Downloaded from British Journal of Nutrition (2019), 122, 1062–1072 doi:10.1017/S0007114519001740 © The Authors 2019 https://www.cambridge.org/core Proteomics study of the effect of high-fat diet on rat liver Jian Sang1,2†, Hengxian Qu1,2†, Ruixia Gu1,2, Dawei Chen1,2, Xia Chen1,2, Boxing Yin1,2, Yingping Huang3, 3 3 1,2 Wenbo Xi , Chunlei Wang and Yujun Huang * . IP address: 1College of Food Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225127, People’s Republic of China 2Key Lab of Dairy Biotechnology and Safety Control, Yangzhou, Jiangsu 225127, People’s Republic of China 3Uni-enterprise (China) Holding Ltd., Kunshan, Jiangsu 215300, People’s Republic of China 170.106.35.76 (Submitted 6 March 2019 – Final revision received 9 July 2019 – Accepted 11 July 2019 – First published online 30 September 2019) , on 30 Sep 2021 at 00:24:24 Abstract Excessive intake of high-energy diets is an important cause of most obesity. The intervention of rats with high-fat diet can replicate the ideal animal model for studying the occurrence of human nutritional obesity. Proteomics and bioinformatics analyses can help us to systematically and comprehensively study the effect of high-fat diet on rat liver. In the present study, 4056 proteins were identified in rat liver by using tandem mass tag. A total of 198 proteins were significantly changed, of which 103 were significantly up-regulated and ninety-five were significantly down- regulated. These significant differentially expressed proteins are primarily involved in lipid metabolism and glucose metabolism processes. The , subject to the Cambridge Core terms of use, available at intake of a high-fat diet forces the body to maintain physiological balance by regulating these key protein spots to inhibit fatty acid synthesis, promote fatty acid oxidation and accelerate fatty acid degradation. The present study enriches our understanding of metabolic disorders induced by high-fat diets at the protein level. Key words: High-fat diet: Proteomics: Bioinformatics: Lipid metabolism Changes in the structure of modern diets and excessive intake of an animal model with an obese phenotype similar to human high-fat, high-energy diets cause disorders in the metabolism of obesity(7). The effects of high-fat diet on rat liver protein expres- fats, proteins and carbohydrates. Excess energy is gradually sion were studied by tandem mass tag combined with LC-MS/ accumulated in the body in the form of fat, which leads to MS. Bioinformatics analysis was used to further screen out the obesity(1). After the formation of obesity, it will further induce significant differential proteins that play a key role and to explore insulin resistance (IR), which in turn increases the incidence the mechanism of the body’s response to high-fat diets. https://www.cambridge.org/core/terms of the metabolic syndrome, diabetes, hypertension and CVD(2). The liver plays a vital role in maintaining the body’s carbohydrate, lipid and protein metabolism balance(3). Materials and methods Obesity-related metabolic disorders are manifested in the liver, Animals and treatment causing excessive deposition of fat in the liver cells, which can lead to fatty liver(4). The pathogenesis of obesity is not fully Eighteen healthy male Sprague–Dawley rats which were understood. It is generally believed that obesity is mainly caused 5 weeks old and weighing about 150 g at the start of the experi- by the combination of genetic factors and environmental ment were purchased from Comparative Medical Center of factors(5). In addition, factors such as endocrine, metabolism Yangzhou University, Jiangsu, China. The animal experiments . and central nervous system are also involved in the pathogenesis conformed the U.S. National Institutes of Health guidelines for https://doi.org/10.1017/S0007114519001740 of obesity(6). the care and use of laboratory animals (NIH publication no. Unlike traditional molecular biology techniques, proteomics 85-23 Rev. 1985) and were approved by the Animal Care enables researchers to define a global overview of protein Committee of the Center for Disease Control and Prevention expression in a specific physical or pathological state. In the (Jiangsu, China). Then, rats were randomly divided into a control present study, rats were treated with a high-fat diet to replicate group (C) and high-fat diet group (HF) (n 9). The control group Abbreviations: GO, gene ontology; HF, high-fat diet group; IR, insulin resistance; KEGG, Kyoto Encyclopedia of Genes and Genomes; UGT, UDP- glucuronosyltransferase. * Corresponding author: Y. Huang, email [email protected] † These authors contributed equally to this work and should be regarded as co-first authors. Downloaded from High-fat diet on rat liver 1063 Table 1. Composition of the diets Filter-aided sample preparation digestion https://www.cambridge.org/core Content Total energy A quantity of 200 μg of proteins for each sample was incorpo- Diet Ingredient (%, w/w) (kJ/100g) rated into 30 μl SDT buffer (4 % SDS, 100 mM dithiothreitol, Low-fat diet Flour 20 690·36 150 mM Tris-HCl, pH 8·0). The detergent, dithiothreitol and other Rice flour 10 low-molecular-weight components were removed using UA Maize 20 buffer (8 M urea, 150 mM Tris-HCl, pH 8·0) by repeated ultrafil- Drum skin 26 μ Soya material 20 tration (microcon units, 10 kDa). Then, 100 l iodoacetamide Fishmeal 2 (100 mM iodoacetamide in UA buffer) was added to block . IP address: Bone flour 2 reduced cysteine residues, and the samples were incubated High-fat diet Lard 10 1548·08 μ Egg powder 10 for 30 min in darkness. The filters were washed with 100 l Cholesterol 1 UA buffer three times and then with 100 μl 100 mM triethylammo- Bile salts 0·2 nium bicarbonate (TEAB) buffer twice. Finally, the protein sus- 170.106.35.76 · Low-fat diet 78 8 pensions were digested with 4 μg trypsin (Promega) in 40 μl TEAB buffer overnight at 37°C, and the resulting peptides were collected as a filtrate. The peptide content was estimated by UV , on was fed a low-fat diet (Table 1). The HF was fed a high-fat diet light spectral density at 280 nm using an extinction coefficient of 30 Sep 2021 at 00:24:24 (Table 1) for 8 weeks. All rats were housed under a 12 h light–12 1·1of0·1 % (g/l) solution which was calculated on the basis of h dark cycle in a controlled room with a temperature of 23°C and the frequency of tryptophan and tyrosine in vertebrate proteins a humidity of 50 % and allowed free access to food and water. After 8 weeks, rats underwent 12 h of fasting prior to being Tandem mass tag labelling anaesthetised and dissected. All rats were euthanised at the μ The peptide mixture (100 g) of each sample was labelled using , subject to the Cambridge Core terms of use, available at anoestrus period, following anaesthesia under 1 % sodium a tandem mass tag reagent according to the manufacturer’s − pentobarbital. Livers were removed and stored at 4 and 80°C instructions (Thermo Fisher Scientific). for subsequent analyses. Peptide fractionation with high pH reversed phase Compliance with ethics guidelines A Pierce high pH reversed-phase fractionation kit (Thermo All institutional and national guidelines for the care and use of Scientific) was used to fractionate tandem mass tag-labelled laboratory animals were followed. digest samples into ten fractions by an increasing acetonitrile step- Haematoxylin–eosin staining gradient elution according to the instructions. Liver samples were fixed in 4 % paraformaldehyde for more than Mass spectrometry 48 h, followed by sectioning after embedding with paraffin. The paraffin section was stained by routine haematoxylin–eosin HPLC. Each fraction was injected for nanoLC-MS/MS analysis. The peptide mixture was loaded onto a reversed-phase trap col- methods. https://www.cambridge.org/core/terms umn (Thermo Scientific Acclaim PepMap100, 100 μm × 2 cm, nanoViper C18) connected to the C18-reversed-phase analytical Protein extraction and normalisation column (Thermo Scientific Easy Column, 10 cm long, 75 μm μ · SDT buffer (4 % (w/v) SDS, 0·1 MDL-dithiothreitol, 100 mM Tris/ inner diameter, 3 m resin) in buffer A (0 1 % formic acid) HCl, pH 7·6) was added to the liver sample and transferred to 2 and separated with a linear gradient of buffer B (84 % acetonitrile · ml tubes with quartz sand (another 1/4 inch ceramic bead MP and 0 1 % formic acid) at a flow rate of 300 nl/min controlled by 6540-424 for tissue samples). The lysate was homogenised by IntelliFlow technology. · – – an MP homogeniser (24 × 2, 6·0m/s,60s,twice).Thehomog- 1 5 h gradient: 0 55 % buffer B for 80 min, 55 100 % buffer B enate was sonicated and then boiled for 15 min. After centrifuged for 5 min, hold in 100 % buffer B for 5 min. · μ . at 14 000 g for 40 min, the supernatant was filtered with 0 22 m https://doi.org/10.1017/S0007114519001740 filters. The filtrate was quantified with the Bicinchoninic Acid LC-MS/MS analysis. LC-MS/MS analysis was performed on a Q Protein Assay Kit (Bio-Rad). The sample was stored at −80°C. Exactive MS (Thermo Scientific) that was coupled with Easy nLC Equivalent amounts of protein from each of the three different rats (Proxeon Biosystems, now Thermo Fisher Scientific) for 90 min were pooled to generate three protein samples for each group. (determined by project proposal). The MS was operated in pos- itive ion mode. Mass spectometry data were acquired using a data-dependent top-ten method dynamically choosing the most SDS-PAGE separation abundant precursor ions from the survey scan (300–1800 m/z) A quantity of 20 μg of proteins for each sample was mixed with for higher-energy C-trap dissociation (HCD) fragmentation.