Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 14, 2015; 21(6): 1784-1793
Published online Feb 14, 2015. doi: 10.3748/wjg.v21.i6.1784
In vitro identification of nonalcoholic fatty liver disease-related protein hnRNPM
Jun-ichi Takino, Kentaro Nagamine, Masayoshi Takeuchi, Takamitsu Hori
Jun-ichi Takino, Kentaro Nagamine, Takamitsu Hori, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hiroshima International University, Hiroshima 737-0112, Japan
Masayoshi Takeuchi, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, Ishikawa 920-0293, Japan
Author contributions: Takeuchi M designed the study and contributed experimental reagents; Takino J and Nagamine K performed the study; Nagamine K analyzed data; Takino J wrote the paper; and Hori T reviewed the paper.
Supported by Grants from the Japan Society for the Promotion of Science, No. 22300264 and No. 25282029.
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: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Masayoshi Takeuchi, PhD, Department of Advanced Medicine, Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada-machi, Kahoku, Ishikawa 920-0293, Japan. takeuchi@kanazawa-med.ac.jp
Telephone: +81-76-2862211 Fax: +81-76-2863652
Received: July 18, 2014
Peer-review started: July 20, 2014
First decision: August 15, 2014
Revised: August 29, 2014
Accepted: October 14, 2014
Article in press: October 15, 2014
Published online: February 14, 2015
Processing time: 208 Days and 3.7 Hours
Abstract

AIM: To study the formation of intracellular glyceraldehyde-derived advanced glycation end products (Glycer-AGEs) in the presence of high concentrations of fructose.

METHODS: Cells of the human hepatocyte cell line Hep3B were incubated with or without fructose for five days, and the corresponding cell lysates were separated by two-dimensional gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Glycer-AGEs were detected with the anti-Glycer-AGEs antibody. Furthermore, the identification of the proteins that are modified by glyceraldehyde in the presence of high concentrations of fructose was conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The protein and mRNA levels were determined by Western blotting and real-time reverse transcription PCR, respectively.

RESULTS: The results of the two-dimensional gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a greater amount of Glycer-AGEs in the sample exposed to high concentrations of fructose than in the control. The detected Glycer-AGEs showed isoelectric points in the range of 8.0-9.0 and molecular weights in the range of 60-80 kDa. The heterogeneous nuclear ribonucleoprotein M (hnRNPM), which plays an important role in regulating gene expression by processing heterogeneous nuclear RNAs to form mature mRNAs, was identified as a modified protein using MALDI-TOF-MS. Increasing the concentration of fructose in the medium induced a concentration-dependent increase in the generated Glycer-AGEs. Furthermore, in an experiment using glyceraldehyde, which is a precursor of Glycer-AGEs, hnRNPM was found to be more easily glycated than the other proteins.

CONCLUSION: The results suggest that glyceraldehyde-modified hnRNPM alters gene expression. This change may cause adverse effects in hepatocytes and may serve as a target for therapeutic intervention.

Keywords: Advanced glycation end-products; Fructose; Glycation; Glyceraldehyde; Heterogeneous nuclear ribonucleoprotein M; Nonalcoholic fatty liver disease; Nonalcoholic steatohepatitis

Core tip: Excessive intake of fructose contributes to the development of nonalcoholic fatty liver disease and to the progression of the disease to nonalcoholic steatohepatitis. Fructose is metabolized to glyceraldehyde, which is a precursor of glyceraldehyde-derived advanced glycation end-products (Glycer-AGEs). We showed that intracellular Glycer-AGEs were formed in the presence of high concentrations of fructose. Additionally, heterogeneous nuclear ribonucleoprotein M (hnRNPM) was identified as one of the target proteins for glycation. These results suggest that the glyceraldehyde-modified hnRNPM resulting from the exposure of the cells to high concentrations of fructose, alters gene expression and causes adverse effects in hepatocytes.