Transcriptomic and Metabonomic Profiling Reveal Synergistic Effects

Aug 24, 2012 - Center for Chinese Medical Therapy and Systems Biology, E-Institute, Shanghai University of Traditional Chinese Medicine, Shanghai 2012...
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Transcriptomic and Metabonomic Profiling Reveal Synergistic Effects of Quercetin and Resveratrol Supplementation in High Fat Diet Fed Mice Mingmei Zhou,†,∇ Shidong Wang,†,§,∇ Aihua Zhao,∥,⊥ Ke Wang,§ Ziquan Fan,† Hongzhou Yang,† Wen Liao,† Si Bao,† Linjing Zhao,∥ Yinan Zhang,⊥ Yongqing Yang,† Yunping Qiu,‡ Guoxiang Xie,‡ Houkai Li,*,‡ and Wei Jia*,†,‡,⊥ †

Center for Chinese Medical Therapy and Systems Biology, E-Institute, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China ‡ Center for Translational Biomedical Research, University of North Carolina at Greensboro, North Carolina Research Campus, Kannapolis, North Carolina 28081, United States ∥ School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China § National Engineering Center for Biochip at Shanghai & Shanghai-MOST Key Laboratory of Health and Disease Genomics, Shanghai, China ⊥ X-omics Center for Metabolic Disease and Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China S Supporting Information *

ABSTRACT: Dietary quercetin and resveratrol have been frequently used in treating various diseases, but the underlying mechanisms are not entirely clear. Here, we report combined transcriptomic and metabonomic profiling that showed that the combined supplementation with quercetin and resveratrol produced synergistic effects on a high-fat diet-induced metabolic phenotype in mice. Histological and phenotypic improvements in serum and hepatic total cholesterol, insulin, fasting blood glucose, and HbA1c were also observed in mice receiving combined quercetin and resveratrol supplementation. This combined quercetin and resveratrol supplementation resulted in significant restoration of gene sets in functional pathways of glucose/lipid metabolism, liver function, cardiovascular system, and inflammation/immunity, which were altered by high fat diet feeding. The integration of transcriptomic and metabonomic data indicated quercetin and resveratrol supplementation enhanced processes of glycolysis and fatty acid oxidation, as well as suppressed gluconeogenesis. These alterations discovered at both the transcriptional and metabolic levels highlight the significance of combined “omics” platforms for elucidating mechanistic pathways altered by dietary polyphenols, such as quercetin and resveratrol, in a synergistic manner. KEYWORDS: resveratrol, quercetin, metabonomics, transcriptomics, GC/MS, high fat diet, fatty liver

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INTRODUCTION The plant-derived polyphenols are largely consumed in the form of fruits, vegetables, wine, and dietary supplements. In recent years, the dietary supplement of polyphenols has generated great interest among the public and scientists. Increasing experimental and clinical evidence has demonstrated the protective effects of polyphenols against a number of metabolic diseases, including fatty liver disease,1,2 obesity,3−5 diabetes mellitus,6,7 cardiovascular disease, 8,9 and even cancer.10−13 Accordingly, the French Paradox phenomenon14 has been attributed to the beneficial effect of dietary polyphenols, which is defined as the low occurrence rate of coronary heart diseases (CHD) in persons consuming large amounts of saturated fat in the diet. Quercetin, one of the most abundant flavonols present in wine, vegetables, and fruits, is © 2012 American Chemical Society

regarded as a powerful bioactive component with antioxidative and anti-inflammatory properties, as well as a regulator of endogenous enzymes.15,16 Experimental studies have indicated that quercetin is effective in lowering serum triglyceride (TG) and total cholesterol (TC) levels and reducing atherosclerotic lesions in high-fat diet (HFD)-induced atherosclerosis rabbits.17 In vitro studies have revealed that quercetin can inhibit de novo fatty acid and TG synthesis in rat hepatocytes, which may account for the protection against lipid accumulation in liver.18 Another plant-derived phenol, resveratrol (trans-3,4′,5-trihydroxystibene), is abundant in red wine and other fruits, and has been widely investigated for its multiple beneficial functions Received: May 30, 2012 Published: August 24, 2012 4961

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(Resveratrol) group fed with HFD and supplemented with 0.4% resveratrol (4 g/kg diet); combined quercertin and resveratrol (Combined) group fed with HFD and supplemented with 0.2% quercetin and 0.2% resveratrol (2 g quercetin + 2 g resveratrol per kg diet). All mice were kept on the respective diets for 26 weeks. One seriously injured mouse in the Resveratrol group was excluded in the following study due to fighting with other mice. At the end of the experiment, all mice were euthanized for blood and liver tissue collection. Samples from six mice in each group were used for metabonomic and transcriptomic analysis, except for mice in the Resveratrol group (n = 5). The animal experiments were conducted under the Guidelines for Animal Experiment of Shanghai University of Traditional Chinese Medicine (Shanghai, China), and the protocol was approved by the institutional Animal Ethics Committee.

against CHD, inflammation, diabetes mellitus, and cancers.19−21 The beneficial effects of resveratrol are believed to be associated with activities including antioxidation, antiplatelet aggregation, and negatively regulatory activity on vascular smooth muscle cells proliferation.19 Recently, resveratrol has also been shown to activate Sirtuin 1(SIRT1), AMP-activated protein kinase (AMPK), and peroxisome proliferative activated receptor gamma coactivator 1 alpha (PGC-1α), causing a calorie restriction-like effect and improving the metabolism in obese subjects.22 Since both quercetin and resveratrol are abundant in fruits and wine products, and both of them possess protective activities against various diseases, the coadministration of quercetin and resveratrol in vivo and in vitro has been reported to be beneficial for various conditions,23−25 suggesting an additive or a synergistic effect of the two polyphenolic compounds. However, given their multifarious properties reported to date, the exact mechanisms underlying the protective effects of quercetin and resveratrol are largely unclear. In recent years, the emerging “omics” technologies, including transcriptomics, proteomics, and metabonomics, have greatly enhanced the capability of investigating the biochemical alterations in response to nutritional or chemical intervention in a biological system. The integration of transcriptomics and metabonomics has being applied in studies on fatty liver disease,26 chemical-induced hepatotoxicity,27 cancers,28−30 diabetes,31,32 and stresses.33 We have also employed the combined transcriptomic and metabonomic approach to investigate the metabolic differences between obesity-prone and obesity-resistant rats, as well as the gene expression of critical enzymes involved in lipid metabolism.34 Our results indicate that the combined transcriptomic and metabonomic approach dramatically increases the scope of the metabolic network identifiable by each platform. Although quercetin and resveratrol have been extensively studied individually with conventional pharmacological and molecular biological means, to the best of our knowledge, there has been no report on a systematic evaluation of the beneficial activities of quercetin and resveratrol using combined “omics” approaches. In this study, we used a combined transcriptomic and metabonomic profiling approach to evaluate the beneficial effects of quercetin and/or resveratrol supplementation in mice fed a HFD. We hypothesized that the supplementation of quercetin and resveratrol together would provide synergistic activity in protecting against HFD-induced fatty liver conditions, which would be identifiable at both the transcriptional and metabolic levels, in addition to biochemical and histological outcome measurements.

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Biochemical Parameters in Serum and Liver

Biochemical parameters in serum were measured with commercial kits according to the manufacturers’ instructions, including TG, TC, low density lipoprotein (LDL), and high density lipoprotein (HDL), leptin, adiponectin, resistin, fasting blood glucose (FBG), interneukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and hemoglobin A 1c (HbA1c). The hepatic lipids were extracted from freshly collected liver tissues with chloroform and methanol (2:1) according to the previously published method.35 The TG and TC commercial kits were used to measure hepatic TG and TC contents, which were normalized to the wet weight of analyzed liver tissues. Glucose Tolerance Test

The glucose tolerance test (GTT) was conducted in the 24th week of the experiment. After 20 h fasting, the fasting blood glucose was measured with a hand-held glucometer (Johnson & Johnson) from whole blood drawn from the tail. After establishment of a stable baseline glucose level, the animals were injected intraperitoneally with 20 g/kg glucose by body weight. Then, whole blood glucose levels were then measured at 30, 60, 90, and 120 min after the glucose injection. Metabolites Extraction and Metabonomic Profile of Liver Tissue

GC/MS-based metabonomic profile was performed on extracts of liver tissues using an established two-step extraction method.36 Briefly, 50 mg of each liver sample was extracted with 500 μL of mixed solvents (mixture of chloroform, methanol, and water; 1:2:1, v/v/v, respectively) and following an extraction with methanol alone. Two internal standard solutions [L-2-chlorophenylalanine (0.3 mg/mL) in water and hepatadecanoic acid (1 mg/mL)] in methanol were added to the extractions and dried by vacuum at room temperature. The dried residue was resuspended in 80 μL of methoxymine in pyridine (15 mg/mL) and incubated at 30 °C for 90 min. The resulting solution was derivatized with 80 μL of N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA) at 70 °C for 60 min. Each 1-μL aliquot of the derivatized solution was injected in spitless mode into an Agilent 6890N gas chromatograph coupled with a Pegasus HT time-of-flight mass spectrometer (Leco Corporation, St Joseph, USA). Separation was achieved on a DB-5 ms capillary column [30 m × 250 μm i.d., 0.25-μm film thickness; (5%-phenyl)-methylpolysiloxane bonded and cross-linked; Agilent J&W Scientific, Folsom, CA, USA] with helium as the carrier gas at a constant flow rate of 1.0 mL/min. The temperature of injection, transfer interface, and ion source

EXPERIMENTAL SECTION

Animal Experiment

A total of 60 7-week-old male C57/6J mice were commercially obtained from Shanghai Laboratory Animal Co., Ltd. (SLAC, Shanghai, China). All animals were kept in a barrier system at a regulated temperature (23−24 °C) and humidity (60 ± 10%) and on a 12/12 h light-dark cycle with lights on at 07:00 a.m. Mice were fed with standard diet chow and provided tap water ad libitum for a 1 week acclimation period. The mice were then randomly divided into five groups (n = 12 for each group): normal diet (Normal) group fed with normal diet; high-fat diet (HFD) group; quercetin (Quercetin) group fed with HFD and supplemented with 0.4% quercetin (4 g/kg diet); resveratrol 4962

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Table 1. Biochemical Parameters of Serum and Livera Normal body weight (g) fat mass index (%) serum triglyceride (mmol/L) serum total cholesterol (mmol/L) LDL (mmol/L) HDL (mmol/L) insulin (mmol/L) leptin (ng/mL) adiponectin (μg/mL) resistin (ng/mL) IL-6 (pg/mL) TNF-α (pg/mL) HbAlc (%) FBG (mmol/L) BG 30 min (mmol/L) BG 60 min (mmol/L) BG 90 min (mmol/L) BG 120 min (mmol/L) hepatic triglyceride (mg/g) hepatic total cholesterol (mg/g)

30.84 2.40 1.25 2.9 0.33 2.09 221.17 22.2 12.12 1.21 3.13 1.21 4.37 5.82 13.19 9.86 9.44 8.63 11.45 2.39

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.05 1.04 0.27 0.43 0.11 0.32 21.22 3.77 1.63 0.2 0.56 0.22 0.36 1.15 2.18 1.77 2.06 1.10 6.62 0.35

HFD 38.38 7.44 1.84 3.65 0.48 2.06 309.67 40.8 12.41 3.54 5.7 2.4 4.88 7.68 19.82 15.04 13.15 12.23 24.08 7.29

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Quercetin

2.65b 1.69b 0.26b 0.39b 0.05c 0.14 39.94b 5.29b 1.06 0.72b 1.25b 0.57b 0.34b 1.3b 2.05b 2.60b 1.98b 2..36b 3.4b 2.93b

35.52 5.62 1.45 3.47 0.42 2.26 264 37.24 12.77 3.02 4.17 2.06 4.82 7.01 17.55 13.66 11.68 10.35 18.67 5.27

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.88e 1.81e 0.25e 0.37 0.11 0.21 48.61 4.39 1.69 0.67 1.53e 0.45 0.3 0.96 1.98e 1.50 1.76 2.02 6.21 1.55e

Resveratrol 34.80 5.68 1.32 3.47 0.4 2.34 247.17 33.61 11.9 2.48 3.81 2.22 4.66 6.58 16.24 12.05 11.27 10.20 22.18 4.08

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.64e 1.85e 0.23d 0.44 0.06 0.19e 55.57e 4.07d 1.67 0.71d 0.78d 0.18 0.23 0.75 2.46e 2.08d 2.00e 1.17e 3.86 0.55d

Combined 34.67 5.90 1.47 2.67 0.36 2.41 233.67 34.07 12.56 2.28 4.03 2.15 4.48 5.81 15.93 12.45 11.16 9.97 18.71 2.59

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.36e 1.17e 0.34e 0.63d 0.13e 0.3e 33.89d 3.57d 1.39 0.45d 0.55d 0.53 0.41d 1.17d 1.51d 1.80d 1.32d 1.23d 3.66 0.75d

a

Significant differences were determined by One-Way ANOVA. bP < 0.01. cP < 0.05, vs Normal group. dP < 0.01. eP < 0.05, vs HFD group. Data are mean ± SD. n = 6. Fat mass index (%): weight of white adipose tissue/body weight × 100%. FBG: fasting blood glucose; BG: blood glucose.

was set to 270 °C, 260 °C, and 200 °C, respectively. The GC temperature programming was set to 2 min isothermal heating at 80 °C, followed by 10 °C/min oven temperature ramps to 180 °C, 5 °C/min to 240 °C, and 25 °C/min to 290 °C, and a final 9 min maintenance at 290 °C. Electron impact ionization (70 eV) at full scan mode (m/z 30−600) was used, with an acquisition rate of 20 spectrum/s.

(Invitrogen, Carlsbad, CA, USA). Genes involved in cholesterol and fatty acid metabolism in the microarray data were selected for quantitative RT-PCR (qRT-PCR) analysis. Reactions were carried out in triplicate for each sample in a 25 μL volume using β-actin (β-Act) as an internal control on an ABI Prism 7300 Sequence Detection System (Applied Biosystems, Foster City, CA) with the SYBR green I qRT-PCR kit (Toyobo, Osaka, Japan). Gene expression was evaluated with threshold cycles. The PCR primer sequences are listed in Table S1.

RNA Preparation

Total RNA was isolated from 29 individual mouse livers using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified with the RNeasy mini kit (QIAGEN GmBH, Germany). The RNA integrity number was qualified to be greater than 7.0 using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA). The numbers of samples for each group (Normal, HFD, Quercetin, Resveratrol, and Combined) were 6, 6, 6, 5, and 6, respectively.

Data Analysis

Raw data of the microarray were log10 transformed and then normalized to yield Z-scores, which in turn were used to calculate a Z-ratio value for each gene between two groups.37 The Z-ratio was calculated as the difference between the observed gene Z-scores for two contrastive groups and divided by the standard deviation associated with the distribution of these differences.38 Genes considered to be significantly changed (Z-ratio values ≥ +1.5 or ≤ −1.5) were further applied to Parametric Analysis of Gene Set Enrichment (PAGE)39 using the disease/phenotype web-PAGE (D/P webpage, http://dpwebpage.nia.nih.gov)40 to identify key biologically functional gene sets derived from the Mouse Genome Informatics (MGI) database.41 A P value was calculated to test the significance of the cumulate Z-score (positive or negative) of an enriched gene set. Note, the directionality of a gene set name simply means that this functional category is altered, rather than representing the real tendency of the present study. For GC/MS data analysis, the sample information, peak retention time, and peak area (quant mass) were included in the final data set. All those known artificial peaks, such as peaks caused by noise, column bleed, and the BSTFA derivatization procedure, were removed from the data set. The resulting data were normalized to an internal standard prior to statistical analysis. The normalized data were mean centered and unit variance scaled during chemometric data analysis in the SIMCA-p 12.0 Software package (Umetrics, Umeå, Sweden).

Microarray Hybridization and Data Acquisition

Twenty-eight RNA samples were individually applied to the Agilent Whole Mouse Genome Oligo Microarray (4×44K) platform (Agilent Technologies, Santa Clara, USA). This microarray contains 41,174 mouse probes representing at least 20,423 mouse unigenes. Subsequent RNA linear amplification and microarray hybridization were conducted per the manufacturer’s protocol. After hybridization, slides were scanned by using an Agilent Microarray Scanner (Agilent Technologies, Santa Clara, USA) with 5 μm resolutions for each slide at photomultiplier tube settings of 100% and 10%. Furthermore, TIFF images were analyzed using Agilent’s feature extraction software (version 9.5.3). Row data were exported to TXT files. The microarray platform was submitted to the Gene Expression Omnibus Web site (http://www.ncbi. nlm.nih.gov/geo) with the accession number GSE38325. Quantitative RT-PCR Analysis

Total RNA from each sample used for the microarray experiment was subjected to reverse transcription using oligo (dT) primers and Superscript II reverse transcriptase 4963

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Figure 1. Representative photos of histological examination of liver tissues. Liver tissues were fixed with 4% paraformaldehyde overnight and stained with hematoxylin and eosin. (A) Normal group; (B) HFD group; (C) Quercetin group; (D): Resveratrol group; (E) Combined group (n = 4, magnification ×200).

lowering levels of serum TG, insulin, IL-6, and hepatic TC; however, serum TG, insulin, leptin, resistin, IL-6, and hepatic TC were decreased to a greater extent in the Resveratrol group. Moreover, a synergistic effect was observed in the Combined group, which was reflected by the greater decreases in serum TC, LDL, insulin, resistin, HbA1c, and hepatic TC compared to quercetin or resveratrol alone (Table 1). To further determine the activity of quercetin and resveratrol in alleviating glucose intolerance, GTT was performed at the end of the 24th week. Compared to the Normal group, HFD induced significant glucose intolerance, which was characterized by higher levels of fasting blood glucose, as well as higher levels of blood glucose at 30, 60, 90, and 120 min after the glucose injection (Table 1). Although the glucose intolerance was attenuated in both Quercetin and Resveratrol groups, the most significant attenuation was observed in the Combined group (Table 1), suggesting that the combined quercetin and resveratrol are synergistic in improving the glucose intolerance condition. In addition, HFD feeding resulted in significant increases in body weight and fat mass index compared to mice in the Normal group, which were attenuated in the three treatment groups. The reduction of body weight and fat mass index was statistically significant in Quercetin, Resveratrol, and Combined groups compared to the HFD group but was not statistically significant among the three treatment groups (Table 1).

The unsupervised multivariate statistic, principal component analysis (PCA) was first used to compare the metabonomic profiles among groups. Differential variables were then selected with the criteria of variable importance in the projection (VIP > 1) in the partial least-squares-discriminant analysis (PLS-DA) model and p < 0.05 in a Student’s t-test. Compound identification was performed by comparing the mass fragments of interesting variables with NIST 05 standard mass spectral databases in NIST MS search 2.0 (NIST, Gaithersburg, MD) software at a similarity score of greater than 70%. The identified differential metabolites were then validated by using available reference compounds. The corresponding fold change shows how these selected differential metabolites varied between groups. The biochemical parameters were compared with oneway analysis of variance (ANOVA), followed by the Student− Newman−Keuls (SNK) or Student’s t-test as indicated. A P < 0.05 was considered statistically significant. Histological Examination

Liver samples were fixed with 4% paraformaldehyde overnight and stained with hematoxylin and eosin as previously described.42

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RESULTS

Biochemical Parameters of Serum and Liver

After 26 weeks’ HFD feeding, typical characteristics of fatty liver and insulin resistance were observed in most mice of the HFD group, which included significant increases in levels of serum TG, TC, LDL, insulin, leptin, resistin, IL-6, TNF-α, HbA1c, FBG, and hepatic TG and TC (Table1). Histological evidence of hepatic steatosis was also observed (Figure 1). However, one mouse in the HFD group showed an obvious difference in phenotypes from the other mice, including a substantially smaller increase of body weight and absence of hepatic steatosis. We suspected that it may be a nonresponder to HFD feeding, and therefore, we excluded it from the following data analysis. We then compared the effects of quercetin and resveratrol on HFD-fed mice. We found that supplementing either quercetin or resveratrol was effective in

Differential Gene Expression Patterns and Functional Annotation by Quercetin or Resveratrol Supplementation

Compared to the Normal group, 135 gene sets were significantly altered by HFD (Figure 2A), while 90, 91, and 98 gene sets were significantly altered in the Quercetin, Resveratrol, and Combined groups compared to the HFD group, respectively (Figure 2B). Among the 135 altered gene sets, approximately 50, 55.7, and 43.4% of gene sets showed identical directions with the HFD group in the Quercetin, Resveratrol, and Combined groups, respectively (Figure 2A). Moreover, compared to the HFD group, the hierarchical cluster revealed that the expression pattern of the altered gene sets in 4964

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nation of quercetin and resveratrol, but not by supplementation with quercetin or resveratrol alone. HFD feeding, as well as the combined supplementation with quercetin and resveratrol, resulted in comprehensive alterations in glucose/lipid metabolism, in which 7 and 10 altered gene sets were enriched, respectively. Six altered gene sets in the Combined group showed a different direction from that in the HFD group. However, only 3 and 2 altered gene sets were observed in the Quercetin and Resveratrol groups, respectively. Similarly, a much more profound alteration in liver function was only observed in the Combined group (Figure 3B−D). Based on the enriched gene sets among groups, a further comparison was performed to identify the specific genes that were significantly altered within the four biologically functional categories of metabolic diseases. We found that very few genes showed overlapping enrichment in these gene sets in the different therapy groups (Figure S1). Most genes altered in the Quercetin group occurred in inflammation/immunity gene sets, while the major of altered genes in the Resveratrol group were clustered in gene sets of the cardiovascular system. However, genes altered in the Combined group were comprehensively distributed in the four functional categories (Figure S1). Validation of Microarray Data with qRT-PCR

Since quercetin and resveratrol are effective in lowering cholesterol and TG levels, we analyzed 23 genes implicated in cholesterol and fatty acid metabolism with qRT-PCR to validate the data obtained by microarray analysis. In general, the expression of most genes analyzed by qRT-PCR was highly consistent with the results from microarray analysis. Compared to the Normal group, various genes relevant to cholesterol biosynthesis and conversion were significantly decreased by HFD feeding, such as 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (Hmgcr), 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (Hmgcs1), farnesyl diphosphate synthetase (Fdps), 7-dehydrocholesterol reductase (Dhcr7), squalene epoxidase (Sqle), cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1), and cytochrome P450, family 8, subfamily b, polypeptide 1 (Cyp8b1). However, most of the changes was effectively reversed by supplementation with quercetin or resveratrol, and especially when both were combined. In addition, the expression of several fatty acids synthesis-related genes was also upregulated in the HFD group, but was decreased in the Quercetin, Resveratrol, and Combined groups, such as very low density lipoprotein receptor (Vldlr), peroxisome proliferator activated receptor γ (Ppar-γ), and sterol regulatory element binding transcription factor 1 (Srebf1) (Table 2).

Figure 2. Altered gene expression patterns in livers from different groups. Parametric analysis of gene-set enrichment (PAGE) was performed on significantly altered genes induced by different treatments. (A) The altered gene sets in the HFD, Quercetin, Resveratrol, and Combined groups are shown with a heatmap compared to the Normal group. Columns represent every gene set that was significantly up-regulated (red) or down-regulated (green) in different groups. (B) The number of altered gene sets is shown with a Venn diagram analysis in the Quercetin, Resveratrol, and Combined groups compared to the HFD group. (C) The hierarchical clustering of significantly altered gene sets with Z-score values in the Quercetin, Resveratrol, and Combined groups compared to the HFD group.

Metabonomic Profile of Liver Tissues

the Combined group was much more different from those of either the Quercetin or Resveratrol group (Figure 2C). Significantly altered expressions of gene sets were functionally annotated in several major metabolic pathways related to liver function, glucose/lipid metabolism, cardiovascular system, and inflammation/immunity. In contrast to the decreased expression of gene sets in the cardiovascular system, the expression of the most identified gene sets related to liver function, glucose/lipid metabolism, and inflammation/immunity was upregulated in the HFD group (Figure 3A). The decreased expression of gene sets in the cardiovascular system by HFD feeding was restored by supplementation with quercetin, resveratrol, or both combined. HFD feeding resulted in comprehensive increases in expression of gene sets related to inflammation/immunity, which was recovered by the combi-

The metabonomic profiles among groups were evaluated with unsupervised statistics, PCA, which showed a trend of separation between the HFD and Normal group, as well as between Quercetin, Resveratrol, Combined, and HFD groups (Figure S2). We then constructed the three-component PLSDA models between the HFD and Normal groups [R2X = 0.58, R2Y = 0.99, Q2(cum) = 0.64], as well as between Quercetin, Resveratrol, Combined, and HFD groups [R2X = 0.91, R2Y = 0.83, Q2(cum) = 0.49; R2X = 0.78, R2Y = 0.99, Q2(cum) = 0.73; R2X = 0.67, R2Y = 0.99, Q2(cum) = 0.76, respectively]. One obvious outlier in the HFD group was excluded from the model because it had a substantially lower body weight compared to others in that group. Figure 4 shows that the score plots of the PLS-DA model of mice from the HFD, Quercetin, 4965

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Figure 3. Enrichment of metabolic disease-related gene sets altered in different groups. Metabolic disease-related gene sets were classified into four functional categories based on the gene set name derived from the MGI database. The altered gene sets were enriched with PAGE analysis as described in the Methods. (A) The altered gene sets between the HFD and Normal groups. (B, C, and D) The altered gene sets in the Quercetin, Resveratrol, and Combined groups compared to the HFD group, respectively.

Resveratrol, and Combined groups were clearly separated from the Normal or HFD group. Based on the criteria of VIP > 1.0 in the PLS-DA model and p < 0.05 in the Student’s t-test, 17 differential metabolites were identified between the HFD and

Normal groups, which showed obvious down-regulation in the HFD group, but were restored to the normal levels by supplementation with quercetin, resveratrol, or both (Figure 5E, Table 3). To further characterize and compare the effects of 4966

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Table 2. Validation of Selected Genes with qRT-PCRa microarray (Z-ratio)

qRT-PCR (fold change)

gene symbol

HFD vs Normal

Quercetin vs HFD

Resveratrol vs HFD

Combined vs HFD

HFD vs Normal

Quercetin vs HFD

Resveratrol vs HFD

Combined vs HFD

Hmgcr Hmgcs1 Idi1 Fdps Dhcr7 Sqle Cyp7a1 Cyp8b1 Ldlr Scarb2 Abcg5 Abcg8 Abcb11 Lpl Vldlr Apoa1 Ppara Pparg Insig2 Nr1h3 Nr1h4 Srebf1 Srebf2

−1.92 −0.16 −0.68 −1.93 −2.39 −5.12 −1.64 −4.48 −0.62 −0.54 1.12 2.44 −1.14 −2.09 3.49 −1.88 0.44 2.85 −1.02 −0.67 −1.01 3.34 −0.32

−0.94 −0.40 −0.38 −0.64 −0.44 −0.21 0.52 1.11 −0.10 −0.06 0.50 −0.77 0.61 2.54 −1.10 1.10 −0.52 −0.32 −0.82 1.28 0.58 −1.29 −1.52

0.42 −0.13 −0.16 0.49 −0.72 1.98 0.35 1.97 −1.44 1.47 −0.42 −1.76 0.28 2.32 −1.18 0.88 −0.87 −1.88 −2.83 0.98 −0.31 −4.11 −0.02

−0.30 1.60 −0.39 1.64 1.72 2.14 −0.76 0.30 0.10 −0.73 1.06 0.09 1.22 2.44 −3.52 1.17 −0.83 −0.98 −1.87 0.42 −0.51 0.08 3.03

−1.93 −2.16 −1.18 −1.12 −3.88b −2.05b −1.70 −5.14c 1.03 −1.16 1.86c −1.00 1.09 −1.54 1.82b −1.52b −1.48 1.383 −1.55 −1.14 1.14 12.94c −1.94b

−1.17 1.31 −1.31 −1.20 2.13 1.53 1.60 1.74 1.27 1.42 −1.12 −1.02 −1.13 2.02b 1.10 1.01 1.04 1.71 −1.15 1.19 −1.06 −3.65c 1.12

1.17 1.07 −1.05 −1.14 1.27 1.77c −1.18 2.22 1.08 1.33 −1.23 −1.19 1.10 1.86b 1.25 −1.15 −1.27 1.20 −1.50 1.21 −1.26 −21.19c 1.01

1.01 1.98 −1.05 1.58 1.98 1.81b −1.24 1.64 1.30 1.38 1.01 −1.04 1.24 1.37 −1.17 1.16 1.28 1.79 −1.11 1.58 1.08 −4.01c −1.29

a qRT-PCR was performed on each sample in triplicate. β-actin was used as an internal control. Z-ratios reflect statistical confidence but do not relate directly to fold-change in microarray data. The expression of genes in qRT-PCR was compared with a Student’s t-test. bP < 0.05. cP < 0.01.

Figure 4. Metabolic profiles depicted by PLS-DA scores plot and heatmap of differential metabolites. The metabolic profiles of liver tissues were depicted by PLS-DA scores plot with GC/MS spectral data. (A) Normal group vs HFD group; (B) Quercetin group vs HFD group; (C) Resveratrol group vs HFD group; (D) Combined group vs HFD group; (E) the heatmap showed the changes of the 17 differential metabolites identified between the HFD and Normal groups, as well as their changes in the Quercetin, Resveratrol, and Combined groups (1: HFD vs Normal; 2: Quercetin vs Normal; 3: Resveratrol vs Normal; 4: Combined vs Normal).

Figure 5. Summarized metabolic pathways between the Combined and HFD groups. Metabolites and genes (italics) in red represent increases in concentration or expression in the Combined group compared to the HFD group, while the green color indicates decreases in concentration or expression. The dashed arrow indicates multiple steps in the pathway. The number besides the gene name represents the corresponding Z-ratio value (described in Data analysis) of the gene between the Combined and HFD groups. Val: valine; Ile: isoleucine; Gly: glycine; Ser: serine; His: histidine.

quercetin and resveratrol on HFD-fed mice, comparisons between the Quercetin, Resveratrol, Combined, and HFD groups were performed, in which 5, 18, and 28 differential metabolites were identified, respectively (Table S2−S4). We found that the majority of differential metabolites in the Quercetin and Resveratrol groups were amino acids, while a set

of other differential metabolites were also observed in the Combined group such as pyruvic acid, lactic acid, succinic acid, malic acid, ribose, nicotinamide, glyceric acid, 1-palmitoylglycerol, stearic acid, uracil, and uridine, suggesting that 4967

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Table 3. Changes of High Fat Diet-Induced Differential Metabolites in Different Groups FC

a

metabolites

RT_min

VIP

p valuea

HFD vs Normal

Quercetin vs Normal

Resveratrol vs Normal

Combined vs Normal

chemical class

2-aminobutyric acid valine succinic acid serine threonine β-alanine oxoproline 4-aminobutyric acid threonic acid glutamic acid phenylalanine hypoxanthine ornithine histidine lysine tyrosine tryptophan

7.15 7.78 9.20 9.80 10.13 10.72 11.93 12.02 12.11 13.14 13.25 15.65 15.76 17.22 17.26 17.50 21.11

1.84 1.87 2.12 1.68 1.68 1.69 1.68 1.84 1.82 1.94 1.69 1.59 1.77 2.09 2.04 1.67 1.83

0.014 0.012 0.002 0.030 0.029 0.029 0.030 0.014 0.015 0.007 0.029 0.043 0.020 0.002 0.004 0.030 0.014

0.37 0.65 0.42 0.66 0.67 0.59 0.60 0.49 0.40 0.61 0.63 0.57 0.53 0.55 0.52 0.66 0.62

0.79 0.93 1.34 0.97 0.97 0.82 1.10 0.90 1.06 1.01 0.88 1.01 0.92 1.02 0.91 0.97 1.00

0.79 1.33 0.98 1.12 1.20 0.98 0.96 0.82 0.59 1.01 1.27 0.69 1.18 1.00 1.11 1.23 1.12

0.55 1.05 1.23 1.14 1.11 0.94 1.33 0.78 1.34 0.99 1.00 0.92 0.82 1.02 0.94 1.15 1.09

amino acid amino acid organic acid amino acid amino acid amino acid amino acid amino acid organic acid amino acid amino acid nucleobase amino acid amino acid amino acid amino acid amino acid

Comparison of differential metabolites between HFD and Normal group with a Student’s t test; FC: fold change; RT: retention time.

supplementation with quercetin and resveratrol together may induce substantially greater metabolic impacts than quercetin or resveratrol alone. On the other hand, 11 amino acids showed similar changes in the Resveratrol and Combined groups compared to the HFD group, whereas only 3 common differential metabolites were observed in the Quercetin and Combined groups including histidine, lysine, and arabinose (Table S2−S4).

quercetin and resveratrol on the HFD-fed mice was effective at both transcriptional and metabolic levels.

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DISCUSSION In this study, we used transcriptomic and metabonomic approaches to systematically compare the effects of quercetin and resveratrol on liver metabolism in HFD-fed mice, in addition to biochemical and histological evaluation. Liver histology and serum biomarkers, such as hepatic and serum cholesterol levels and insulin sensitivity, were improved to a greater extent with quercetin and resveratrol in combination compared to either supplement alone. Moreover, the transcriptomic and metabonomic results showed a much broader range of alterations in gene sets and metabolite when quercetin and resveratrol were combined compared to quercetin and resveratrol treatment alone, suggesting synergistic effects of the two polyphenols. Both quercetin and resveratrol have long been recognized as important components in wine, fruits, and vegetables that protect against cardiovascular diseases and certain cancers through multiple mechanisms of action. Enhanced activity has been observed in combining resveratrol and quercetin in the inhibition of adipogenesis and induction of apoptosis in adipocytes;25 however, few reports have systemically evaluated the synergistic effects of combined resveratrol and quercetin supplementation. It has been reported that quercetin or resveratrol alone can inhibit TG synthesis in rat hepatocytes;18,43 however, no synergistic effects of quercetin and resveratrol were observed in lowering hepatic TG levels and body weight in our study. Although the doses of both quercetin and resveratrol in the Combined group were half of that in the Quercetin or Resveratrol groups, our results showed much more profound metabolic impact when the supplements were combined, which is consistent with the larger number of restored gene sets in liver function, glucose/lipid metabolism, cardiovascular system, and inflammation/immunity by transcriptomic analysis. Although quercetin and resveratrol have been extensively studied for their preventive and therapeutic effects on various disorders, and even cancers, the complexity of their biological

Integration of Metabonomic and Transcriptomic Results

To further characterize the connections between transcriptomic and metabonomic data, we integrated the main phenotypes and metabolic profiles with the alterations of gene expression between the Combined and HFD groups. We observed that the significant reduction in glucose and TG levels in the Combined group was accompanied with more up-regulation of metabolites such as pyruvate, lactate, glyceric acid, stearic acid, 1palmitoylglycerol, β-alanine, intermediates of the tricarboxylic acid (TCA) cycle, and several glucogenic amino acids. Most of these differential metabolites are implicated in glucose/lipid metabolism. We then screened the genes involved in modulating this metabolic process, within the transcriptomic data. Several glucose/lipid metabolism-related genes with significantly different expression between the Combined and HFD group were identified. Genes with a significant increase in expression included lipoprotein lipase (Lpl), acyl-CoA synthetase long-chain family member 6 (Acsl6), acetyl-CoA acyltransferase 1 A(Acaa1a), acetyl-CoA acyltransferase 1 B (Acaa1b), enoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh), ATP-binding cassette, subfamily D, member 3 (Abcd3), and lactate dehydrogenase (Ldh). Genes with a significant decrease in expression included succinate dehydrogenase A (Sdha), succinate dehydrogenase C (Sdhc), succinateCoA ligase, GDP-forming, beta subunit (Suclg2), and malate dehydrogenase 1(Mdh1) (Figure 5). These differentially expressed genes are mainly implicated in the regulation of lipid lipolysis, fatty acid oxidation, and the TCA cycle. Accordingly, the integration of transcriptomic and metabonomic data suggested that the combined supplementation with 4968

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quercetin and resveratrol between our study and previous reports may be associated with a difference in the models used. On the other hand, our results also demonstrated that combined supplementation with quercetin and resveratrol resulted in an increase in lipid catabolism and fatty acid oxidation on HFD-fed mice, as evidenced by a significant decrease in serum TG, and increase of 1-palmitoylglycerol, glyceric acid, and stearic acid. These findings were in agreement with the up-regulation in the expression of several lipid metabolism-related genes. Moreover, stearic acid has also been reported to have a protective effect against cholestasis-induced liver injury, which is associated with its anti-inflammatory property in rats.55 We hypothesized that the effect on the regulation of the inflammatory response may be associated with an increase in stearic acid levels in the Combined group. Resveratrol has been reported to up-regulate gluconeogenic gene expression in primary hepatocytes,56 while a quercetinmediated inhibitory effect on gluconeogenesis was also observed on rat liver slices.57 The metabolic profile from the current study showed significantly higher levels of glucogenic amino acids in the Combined group, and most of them were also present in the Resveratrol group but not in the Quercetin group. Although the expression of the main gluconeogenic genes, including glucose-6-phosphatase (G6p) and phosphoenolpyruvate carboxykinase (Pepck), was not different between the Resveratrol, Combined, and HFD groups in our transcriptomic profile, we speculated that the glucose-lowering effect in the Resveratrol and Combined groups may be partially due to the suppression of gluconeogenesis by resveratrol. The inconsistency between gene expression and metabolites fluctuation is possible because of the sequential biochemical changes from mRNA, protein expression, and metabolic alteration.58 A similar phenomenon was also observed in the alteration of intermediates of the TCA cycle and the expression of corresponding regulatory genes. As a result, the integrated pathways of glycolysis, fatty acid oxidation, and the TCA cycle with transcriptomic and metabonomic data indicated that combined supplementation of quercetin and resveratrol enhanced glycolysis and fatty acid oxidation in liver. The synergistic effect of quercetin and resveratrol in protecting HFD-induced fatty liver and metabolic disorders could be delineated by combining transcriptomics and metabolomics at both transcriptional and metabolic levels. In this study, we obtained transcriptomic and metabonomic profiles of liver metabolism resulting from exposure to quercetin, resveratrol, or both together. However, the limitations of the current study should also be noted. First, we only used the GC/MS-based metabolomics platform to characterize the metabolic profile of liver tissue. The established protocol for sample preparation and derivatization before instrumental analysis may cause some loss in metabolites information, which may account for the limited number of identified metabolites in the current report. A LC/MS-based metabonomics platform is needed in further studies. Second, we only analyzed the liver tissues with transcriptomics and metabonomics in this study. Given the comprehensive impacts of chronic HFD feeding, the simultaneous analysis of other metabolic tissues such as white and brown adipose tissues, as well as skeletal muscles, is necessary for uncovering the mechanism underlying the multitargeting effects of quercetin and resveratrol. Finally, additional mechanistic studies will be conducted to understand the alterations identified in tran-

functions and systemic responses within mammals hinders recognition of the mechanisms involved using conventional methods. In the current study, four common metabolites were found to be differentially expressed between the HFD and Normal groups, as well as the Quercetin, Resveratrol, and HFD groups, including 4-amniobutyric acid, ornithine, histidine, and lysine. These metabolites were decreased in the HFD group but were restored to normal levels by quercetin or resveratrol treatment. A recent study showed that dietary supplement of 4aminobutyric acid improves glucose tolerance and insulin sensitivity by inhibiting inflammation in HFD-fed mice.44 Accordingly, we hypothesize that 4-aminobutyric acid may play an important role in mediating the anti-inflammatory effect of quercetin and resveratrol. Moreover, HFD feeding resulted in a clear up-regulation of most gene sets implicated in the inflammation/immunity pathway, while the restoration of the expression of these gene sets was only observed in the Combined group. Although there was no significant difference in the levels of serum inflammatory cytokines, IL-6 and TNF-α, among the Quercetin, Resveratrol, and Combined groups, the number and trend of altered gene sets involved in inflammation/immunity in the Quercetin and Resveratrol groups were similar to the HFD group, suggesting that the combined supplementation with quercetin and resveratrol had a synergistic effect in attenuating the HFD-induced inflammation/immunity disorder. The levels of both histidine and lysine in liver tissue were reduced in the HFD group and were restored by supplementation with quercetin, or resveratrol in our study. It has been reported that the serum levels of several amino acids, including histidine and lysine, are lower in obese subjects,45 and dietary supplementation with histidine or lysine can alleviate HFD-induced hepatic steatosis, as well as fat accumulation in mice.46,47 In this study, we did not measure the levels of these amino acids in serum. However, it is possible that the relatively higher levels of histidine and lysine in the livers of mice from the Quercetin, Resveratrol, and Combined groups compared to the HFD group may inhibit de novo lipogenesis. Long-term HFD feeding is one of the leading causes of dysfunction in glucose metabolism and insulin resistance,48 while quercetin and resveratrol have been well established as effective supplements for improving insulin sensitivity.16,49−51 In this study, we found that the combined supplementation with quercetin and resveratrol was more efficacious for improving glucose tolerance and insulin sensitivity than quercetin or resveratrol alone. Similar findings were observed in both transcriptomic and metabonomic results, in which a substantially greater number of gene sets involved in glucose/ lipid metabolism was altered. In addition, a greater number of differential metabolites involved in glycolysis, TCA cycle, and fatty acid metabolism were identified in the Combined group compared to the Quercetin and Resveratrol groups. Although we did not detect a significant difference in the expression of genes modulating glycolysis between the HFD and Combined group, the higher level of pyruvate, and lactate, as well as obvious decrease of glucose in the Combined group suggested that glycolysis was up-regulated. The higher level of lactate was only observed in the Combined group compared to the HFD group, which was accompanied with an up-regulation of Ldh gene expression. Resveratrol and quercetin have been reported to inhibit glucose metabolism and lactate production in cancer cells52,53 or rat liver,54 and therefore, we suspected that the inconsistency in the regulation on glucose metabolism by 4969

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scription and metabolism as a result of quercetin and resveratrol supplementation.

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ASSOCIATED CONTENT

S Supporting Information *

PCR primer sequences and metabonomic profiles among groups. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*W.J.: phone, 704-250-5803; fax, 704-250-5809; e-mail, w_jia@ uncg.edu. H.L.: phone, 704-250-5805; fax, 704-250-5809; email, [email protected]. Author Contributions ∇

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Basic Research Program of China (2007CB914700), the Innovation Program of the Shanghai Municipal Education Commission (09YZ119), and E-institutes of Shanghai Municipal Education Commission (E03008). We appreciated Yang Gao, Junsong Han, and Ting Wei at the National Engineering Center for Biochip in Shanghai for their technical assistance in transcriptomic experiments.

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dx.doi.org/10.1021/pr3004826 | J. Proteome Res. 2012, 11, 4961−4971