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. 2021 Jul 20;41(7):1073–1078. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2021.07.16

Exendin-4通过促进自噬减少非酒精性脂肪性肝病细胞模型的脂质沉积

Exendin-4 promotes autophagy to relieve lipid deposition in a NAFLD cell model by activating AKT/mTOR signaling pathway

Chuman LIN 1,2,3, Junbo FANG 1,3, Qianru XIANG 1, Rui ZHOU 3,*, Li YANG 1,*
PMCID: PMC8329682  PMID: 34308859

Abstract

Objective

To investigate the effect of exendin-4 on lipid deposition in hepatocytes and explore its possible mechanism for treatment of nonalcoholic fatty liver disease (NAFLD).

Methods

Human normal hepatocyte line LO2 and hepatoma cell line HepG2 were treated with palmitic acid (PA) to mimic hepatocyte steatosis or with combined treatments with PA+exendin-4 or PA+exendin-4+3BDO. Lipid deposition and proliferation of the two cell lines following treatment with PA or PA+exendin-4 were detected using Oil Red O staining and CCK8 assay, and the expression of p-mTOR, m-TOR, p-AKT, AKT and autophagy-related proteins LC3-Ⅰ/Ⅱ and p62 were detected with Western blotting; the expression of GLP-1R was detected with both Western blotting and immunofluorescence assay. The expression of LC3-Ⅰ/Ⅱ and p62 in the cells following treatment with PA+exendin-4 and PA+exendin-4+3BDO was detected with Western blotting.

Results

Lipid deposition in the two cell lines increased significantly after PA treatment, but was alleviated by co-treatment with exendin-4. PA treatment significantly inhibited the proliferation of the two cell lines (P < 0.01), and this inhibitory effect was obviously attenuated by exendin-4 (P < 0.05). Immunofluorescence assay showed that both LO2 and HepG2 cells expressed GLP-1R. The expression of p-mTOR was significantly lower and that of p-AKT was higher in cells treated with PA+exendin-4 than in PA-treated cells. Exendin-4 also down-regulated the autophagy-associated protein p62 and up-regulated the expression of LC3-Ⅱ in PA-treated cells, and this effect was obviously reversed by 3BDO.

Conclusion

Exendin-4 may activate the AKT-mTOR signal pathway to promote autophagy via its direct action on GLP-1R. Exendin-4 can also alleviate lipid deposition and promote proliferation of PA-treated hepatocytes, suggesting its important role in PA-induced lipid deposition in hepatocytes.

Keywords: GLP-1, nonalcoholic fatty liver disease, autophagy, AKT-mTOR signaling pathway

非酒精性脂肪性肝病(NAFLD)是目前世界上最常见的慢性肝病，并且与世界范围内肥胖人数的增加息息相关。目前全球NAFLD的患病率约为24%，但是由于检测手段的局限性，其患病率常常被低估[1, 2]。NAFLD与多种代谢性疾病密切相关，包括肥胖、胰岛素抵抗、2型糖尿病和血脂异常等[3, 4]。然而，目前除了对生活方式的干预以及减肥之外，尚无明确有效的药物治疗方法[5]。已有研究证明，胰高血糖素样肽-1(GLP-1)受体在人肝细胞中表达，提示GLP-1可以直接作用于肝细胞上的GLP-1R进而发挥其功能；也有文献报道，在NAFLD中，GLP-1R的表达下调，提示针对GLP-1R的治疗可能有效，因此，我们对GLP-1在NAFLD模型中的作用进行了研究。以期为NAFLD的治疗提供新的思路

GLP-1是一种由肠道L-细胞分泌的内分泌激素[6]。它在哺乳动物中具有多种功能，包括促进胰岛素分泌，抑制胰高血糖素的释放，减缓胃排空，最大限度地减少胰岛素介导的葡萄糖摄取[7]。GLP-1受体激动剂艾塞那肽(Exendin-4)作为一类新型的抗糖尿病药物，已在临床上用于治疗2型糖尿病。最近的研究表明GLP-1受体(GLP-1R)在人肝细胞上表达，提示GLP-1可以直接作用于肝细胞上的GLP-1R进而发挥其功能[8]。此外，越来越多的证据表明，它们能有效降低脂质负荷和游离脂肪酸(FFA)诱导的肝细胞凋亡[8-10]。然而，GLP-1改善肝细胞脂肪变性的确切机制和所涉及的信号通路尚不完全清楚。我们前期的研究证明，在肾脏中，GLP-1通过调节AMPK/mTOR通路激活自噬改善糖尿病肾病[11]，因此，我们猜想：在NAFLD中，GLP-1发挥了相似的作用，以改善NAFLD。

自噬是一种细胞自身产生的应激适应性反应应答，其能够通过降解细胞毒性蛋白和细胞器，重新利用细胞内能量资源以应对营养物质耗竭[12]。自噬功能失调与多种疾病的发病机制有关，包括NAFLD[13]，糖尿病肾病[14]，癌症[15]和神经退行性疾病[16]等。已有研究表明，GLP-1受体激动剂Exendin-4可通过哺乳动物雷帕霉素靶蛋白/Unc-51-样激酶1(mTOR/ULK1)依赖性自噬减轻葡萄糖毒性引起的心脏损伤[17]；Exendin-4还通过增加自噬流和恢复溶酶体功能而保护胰腺β细胞免于死亡[18]；另外，Exendin-4还通过促进大鼠脊髓损伤后的自噬和抑制神经元凋亡而促进运动功能的恢复[19]。

有研究证明，Exendin-4可通过增强线粒体自噬，减轻氧化应激损伤，具有肝脏保护作用，但未在细胞水平上探索具体机制[20]。本实验通过复制NAFLD细胞模型，观察GLP-1受体激动剂Exendin-4对NAFLD细胞模型的作用，并阐明了其可能的潜在机制，为改善NAFLD的临床实践提供理论基础。

1. 材料和方法

1.1. 实验材料

人正常肝细胞LO2和肝癌细胞HepG2细胞株购自中国科学院上海细胞生物学研究所。胎牛血清和DMEM细胞培养基(Gibco)。CCK8(cell counting kit-8)试剂盒(全式金生物)。棕榈酸钠(PA，sigma)。Exendin-4、3BDO(mTOR激活剂)(MedChemExpress)。油红O试剂盒(索莱宝)。GLP-1R抗体(博奥森生物)，AKT、p-AKT、mTOR、P-mTOR、p62、LC3-Ⅰ/Ⅱ、GAPDH抗体(CST)。Alexa Fluor594标记山羊抗兔IgG二抗(赛默飞世尔科技)。4', 6-二脒基-2-苯基吲哚(DAPI)(碧云天)。

1.2. 方法

1.2.1. 细胞培养及肝细胞脂肪变性模型建立

LO2及HepG2细胞均在含10%胎牛血清的DMEM培养基，37 ℃、5% CO2培养箱内培养。在终浓度为0.3 µmol/L的棕榈酸钠(PA)作用下，诱导肝细胞脂肪变性。细胞分组：(1)正常对照组：使用含10%胎牛血清的DMEM培养基培养，无其他任何处理；(2)PA组：0.3 µmol PA，处理24 h；(3) PA+Exendin-4组：PA(0.3 µmol)、Exendin-4(100 nmol) 共同处理24 h。按照油红O试剂盒进行脂质染色。

1.2.2. CCK-8试验检测细胞增殖

将LO2及HepG2两种细胞用胰酶消化成单细胞后计数，按照每组检测6次，每次5个复孔，每孔初始1000个细胞计算各组的总加细胞液量，加至96孔板中。分别按照上述分组处理，并置于培养箱中培养，分别于0、1、2、3、4、5 d，向对应孔加100 µL的CCK8试剂(原液：DMEM培养基=1∶9)，立即放回孵箱，避光，反应2 h。结束后，酶标仪检测每孔中液体的吸光度(A450 nm)。

1.2.3. 细胞免疫荧光

将LO2及HepG2细胞铺至共聚焦小皿中，待细胞贴壁后，用PBS洗涤，4%多聚甲醛固定10 min，PBS洗涤，0.5% Triton处理15 min，PBS洗涤后在室温下用5%山羊血清孵育1h以阻断抗体的非特异性结合，随后在4 ℃下与GLP-1R抗体孵育过夜。第2天用PBS洗涤后，使用Alexa Fluor594标记山羊抗兔IgG荧光二抗避光孵育1 h，最后用DAPI对细胞核进行染色。用激光共聚焦显微镜捕获图像。

1.2.4. Western blot检测蛋白表达

提取细胞总蛋白，加入上样缓冲液，99 ℃下变性5 min，取等量蛋白进行10% 聚丙烯酰胺凝胶电泳(90 V，30 min；120 V，60 min)分离蛋白，湿法(200 mA，60 min)转印至PVDF膜；用含5% 牛血清白蛋白(BSA)封闭液室温封闭1 h；一抗GLP-1R、AKT、p-AKT、mTOR、p-mTOR、p62、LC3-Ⅰ/Ⅱ、GAPDH，分别置于4 ℃孵育过夜；PBST液洗膜3次，10 min/洗；二抗羊抗兔IgG、羊抗鼠IgG分别室温孵育1 h；PBST液洗膜5次，5 min/洗；ECL发光试剂盒显影；Bio-Rad凝胶成像系统获取图像。

1.2.5. 统计学方法

实验数据均采用SPSS26统计软件对检测结果进行统计学分析，定量资料以均数±标准差表示。P < 0.05认为差异具有统计学意义。

2. 结果

2.1. Exendin-4减少肝细胞内脂质沉积

与NC组相比，PA处理组细胞内脂滴明显增多，而Exendin-4可部分逆转PA的作用，减少细胞内脂质沉积(图 1)。

1.

1

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Exendin-4缓解肝正常细胞LO2和肝癌细胞HepG2细胞内脂质沉积

Exendin-4 relieves lipid deposition both in normal liver cell line LO2 and hepatoma cell line HepG2.

2.2. Exendin-4促进脂质沉积细胞增殖

PA处理后，细胞的增殖受到明显的抑制，而Exendin-4的加入可逆转PA对细胞的抑制作用(图 2)。

2.

<sup>**</sup><italic>P</italic> < 0.001, <sup>***</sup><italic>P</italic> < 0.001 <italic>vs</italic> the NC group; <sup>#</sup><italic>P</italic> < 0.05, <sup>##</sup><italic>P</italic> < 0.01 <italic>vs</italic> the PAgroup.

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Exendin-4促进肝正常细胞LO2和肝癌细胞HepG2的细胞增殖

Exendin-4 promotes proliferation of both LO2 and HepG2 cells (Mean ± SD).

2.3. GLP-1R表达于LO2及HepG2

Western blot结果显示，在人正常肝细胞LO2及肝癌细胞HepG2中均存在GLP-1R的表达(图 3)

3.

3

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肝正常细胞LO2和肝癌细胞HepG2中均有GLP-1受体表达

GLP-1 receptor is expressed in both LO2 and HepG2 cells.

2.4. Exendin-4通过AKT-mTOR信号通路促进自噬

Exendin-4处理后，p-mTOR的表达较PA组下调，p-AKT的表达较PA组上调。WB检测p62及LC3-Ⅰ/Ⅱ的表达，结果显示，Exendin-4可下调p62，上调LC3-Ⅱ的表达(图 4)。

4.

4

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Exendin-4通过AKT-mTOR信号通路促进肝正常细胞LO2和肝癌细胞HepG2的细胞自噬

Exendin-4 promotes autophagy of LO2 and HepG2 cells viaAKT-mTOR signaling pathway.

2.5. mTOR激活剂3BDO逆转Exendin-4的自噬促进作用

在PA诱导的LO2和HepG2细胞中，给予100 nmol/L Exendin-4处理的同时给予mTOR激活剂Exendin-4可下调p62，上调LC3-Ⅱ的表达。而在Exendin-4基础上给予3BDO干预其p62表达量上调，LC3-Ⅱ表达量下调(图 5)。

5.

5

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mTOR激活剂3BDO能够逆转Exendin-4的自噬促进作用

mTOR agonist 3BDO can reverse the autophagypromoting effect of exendin-4.

3. 讨论

NAFLD是全世界常见的肝脏疾病，其中包括非酒精性脂肪肝与非酒精性脂肪性肝炎。脂肪肝可能不会严重干扰肝功能，但是，脂肪性肝炎合并炎症和纤维化可发展为肝损害，并伴有许多并发症[21]。高达80%的肥胖者患有NAFLD，而20%的正常体质量的人也有患上这种疾病的风险[1]。在中国，约有50%~70%的2型糖尿病患者，他们也更容易患NAFLD。不幸的是，目前还没有完全证实的治疗NAFLD的药物，肝移植是治疗终末期NAFLD的唯一选择。因此，临床上迫切需要一种新的治疗NAFLD的新策略。已有研究报道，GLP-1R表达于人或小鼠的多种组织细胞中，如脑[22]、肝[23]、肾[24]、心脏[25]等，也有报道该受体在NAFLD中表达下调[26]，因此我们有理由认为，针对该受体的药物可能成为临床上治疗NAFLD的新靶点。在我们的研究中，通过Western blot检测到LO2及HepG2两种细胞中均存在GLP-1R，免疫荧光实验表明GLP-1R主要定位于细胞膜及细胞浆，证实了给GLP-1R在肝细胞中的表达，提示了针对该靶点治疗的可行性。在本研究中，由PA诱导的LO2和HepG2的NAFLD细胞模型中，经Exendin-4处理后，油红O实验提示PA诱导的细胞内脂质沉积得到部分逆转，并且NAFLD模型细胞的增殖能力得到促进，表明其在GLP-1介导的脂肪肝缓解中的作用。提示GLP-1R激动剂药物，如新型的抗糖尿病药物艾塞那肽等，对NAFLD的治疗可能有效。意义更为重大的是，本实验同时也为糖尿病患者合并NAFLD的治疗、糖尿病患者的NAFLD预防提供了新的临床实践指南。然而，GLP-1R介导脂肪肝缓解的胞内机制尚不清楚，亟待进一步阐明。

Akt/mTOR是参与调节细胞增殖、转移、细胞周期和凋亡的重要的信号通路[27]。AKT也称为蛋白激酶B(PKB)，是该信号通路的中心节点。由于Akt/mTOR通路可能参与上述几个重要过程，Exendin-4可能通过AKT/mTOR信号通路发挥作用。自噬依赖自噬体的形成，自噬体具有双膜结构，随后可以与溶酶体结合形成自溶体，是一种自我消化的过程，也被认为是消除受损细胞器和生物大分子以维持细胞稳态的重要过程[28]，但是自噬对经Exendin-4处理的NAFLD模型的增殖的影响尚不清楚。AKT/mTOR信号转导通路已被证实与自噬有关[29]，但该通路是否参与GLP-1R介导的自噬过程尚不清楚。因此，我们通过实验进一步验证Exendin-4能否通过GLP-1R激活AKT，抑制mTOR信号通路，进而促进细胞自噬以减少细胞内脂质沉积，本课题利用Western blot检测Exendin-4处理后的NAFLD细胞中AKT、mTOR、p62及LC3-Ⅰ/Ⅱ蛋白表达及激活情况。我们发现，p-AKT在Exendin-4处理后表达上调而pmTOR表达下调，Exendin-4在两种细胞中均可增加LC3-Ⅱ的表达，而mTOR激活剂3BDO能够逆转这一作用，而Exendin-4使PA处理的LO2中p62表达上调，HepG2中表达下调，而3BDO均能够上调p62的表达。近来，人们愈发认识到，p62与自噬过程密切相关。p62通过泛素相关结构域与泛素化蛋白结合，并将其递送至自噬体进行降解，因此，p62的细胞水平被认为是自噬通量的标志[30]。LC3是一种存在于胞质的泛素样蛋白[31]，在发生自噬时会与自噬小体膜上的磷酸酰乙醇胺共价结合[32]，因此其被认为是自噬小体的标志[31]，自噬过程中，脂质结合导致可溶形式的LC3(LC3-Ⅰ)转化为LC3自噬囊泡相关形式(LC3-Ⅱ)[33]，因此LC3Ⅰ/Ⅱ常被用来衡量细胞自噬水平。然而p62与LC3在GLP-1R介导的改善NAFLD脂质沉积中是否发挥作用尚不清楚。在我们的研究中，证实了GLP1受体被激活后，胞内相关自噬标记分子表达水平发生了前述变化，促进了细胞自噬。虽然PA+Exendin-4组中LO2与HepG2的p62水平变化不一致，但是LC3-Ⅱ的表达变化一致仍能够说明细胞自噬水平升高，因此后续仍需要对p62进行更为深入的研究，如对可溶性与不可溶性的p62区分检测，以进一步明确Exendin-4对细胞自噬的促进作用。综上所述，我们的研究进一步证实Exendin-4作用于GLP-1R并通过AKT-mTOR信号通路，激活细胞自噬。

Exendin-4改善肝疾病的机制已有许多研究，如Exendin-4通过抑制单核/巨噬细胞社区人氧化低密度脂蛋白进而降低肝脏的炎症反应[34]、或通过降低肝细胞的氧化水平以改善脂肪变性[35]、而在缺血再灌注损伤引起的肝细胞脂肪变性中，Exendin-4却可以通过降低细胞自噬水平改善脂肪变性[36]。我们的另一项研究发现GLP-1能够通过GLP-1R-PKA-AMPK途径上调PPARα，进而缓解肝脂肪变性和肝损伤，这些都是Exendin-4治疗NAFLD的可能机制。因此我们认为，在NAFLD中，GLP-1发挥的作用亟待进一步的研究。

综上所述，GLP-1R激动剂Exendin-4可能通过直接作用于细胞上GLP-1R，激活AKT-mTOR信号通路进而促进自噬，减少NAFLD细胞模型脂质沉积，并促进细胞增殖，提示临床上使用Exendin-4药物，艾塞那肽等对NAFLD的治疗有效。

Biographies

林楚曼，硕士研究生，E-mail: lin15622762077@163.com

方俊博，本科，E-mail: 2652247998@qq.com

Funding Statement

国家自然科学基金（81270966）；广东省自然科学基金（2014A030310036，2018A030313609，2017A030313519）；南方医科大学临床研究育苗项目（LC2016YM007）；南方医科大学省级大学生创业创新训练项目（S201912121056）

Supported by National Natural Science Foundation of China (81270966)

Contributor Information

林 楚曼 (Chuman LIN), Email: lin15622762077@163.com.

方 俊博 (Junbo FANG), Email: 2652247998@qq.com.

周 蕊 (Rui ZHOU), Email: yaruisunny@sina.com.

杨 力 (Li YANG), Email: yangli19762009@163.com.

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