HFD, n=22). Rats in ND group were fed with standard lab chow diet with the energy contents as follows: 65.5% calories from carbohydrate, 10.3% calories from fat, and 24.4% calories from protein with total energy of 348kcal/100 g. Rats in HFD group were fed with a high fructose diet with the same energy contents as normal diet; in which carbohydrates were partially substituted with fructose(fructose provided 60% calories). All rats were fed with the same energy each day. The diet intake amount was recorded every day and weight was recorded every week. After eight weeks of feeding, six rats were randomly chosen from each group to be sacrificed. Liver tissues were collected immediately and sent to histopathological examination to prove the model establishment of liver fat deposition in rats. Then rats in HFD were randomly divided into two subgroups: HFD(n=8) and high fructose diet with exenatide intervention(HFD+Ex, n=8). Rats in HFD+Ex group were received subcutaneous injection with exenatide(10ug/kg) twice a day; while the rats in ND and HFD received normal saline injection with the equal volume of exenatide. The drug dosage was recalculated every week according to the weight change. After eight weeks of feeding with high fructose diet and four weeks of drug intervention, intraperitoneal glucose test(IPGTT) and hyperinsulinemic-euglycemic clamp test were performed. The serum was collected to measure fasting blood glucose(FBG), total cholesterol(TC), triglyceride(TG), alanine aminotransferase(ALT) and aspartate aminotransferase(AST) by automatic biochemical analyzer. The fasting serum insulin(FINS) and free fatty acid(FFA) were determined by ELISA. Liver tissues were collected for the TG contents measurement in liver and oil red O staining.Results:1 Model establishment of liver fat deposition in rats induced by high fructose diet.1) Basic data comparison: Compared with ND group, the weight, liver index, fasting blood glucose(FBG), fasting insulin(FINS), TG, FFA, and hepatic TG contents were significant higher in HFD group(P<0.05). There was no significant difference in TC 、ALT and AST between two groups(P>0.05).2) Results of intraperitoneal glucose test: Compared with ND group,blood glucose at 0’, 5’, 30’, and 60’ was significantly higher in HFD group(P<0.05). The glucose area under the curve(AUCglu) was significantly higher in HFD group than that in ND group(P<0.05).3) Compared with ND, the glucose infusion rate(GIR) was significant decreased in HFD(P<0.05).4) Histology change in liver tissues: After the oil red O staining, the liver cell cytoplasm in ND was pale blue without red lipid droplet. While there was a lot of red lipid droplets in liver cytoplasm in HFD, indicating liver cell fat deposition and steatosis.2 Changes after exenatide intervention.1) Basic data comparison: Compared with ND, the weight, liver index, FBG, FINS, TG, FFA, ALT and hepatic TG contents were significant higher in HFD(P<0.05). After exenatide intervention, the abovementioned indexes in HFD+Ex group were significantly decreased when compared with HFD(P<0.05). No significant differences were found in TC and AST among ND, HFD, HFD+Ex groups.2) Results of intraperitoneal glucose test: Compared with ND group,blood glucose at 0’, 5’, 30’, 60’ and 120’ was significantly higher in HFD group(P<0.05). Compare with HFD, blood glucose at 0’, 30’, 60’ and 120’ was significantly decreased in HFD+Ex group(P<0.05). AUCglu was significantly higher in HFD group than that in ND group(P<0.05). After exenatide intervention, AUCglu in HFD+Ex group was significantly decreased when compared with HFD(P<0.05).3) Compared with ND, the GIR was significant decreased in HFD(P<0.05). After exenatide intervention, the GIR in HFD+Ex group increased significantly compared with HFD(P<0.05).4) Histology change in liver tissues among three groups: After the oil red O staining, the liver cell cytoplasm in ND was pale blue without red lipid droplet. There are a lot of red lipid droplets in liver cytoplasm in HFD. The amount of red lipid droplets was decreased in HFD+Ex after exenatide intervention.Conclusions:1 High fructose diet induced liver fat deposition in rats, led to hyperlipidemia and insulin resistance.2 GLP-1 receptor agonist attenuated liver fat deposition induced by high fructose diet in rats, caused weight loss and improved glucose tolerance, hyperlipidemia and insulin resistance. Part Two Effect of GLP-1 receptor agonist on gene and protein expressionin de novo lipogenesis pathway in rat liverObjective: To investigate gene and protein expressions of acetyl-Co A carboxylase(ACC), fatty acid synthase(FAS), stearoyl-Co A desaturase 1(SCD-1) and sterol regulatory element binding protein 1(SREBP-1) in de novo lipogenesis pathway in liver after exenatide intervention, and to explore mechanisms of GLP-1 receptor agonist in improving liver fat deposition from the perspective of lipid metabolism.Methods: Animal models and sample collection were illustrated in part one. Quantitative real-time PCR was used to measure the m RNA levels of ACC, FAS, SCD-1 and SREBP-1. Western blot was used to determine the protein expressions of ACC, FAS, SCD-1 and SREBP-1.Results:1 Comparison of expressions of key enzymes ACC, FAS and SCD-1 among there groups.Compared with ND group, the m RNA and protein expressions of ACC, FAS and SCD-1 were significant higher in liver tissues in HFD group(P<0.05). Exenatide intervention significantly decreased the m RNA and protein expressions of ACC, FAS and SCD-1 in HFD+Ex group when compared with HFD group(P<0.05).2 Comparison of expressions of SREBP-1 among three groups. Compared with ND group, the m RNA and protein expression of SREBP-1 was significant higher in liver tissues in HFD group(P<0.05). Exenatide intervention significantly decreased the m RNA and protein expression of SREBP-1 in HFD+Ex group when compared with HFD group(P<0.05).Conclusion:1 In rats fed with high fructose diet, the gene and protein expressions of ACC, FAS, SCD-1 and SREBP-1 in de novo lipogenesis pathway in liver were increased, indicating that high fructose diet promoted de novo lipogenesis.2 Exenatide decreased the gene and protein expressions of ACC, FAS, SCD-1 and SREBP-1, demonstrating that GLP-1 receptor agonist attenuated liver fat deposition through inhibiting de novo lipogenesis. Part Three Effect of GLP-1 receptor agonist on β-catenin expression inrat liverObjective: To determine β-catenin expression changes and nuclear translocation after GLP-1 receptor agonist intervention, and to clarify weather β-catenin participated in GLP-1 improving fat deposition in liver.Methods: Animal models and sample collection were illustrated in part one. Quantitative real-time PCR was used to measure the β-catenin m RNA expression. Western blot was used to determine the total β-catenin protein expressions and endonuclear β-catenin protein expression. Translocation of β-catenin was determined by immunohistochemistry.Results:1 The m RNA expression of β-catenin among three groups. Compared with ND group, the m RNA expression of β-catenin was significant lower in liver tissues in HFD group(P<0.05). Exenatide intervention significantly increased m RNA expression of β-catenin in HFD+Ex group when compared with HFD group(P<0.05).2 The protein expression of β-catenin among three groups. The β-catenin protein expression in liver tissues in HFD group was significant lower than that in ND group(P<0.05). After exenatide intervention, the protein expression of β-catenin was significantly increased in HFD+Ex group when compared with HFD group(P<0.05).3 The endonuclear protein expression of β-catenin among three groups. Compared with ND group, the endonuclear protein expression of β-catenin was significant lower in liver tissues in HFD group(P<0.05). Exenatide intervention significantly increased endonuclear protein expression of β-catenin in HFD+Ex group when compared with HFD group(P<0.05).4 Immunohistochemistry results of β-catenin among three groups. After exenatide intervention, β-catenin staining was increased in nuclear in HFD+Ex group, indicating increased β-catenin translocation from cytoplasm to nuclear.Conclusions:1 The gene and protein expressions of β-catenin, and endonuclear protein expressions of β-catenin were decreased in liver tissues of rat fed with high fructose diet.2 GLP-1 receptor agonist increased the gene and protein expressions of β-catenin,and endonuclear protein expressions of β-catenin, which may induce β-catenin expression and translocation.3 GLP-1 receptor agonist may improve liver fat deposition through β-catenin activation. Part Four Investigation of mechanisms of GLP-1 receptor agonist onimproving liver fat deposition through regulating β-cateninexpressionObjective: Cell transfection was applied to targeted intervene β-catenin expression in Hep G2 cells to observe changes of fat deposition and de novo lipogenesis, to explore whether β-catenin regulate de novo lipogenesis, and to reveal relationship between GLP-1 and β-catenin in fat deposition.Methods:1 Hep G2 cells were cultured with fructose plus Exendin-4 and intervened with β-catenin si RNA. In the β-catenin si RNA study, five groups were concluded: control group(Con), high fructose group(HF), high fructose plus Exendin-4 group(HF+Ex4), high fructose plus Exendin-4 plus control si RNA group(HF+Ex4+Si-control) and high fructose plus Exendin-4 plus β-catenin si RNA group(HF+Ex4+ Si-β-catenin). TG content in Hep G2 cells and oil red O staining was used to determine cell fat deposition. Quantitative real-time PCR was used to measure m RNA expressions of β-catenin, SREBP-1, ACC, FAS and SCD-1. Western blot was used to determine the protein expression of β-catenin, endonuclear β-catenin, SREBP-1, ACC, FAS and SCD-1.2 Hep G2 cells were cultured with fructose plus Exendin-4 and intervened with β-catenin overexpression plasmid. In the β-catenin overexpression plasmid study, there are five groups: control group(Con), high fructose group(HF), high fructose plus Exendin-4 group(HF+Ex4), high fructose plus Exendin-4 plus control empty plasmid group(HF+Ex4+ p EGFP-N1) and high fructose plus Exendin-4 plus β-catenin overexpression plasmid group(HF+Ex4+p EGFP-N1-h CTNNB1).The testing indexes as above were measured.Results:1 Establishment of fat deposition model in Hep G2 cell in vitro.The TG content in HF group was significant higher than that in Con group(P<0.05) after 24 hours culture. Red staining area increased significantly in HF group than in the Con group(P<0.05), indicating successful establishment of cell fat deposition model in vitro induced by high fructose.2 Effects of targeted intervention on β-catenin in Hep G2 cell.1) After transfected with β-catenin si RNA in Hep G2 cells cultured with fructose plus Exendin-4, the m RNA and protein expressions of β-catenin were significantly decreased in HF+Ex4+Si-β-catenin group when compared with HF+Ex4 group(P<0.05). Endonuclear β-catenin protein expression also significantly decreased, indicating that β-catenin expression was suppressed. There was no significant difference in β-catenin expression between HF+Ex4+Si-control group and HF+Ex4 group(P>0.05).2) After transfected with β-catenin overexpression plasmid in Hep G2 cells cultured with fructose plus Exendin-4, the m RNA and protein expressions of β-catenin in HF+Ex4+p EGFP-N1-h CTNNB1 group were significant higher than those in HF+Ex4 group(P<0.05). Endonuclear β-catenin protein expression also significantly increased, indicating that β-catenin expression was upregulated. There was no significant difference in β-catenin expression between HF+Ex4+p EFFP-N1 group and HF+Ex4 group(P>0.05).3) Compared with Con group, the m RNA and protein expressions of β-catenin and endonuclear protein expressions of β-catenin were significantly decreased in HF group(P<0.05) either in upregulation or in downregulation study. After Exendin-4 intervention, the m RNA and protein expressions of β-catenin and endonuclear protein expressions of β-catenin were significantly increased in HF+ Ex4 group when compared with HF group(P<0.05).3 Effects of targeted intervention of β-catenin on fat deposition in Hep G2 cell.1) The TG content in HF group was significant higher than that in Con group either in upregulation or in downregulation study(P<0.05), and the oil red O staining area increased in HF group. Compared with HF group, the TG content was significantly decreased in HF+ Ex4 group after Exendin-4 intervention(P<0.05), and the oil red O staining area decreased.2) After transfected with β-catenin si RNA in Hep G2 cells cultured with fructose plus Exendin-4, the TG content in HF+Ex4+Si-β-catenin group was significant higher than that in HF+Ex4 group(P<0.05), and the oil red O staining area significantly increased. There was no significant difference in TG content between HF+Ex4+Si-control group and HF+Ex4 group(P>0.05).3) After transfected with β-catenin overexpression plasmid in Hep G2 cells cultured with fructose plus Exendin-4, there was no significant difference in the TG content between HF+Ex4+p EGFP-N1-h CTNNB1 group and HF+Ex4 group(P>0.05), although a decrease trend of TG content and oil red O staining area in HF+Ex4+p EGFP-N1-h CTNNB1 group. There was no significant difference in TG content between HF+Ex4+p EGFP-N1 group and HF+Ex4 group(P>0.05). Compared with HF group, the TG content was significantly decreased in HF+Ex4+p EGFP-N1-h CTNNB1 group(P<0.05), and the oil red O staining area significantly decreased.4 Effects of targeted intervention of β-catenin on upstream regulatory factors and key enzymes in de novo lipogenesis pathway in Hep G2 cell.1) The m RNA and protein expressions of SREBP-1, ACC, FAS and SCD-1 in HF group were significant higher than those in Con group either in upregulation or in downregulation study(P<0.05). Compared with HF group, the m RNA and protein expressions of SREBP-1, ACC, FAS and SCD-1 were significantly decreased in HF+Ex4 group after Exendin-4 intervention(P<0.05).2) After successfully transfected with β-catenin si RNA in Hep G2 cells cultured with fructose plus Exendin-4, the m RNA and protein expressions of SREBP-1, ACC, FAS and SCD-1 in HF+Ex4+Si-β-catenin group was significant higher than those in HF+Ex4 group(P<0.05). There were no significant differences in m RNA and protein expressions of SREBP-1, ACC, FAS and SCD-1 between HF+Ex4+Si-control group and HF+Ex4 group(P>0.05).3) After successfully transfected with β-catenin overexpression plasmid in Hep G2 cells cultured with fructose plus Exendin-4, the m RNA and protein expressions of SREBP-1, ACC, and SCD-1 and FAS m RNA expression in HF+Ex4+ p EGFP-N1-h CTNNB1 group were significantly lower than those in HF+Ex4 group(P<0.05). No significant difference was observed in FAS protein expression(P>0.05), although a decrease trend in HF+Ex4+p EGFP-N1-h CTNNB1 group. There were no significant differences in in m RNA and protein expressions of SREBP-1, ACC, FAS and SCD-1 between HF+Ex4+p EGFP-N1 group and HF+Ex4 group(P>0.05).Conclusions:1 GLP-1 receptor agonist Exendin-4 attenuated fat deposition in Hep G2 cell cultured with high fructose through regulating SREBP-1 expression to regulate the key enzymes expression of ACC、FAS、SCD-1 to improve intracellular fat deposition.2 β-catenin may be the key molecules for GLP-1 receptor agonist regulating the expression of ACC、FAS、SCD-1 and SREBP-1 and improving intracellular fat deposition. |
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