Hepatic protein Carbonylation profiles induced by lipid accumulation and oxidative stress for investigating cellular response to non-alcoholic fatty liver disease in vitro

Abstract

Background Non-alcoholic fatty liver disease (NAFLD) is caused by excessive accumulation of fat within the liver, leading to further severe conditions such as non-alcoholic steatohepatitis (NASH). Progression of healthy liver to steatosis and NASH is not yet fully understood in terms of process and response. Hepatic oxidative stress is believed to be one of the factors driving steatosis to NASH. Oxidative protein modification is the major cause of protein functional impairment in which alteration of key hepatic enzymes is likely to be a crucial factor for NAFLD biology. In the present study, we aimed to discover carbonylated protein profiles involving in NAFLD biology in vitro. Methods Hepatocyte cell line was used to induce steatosis with fatty acids (FA) in the presence and absence of menadione (oxidative stress inducer). Two-dimensional gel electrophoresis-based proteomics and dinitrophenyl hydrazine derivatization technique were used to identify carbonylated proteins. Sequentially, in order to view changes in protein carbonylation pathway, enrichment using Funrich algorithm was performed. The selected carbonylated proteins were validated with western blot and carbonylated sites were further identified by high-resolution LC-MS/MS. Results Proteomic results and pathway analysis revealed that carbonylated proteins are involved in NASH pathogenesis pathways in which most of them play important roles in energy metabolisms. Particularly, carbonylation level of ATP synthase subunit α (ATP5A), a key protein in cellular respiration, was reduced after FA and FA with oxidative stress treatment, whereas its expression was not altered. Carbonylated sites on this protein were identified and it was revealed that these sites are located in nucleotide binding region. Modification of these sites may, therefore, disturb ATP5A activity. As a consequence, the lower carbonylation level on ATP5A after FA treatment solely or with oxidative stress can increase ATP production. Conclusions The reduction in carbonylated level of ATP5A might occur to generate more energy in response to pathological conditions, in our case, fat accumulation and oxidative stress in hepatocytes. This would imply the association between protein carbonylation and molecular response to development of steatosis and NASH. Electronic supplementary material The online version of this article (10.1186/s12953-019-0149-9) contains supplementary material, which is available to authorized users.

Ferulic Acid Ameliorates Atherosclerotic Injury by Modulating Gut Microbiota and Lipid Metabolism

“…Second, FA treatment significantly suppressed redundant lipid deposition in the liver, which is consistent with previous studies ( Ma et al, 2019 ). Lipid accumulation in hepatocytes causes lipid peroxidation and increases oxidative stress ( Chienwichai et al, 2019 ), which is an important mechanism in liver injury ( Li et al, 2015 ; Dey et al, 2020 ). The antioxidant effect of FA has also been identified in other studies ( Bumrungpert et al, 2018 ).…”

Oxidative Stress Parameters in the Liver of Growing Male Rats Receiving Various Alcoholic Beverages

“…Numerous studies in rats or mice have proved the intensified oxidation of lipids, which was assessed based on the level of TBARS, after the administration of ethanol at a concentration of 18-30% [36][37][38][39][40][41], but they also showed an increased level of protein carbonyl groups in the liver as a result of the intensification of oxidative stress by ethanol consumption [40][41][42]. According to some authors, the process of protein oxidation may be of great importance in the pathogenesis of ALD [41], as well as in the pathogenesis of the non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steato-hepatitis (NASH) [43]. In the present study, the highest level of protein carbonyl groups observed in the liver was attributed to beer consumption, which may once again suggest the adverse effect of some components present in beer, or the higher vulnerability of growing organisms to those components.…”

α-Mangosteen as An Oxidative Inhibitor in Hepatocellular Carcinoma

“…The subsequent oxidative stress not only causes cell damage but also contributes lipids and proteins oxidation, including protein carbonylation. Alter of protein carbonyl formation correlated with free radicals production within HepG2 cells (Chienwichai, et al, 2019).…”