Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Mar 14, 2020; 26(10): 1029-1041
Published online Mar 14, 2020. doi: 10.3748/wjg.v26.i10.1029
Mesencephalic astrocyte-derived neurotrophic factor ameliorates steatosis in HepG2 cells by regulating hepatic lipid metabolism
Miao He, Cong Wang, Xiao-Hong Long, Jia-Jia Peng, Dong-Fang Liu, Gang-Yi Yang, Michael D Jensen, Li-Li Zhang
Miao He, Cong Wang, Xiao-Hong Long, Jia-Jia Peng, Dong-Fang Liu, Gang-Yi Yang, Li-Li Zhang, Department of Endocrinology, the Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China
Michael D Jensen, Endocrine Research Unit, Mayo Clinic, Rochester, MN 55905, United States
Author contributions: He M and Wang C performed the majority of the experiments and contributed to the analysis of the data and the drafting of the manuscript; Long XH and Peng JJ contributed to the analysis of the data; Liu DF and Yang GY revised and approved the final version of the manuscript; Zhang LL contributed to the study design, the drafting of the manuscript, and critical discussion and approved the final version of the manuscript; Jensen MD revised, discussed, and approved the final version of the manuscript.
Supported by National Natural Science Foundation of China, No. 81300702 and No. 81501199; Natural Science Foundation Project of Chongqing CSTC, No. cstc2018jcyjAX0210 and No. cstc2017jcyjAX0016; and the Kuanren Talents Program of the Second Affiliated Hospital of Chongqing Medical University.
Institutional animal care and use committee statement: This study protocol was reviewed and approved by the Animal Care and Use Ethics Committee of Chongqing Medical University, Chongqing, China (protocol No. 2019-48). All animal experiments conformed to the accepted principles for the care and use of laboratory animals (license No. SYXK 2018-0003).
Conflict-of-interest statement: All the authors have no conflicts of interest related to the manuscript.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Li-Li Zhang, MD, PhD, Associate Professor, Doctor, Postdoc, Research Fellow, Department of Endocrinology, the Second Affiliated Hospital, Chongqing Medical University, 74 Linjiang Road, Yuzhong District, Chongqing 400010, China.
Received: November 21, 2019
Peer-review started: November 21, 2019
First decision: January 7, 2020
Revised: February 15, 2020
Accepted: February 21, 2020
Article in press: February 21, 2020
Published online: March 14, 2020

Nonalcoholic fatty liver disease (NAFLD) is a global metabolism-associated liver disease. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a newly discovered secreted protein that is involved in metabolic homeostasis. However, much remains to be discovered about its function in hepatic lipid metabolism; thus, we assessed whether MANF could regulate hepatic metabolism.


To establish in vivo and in vitro NAFLD models to explore the role of MANF in hepatic lipid metabolism.


HepG2 cells treated with free fatty acids (FFAs) and ob/ob mice were used as NAFLD models. Liver tissues collected from wild type and ob/ob mice were used to detect MANF expression. Cells were treated with FFAs for different durations. Moreover, we used lentiviral constructs to establish overexpression and knockdown cell models in order to interfere with MANF expression levels and observe whether MANF influences hepatic steatosis. Western blot analysis and quantitative real-time PCR were used to detect protein and gene expression, and oil red O staining was used to visualize intracellular lipid droplets.


Hepatic MANF protein and mRNA expression in wild type mice were 10-fold and 2-fold higher, respectively, than those in ob/ob mice. The MANF protein was temporarily increased by 1.3-fold after stimulation with FFAs for 24 h and gradually decreased to 0.66-fold that of the control at the 72 h time point in HepG2 cells. MANF deficiency upregulated the expression of genes involved in fatty acid synthesis, cholesterol synthesis, and fatty acid uptake and aggravated HepG2 cell steatosis, while MANF overexpression inhibited fatty acid synthesis and uptake and cholesterol synthesis, and rescued HepG2 cells from FFAs-induced steatosis. Furthermore, a significant decrease in triglyceride levels was observed in the MANF overexpression group compared with the control group (0.4288 ± 0.0081 mmol/g vs 0.3746 ± 0.0121 mmol/g, P < 0.05) upon FFAs treatment. There was also a 17% decrease in intracellular total cholesterol levels between the MANF overexpression group and the control group (0.1301 ± 0.0059 mmol/g vs 0.1088 ± 0.0009 mmol/g, P < 0.05) upon FFAs treatment. Moreover, MANF suppressed lipid deposition in HepG2 cells.


Our findings indicate that MANF improves the phenotype of liver cell steatosis and may be a potential therapeutic target in hepatic steatosis processes.

Keywords: Mesencephalic astrocyte-derived neurotrophic factor, Nonalcoholic fatty liver disease, Hepatic steatosis, Lipogenesis, In vitro, HepG2

Core tip: We first uncovered an important function of mesencephalic astrocyte-derived neurotrophic factor (MANF) in the pathogenesis of nonalcoholic fatty liver disease. We found that MANF exerts a significant effect on hepatic fatty metabolism. This study suggests for the first time that MANF expression was increased at an early stage and gradually decreased afterward under high free fatty acids stimulation in HepG2 cells. Moreover, the results from gain-and-loss functional experiments showed that loss of MANF accelerated lipogenesis and aggravated HepG2 cell steatosis, while MANF overexpression inhibited lipogenesis and rescued HepG2 cell steatosis from free fatty acids treatment.