Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 7, 2016; 22(25): 5769-5779
Published online Jul 7, 2016. doi: 10.3748/wjg.v22.i25.5769
Urotensin II-induced insulin resistance is mediated by NADPH oxidase-derived reactive oxygen species in HepG2 cells
Ying-Ying Li, Zheng-Ming Shi, Xiao-Yong Yu, Ping Feng, Xue-Jiang Wang
Ying-Ying Li, Xiao-Yong Yu, Xue-Jiang Wang, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China
Ying-Ying Li, Xiao-Yong Yu, Xue-Jiang Wang, Beijing Key Laboratory for Cancer Invasion and Metastasis Research, Capital Medical University, Beijing 100069, China
Zheng-Ming Shi, Department of General Surgery, Beijing Jishuitan Hospital, Beijing 100069, China
Ping Feng, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China
Author contributions: Li YY and Shi ZM performed the experiments; Wang XJ contributed reagents/materials/analysis tools; Li YY, Yu XY and Feng P analyzed the data; Wang XJ conceived and designed the research; Li YY wrote the paper; all authors read and approved the final manuscript.
Supported by National Natural Science Foundation of China, No. 81272757 and the Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipality, No. IDHT20150502.
Conflict-of-interest statement: The authors declare no conflicts of interest.
Data sharing statement: No additional unpublished data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (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:
Correspondence to: Xue-Jiang Wang, Professor, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Capital Medical University, No. 10 Xitoutiao, You An Men, Beijing 100069, China.
Telephone: +86-10-83911434 Fax: +86-10-83911434
Received: March 2, 2016
Peer-review started: March 4, 2016
First decision: April 1, 2016
Revised: April 12, 2016
Accepted: May 4, 2016
Article in press: May 4, 2016
Published online: July 7, 2016

AIM: To investigated the effects of urotensin II (UII) on hepatic insulin resistance in HepG2 cells and the potential mechanisms involved.

METHODS: Human hepatoma HepG2 cells were cultured with or without exogenous UII for 24 h, in the presence or absence of 100 nmol/L insulin for the last 30 min. Glucose levels were detected by the glucose-oxidase method and glycogen synthesis was analyzed by glycogen colorimetric/fluorometric assay. Reactive oxygen species (ROS) levels were detected with a multimode reader using a 2′,7′-dichlorofluorescein diacetate probe. The protein expression and phosphorylation levels of c-Jun N-terminal kinase (JNK), insulin signal essential molecules such as insulin receptor substrate -1 (IRS-1), protein kinase B (Akt), glycogen synthase kinase-3β (GSK-3β), and glucose transporter-2 (Glut 2), and NADPH oxidase subunits such as gp91phox, p67phox, p47phox, p40phox, and p22phox were evaluated by Western blot.

RESULTS: Exposure to 100 nmol/L UII reduced the insulin-induced glucose consumption (P < 0.05) and glycogen content (P < 0.01) in HepG2 cells compared with cells without UII. UII also abolished insulin-stimulated protein expression (P < 0.01) and phosphorylation of IRS-1 (P < 0.05), associated with down-regulation of Akt (P < 0.05) and GSK-3β (P < 0.05) phosphorylation levels, and the expression of Glut 2 (P < 0.001), indicating an insulin-resistance state in HepG2 cells. Furthermore, UII enhanced the phosphorylation of JNK (P < 0.05), while the activity of JNK, insulin signaling, such as total protein of IRS-1 (P < 0.001), phosphorylation of IRS-1 (P < 0.001) and GSK-3β (P < 0.05), and glycogen synthesis (P < 0.001) could be reversed by pretreatment with the JNK inhibitor SP600125. Besides, UII markedly improved ROS generation (P < 0.05) and NADPH oxidase subunit expression (P < 0.05). However, the antioxidant/NADPH oxidase inhibitor apocynin could decrease UII-induced ROS production (P < 0.05), JNK phosphorylation (P < 0.05), and insulin resistance (P < 0.05) in HepG2 cells.

CONCLUSION: UII induces insulin resistance, and this can be reversed by JNK inhibitor SP600125 and antioxidant/NADPH oxidase inhibitor apocynin targeting the insulin signaling pathway in HepG2 cells.

Keywords: Urotensin II, Insulin resistance, NADPH oxidase, Reactive oxygen species, HepG2 cells

Core tip: We report our results on urotensin II (UII) in a newly developed insulin resistance model and the possible mechanisms involved. Exposure to UII may contribute to oxidative damage via the NADPH oxidase pathway and enhance the phosphorylation of c-Jun N-terminal kinase, which is associated with insulin signal transduction pathways. These may be the underlying mechanisms of UII-mediated insulin resistance in HepG2 cells. These findings confirm the important role of UII in hepatic insulin resistance, which shed light on new insight into hepatic insulin resistance.