Comparative Proteomics Analysis Reveals L-Arginine Activates Ethanol Degradation Pathways in HepG2 Cells.

Guokai Yan,Baisheng Long,Qiwen Fan,Zhichang Wang,Changqing Chen,Lu Liu,Agung Purnomoadi,Xingya Yang,Xianghua Yan,Joelal Achmadi,Retno Lestari,Jun Hu,Xiuzhi Li,Jie Yu,Xiaozhen Guo

doi:10.1038/srep23340

Abstract

L-Arginine (Arg) is a versatile amino acid that plays crucial roles in a wide range of physiological and pathological processes. In this study, to investigate the alteration induced by Arg supplementation in proteome scale, isobaric tags for relative and absolute quantification (iTRAQ) based proteomic approach was employed to comparatively characterize the differentially expressed proteins between Arg deprivation (Ctrl) and Arg supplementation (+Arg) treated human liver hepatocellular carcinoma (HepG2) cells. A total of 21 proteins were identified as differentially expressed proteins and these 21 proteins were all up-regulated by Arg supplementation. Six amino acid metabolism-related proteins, mostly metabolic enzymes, showed differential expressions. Intriguingly, Ingenuity Pathway Analysis (IPA) based pathway analysis suggested that the three ethanol degradation pathways were significantly altered between Ctrl and +Arg. Western blotting and enzymatic activity assays validated that the key enzymes ADH1C, ALDH1A1, and ALDH2, which are mainly involved in ethanol degradation pathways, were highly differentially expressed, and activated between Ctrl and +Arg in HepG2 cells. Furthermore, 10 mM Arg significantly attenuated the cytotoxicity induced by 100 mM ethanol treatment (P < 0.0001). This study is the first time to reveal that Arg activates ethanol degradation pathways in HepG2 cells.

Highlights

To gain a comprehensive understanding of the effect of amino acids deprivation and/or supplementation, 2-dimensional gel electrophoresis (2-DE) based proteomics approaches have been commonly applied in functional studies of amino acids
In an effort to identify the differentially expressed proteins related to Arg supplementation in HepG2 cells, we employed the isobaric tags for relative and absolute quantification (iTRAQ)-based comparative proteomic approach
As liver serves as a main organ in ethanol metabolism, ethanol is largely metabolized to acetaldehyde by alcohol dehydrogenases (ADH) in the cytosol of liver cells[19]

Summary

Introduction

To gain a comprehensive understanding of the effect of amino acids deprivation and/or supplementation, 2-dimensional gel electrophoresis (2-DE) based proteomics approaches have been commonly applied in functional studies of amino acids. Lenaerts et al.[14] analyzed the effect of glutamine on protein profile of Caco-2 cells using a strategy combining 2-DE and MALDI-TOF-MS and identified 14 differentially expressed proteins specially altered by glutamine[14]. Lenaerts et al.[16] explored the protein profiles induced by Arg deficiency in either preconfluent or postconfluent Caco-2 cells and found that Arg deprivation leads to the decreased cell proliferation and heat shock protein expression, and the enhanced cell susceptibility to apoptosis[16]. Our data indicated that ethanol degradation pathways were significantly activated in Arg-supplemented HepG2 cells, suggesting the potential role of Arg on ethanol degradation in HepG2 cells

Methods

Results

Conclusion