G蛋白偶联受体MAS对肝脏糖脂代谢的影响

doi:10.3969/j.issn.1006-7795.2019.03.016

摘要/Abstract

摘要： 目的探究G蛋白偶联受体MAS （MAS receptor，MASR）对肝脏糖脂代谢的影响。方法采用MAS腺病毒感染人肝癌细胞株HepG2细胞，再加入毒胡萝卜素（thapsigargin，Tg）诱导内质网应激，检测HepG2细胞内三酰甘油（triglyceride，TG）、总胆固醇（total cholesterol，TC）及糖原（glycogen）含量；Real-time PCR检测HepG2细胞内内质网应激、细胞凋亡以及糖脂代谢相关基因mRNA水平；Western blotting法检测、葡萄糖-6-磷酸酶（glucose-6-phosphatase，G6pase）、磷酸烯醇式丙酮酸羧激酶（phosphoenolpyruvate carboxykinase，PEPCK）、脂肪酸合成酶（fatty acid synthase，FAS）、乙酰辅酶A羧化酶α（acetyl CoA carboxylase α，ACCα）、磷酸化乙酰辅酶A羧化酶α（phosphorylated-acetyl CoA carboxylase α，p-ACCα）、蛋白水平。结果内质网应激诱导MAS基因的表达；MAS过表达明显降低内质网应激相关基因C/EBP同源蛋白（C/EBP homologous protein，CHOP）、葡萄糖调节蛋白78（glucose regulated protein 78，GRP78）、活化转录因子6（activating transcription factor6，ATF6）、X盒结合蛋白1（X box-binding protein1，Xbp1）和肌醇需求酶1（inositol requiring protein-1，IRE1）的表达，同时，细胞凋亡相关基因Bax和Caspase-12表达明显降低。MAS过表达降低Tg诱导的HepG2细胞内三酰甘油，此外HepG2细胞内糖原浓度升高。同时，在分子水平，糖异生关键酶G6pase、PEPCK在mRNA及蛋白水平表达下调，脂质合成相关的FAS和ACCα表达也下调，差异均有统计学意义。结论 MAS改善HepG2细胞糖脂代谢，可能与MAS对肝脏内质网应激和凋亡的保护作用有关。

关键词: MAS受体, 内质网应激, 细胞凋亡, 肝脏糖脂代谢

Abstract: Objective To explore effects of G protein-coupled MAS receptor (MASR) on glycolipid metabolism in human liver. Methods Human hepatocyte cell line HepG2 was infected with MAS adenovirus. Endoplasmic reticulum stress was induced by adding thapsigargin (Tg) to detect triglyceride (TG), total cholesterol (TC) and glycogen content in HepG2 cells. Real-time PCR was used to detect the mRNA levels of endoplasmic reticulum stress-related apoptosis genes and glucose/lipid metabolism related genes in HepG2 cells; Western blotting method was performed for the detection of glucose-6-phosphatase (G6pase), phosphoenol phosphoenolpyruvate carboxykinase (PEPCK), fatty acid synthase (FAS), acetyl-CoA carboxylase α (ACCα), phosphorylated acetyl CoA carboxylase α (p-ACCα); protein level expression.Results Endoplasmic reticulum stress induced the expression of MAS gene, while MAS overexpression significantly decreased the expression of endoplasmic reticulum stress-related genes including C/EBP homologous protein (CHOP), glucose regulated protein (GRP78), activating transcription factor 6 (ATF6), X box-binding protein 1 (Xbp1) and inositol requiring protein 1(IRE1). The expression levels of apoptosis-related genes Bax and Caspase-12 were obviously reduced. MAS overexpression reduced Tg-induced intracellular triglyceride levels, in addition to elevated levels of glycogen in HepG2 cells. At the same time, at the molecular level, the expression of G6pase and PEPCK were down-regulated both in mRNA and protein levels, and the expressions of FAS and ACCα related to lipid synthesis were also down-regulated. Conclusion MAS can improve the glucose and lipid metabolism in HepG2 cells, which may be related to the protective effect of MAS on hepatic endoplasmic reticulum stress and apoptosis.

Key words: MAS receptor, endoplasmic reticulum stress, apoptosis, hepatic glycolipid metabolism

中图分类号:

张怡尘, 曹曦, 杨金奎. G蛋白偶联受体MAS对肝脏糖脂代谢的影响[J]. 首都医科大学学报, 2019, 40(3): 409-416.

Zhang Yichen, Cao Xi, Yang Jinkui. Effect of G protein-coupled receptor MAS on hepatic glycolipid metabolism[J]. Journal of Capital Medical University, 2019, 40(3): 409-416.

参考文献

[1] Hu C, Jia W. Diabetes in China:epidemiology and genetic risk factors and their clinical utility in personalized medication[J]. Diabetes, 2018, 67(1):3-11.
[2] Fruci B, Giuliano S, Mazza A, et al. Nonalcoholic fatty liver:a possible new target for type 2 diabetes prevention and treatment[J]. Int J Mol Sci, 2013, 14(11):22933-22966.
[3] Bader M,Santos R A, Unger T, et al. New therapeutic pathways in the RAS[J]. J Renin Angiotensin Aldosterone Sys, 2012,13(4):505-508.
[4] Balakumar P, Jagadeesh G. A century old renin-angiotensin system still grows with endless possibilities:AT1 receptor signaling cascades in cardiovascular physiopathology[J]. Cell Signal, 2014, 26(10):2147-2160.
[5] Santos R A,Simoese e Silva A C, Maric C, et al. Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas[J]. Proc Natl Acad Sci U S A, 2003, 100(14):8258-8263.
[6] Sampaio W O,Souza dos Santos R A, Faria-Silva R, et al. Angiotensin-(1-7) through receptor Mas mediates endothelial nitric oxide synthase activation via Akt-dependent pathways[J]. Hypertension, 2007, 49(1):185-192.
[7] Becker L K, Etlvino G M, Walther T, et al. Immunofluorescence localization of the receptor Mas in cardiovascular-related areas of the rat brain[J]. Am J Physiol Heart Circ Physiol, 2007,293(3):H1416-H1424.
[8] Vaz-Silva J, Tavares R L, Ferreira M C, et al. Tissue specific localization of angiotensin-(1-7) and its receptor Mas in the uterus of ovariectomized rats[J]. J Mol Histol, 2012, 43(5):597-602.
[9] Vaajanen A,Kalesnykas G, Vapaatalo H, et al. The expression of Mas-receptor of the renin-angiotensin system in the human eye[J]. Graefes Arch Clin Exp Ophthalmol, 2015, 253(7):1053-1059.
[10] Cao X,Yang F Y, Yin Z, et al. The ACE2/Ang-(1-7)/Mas axis can inhibit hepatic insulin resistance[J]. Mol Cell Endocrinol, 2014, 393(1-2):30-38.
[11] Cao X,Yang F, Shi T, et al. Angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis activates Akt signaling to ameliorate hepatic steatosis[J]. Sci Rep, 2016, 6:21592.
[12] Marciniak S J, Ron D. Endoplasmic reticulum stress signaling in disease[J]. Physiol Rev, 2006,86(4):1133-1149.
[13] Kadowaki H, Nishitoh H. Signaling pathways from the endoplasmic reticulum and their roles in disease[J]. Genes (Basel), 2013,4(3):306-333.
[14] Yu C,Cui, S, Zong C, et al. The orphan nuclear receptor NR4A1 protects pancreatic β-cells from endoplasmic reticulum (ER) stress-mediated apoptosis[J]. J Biol Chem, 2015, 290(34):20687-20699.
[15] Waris G, Tardif K D, Siddiqui A. Endoplasmic reticulum (ER) stress:hepatitis C virus induces an ER-nucleus signal transduction pathway and activates NF-kappaB and STAT-3[J]. Biochem Pharmacol, 2002, 64(10):1425-1430.
[16] Zhang J, Li Y, Jiang S, et al. Enhanced endoplasmic reticulum SERCA activity by overexpression of hepatic stimulator substance gene prevents hepatic cells from ER stress-induced apoptosis[J]. Am J Physiol Cell Physiol, 2014, 306(3):C279-290.
[17] Shi F H,Wu Y, Dai D Z, et al. Hepatosteatosis and hepatic insulin resistance are blunted by argirein, an anti-inflammatory agent, through normalizing endoplasmic reticulum stress and apoptosis in diabetic liver[J]. J Pharm Pharmacol, 2013, 65(6):916-927.
[18] Mota M, Banini B A, Cazanave S C, et al. Molecular mechanisms of lipotoxicity and glucotoxicity in nonalcoholic fatty liver disease[J]. Metabolism, 2016,65(8):1049-1061.
[19] Arumugam S, Thalavarayan R A, Palaniyandi S S, et al. Candesartan cilexetil protects from cardiac myosin induced cardiotoxicity via reduction of endoplasmic reticulum stress and apoptosis in rats:involvement of ACE2-Ang (1-7)-mas axis[J]. Toxicology, 2012, 291(1-3):139-145.
[20] Uhal B D, Nguyen H, Dang M, et al. Abrogation of ER stress-induced apoptosis of alveolar epithelial cells by angiotensin 1-7[J]. Am J Physiol Lung Cell Mol Physiol, 2013, 305(1):L33-L41.
[21] Santos S H, Giani J F, Burghi V, et al. Oral administration of angiotensin-(1-7) ameliorates type 2 diabetes in rats[J]. J Mol Med (Berl), 2014, 92(3):255-265.
[22] Santos S H, Braga J F, Mario E G, et al. Improved lipid and glucose metabolism in transgenic rats with increased circulating angiotensin-(1-7)[J]. Arterioscler Thromb Vasc Biol, 2010, 30(5):953-961.
[23] Zhang F,Liu C, Wang L, et al. Antioxidant effect of angiotensin (17) in the protection of pancreatic beta cell function[J]. Mol Med Rep, 2016,14(3):1963-1969.
[24] Santos R A S, Sampaio W O, Alzamora A C, et al. The ACE2/Angiotensin-(1-7)/MAS axis of the renin-angiotensin system:focus on angiotensin-(1-7)[J]. Physiol Rev, 2018, 98(1):505-553.
[25] Liu C, LV X H, Li H X, et al. Angiotensin-(1-7) suppresses oxidative stress and improves glucose uptake via Mas receptor in adipocytes[J]. Acta Diabetol, 2012, 49(4):291-299.
[26] Santos S H, Fernandes L R, Mario E G, et al. Mas deficiency in FVB/N mice produces marked changes in lipid and glycemic metabolism[J]. Diabetes, 2008, 57(2):340-347.
[27] Pinheiro S V B, Ferreira A J, Kihen G T, et al. Genetic deletion of the angiotensin-(1-7) receptor Mas leads to glomerular hyperfiltration and microalbuminuria[J]. Kidney Int, 2009, 75(11):1184-1193.