-
非酒精性脂肪肝(NAFLD)与胰岛素抵抗、糖尿病、代谢综合征和心血管疾病发病密切有关[1],严重时可能发展为肝硬化、肝癌。生活方式干预为治疗的首选,目前仍缺乏较为有效的治疗药物。胰高血糖素样肽-1(GLP-1)是一种由小肠L细胞分泌的具有刺激胰岛素分泌功能的激素,能调节食物摄入、胃肠蠕动、体液稳态和脂质代谢,刺激细胞增殖,减少炎症和细胞凋亡[2]。利拉鲁肽(LG)是GLP-1的类似物,2009年首次获得许可用于肥胖2型糖尿病(T2DM)病人的血糖控制。LG具有抑制中枢食欲、延迟胃排空时间和诱导剂量依赖性体质量减轻的特性,以往的研究表明LG可以通过非减肥的方式减轻高脂饮食(HFD)诱导的肝脏脂质积聚[3]。LG成为NAFLD病人考虑的一种有希望的治疗选择,然而,LG对NAFLD影响的机制尚不清楚。
腺苷酸活化蛋白激酶(AMPK)/雷帕霉素靶蛋白(mTOR)是调节自噬的主要通路[4],激活AMPK / mTOR通路调控自噬是一种重要的保护机制[5]。研究[6]表明,LG可以减轻肝脏脂肪变性,减少氧化应激,并激活自噬。LG除了改善T2DM和NAFLD病人的血糖控制外,还能降低体质量、肝内脂肪和内脏脂肪含量[7]。本研究基于AMPK/mTOR通路探讨LG在肝细胞脂肪变性的影响,观察自噬调节蛋白的变化。
-
结果显示,3组之间总体均数差异有统计学意义,其中模型组与对照组比较,小鼠血清ALT、AST、LDL、TC、TG升高; LG组与模型组相比,血清ALT、AST、LDL、TC、TG下降,差异具有统计学意义(P < 0.05~P < 0.01)(见表 1)。
分组 n ALT/(U/L) AST/(U/L) LDL/(mmo/L) TC/(mmol/L) TG/(mmol/L) 对照组 10 11.33±1.75 31.04±4.26 0.44±0.09 2.04±0.17 0.34±0.05 模型组 10 107.80±7.33** 63.49±8.90** 1.04±0.15** 5.02±0.55** 1.22±0.18** LG组 10 40.40±3.05**## 40.27±6.02**## 0.57±0.12*## 3.57±0.45**## 0.46±0.11*## F — 1 111.98 62.79 68.54 126.41 144.35 P — < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 MS组内 — 22.022 44.523 0.015 0.176 0.016 q检验:与对照组相比*P < 0.05,**P < 0.01;与模型组相比##P < 0.01 表 1 各组小鼠ALT、AST、LDL、TC、TG比较(x±s)
-
结果显示,对照组小鼠肝细胞排列规则、细胞核清晰、细胞质均匀、基本无脂肪变性,模型组肝细胞排列紊乱、细胞膨胀、可见大量脂肪空泡形成,LG组较模型组相比,肝细胞排列较其整齐,脂肪空泡明显减少。说明LG能改善肝细胞脂肪变性(见图 1)。
-
结果显示,对照组小鼠肝组织细胞中偶见橘黄色脂滴,高脂喂养后模型组小鼠肝组织细胞可见大量橘黄色脂滴,融合成片,而LG干预后脂滴含量明显减少(见图 2)。
-
结果显示,与对照组相比,模型组p-AMPK、LC3B、Beclin1的蛋白表达下降,p-mTOR表达升高;而LG组与模型组相比,p-AMPK、LC3B、Beclin1的蛋白表达水平均升高,p-mTOR表达下降,差异均有统计学意义(P < 0.05~P < 0.01)(见图 3、表 2)。
分组 n p-AMPK/AMPK p-mTOR/TOR LC3B Beclin1 对照组 10 1.10±0.14 0.48±0.09 0.37±0.10 0.79±0.20 模型组 10 0.54±0.08** 0.98±0.09** 0.26±0.03* 0.64±0.09* LG组 10 0.79±0.14**## 0.82±0.12**## 1.14±0.17**## 1.25±0.12**## F — 53.48 61.17 175.35 48.84 P — < 0.01 < 0.01 < 0.01 < 0.01 MS组内 — 0.015 0.010 0.013 0.020 q检验:与对照组相比*P < 0.05,**P < 0.01;与模型组相比##P < 0.01 表 2 各组小鼠肝细胞内p-AMPK、p-mTOR、LC3B、Baclin1蛋白水平的比较(x±s)
-
结果显示,模型组HepG2细胞可见极少量的LC3B的表达,LG组细胞内LC3B的表达较模型组升高,而AMPK抑制剂组LC3B的表达较LG组减少(见图 4)。
-
结果显示,和模型组相比,LG组p-AMPK、LC3B、Beclin1的蛋白表达升高,而应用AMPK抑制剂Compound C后上述指标表达较LG组受抑制下降(P < 0.05~P < 0.01);此外,和模型组相比,p-mTOR在LG组表达降低,应用AMPK抑制剂Compound C后表达较LG组增加,差异均有统计学意义(P < 0.05~P < 0.01)(见图 5、表 3)。
分组 p-AMPK/AMPK p-mTOR/ mTOR LC3B Beclin1 模型组 0.70±0.05 0.96±0.12 0.70±0.03 0.52±0.03 LG组 1.01±0.07* 0.52±0.05** 1.03±0.11** 1.06±0.10** AMPK抑制剂组 0.79±0.17# 0.68±0.04**# 0.70±0.05## 0.79±0.03**## F 6.39 24.39 20.56 57.39 P < 0.05 < 0.01 < 0.01 < 0.01 MS组内 0.012 0.006 0.005 0.004 q检验:与模型组相比*P < 0.05,**P < 0.01;与LG组相比#P < 0.05,##P < 0.01 表 3 各组HepG2细胞p-AMPK、p-mTOR、LC3B、Baclin1蛋白水平的比较(x±s)
利拉鲁肽通过AMPK/mTOR信号通路诱导自噬改善肝脂肪变性
Liraglutide induces autophagy through AMPK / mTOR signaling pathway and improves hepatocyte steatosis
-
摘要:
目的探讨腺苷酸活化蛋白激酶(AMPK)/雷帕霉素靶蛋白(mTOR)通路在利拉鲁肽(LG)改善肝细胞脂肪变性中的作用及机制。 方法动物实验:将小鼠分为对照组、模型组和LG组,各10只,对照组普通饲料喂养, 另2组高脂饲料喂养建立肝脂肪变性模型。LG组应用LG腹腔注射0.6 mg·kg-1·d-1,另2组应用等量0.9%氯化钠溶液,连续4周。检测小鼠血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、低密度脂蛋白(LDL)、胆固醇(TC)、三酰甘油(TG);应用HE染色和油红O染色观察肝细胞形态、脂肪变性和脂滴情况;应用蛋白质印迹(Western blotting)法检测p-AMPK、p-mTOR及自噬蛋白LC3B、Beclin1表达情况。细胞实验:应用棕榈酸(PA)诱导建立HepG2肝细胞脂肪变性模型,分为模型组(PA)、LG组(PA+LG)和AMPK抑制剂组(PA+LG+Compound C)。应用免疫荧光检测自噬相关蛋白LC3B的表达;应用Western blotting法检测p-AMPK、p-mTOR、LC3B、Beclin1的蛋白表达情况。 结果动物实验:模型组ALT、AST、LDL、TC、TG升高,应用LG后下降,差异均有统计学意义(P < 0.05~P < 0.01);HE染色、油红O染色发现,模型组肝细胞可见大片脂肪变性、大量融合脂滴,LG组脂变减轻、脂滴含量减少。Western blotting结果显示,模型组p-AMPK、LC3B、Beclin1的蛋白表达下降,而LG组中升高,模型组p-mTOR升高,而LG组中降低(P < 0.05~P < 0.01)。细胞实验:免疫荧光染色LC3B发现,LG组较模型组增加,而AMPK抑制剂组,LC3B的表达受到抑制;Western blotting结果示:和模型组相比,LG组p-AMPK、LC3B、Beclin1的蛋白表达升高,而应用AMPK抑制剂后表达受抑制,而p-mTOR相反, 差异均有统计学意义(P < 0.05~P < 0.01)。 结论LG通过AMPK/mTOR通路来改善肝细胞脂肪变性,自噬可能参与其中。 -
关键词:
- 肝脂肪变性 /
- 利拉鲁肽 /
- 腺苷酸活化蛋白激酶/雷帕霉素靶蛋白通路 /
- 自噬
Abstract:ObjectiveTo investigate the role and possible mechanism of adenylate activated protein kinase(AMPK)/rapamycin target protein(mTOR) pathway in the improvement of liraglutide(LG) on hepatocyte steatosis. MethodsIn animal experiment, the mice were randomly divided into control group, model group and LG group, with 10 mice in each group.The mice in the control group were fed with ordinary diet, and the mice in other groups were fed with high-fat diet to construct hepatic steatosis model.In the LG group, the mice were injected with LG at 0.6 mg·kg-1·d-1 intraperitoneally, and in the other two groups, the mice were treated with the same amount of 0.9% sodium chloride solution for 4 weeks.The levels of serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), low density lipoprotein(LDL), cholesterol(TC) and triglyceride(TG) were detected; the changes of morphology, steatosis and lipid droplets of hepatocytes were observed through HE and oil red O staining methods.The expressions of p-AMPK, p-mTOR, autophagy-related proteins LC3B and Beclin1 were detected by Western blotting.In cell experiment, the palmitic acid(PA) induced steatosis model in HepG2 hepatocytes was established, the cells were equally divided into model group(PA), LG Group(PA+LG) and AMPK inhibitor group(PA+LG+compound C).The expression of autophagy-related protein LC3B was detected by immunofluorescence; the protein expressions of p- AMPK, p-mTOR, LC3B and Beclin1 were detected by Western blotting. ResultsIn contrast to control group, the levels of ALT, AST, LDL, TC and TG in model group were increased significantly, and decreased significantly after LG treatment (P < 0.05 to P < 0.01);HE and oil red O staining showed that in the model group, there had large areas of steatosis and a large number of fused lipid droplets.The steatosis and lipid droplets content was decreased significantly in the LG group.Western blotting results showed that the protein expressions of p-AMPK, LC3B and Beclin1 were decreased significantly in model group, but increased in LG group, p-mTOR was increased in model group, and decreased in LG group(P < 0.05 to P < 0.01).Cell experiment: immunofluorescence staining showed that LC3B expression in LG group was significantly higher than that in model group, while LC3B expression was inhibited in AMPK inhibitor group.Western blotting results showed that compared with the model group, the protein expressions of p-AMPK, LC3B and Beclin1 in LG group were significantly increased, but the expressions were inhibited after the application of AMPK inhibitor, while p-mTOR expression was on the contrary, which had statistically significance(P < 0.05 to P < 0.01). ConclusionsLiraglutide improved hepatocyte steatosis through AMPK/mTOR pathway, and autophagy may be involved. -
表 1 各组小鼠ALT、AST、LDL、TC、TG比较(x±s)
分组 n ALT/(U/L) AST/(U/L) LDL/(mmo/L) TC/(mmol/L) TG/(mmol/L) 对照组 10 11.33±1.75 31.04±4.26 0.44±0.09 2.04±0.17 0.34±0.05 模型组 10 107.80±7.33** 63.49±8.90** 1.04±0.15** 5.02±0.55** 1.22±0.18** LG组 10 40.40±3.05**## 40.27±6.02**## 0.57±0.12*## 3.57±0.45**## 0.46±0.11*## F — 1 111.98 62.79 68.54 126.41 144.35 P — < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 MS组内 — 22.022 44.523 0.015 0.176 0.016 q检验:与对照组相比*P < 0.05,**P < 0.01;与模型组相比##P < 0.01 表 2 各组小鼠肝细胞内p-AMPK、p-mTOR、LC3B、Baclin1蛋白水平的比较(x±s)
分组 n p-AMPK/AMPK p-mTOR/TOR LC3B Beclin1 对照组 10 1.10±0.14 0.48±0.09 0.37±0.10 0.79±0.20 模型组 10 0.54±0.08** 0.98±0.09** 0.26±0.03* 0.64±0.09* LG组 10 0.79±0.14**## 0.82±0.12**## 1.14±0.17**## 1.25±0.12**## F — 53.48 61.17 175.35 48.84 P — < 0.01 < 0.01 < 0.01 < 0.01 MS组内 — 0.015 0.010 0.013 0.020 q检验:与对照组相比*P < 0.05,**P < 0.01;与模型组相比##P < 0.01 表 3 各组HepG2细胞p-AMPK、p-mTOR、LC3B、Baclin1蛋白水平的比较(x±s)
分组 p-AMPK/AMPK p-mTOR/ mTOR LC3B Beclin1 模型组 0.70±0.05 0.96±0.12 0.70±0.03 0.52±0.03 LG组 1.01±0.07* 0.52±0.05** 1.03±0.11** 1.06±0.10** AMPK抑制剂组 0.79±0.17# 0.68±0.04**# 0.70±0.05## 0.79±0.03**## F 6.39 24.39 20.56 57.39 P < 0.05 < 0.01 < 0.01 < 0.01 MS组内 0.012 0.006 0.005 0.004 q检验:与模型组相比*P < 0.05,**P < 0.01;与LG组相比#P < 0.05,##P < 0.01 -
[1] ARAB JP, ARRESE M, TRAUNER M. Recent insights into the pathogenesis of nonalcoholic fatty liver disease[J]. Annu Rev Pathol, 2018, 13(1): 321. doi: 10.1146/annurev-pathol-020117-043617 [2] MVLLER TD, FINAN B, BLOOM SR, et al. Glucagon-like peptide 1(GLP-1)[J]. Mol Metab, 2019, 30: 72. doi: 10.1016/j.molmet.2019.09.010 [3] LYU J, IMACHI H, FUKUNAGA K, et al. Role of ATP-binding cassette transporter A1 in suppressing lipid accumulation by glucagon-like peptide-1 agonist in hepatocytes[J]. Mol Metab, 2020, 34: 16. doi: 10.1016/j.molmet.2019.12.015 [4] ZHAO B, QIANG L, JOSEPH J, et al. Mitochondrial dysfunction activates the AMPK signaling and autophagy to promote cell survival[J]. Genes Dis, 2016, 3(1): 82. doi: 10.1016/j.gendis.2015.12.002 [5] WANG H, LIU Y, WANG D, et al. The upstream pathway of mtor-mediated autophagy in liver diseases[J]. Cells, 2019, 8(12): 1597. doi: 10.3390/cells8121597 [6] TONG W, JU L, QIU M, et al. Liraglutide ameliorates non-alcoholic fatty liver disease by enhancing mitochondrial architecture and promoting autophagy through the SIRT1/SIRT3-FOXO3a pathway[J]. Hepatol Res, 2016, 46(9): 933. doi: 10.1111/hepr.12634 [7] YAN J, YAO B, KUANG H, et al. Liraglutide, sitagliptin, and insulin glargine added to metformin: the effect on body weight and intrahepatic lipid in patients with type 2 diabetes mellitus and nonalcoholic fatty liver disease[J]. Hepatology, 2019, 69(6): 2414. doi: 10.1002/hep.30320 [8] 中华医学会内分泌学分会, 中华医学会糖尿病学分会. 中国成人2型糖尿病合并非酒精性脂肪性肝病管理专家共识[J]. 中华内分泌代谢杂志, 2021, 37(7): 589. doi: 10.3760/cma.j.cn311282-20210105-00016 [9] HAZLEHURST JM, WOODS C, MARJOT T, et al. Non-alcoholic fatty liver disease and diabetes[J]. Metabolism, 2016, 65(8): 1096. doi: 10.1016/j.metabol.2016.01.001 [10] NIEDERSEER D, WERNLY S, BACHMAYER S, et al. Diagnosis of non-alcoholic fatty liver disease (NAFLD) is independently associated with cardiovascular risk in a large austrian screening cohort[J]. J Clin Med, 2020, 9(4): 1065. doi: 10.3390/jcm9041065 [11] SU K, YI B, YAO BQ, et al. Liraglutide attenuates renal tubular ectopic lipid deposition in rats with diabetic nephropathy by inhibiting lipid synthesis and promoting lipolysis[J]. Pharmacol Res, 2020, 156: 104778. [12] RONDINELLI M, ROSSI A, GANDOLFI A, et al. Use of liraglutide in the real world and impact at 36 months on metabolic control, weight, lipid profile, blood pressure, heart rate, and renal function[J]. Clin Ther, 2017, 39(1): 159. doi: 10.1016/j.clinthera.2016.11.001 [13] RUSSELL-JONES D. Molecular, pharmacological and clinical aspects of liraglutide, a once-daily human GLP-1 analogue[J]. Mol Cell Endocrinol, 2009, 297(1): 137. [14] TAHER J, BAKER CL, CUIZON C, et al. GLP-1 receptor agonism ameliorates hepatic VLDL overproduction and de novo lipogenesis in insulin resistance[J]. Mol Metab, 2014, 3(9): 823. doi: 10.1016/j.molmet.2014.09.005 [15] HAO T, CHEN H, WU S, et al. LRG ameliorates steatohepatitis by activating the AMPK/mTOR/SREBP1 signaling pathway in C57BL/6J mice fed a highfat diet[J]. Mol Med Rep, 2019, 20(1): 701. [16] 沈敏燕, 胡曦, 朱静, 等. 黄连碱促进肝细胞自噬及胆固醇外流改善脂肪肝的脂质蓄积[J]. 蚌埠医学院学报, 2021, 46(4): 421. [17] ZHANG E, YIN S, SONG X, et al. Glycycoumarin inhibits hepatocyte lipoapoptosis through activation of autophagy and inhibition of ER stress/GSK-3-mediated mitochondrial pathway[J]. Sci Rep, 2016, 6: 38138. [18] 张强, 刘勤, 牛春燕. 利拉鲁肽减轻脂毒性肝细胞损伤并促进自噬改善非酒精性脂肪肝[J]. 中华肝脏病杂志, 2021, 29(5): 456. [19] FANG Y, JI L, ZHU C, et al. Liraglutide alleviates hepatic steatosis by activating the tfeb-regulated autophagy-lysosomal pathway[J]. Front Cell Dev Biol, 2020, 8: 602574. [20] ALTAMIRANO J, QI Q, CHOUDHRY S, et al. Non-invasive diagnosis: non-alcoholic fatty liver disease and alcoholic liver disease[J]. Transl Gastroenterol Hepatol, 2020, 5: 31. [21] ZHANG S, MAO Y, FAN X. Inhibition of ghrelin oacyltransferase attenuated lipotoxicity by inducing autophagy via AMPK-mTOR pathway[J]. Drug Design, Develop Ther, 2018, 12(4): 873. [22] 徐玲, 马红艳, 杨军, 等. 高脂饮食通过p-AMPK/mTOR信号通路下调小鼠肝细胞自噬水平[J]. 基础医学与临床, 2018, 38(1): 37.