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Article Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Effects of Stigmasterol and β‑Sitosterol on Nonalcoholic Fatty Liver Disease in a Mouse Model: A Lipidomic Analysis Simin Feng,†,‡,§ Ling Gan,† Chung S. Yang,‡ Anna B. Liu,‡ Wenyun Lu,⊥ Ping Shao,† Zhuqing Dai,∥ Peilong Sun,*,† and Zisheng Luo*,§ †

Department of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310014, People’s Republic of China Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, United States § Zhejiang University, College of Biosystems Engineering and Food Science, Key Laboratory of Agro-Products Postharvest Handling Ministry of Agriculture, Zhejiang Key Laboratory for Agri-Food Processing, Hangzhou 310058, People’s Republic of China ∥ Institute of Agro-product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, People’s Republic of China ⊥ Department of Chemistry & Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544, United States ‡

S Supporting Information *

ABSTRACT: To study the effects of stigmasterol and β-sitosterol on high-fat Western diet (HFWD)-induced nonalcoholic fatty liver disease (NAFLD), lipidomic analyses were conducted in liver samples collected after 33 weeks of the treatment. Principal component analysis showed these phytosterols were effective in protecting against HFWD-induced NAFLD. Orthogonal projections to latent structures−discriminate analysis (OPLS-DA) and S-plots showed that triacylglycerols (TGs), phosphatidylcholines, cholesteryl esters, diacylglycerols, and free fatty acids (FFAs) were the major lipid species contributing to these discriminations. The alleviation of NAFLD is mainly associated with decreases in hepatic cholesterol, TGs with polyunsaturated fatty acids, and alterations of free hepatic FFA. In conclusion, phytosterols, at a dose comparable to that suggested for humans by the FDA for the reduction of plasma cholesterol levels, are shown to protect against NAFLD in this long-term (33-week) study. KEYWORDS: stigmasterol, β-sitosterol, fatty liver, mice, cholesterol, lipidomics

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INTRODUCTION Similar to cholesterol, phytosterols are made of a tetracyclic cyclopenta [α] phenanthrene ring and a long flexible side chain at the C-17 carbon atom.1 More than 200 different phytosterols have been studied, and campesterol, stigmasterol, and βsitosterol are the most representative phytosterols.2 For example, β-sitosterol and stigmasterol have been shown in our previous work as the two major phytosterols in sugar cane.3−5 The daily dietary intake of phytosterols was estimated to be 140−375 mg in different countries and regions.6 A daily intake of 2.0 to 2.5 g phytosterols was recommended for lowering serum total cholesterol and low-density lipoprotein cholesterol levels.7,8 To achieve this level to gain the beneficial effects of phytosterols, the use of dietary supplements is needed. The cholesterol-lowering effects of phytosterols are mainly due to their ability of inhibiting cholesterol intestinal absorption and regulating proteins involved in cholesterol metabolism.9 Their possible effect in attenuating nonalcoholic fatty liver disease (NAFLD) is not well studied. NAFLD is a high-prevalence disease with a wide spectrum of liver damage stages, ranging from isolated hepatic steatosis without liver inflammation to nonalcoholic steatohepatitis (NASH).10 In the Western countries, the prevalence of NAFLD in the general population ranges from 15 to 39%.11 The underlying mechanisms of disease progression are not completely defined. There is no established treatment for © XXXX American Chemical Society

NAFLD, even though cholesterol-lowering drugs have been suggested as effective therapy for NAFLD.12 Many phytochemicals from different plants are reported to have preventive effects on NAFLD.13−15 The histological hallmark of NAFLD is the accumulation of triacylglycerol (TG)-rich lipid droplets within hepatocytes as well as the liver damage caused by oxidative stress and inflammation. Changes of other lipid classes were also shown to be related to the progress of NAFLD.16−18 Phytosterol esters were reported to attenuate hepatic steatosis in the NAFLD model.19 Our previous study showed that stigmasterol and β-sitosterol had anti-inflammatory effects in a colitis model.20 The effects of different phytosterols on specific lipid classes, such as hepatic cholesterol, TG, and free fatty acids (FFAs), in the progression of NAFLD are not known. Lipidomics is an effective and sensitive approach for the determination of biological responses by investigating the level of individual lipid species. The analysis of lipidomic changes could provide valuable information in studying NAFLD.18,21−23 The lipidomic analysis allows simultaneous identification of individual lipid species that were affected by phytosterols, Received: December 29, 2017 Revised: March 22, 2018 Accepted: March 23, 2018

A

DOI: 10.1021/acs.jafc.7b06146 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Effects of stigmasterol and β-sitosterol on body weight (a), dietary consumption (b), liver morphology (c), and liver histology (d). The data are shown as mean ± SD (n = 5 for G1 and n = 10 for G2−4). * indicates statistical difference from the HFWD group (p < 0.05) with ANOVA analysis. Multiple comparison between the groups was performed using Duncan’s new multiple range test. 2500 kcal), then 2 g/500 g = 0.4%. Therefore, we used a level of 0.4% of phytosterols in the diet in this study. Experimental Design. After 1 week of acclimation on chow diet, the mice were randomly allocated into four groups and maintained on CD (G1, n = 10), HFWD (G2, n = 20), HFWD+stigma (G3, n = 20), and HFWD+β-sito (G4, n = 20). Body weight and diet consumption were monitored weekly. After 17 weeks of treatment, half of the mice in each group without fasting were euthanized by CO2 asphyxiation (for a separate study). The remaining mice were sacrificed in week 33 and used for this study. The liver was quickly collected and weighed, and half of the small lobe was cut and fixed in 10% buffered formalin for histopathological analyses. Other liver tissue was quickly clamped with a Wollenberg clamp at liquid nitrogen temperature and stored at −80 °C for lipidomic analyses. Interscapular brown adipose tissue and visceral white adipose tissues (ATs) (mesenteric, epididymal, and retroperitoneal depots) were collected, weighed, and stored at −80 °C. Histological Examination of Liver Tissue. The formalin-fixed liver tissue samples were first embedded in paraffin. Then, the paraffin was sectioned serially at 4 μm thickness. At last the sectioned paraffin was stained with hematoxylin and eosin. Biochemical Analysis of Liver and Serum Samples. ALT Discrete Pak kit (Catachem, Bridgeport, CT) was used to determine serum levels of alanine aminotransferase (ALT). Serum lipid levels, total cholesterol, and TG levels were determined using commercial kits (Pointe Scientific, Canton, MI) according to manufacturer’s instruction. Hepatic cholesterol lipids were extracted from liver samples according to the method described by Manley et al.26 Total cholesterol levels were measured as described above. Lipid Extraction. A CryoMill machine (Retsch, Germany) with a stainless ball at liquid nitrogen temperature was used to pulverize frozen liver tissue samples (∼30 mg). Then, 1 mL of 0.1 M HCl in 50:50 methanol/H2O was added to the pulverized tissue powder; the mixture was vortexed and then placed in a −20 °C freezer for 30 min. Then, 0.5 mL of chloroform was added and vortexed to mix well, and the mixture was placed on ice for 10 min. Samples were centrifuged at 15 000 RCF for 10 min and the chloroform phase at the bottom was transferred to a glass vial with a Hamilton syringe. About 0.5 mL of chloroform was added to the remaining material and extracted for the second time. The extracts were combined and dried under a stream of nitrogen. Then, a mixture of methanol/chloroform/2-propanol (1:1:1 v/v/v) using a ratio of 1 mL of solvent per 25 mg of initial tissue

thereby providing specific insight into the metabolic perturbations from phytosterols. In this study, the effects of two major phytosterols, namely, stigmasterol and β-sitosterol, in alleviating high-fat Westernstyle diet (HFWD)-induced NAFLD at 33 weeks in a mouse model were investigated. The goal of this study was to define the changes in hepatic lipid composition induced by the two phytosterols. We hypothesized that these phytosterol treatments prevent NAFLD mainly by altering hepatic levels of cholesterol, TG, and FFA. We use lipidomic analysis to identify specific lipids and lipid pathways that were related to the alleviation of NAFLD.

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MATERIALS AND METHODS

Animals and Diet. 78 week old male C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Animal studies were performed under protocol 02-027, approved by the Institutional Animal Care and Use Committee at Rutgers University (Piscataway, NJ). All animal studies and biochemical analyses were conducted at Rutgers University under the supervision of C. S. Yang. All efforts were made to minimize the suffering of experimental animals. Mice were housed in plastic shoe-box cages (five per cage) in our animal facility under standard conditions (lighting regimen of 12 h light−dark cycle, temperature 24 to 25 °C, and humidity 70−75%). All mice were with free access to diet and water. The rodent diets were control diet (CD), HFWD, HFWD containing 0.4% stigmasterol (HFWD+stigma), and HFWD containing 0.4% β-sitosterol (HFWD +β-sito) (prepared by Research Diets, New Brunswick, NJ). This HFWD was modified from the Western-style diet of Newmark et al.24 with 60% calories from fat. The HFWD contains lower amounts of calcium, vitamin D3, choline, folate, and fiber and a higher level of fat than the normal AIN76A diet. The CD was modified from the AIN76A diet with 10% of the calories from fat. The compositions of the HFWD, which we previously used to induce metabolic syndrome in mice, have been published.25 Stigmasterol (95% pure) and βsitosterol (95% pure) were obtained from Sigma-Aldrich (St. Louis, MO). The FDA suggests that a daily intake of 2 g phytosterols is needed to reduce cardiovascular risk and lower low-density lipoprotein cholesterol.7 A person consumes 500 g of food in dry weight (2000 to B

DOI: 10.1021/acs.jafc.7b06146 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. Effects of stigmasterol and β-sitosterol on liver weight (a), hepatic cholesterol level (b), brown adipose tissue weight (c), mesenteric adipose tissues (d), epididymal adipose tissues (e), retroperitoneal adipose tissues (f), circulating total cholesterol (g), circulation triacylglycerols (h), and ALT levels (i). The data are shown as mean ± SD (n = 5 for G1 and n = 10 for G2−4). a,b,c different letters indicate significant difference in week 33 by analysis of variance (ANOVA; p < 0.05). Multiple comparisons between the groups were performed using Duncan’s new multiple range test. weight was added to redissolve the extracts. The lipid extraction and subsequent lipidomic analysis were performed by W. Lu at Princeton University. Lipidomic Analysis by UPLC-ESI-QTOF Mass Spectrometry. A 1290 Infinity UHPLC system coupled to Agilent 6550 iFunnel QTOF mass spectrometer was used to analyze the lipids and fatty acids. To cover both the positive charged (ESI+) and negative charged (ESI−) species, each sample was analyzed twice using the same LC gradient but with different mass spectrometer ionization modes. The LC separation was performed on an Agilent Poroshell 120 EC-C18 column (150 × 2.1 mm, 2.7 μm particle size) with a flow rate of 150 μL/min. Solvent A was 1 mM ammonium acetate + 0.2% acetic acid in water/methanol (90:10). Solvent B was 1 mM ammonium acetate + 0.2% acetic acid in methanol/2-propanol (2:98). The solvent gradient in volume ratios was as follows: 0−2 min, 25% B; 2−4 min, 25 to 65% B; 4−16 min, 65 to 100% B; 16−20 min, 100% B; 20−21 min, 100 to 25% B; 21−27 min, 25% B. Data Processing. Data obtained by QTOF were first converted into mzXML format and then processed using MAVEN software (http://maven.princeton.edu) to obtain the signal intensity of ∼200 lipid species. The data were filtered by interquantile range and normalized to the total intensity for further analysis. Principal component analysis (PCA) was conducted with Statistical Package for Social Science program (SPSS 16.0, Chicago, IL). The 3D data containing the peak number, sample name, and normalized peak area were fed to the SIMCA14 software package (Umetrics, Umea, Sweden) for orthogonal projections to latent structures−discriminate analysis (OPLS-DA) and S-plot analysis. Variable importance projection (VIP) values exceeding 1.0 and P values of one-way ANOVA (p < 0.05) were selected as changed metabolites. Commercial databases, MetaboAnalyst 3.0 (http://www.metaboanalyst.ca), were used for qualitative analysis and to search for the metabolites in the lipid biosynthesis pathway.27 Statistical Analysis. Data were expressed as mean ± standard deviation and analyzed using one-way ANOVA followed by Duncan’s

new multiple range test among multiple groups using SPSS 16.0 (Chicago, IL). In special situations, Student’s t test was used to determine the difference between two groups. All of the tests were set at a significance level of p < 0.05.

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RESULTS General Observation. Food consumption and body weight were monitored weekly throughout the experiment. Because of the HFWD treatment, the body weight of the mice from the HFWD (G2), HFWD+stigma (G3), and HFWD+β-sito (G4) groups was significantly higher than that of the CD (G1) groups. The body weight of mice from G3 was significantly lower than that in the HFWD group (G2) during weeks 8−16 (Figure 1a). However, the lowering body weight effects of stigmasterol treatment disappeared after week 17 (Figure 1a). β-Sitosterol treatment (G4) slightly decreased body weight compared with G2, but the changes were not statistically significant. There was almost no difference in diet consumption of animals among G2, G3, and G4 in the first 16 weeks, except for week 8, in which the diet consumption was significantly lower in G3 than G2 (Figure 1b). From weeks 17 to 19 and week 31, the diet consumption of mice from G3 appeared to be higher than that from G2. The diet consumption of mice from G4 was significantly higher than that from G2 at weeks 23, 24, 26, 27, 30, and 31. Figure 1c shows that in comparison with the control group (G1), the macroscopic liver appearance of the HFWD group (G2) indicated hepatomegaly in weeks 17 and 33. The data indicate that stigmasterol and β-sitosterol treatment alleviate the hepatomegaly, and stigmasterol was more effective than βsitosterol. The protective effect appeared stronger in week 17 C

DOI: 10.1021/acs.jafc.7b06146 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. Hepatic triacylglycerols that significantly changed by phytosterol treatment in lipidomics analysis. Peak area is presented as mean ± standard deviation. (n = 4 for G1 and n = 9 for G2−4). a,b,c different letters indicate significant difference by analysis of variance (ANOVA; p < 0.05). Multiple comparisons between the groups were performed using Duncan’s new multiple range test.

Table 1. Lipidomics Analysis (Negative Mode) of Lipid Abundance in Liver Samples in Week 33a lipid species

G1 CD (×105)

FFA(C16:0) FFA(C16:1) FFA(C18:0) FFA(C18:1) FFA(C18:2) FFA(C18:3) FFA(C20:1) FFA(C20:2) FFA(C20:3) FFA(C20:4) FFA(C22:0) FFA(C22:1) FFA(C22:2) FFA(C22:3) FFA(C22:4) FFA(C22:5) FFA(C22:6) FFA(C24:0) FFA(C24:1) FFA(C24:4) FFA(C24:5) FFA(C24:6) FFA(C26:0) FFA(C28:0) FFA(C30:0) FFA(C32:0) FFA(C34:0)

± 49.65 ± 4.89a ± 31.63a ± 29.28a ± 10.78a ± 0.96a ± 1.21a ± 0.53a ± 1.13a ± 76.46a ± 0.40a ± 0.35a ± 0.05a ± 0.07a ± 0.33a ± 0.42a ± 4.29a ± 0.33a ± 0.18a ± 0.04a ± 0.11a ± 0.19a ± 0.23a ± 0.14a ± 0.15a ± 0.37a ± 0.14a

88.23 4.62 90.63 39.10 15.15 0.96 1.75 0.78 2.15 13.8 1.29 0.59 0.06 0.16 0.86 1.14 10.80 1.05 0.71 0.07 0.22 0.36 0.39 0.47 0.40 0.32 0.29

a

G2 HFWD (×105) 80.81 3.22 99.04 51.95 20.13 1.04 2.78 1.39 3.93 18.5 1.86 0.55 0.07 0.29 2.54 1.84 15.55 1.54 0.84 0.18 0.42 0.43 1.26 0.71 1.55 0.70 0.65

G3 HFWD+Stigma (×105)

± 33.79 ± 0.78a ± 51.65a ± 10.60a ± 1.91a ± 0.13a ± 0.66a ± 0.35a ± 0.84b ± 73.59a ± 0.25b ± 0.12a ± 0.04ab ± 0.16ab ± 1.11b ± 0.63ab ± 4.53ab ± 0.21b ± 0.20a ± 0.06a ± 0.22ab ± 0.22ab ± 0.41b ± 0.14b ± 0.22c ± 0.13b ± 0.06c a

103.85 13.04 81.33 123.26 44.37 2.73 5.47 2.61 5.71 29.31 1.86 0.58 0.13 0.50 4.19 2.67 23.71 1.53 0.84 0.26 0.62 0.82 1.28 0.61 1.11 0.63 0.55

± 38.82 ± 3.69b ± 41.30a ± 28.95b ± 12.36b ± 0.77b ± 1.63b ± 0.69b ± 1.63c ± 7.89b ± 0.16b ± 0.15a ± 0.06b ± 0.22a ± 1.37c ± 0.76b ± 8.84c ± 0.16b ± 0.19a ± 0.13ab ± 0.23b ± 0.30c ± 0.30b ± 0.07ab ± 0.24b ± 0.26b ± 0.09bc a

G4 HFWD+β-Sito (×105) 106.9 9.68 95.34 102.22 39.57 2.14 4.79 2.16 4.90 25.97 1.96 0.58 0.12 0.47 3.55 2.43 23.12 1.62 0.84 0.44 0.67 0.72 1.43 0.61 1.13 0.69 0.52

± 932.94a ± 4.83b ± 24.07a ± 32.48b ± 13.68b ± 0.70b ± 1.52b ± 0.75b ± 1.22bc ± 3.82b ± 0.27b ± 0.12a ± 0.06ab ± 0.28b ± 1.38bc ± 0.85b ± 6.82bc ± 0.28b ± 0.29a ± 0.28b ± 0.47b ± 0.39bc ± 0.47b ± 0.15ab ± 0.35b ± 0.27b ± 0.11b

a Data are shown as mean and standard error of peak area, n = 4 for G1 and n = 9 for G2, G3, and G4. The bold scripts indicate that there is a significant difference (p < 0.05, ANOVA) of lipid abundance in G1, G3, or G4 compared with G2. Mean values within a row with different letters (a, b, c) were significantly different (p < 0.05, ANOVA). Multiple comparison between the groups was performed using Duncan’s new multiple range test.

than in week 33. In the histological analysis of liver tissue, the liver from G1 was histologically normal (Figure 1d, G1). HFWD induced fatty liver with large areas of microvesicular steatosis, regions of evolving macrovesicular fatty change, hepatocellular hypertrophy, and some inflammatory foci in the liver tissue (Figure 1d, G2). Stigmasterol treatment significantly decreased the area of macrovesicular steatosis, microvesicular steatosis, and hepatocellular hypertrophy (Figure 1d, G3). However, β-sitosterol was less effective compared with stigmasterol (Figure 1d, G4). Biochemical Analysis and Liver and Adipose Tissue Weight. At week 33, the mice were sacrificed; adipose tissue and liver were collected and weighed (Figure 2). Compared with G1, HFWD significantly increased liver weight (Figure

2a), adipose tissue weight (Figure 2c−f), and hepatic cholesterol levels (Figure 2b). Compared with G2, both phytosterols significantly increased the retroperitoneal adipose tissue weight (Figure 2f) and decreased the hepatic cholesterol level. Stigmasterol or β-sitosterol treatment significantly decreased the serum total cholesterol levels (Figure 2g) but had no significant effect on serum TG levels (Figure 2h). The serum ALT levels were higher in G2 than G1, suggesting the induction of liver damage by HFWD. Stigmasterol was effective in preventing the elevation of ALT levels, but β-sitosterol was not effective (Figure 2i). Lipidomics Analysis of Liver Samples in Week 33. Four samples from G1 and nine samples from G2, G3, and G4 were analyzed with UPLC-ESI-QTOF in both negative mode and D

DOI: 10.1021/acs.jafc.7b06146 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry positive mode. To investigate the possible alteration of lipid species by phytosterols, species-level analyses of lipids from liver samples were conducted and compared by ANOVA. About 149 different lipid species were identified in the positive mode (Supplementary Table S1). Compared with G1, HFWD treatment (G2) significantly altered 107 species, including cholesteryl ester (CE), ceramides (CMs), diacylglycerols (DGs), phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), sphingomyelins (SMs), sphingosine, and TG. Among the 50 TG species analyzed, 35 TGs were significantly increased by HFWD. However, TGs with a lower number of double bonds, namely, TG (46:0, 48:0, 48:1, 50:1 and 58:2), were significantly decreased by HFWD treatment. Stigmasterol treatment significantly decreased TG with polyunsaturated fatty acids (50:6, 52:5, 52:6, 52:7, 52:8, 54:6, 54:8, 56:10, 56:4, and 56:9). β-Sitosterol had similar effects and significantly decreased TG (46:3, 48:3, 48:4, 50:4, 50:6, 52:5, 52:6, 52:7 and 56:4) (Figure 3). Among the 17 DG species analyzed, 16 DGs were significantly increased by HFWD. However, both phytosterols had no effect in altering the DG levels. Among the 37 PC species analyzed, 16 PCs were significantly decreased by HFWD. However, PCs (34:0, 38:3, 38:4, 40:2, 40:6, 40:7, and 42:9) were significantly increased by HFWD treatment. Both phytosterols had little effect on altering the PC levels. βSitosterol significantly decreased PC (38:2 and 38:3) and increased PC (40:2) compared with G2. Stigmasterol only significantly decreased PC (34:3) levels. HFWD treatment significantly decreased 8 of the 12 PE species analyzed, whereas both phytosterols had no effect on altering the PE levels. HFWD treatment significantly increased one CE, four CM, four PE, and four SM species and decreased the other three CE and four CM species compared with G1. Both phytosterols had no effect on altering the levels of these lipid species. We identified a total of 23 different FFAs in the negative mode (Table 1). Among the 23 FFAs analyzed, HFWD treatment increased many saturated FFAs. Both phytosterols effectively increased the unsaturated FFA levels compared with G2. Interestingly, stigmasterol significantly increased levels of FFA (C16:1, C18:1, C18:2, C18:3, C20:1, C20:3, C20:4, C22:4, C22:6, and C24:6) by almost two-fold (Table 1). βSitosterol also significantly increased levels of FFA (C16:1, C18:1, C18:2, C18:3, C20:1, C20:2, C20:3, C20:4, C22:4) compared with with G2. Both phytosterols significantly decreased the saturated FFA (C30:0) compared with G2. PCA is an unsupervised multivariable statistical method to discriminate the metabolic pattern among different groups in a multivariate pattern. In Figure 4a,b, hepatic lipid species, in positive and negative modes, of the four groups were displayed by score plots in 2D space. The clusters of lipid species (positive modes) from the CD group were clearly separated from those of the HFWD treatment groups. The first principal component factor efficiently separated the G3 samples from G2 samples (with some overlap) and located the profile of G3 between G1 and G2. However, the β-sitosterol treatment samples (G4) were not significantly distinct from G2 samples. Three principal component factors together explain >68.3% of the variance. In the negative mode, the global profiles among G3 and G2 were highly distinct. The first and second principal component factors did not separate G1, G2, G3, and G4. However, the third principal component factor efficiently separated the G3 samples from G2 samples and located the profile of G3 between G1 and G2. The three principal

Figure 4. PCA score plot based on the hepatic metabolic profiling among four groups in (a) positive and (b) negative modes.

component factors together explain >76.9% of the variance (Figure 4b). These results showed that stigmasterol treatment can prevent the lipid alteration induced by HFWD and βsitosterol was less effective. OPLS-DA for Discrimination of HFWD, Stigmasterol, and β-Sitosterol Treatment Groups. To obtain a higher level of group separation and for a better understanding of the global lipid alterations among G2, G3, and G4, OPLS-DA analysis was conducted. As presented in Figure 5a,b, clear separation between G2 and G3 or G4 was observed in the score plots of OPLS-DA models, indicating lipid perturbations by both phytosterols. VIP analysis was employed to select potential biomarkers for distinguishing HFWD and phytosterol-treated groups. In general, the candidate metabolites with VIP value >1 and p < 0.05 (between G2 and G3 or G4) were considered potential biomarkers with an above average influence. The lipid species with VIP values >2.0 in the S-plot, namely, TGs (52:5, 50:3, 52:6, 50:2, 54:7, 54:6, 56:9, 56:6, 48:2, 50:4, 56:8, 54:8, and 56:3) and PC (36:4) (Figure 4c), together with the other 20 species with VIP values >1 and cholesterol, were screened out as potential markers for stigmasterol effects. About 11 TG, 1 CE, 4 PC, and 1 DG species (Figure 4d), together with other 17 species with VIP values >1 and cholesterol, were screened out as potential markers for β-sitosterol. The permutation test was conducted to check the overfitting of OPLS-DA after modeling the data. The permuted R2 values to the left of the intercept (stigmasterol: R2 = 0.590; βsitosterol: R2 = 0.571) were lower than the original point to the right, and the Q2 regression line has a negative intercept (stigmasterol: Q2 = −1.01; β-sitosterol: Q2 = −1.18) (Figure 4e,f). These indicated that the OPLS-DA models were valid without over fitting. E

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Figure 5. OPLS-DA models applied to HFWD, stigmasterol and β-sitosterol treatment groups. (a,b) In the score plot, mice from HFWD and stigmasterol and β-sitosterol treatment group were well separated. (c,d) Using a statistically significant threshold of variable confidence ∼0.6 and VIP value >2.0 in the S-plot, a number of lipid species were screened out as potential markers of stigmasterol and β-sitosterol treatment. (e,f) Permutation test of the OPLS-DA models. Stigmasterol: the intercepts of R2 = 0.590, Q2 = −1.01; β-sitosterol: R2 = 0.571, Q2 = −1.18.

Table 2. Results from Pathway Analysis pathway name 1. 2. 3. 4. 5. 6. 7. 8.

glycerophospholipid metabolism linoleic acid metabolism steroid biosynthesis α-linolenic acid metabolism primary bile acid biosynthesis glycosylphosphatidylinositol (GPI)-anchor biosynthesis glycerolipid metabolism arachidonic acid metabolism

match status

p

−log(p)

Holm p

FDR

impact

2/30 1/6 1/35 1/9 1/46 1/14 1/18 1/36

0.004 0.021 0.025 0.031 0.032 0.049 0.062 0.121

5.481 3.862 3.701 3.461 3.428 3.026 2.781 2.113

0.341 1.000 1.000 1.000 1.000 1.000 1.000 1.000

0.342 0.858 1.000 0.858 1.000 0.984 0.984 1.000

0.231 0.000 0.053 0.000 0.037 0.044 0.107 0.000

Pathway Analysis. MetaboAnalyst 3.0 was used to identify and visualize the most relevant metabolic pathways in the

mouse. Pathway analysis showed that the above metabolites were in the biosynthetic pathways of glycerophospholipids, F

DOI: 10.1021/acs.jafc.7b06146 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry linoleic acid, steroids, α-linolenic acid, primary bile acids, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. A summary of the parameters obtained in the pathway analysis is shown in Table 2.

absorption and increased MUFA and PUFA levels. PCA analysis of lipidomic data (negative mode) showed that hepatic FFA species of stigmasterol-treated mice were located near the CD group in third principal component factor dimension [p3], suggesting that stigmasterol prevented the FFA changes caused by HFWD. As for the increase in MUFA and PUFA, pathway analysis showed that the linoleic acid metabolism and αlinolenic acid metabolism were involved (Table 2). The detailed mechanisms by which phytosterols modulate FFA composition and alleviate NAFLD remain to be investigated. The HFWD was used to mimic dietary risk factors for NAFLD. The low (but still adequate) choline and folate status is expected to decrease hepatic levels of PC and enhance lipid disposition and promote fatty liver disease. However, in the absence of a control group with normal AIN dietary levels of choline/folate high-fat diet in our study, the effect of the low choline/folate status on the development of NAFLD is unclear. Laos et al. found that soybean phytosterols decreased body weight in a 60-day experiment.28 However, another study showed that phytosterols or their esters have no effects on decreasing body weight in 12 weeks.19 In the present study, both phytosterols did not significantly decrease the body weight at 33 weeks. These reflected the biological features of different animal models. Both phytosterols significantly increased the retroperitoneal AT weight in week 33. It was proposed that organisms possess a maximum capacity for white adipose tissue expansion, and failure in the capacity for adipose tissue expansion may underlie the development of inflammation.44 The inflammation in adipose tissue will promote the pathogenesis of NAFLD.45 The effects of increasing AT capacity may also contribute to attenuating NAFLD. In conclusion, our results demonstrated that treatment of mice with phytosterols for 33 weeks alleviated the metabolic abnormality and NAFLD induced by HFWD. These beneficial effects are related to the lowering of cholesterol, TG, and some FFA. Most of the effects are likely due to decreased absorption of these lipid species. The pathway analysis showed that biosynthetic pathways, involving glycerophospholipids, linoleic acid, α-linolenic acid, steroids, and primary bile acids, are associated with the alleviation of NAFLD by phytosterols. Detailed mechanisms of action of these phytosterols remain to be further investigated.

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DISCUSSION Marked alterations of hepatic lipids are important pathophysiological hallmarks of fatty liver disease.10 In this study, a lipidomic approach was used to define the types and the amounts of lipids that were altered in a mouse NAFLD model. The effects of specific phytosterols on lipid metabolism and HFWD-induced NAFLD were investigated after the mice were treated for 33 weeks. Our study showed that both phytosterols decreased the hepatic and circulating cholesterol levels. This is consistent with many previous reports showing that phytosterols can decrease the circulating cholesterol.9 It is possible that phytosterols decreased the absorption or bile acid synthesis and lowered cholesterol absorption, which alleviates NAFLD.28,29 It has been reported that hepatic accumulation of free cholesterol is a contributing factor to the pathogenesis of NAFLD.30 Matsuzawa et al. and other researchers reported that cholesterol and FFA worked together, but not individually, in the pathogenesis of steatohepatitis.31,32 This concept is consistent with the pathway analysis, showing that steroid and primary bile acid biosyntheses were involved in the change of hepatic cholesterol contents. The presently observed hepatic cholesterol-lowering effect of phytosterols should contribute to the alleviation of NAFLD. Lipidomic studies of the human liver have shown that NAFLD is associated with an increase in major classes of lipids, including DG and TG, and a decrease in PC.21 Similar results were observed in our HFWD treatment mice. In this study, most TG species were increased by HFWD. Both phytosterols significantly decreased many TGs with polyunsaturated fatty acids (PUFAs) but had no effect on TGs with monounsaturated fatty acids (MUFAs) or saturated fatty acids. The prevention of hepatic TG accumulation may contribute to the attenuating NAFLD effects,33 and the reduction of TG by phytosterols has received much attention in recent years.28,34,35 Our results from the PCA analysis of the hepatic lipidomic data (positive mode) showed that phytosterol treatments slightly separated the cluster from HFWD groups, consistent with the analysis in Supplementary Table S1, showing that phytosterols did not significantly change the levels of PC, CM, DG, and SM compared with G2 (p > 0.05, ANOVA). According to lipidomic analysis (Table 1) and PCA analysis (Figure 4b), metabolite profiles between G3 and G4 are quite different. Stigmasterol may be more effective than β-sitosterol in alleviating NAFLD because stigmasterol, but not β-sitosterol, has been reported to be an antagonist of farnesoid X receptor (FXR),36 which may reduce circulating CM, alter lipid de novo synthesis, and protect against NAFLD.37 The interaction of stigmasterol with FXR in animals on a high-fat diet remains to be further studied. It is widely believed that FFA contributes to the pathogenesis of NAFLD; however, the roles of saturated and unsaturated FFA are unclear.38−40 Puri et al. reported that hepatic FFA content stayed the same during NAFLD development.21 However, low hepatic PUFA content was observed in NASH patients41 and in mouse models.42,43 In our study, HFWD significantly increased the saturated FFA in the liver, possibly due to the high lard content in the diet, while having little effect on unsaturated FFA. Both phytosterols effectively decreased the saturated fatty acids possibly because of decreased fat

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b06146.

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Supplementary Table S1. Lipidomics analysis (positive mode) of lipid abundance in liver samples in week 33. (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Z.L.: Tel: +86-571-88982175. E-mail: [email protected]. *P.S.: Tel: +86-571-88320388. E-mail: [email protected]. ORCID

Simin Feng: 0000-0001-8885-5801 Chung S. Yang: 0000-0001-6713-4837 Zisheng Luo: 0000-0001-8232-9739 G

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The research was supported by the Key Research and Development Program of Zhejiang Province (2018C02049) and Hangzhou Science and Technology Development Program (20170432B24). The work in the United States was supported by grants from the U.S. National Institutes of Health CA120915 (to C.S.Y.) and CA211437 (to W.L.) and shared facilities funded by CA72720 and ES05022 as well as the John L. Colaizzi Chair Endowment fund (to C.S.Y.). This work was also supported by General Financial Grant from the China Postdoctoral Science Foundation 2017M621970. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

We thank the personnel of Laboratory Animal Service in the Department of Chemical Biology at Rutgers University for taking care of our research mice.

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ABBREVIATIONS USED AT, adipose tissue; CE, cholesteryl ester; CM, ceramide; DG, diacylglycerol; FFA, free fatty acid; HFWD, high-fat Westernstyle diet; LFD, low-fat diet; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; OPLS-DA, orthogonal projections to latent structures−discriminate analysis; PCA, principal component analysis; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PUFA, polyunsaturated fatty acid; SM, sphingomyelin; TG, triacylglycerol; VIP, variable importance projection

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