胰高血糖素样肽-1受体激动剂改善高果糖饮食诱导的胰岛素抵抗大鼠肝脏脂质沉积机制研究

Mechanism of Glucagon-like Peptide-1 Receptor Agonist Improving Liver Lipid Deposition in a Rat Model of Insulin Resistance Induced by High-fructose Diet

背景 非酒精性脂肪性肝病发病率逐年升高但无特效药物,临床和基础研究显示降糖药物胰高血糖素样肽-1(GLP-1)受体激动剂能改善肝脏脂质沉积,但具体机制不明确。目的 探讨GLP-1受体激动剂改善高果糖诱导的胰岛素抵抗大鼠肝脏脂质沉积的机制。方法 2016年1—4月选取Wistar大鼠36只随机分为对照(ND)组和造模组,ND组给予普通饲料、造模组给予高果糖饲料喂养,8周后行高胰岛素-正葡萄糖钳夹实验证实造模组胰岛素抵抗形成,继续将造模组大鼠随机分为高果糖(HFD)亚组和高果糖+艾塞那肽(HFD+Ex)亚组,HFD+Ex亚组给予艾塞那肽注射液腹部皮下注射4周后,观察糖脂水平、胰岛素抵抗、肝脏脂质沉积、β-catenin表达和核转位以及脂质合成通路因子的变化。进一步用转染技术在HepG2细胞用小干扰RNA抑制β-catenin的表达观察细胞脂质沉积和脂质合成通路相关因子的变化,将HepG2细胞用25 mmol/L果糖和100 nmol/L exendin-4处理,未转染的细胞用作对照,全部细胞分为正常对照(Con)组、高果糖(HF)组、高果糖+exendin-4(HF+Ex4)组、高果糖+exendin-4+对照siRNA(HF+Ex4+Si-control)组、高果糖+exendin-4+β-catenin siRNA(HF+Ex4+Si-β-catenin)组。实验结束后收集大鼠体质量、肝指数、三酰甘油(TG)、肝脏TG、总胆固醇(TC)、游离脂肪酸(FFA)、丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、空腹血糖(FBG)、空腹胰岛素(FINS)、葡萄糖曲线下面积(AUC_(glu))、葡萄糖输注速率(GIR)、肝脏油红O染色,并测定大鼠肝脏及HepG2细胞固醇调节元素结合蛋白1(SREBP-1)和下游脂质合成的关键酶脂肪酸合成酶(FAS)、乙酰辅酶A羧化酶(ACC)、硬脂酰CoA脱饱和酶1(SCD-1)以及β-catenin的蛋白表达水平。结果 (1)高果糖喂养8周后造模组大鼠体质量、肝指数、肝脏TG水平均高于ND组,GIR低于ND组(P<0.05);药物干预4周后HFD亚组大鼠体质量、肝指数、TG、FFA、ALT、FBG、FINS、AUC_(glu)高于ND组,GIR低于ND组(P<0.05);HFD+Ex亚组大鼠体质量、肝指数、FFA、ALT、FBG、FINS、AUC_(glu)低于HFD亚组,GIR高于HFD亚组(P<0.05)。(2)HFD亚组大鼠肝脏TG水平高于ND组(P<0.05),油红O染色肝细胞内可见大量红色脂滴聚集;HFD+Ex亚组大鼠肝脏TG水平低于HFD亚组(P<0.05),肝细胞内红色脂滴减少。(3)HFD亚组大鼠肝脏SREBP-1、FAS、SCD-1、ACC蛋白表达均高于ND组(P<0.05);HFD+Ex亚组大鼠肝脏SREBP-1、FAS、SCD-1、ACC蛋白表达均低于HFD亚组(P<0.05)。(4)HFD亚组大鼠肝脏β-catenin的总蛋白及核内蛋白表达低于ND组(P<0.05);HFD+Ex亚组大鼠肝脏β-catenin的总蛋白及核内蛋白表达高于HFD亚组(P<0.05)。(5)HF+Ex4组、HF+Ex4+Si-control组HepG2细胞β-catenin总蛋白、核内蛋白表达均高于HF组,TG水平低于HF组(P<0.05);HF+Ex4+Si-β-catenin组HepG2细胞β-catenin总蛋白、核内蛋白表达低于HF+Ex4组,TG水平高于HF+Ex4组(P<0.05)。(6)HF+Ex4组、HF+Ex4+Si-control组HepG2细胞SREBP-1、ACC、FAS、SCD-1蛋白表达均低于HF组(P<0.05);HF+Ex4+Si-β-catenin组HepG2细胞SREBP-1、ACC、FAS、SCD-1蛋白表达高于HF+Ex4组(P<0.05)。结论 GLP-1受体激动剂可能通过调控β-catenin表达改善胰岛素抵抗大鼠肝脏脂质沉积,是治疗非酒精性脂肪性肝病的潜在新药,β-catenin可能是药物治疗的重要靶标。

Background The incidence rate of nonalcoholic fatty liver disease is increasing year by year,but there is still no effective cure.Clinical and basic studies show that a type of hypoglycemic drug,namely glucagon-like peptide-1(GLP-1)receptor agonists can improve liver lipid deposition,but the specific mechanism is unknown.Objective To explore the mechanism of GLP-1 receptor agonists improving liver lipid deposition in a rat model of insulin resistance induced by highfructose diet.Methods This experiment was carried out from January to April 2016.Thirty-six Wistar rats were randomly divided into a control group(ND)receiving a normal diet and a model group receiving a high-fructose diet.After 8 weeks,a hyperinsulinemic-euglycemic clamp test was performed in the model group to verify the formation of insulin resistance.The rats in the model group were further randomized into a high-fructose(HFD)subgroup and a high fructose with exenatide(HFD+Ex)subgroup.The changes of glucose and lipid levels,insulin resistance,liver lipid deposition,the expression and nuclear translocation ofβ-catenin and lipid synthesis pathway related factors were observed in HFD+Ex subgroup at four weeks after receiving subcutaneous abdominal injection of exenatide injection.Further changes in cell lipid deposition and lipid synthesis pathway related factors were observed after inhibiting the expression ofβ-catenin with small interfering RNA(siRNA)by transfection techniques in HepG2 cells.HepG2 cells were treated with 25 mmol/L fructose,100 nmol/L exendin-4,and nontransfected HepG2 cells were used as controls.ALL of the cells were divided into normal control group(Con),high-fructose(HF)group,high fructose with exendin-4(HF+Ex4)group,high fructose with exendin-4 and control siRNA(HF+Ex4+Si-control)group,and high fructose with exendin-4 andβ-catenin siRNA(HF+Ex4+Si-β-catenin)group.After the experiment,the rats'weight and liver index,serum concentrations of triglyceride(TG),total cholesterol(TC),free fatty acid(FFA),alanine aminotransferase(ALT),aspartate aminotransferase(AST),fasting blood glucose(FBG),fasting insulin(FINS)and liver TG concentration were measured,and the area under the plasma glucose curve(AUC_(glu)),and glucose infusion rate(GIR)were calculated,and lipid droplets in liver tissues were observed using Oil Red O staining.The protein expression levels of sterol regulatory element binding protein 1(SREBP-1)and the key enzymes for downstream lipid synthesis,fatty acid synthase(FAS),acetyl coenzyme A carboxylase(ACC),stearoyl-CoA desaturase 1(SCD-1)andβ-catenin of liver tissues and HepG2 cells were also measured.Results(1)After 8-week high-fructose feeding,the model group had significantly higher weight,liver index and liver TG concentration,and lower GIR than the ND group(P<0.05).After 4 weeks of drug intervention,HFD subgroup demonstrated higher weight,liver index,TG,FFA,ALT,FBG,FINS and AUC_(glu),and lower GIR than the ND group(P<0.05).HFD+Ex subgroup showed lower weight,liver index,FFA,ALT,FBG,FINS,and AUC_(glu),and higher GIR than HFD subgroup(P<0.05).(2)Compared with ND group,HFD subgroup demonstrated higher concentration of TG in the liver(P<0.05),and a large number of red lipid droplets in liver cells.HFD+Ex subgroup had lower concentration of TG in the liver(P<0.05)and reduced red lipid droplets in liver cells compared with HFD subgroup.(3)Compared with ND group,the expression of SREBP-1,FAS,SCD-1 and ACC in liver of rats in HFD subgroup increased(P<0.05).Compared with HFD subgroup,the protein expression of SREBP-1,FAS,SCD-1 and ACC in HFD+Ex subgroup decreased(P<0.05).(4)Compared with ND group,the expression levels of total protein and nuclear protein ofβ-catenin in liver of rats in HFD subgroup were significantly decreased(P<0.05).Compared with HFD subgroup,the expression levels of total protein and nuclear protein ofβ-catenin increased in HFD+Ex subgroup(P<0.05).(5)Compared the HepG2 cells treated with HF,HF+Ex4 group had higher expression levels of total protein and nuclear protein ofβ-catenin and lower levels of serum TG,and so did HF+Ex4+Sicontrol group(P<0.05).Compared with HF+Ex4 group,HF+Ex4+Si-β-catenin group had down-regulated expression of total protein and nuclear protein ofβ-catenin(P<0.05).The levels of serum TG of HepG2 cells in HF+Ex4+Si-β-catenin group was higher than that in HF+Ex4 group(P<0.05).(6)HF+Ex4 group had lower protein expression levels of SREBP-1,ACC,FAS,and SCD-1 of HepG2 cells than HF group,and so did the HF+Ex4+Si-control group(P<0.05).The protein expression levels of SREBP-1,ACC,FAS,and SCD-1 of HepG2 cells in HF+Ex4+Si-β-catenin group were higher than those in HF+Ex4 group(P<0.05).Conclusion GLP-1 receptor agonists may regulateβ-catenin expression to improve liver lipid deposition in rats with insulin resistance,which are potential new drugs for nonalcoholic fatty liver disease.β-catenin may be an important target for drug treatment.