Article pubs.acs.org/JAFC
Mass-Spectrometry-Based Serum Metabolomics of a C57BL/6J Mouse Model of High-Fat-Diet-Induced Non-alcoholic Fatty Liver Disease Development Yi-Syuan Lai,† Wei-Cheng Chen,† Tien-Chueh Kuo,‡,§ Chi-Tang Ho,†,◆ Ching-Hua Kuo,§,∥,⊥ Yufeng J. Tseng,‡,§,⊥,# Kuan-Hung Lu,† Shih-Hang Lin,† Suraphan Panyod,† and Lee-Yan Sheen*,†,∇,○
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Institute of Food Science and Technology, ‡Graduate Institute of Biomedical Electronic and Bioinformatics, §The Metabolomics Core Laboratory, ∥Department of Pharmacy, ⊥Center of Genomic Medicine, #Department of Computer Science and Information Engineering, ∇National Center for Food Safety Education and Research, and ○Center for Food and Biomolecules, National Taiwan University, Taipei 10617, Taiwan ◆ Department of Food Science, Rutgers University, New Brunswick, New Jersey 08901, United States ABSTRACT: Obesity, dyslipidemia, insulin resistance, oxidative stress, and inflammation are key clinical risk factors for the progression of non-alcoholic fatty liver disease (NAFLD). Currently, there is no comprehensive metabolic profile of a wellestablished animal model that effectively mimics the etiology and pathogenesis of NAFLD in humans. Here, we report the pathophysiological and metabolomic changes associated with NAFLD development in a C57BL/6J mouse model in which NAFLD was induced by feeding a high-fat diet (HFD) for 4, 8, 12, and 16 weeks. Serum metabolomic analysis was conducted using ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC− QTOF−MS) and gas chromatography−mass spectrometry (GC−MS) to establish a metabolomic profile. Analysis of the metabolomic profile in combination with principal component analysis revealed marked differences in metabolites between the control and HFD group depending upon NAFLD severity. A total of 30 potential biomarkers were strongly associated with the development of NAFLD. Among these, 11 metabolites were mainly related to carbohydrate metabolism, hepatic biotransformation, collagen synthesis, and gut microbial metabolism, which are characteristics of obesity, as well as significantly increased serum glucose, total cholesterol, and hepatic triglyceride levels during the onset of NAFLD (4 weeks). At 8 weeks, 5 additional metabolites that are chiefly involved in perturbation of lipid metabolism and insulin secretion were found to be associated with hyperinsulinemia, hyperlipidemia, and hepatic steatosis in the mid-term of NAFLD progression. At the end of 12 and 16 weeks, 14 additional metabolites were predominantly correlated to abnormal bile acid synthesis, oxidative stress, and inflammation, representing hepatic inflammatory infiltration during NAFLD development. These results provide potential biomarkers for early risk assessment of NAFLD and further insights into NAFLD development. KEYWORDS: non-alcoholic fatty liver disease, high-fat diet, metabolomics, UHPLC−QTOF−MS, GC−MS
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INTRODUCTION
Metabolomics has emerged as a powerful approach for identifying metabolite biomarkers associated with certain phenotypes in systems biology and enables comprehensive quantitative and qualitative analyses to evaluate the dynamic alterations of low-molecular-weight compounds in biological samples during genetic modifications or response to pathophysiological stimuli.2 To date, metabolomic profiling has been carried out to explore potential biomarkers and metabolic pathways involved in the development of NAFLD in various animal models, including methionine-choline-deficient (MCD)-diet-induced mice,3 LDLr−/− mice,4 glycine Nmethyltransferase knockout (GNMT-KO) mice,5 and 129S6/ SvEv mice.6 A proton nuclear magnetic resonance (1H NMR) metabonomics approach was used to investigate the timerelated biochemical perturbations in the serum of C57BL/6 mice fed the MCD diet. Four potential biomarkers, serum
Non-alcoholic fatty liver disease (NAFLD) is recognized as the hepatic manifestation of metabolic syndrome, closely associated with obesity, dyslipidemia, and insulin resistance. NAFLD comprises a wide spectrum of histopathological changes observed in liver diseases. The early stage involves simple hepatic steatosis, owing to triacylglycerol deposition, resulting from excessive accumulation of free fatty acids (FFAs) within heptatocyes. In the middle stage, hepatic FFA oxidation is stimulated in the mitochondria, microsomes, and/or peroxisomes to exacerbate reactive oxygen species (ROS) production, resulting in non-alcoholic steatohepatitis (NASH). Dying hepatocytes release various chemical mediators, such as ROS, chemokines, or cytokines, that activate hepatic stellate cells (HSCs), which may eventually progress to liver fibrosis, cirrhosis, or cancer in the late stage of NAFLD.1 However, the metabolic mechanisms involved in the complex pathophysiological processes and progression of NAFLD remain unclear. Therefore, high-throughput profiling techniques are needed. © 2015 American Chemical Society
Received: Revised: Accepted: Published: 7873
June 8, 2015 August 11, 2015 August 11, 2015 August 11, 2015 DOI: 10.1021/acs.jafc.5b02830 J. Agric. Food Chem. 2015, 63, 7873−7884
Article
Journal of Agricultural and Food Chemistry
Serological Analyses. Whole blood was immediately collected by cardiac puncture and then allowed to clot. After centrifugation at 1000g for 10 min at 4 °C, the resultant serum samples were separated and stored individually at −80 °C for subsequent analyses. Serum chemistry for aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), and TG levels were subjected to biochemical testing with an automatic chemistry analyzer (SPOTCHEM EZ SP-4430, ARKRAY, Inc., Kyoto, Japan). The serum FFA concentration was measured using the enzyme-based FFA Quantification Kit (BioVision, San Francisco, CA), and the insulin level was determined using an enzyme-linked immunosorbent assay kit (Mercodia AB, Uppsala, Sweden). Histopathological Examination. Liver and fat pads (subcutaneous, epididymal, and perirenal fat) were harvested, weighed, and photographed. Liver pathological analysis and semi-quantitative evaluation was carried out as previously described.9 Briefly, paraffinembedded and frozen sections of liver tissue were routinely processed for hematoxylin and eosin (H&E) and Oil Red O staining, respectively. Each H&E section was then scored for steatosis with morphometric analysis by an experienced pathologist. The degree of steatosis was graded from 1 to 5 according to severity: 1, minimal (
