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Am J Physiol Gastrointest Liver Physiol 291: G63-G72, 2006. First published February 2, 2006; doi:10.1152/ajpgi.00565.2005
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LIVER AND BILIARY TRACT

Hepatic cytochrome P-450 reductase-null mice show reduced transcriptional response to quercetin and reveal physiological homeostasis between jejunum and liver

David M. Mutch,1,* Vanessa Crespy,1,* Jennifer Clough,1 Colin J. Henderson,2 Sofiane Lariani,1 Robert Mansourian,1 Julie Moulin,1 C. Roland Wolf,2 and Gary Williamson1

1Nestlé Research Center, Lausanne, Switzerland; and 2Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, Dundee, United Kingdom

Submitted 15 December 2005 ; accepted in final form 26 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using mice deficient in hepatic cytochrome P-450 oxidoreductase (POR), which disables the liver cytochrome P-450 system, we examined the metabolism and biological response of the anticarcinogenic flavonoid, quercetin. Profiling circulating metabolites revealed similar profiles over 72 h in wild-type (WT) and POR-null (KO) mice, showing that hepatic P450 and reduced biliary secretion do not affect quercetin metabolism. Transcriptional profiling at 24 h revealed that two- to threefold more genes responded significantly to quercetin in WT compared with KO in the jejunum, ileum, colon, and liver, suggesting that hepatic P450s mediate many of the biological effects of quercetin, such as immune function, estrogen receptor signaling, and lipid, glutathione, purine, and amino acid metabolism, even though quercetin metabolism is not modified. The functional interpretation of expression data in response to quercetin (single dose of 7 mg/animal) revealed a molecular relationship between the liver and jejunum. In WT animals, amino acid and sterol metabolism was predominantly modulated in the liver, fatty acid metabolism response was shared between the liver and jejunum, and glutathione metabolism was modulated in the small intestine. In contrast, KO animals do not regulate amino acid metabolism in the liver or small intestine, they share the control of fatty acid metabolism between the liver and jejunum, and regulation of sterol metabolism is shifted from the liver to the jejunum and that of glutathione metabolism from the jejunum to the liver. This demonstrates that the quercetin-mediated regulation of these biological functions in extrahepatic tissues is dependent on the functionality of the liver POR. In conclusion, using a systems biology approach to explore the contribution of hepatic phase 1 detoxification on quercetin metabolism demonstrated the resiliency and adaptive capacity of a biological organism in dealing with a bioactive nutrient when faced with a tissue-specific molecular dysfunction.

metabolite; microarray; nutrigenomics; flavonoid; global error assessment; pathway; systems biology; bioavailability

FLAVONOIDS ARE A GROUP of phenolic compounds that occur naturally in fruits and vegetables (22). Their ingestion has been correlated with a multitude of biological functions, including anti-oxidant activity, anti-inflammatory and anticarcinogenic properties, and antiproliferative effects (38). Recent work has implicated several of these compounds in the modulation of lipid biomarkers of cardiovascular disease, such as total cholesterol, low-density lipoprotein (LDL) levels, and various apolipoproteins (20). Thus, to understand the means by which these dietary molecules are capable of mediating disparate biological functions, it is essential to define the molecular players responsible for regulating their metabolism and effects throughout an organism.

To date, the great majority of studies aimed at identifying the molecular players mediating flavonoid bioactivity have profiled gene expression in cultured cell lines (1, 7, 13, 19, 34, 36). Recently, van Erk et al. (34) reported the first in vitro comprehensive analysis of gene expression for quercetin, a well-known and commonly consumed flavonoid (34). The authors coupled gene expression profiling with functional assays and demonstrated the anticarcinogenic and antiproliferative effects on Caco-2 cells; however, it remains unclear to what extent in vitro results can be transposed to an in vivo context (14). Furthermore, many studies based in cultured cell systems use polyphenols in forms (i.e., aglycones) that are not normally found in the blood and peripheral tissues, suggesting that different biological activities may be observed if using the appropriate physiological compounds (10, 14).

In an attempt to decipher the contribution of the hepatic xenobiotic metabolism system on the circulating quercetin metabolite profile and on biological response, we used a mouse model deficient in liver cytochrome (CYP) P-450 oxidoreductase (POR) (12). This model, which enabled the systematic decoupling of the hepatic P450 detoxification system from that of the small intestine, was deemed appropriate for exploring quercetin metabolism for the following reasons. First, the hepatic CYP P450 system contributes to the detoxification and activation of a large number of endogenous and exogenous (e.g., ingested) compounds. Furthermore, quercetin and its conjugates have previously been demonstrated to interact and modulate both directly and indirectly the expression and activity of several hepatic monooxygenases (5, 25, 31, 39). Secondly, the conditional deletion of POR results in a loss of function of CYP7A1, the rate-limiting enzyme for the conversion of cholesterol to primary bile acids, and a corresponding reduction of ~90% in the production and secretion of bile acids (12), thereby enabling the influence of bile on the absorption of quercetin to be determined. Therefore, the goals of this study were to determine 1) the influence of a reduced bile content on the absorption of quercetin, 2) the extent to which liver phase 1 xenobiotic metabolism contributes to the circulating metabolite profile for quercetin, and 3) whether the transcriptional signatures of several tissues (liver, jejunum, ileum, and colon) following quercetin consumption would unravel the bioactive nature of this flavonoid. The results revealed that, whereas the ileum and colon appear to respond uniquely to quercetin, a molecular relationship between the jejunum and liver was identified, exemplifying the robustness of the biological organism and its ability to achieve physiological homeostasis in response to xenobiotics despite the functional elimination of the hepatic P450 system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Diet

All animals were adapted to the RM3 (E) 801710 Soya-free powdered diet (B.S & S., Scotland, UK) over a period of 14 days, as prepared by CXR Biosciences (Dundee, Scotland). Quercetin (Sigma-Aldrich, Buchs, Switzerland) was added separately to the semipurified diets at two concentrations/doses: 1,200 ppm ("low dose," 0.12%, 1.2 g/kg) and 6,200 ppm ("high dose," 0.62%, 6.2 g/kg). CXR Biosciences reared 65 male cytochrome P-450 reductase-null (KO) mice and 65 wild-type (WT) C57BL/6 mice, all aged between 6–8 wk. Animals were housed three per cage, where both temperature and relative humidity were maintained within a range of 19–23°C and 40–70%, respectively. Twelve-hour periods of light were cycled with 12-h periods of darkness.

For each strain of mouse, the following experimental design was used: a control group (25 mice) receiving powdered RM3 diet ad libitum, a group (15 mice) receiving a low dose of quercetin (1.4 mg/mouse), and a group (25 mice) receiving a high dose of quercetin (7 mg/mouse). The experimental diet was administered on day 15, following a 14-day adaptation period to the RM3 diet. Test groups had access to the quercetin-enriched diets for a period of 4 h, while control animals received powdered RM3 diet ad libitum for an equivalent period of time. Following administration of the experimental diet, animals were fasted for 20 h prior to having access to the control-powdered diet again. Animals were killed after 4, 24, or 72 h. Body weight (days 1, 5, and 10) and food consumption (days 5, 10, and 15) were recorded.

Plasma Samples

Mice were anaesthetized with pentobarbital (70 mg/kg, ip) according to procedures approved by the University of Dundee Ethical Review Committee, and all animal work was carried out in accordance with the Animal (Scientific Procedures) Act. Blood was obtained by cardiac puncture and collected into lithium/heparin-coated tubes and centrifuged. Plasma was removed into new Eppendorf tubes, and an ascorbate-EDTA solution (0.4 M Na2PO4 containing 20% ascorbic acid and 0.1% EDTA, pH 3.6) was added to prevent coagulation. Samples were dispensed into tubes and flash-frozen in liquid nitrogen for polyphenol analysis.

Tissue Samples

Gut sections (jejunum, ileum, and colon) and livers from mice at the 24-h time point (n = 9 for both WT and KO mice) were isolated for RNA extraction and flash-frozen in liquid nitrogen by CXR Biosciences. The gut was removed from the above mice, and the small intestine (below the stomach to above the cecum) was divided into three sections corresponding to the duodenum, jejunum, and ileum. The colon was harvested from below the caecum and above the rectum. Gut sections were rinsed with PBS (pH 7.4), and the epithelial layer was scraped off for RNA processing. The gall bladder was removed and discarded, and the liver harvested. Whole livers from the above animals were processed for RNA extraction.

Gene Expression Analysis

RNA was extracted from the various tissue samples (gut sections were scraped to isolate epithelial cells; whole livers) using 1 ml TRI Reagent (Sigma, Dorset, UK) and Qiagen (Qiagen, West Sussex, UK) Mini columns (colon), 2 ml TRI Reagent and Qiagen Midi columns (ileum and jejunum), or 12.5 ml TRI Reagent and Qiagen Maxi columns (whole livers) as specified by the manufacturer. All samples were treated with DNase to remove any contaminating DNA. RNA was eluted using 50 µl (colon), 150 µl (ileum and jejunum), or 1.2 ml (liver) water. The quality of the RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Where RNA profiles contained extra peaks or were deemed anomalous, RNA was cleaned again using Qiagen columns. The quality of RNA and the level of degradation varied between tissue types; however, RNA samples destined for microarray analysis were only accepted and pooled into three groups if no aberrant signs of degradation (e.g., multiple peaks) were observed. The comprehensive gene expression profiles of the liver, jejunum, ileum, and colon were analyzed and were labeled L1–L12, J1–J12, I1–I12, and C1–C12, respectively. In all cases, except those listed, RNA from three mice was pooled. The following pools consisted of RNA derived from only two mice: J1, I5, I6, I8, C3, C4, C6, C7, and C9.

The protocols detailing RNA preparation for microarray analysis, including cRNA preparation, hybridization, and scanning, have been described elsewhere (24). The only exception to the protocol was the pooling of three cRNA samples (L2, L3, and J3) due to low yields of cRNA synthesis. Samples were hybridized to the murine 430A GeneChip (Affymetrix, Santa Clara, CA), which consists of 14,000 full-length, well-characterized genes. A mathematical method, termed the global error assessment (GEA) model, was applied to the raw GeneChip data for the selection of differentially regulated genes (as described in Ref. 24). Transcriptional data considered significantly modulated in this study met two criteria: 1) when considering global treatment effects (i.e., all control mice, i.e., WT + KO = 6, and all mice fed quercetin, i.e., WT + KO = 6) the alpha value was set at 0.01, and 2) when considering the treatment effects for each type of mouse, the alpha value was set at 0.001. The complete data set is publicly available in the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) through the following accession number:GSE4262. A glossary of the gene symbols used throughout the text can be found in Supplemental Table S1 (available at the American Jounal of Physiology-Gastrointestinal and Liver Physiology web site).1

Data Analysis

Pathway analysis was performed using Ingenuity Pathways Analysis software (http://www.Ingenuity.com). In brief, a data set containing gene identifiers and their corresponding expression values was uploaded as an Excel spreadsheet using the template provided in the application. Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base database, which was then used as the starting point for generating biological networks. Gene ontology (GO) clustering was performed as previously described (28). Principal component analysis (PCA) was performed on the preprocessed data set (normalized and log-transformed) consisting of 14,000 probe sets. The PCA calculation was based on the singular value decomposition of a data matrix containing Ns rows and Ng columns, where Ns corresponds to the numbers of arrays (samples) and Ng the number of genes (variables). Computing and analysis was performed using "R" (http://www.r-project.org) and Bioconductor (http://www.bioconductor.org) software.

HPLC Analysis

Sample preparation. Plasma (175 µl) was mixed with 20 µl acetic acid (0.58 M), 10 µl of internal standard (rhamnetin, final concentration 1 µM) dissolved in DMSO, and 10 µl beta-glucuronidase/sulfatase from Helix pomatia type H-5 (Sigma). The samples were incubated for 30 min at 37°C. Methanol (500 µl) and HCl (200 mM) was added to stop the enzymatic reaction. After centrifugation for 5 min at 4°C and 15,000 rpm, the supernatants were transferred to Pyrex tubes and dried under nitrogen. Once the samples were dry, 175 µl of phase B was added. The liquid was then transferred to a tube, and this was centrifuged once more with the same conditions. Fifty microliters of the supernatant was injected in an HPLC system to be analyzed.

Chromatographic conditions. HPLC was performed using an Agilent Technologies Hypersil BDS-C18 4.6 x 150 mm, 5 µm column. Mobile phase A consisted of 15% acetonitrile in 30 mmol/l NaH2PO4 at pH 3.0, and the mobile phase B consisted of 40% acetonitrile in 30 mM NaH2PO4 at pH 3.0. The separation was performed at 39°C. The flow rate was at 0.6 ml/min with a linear gradient between 5 and 20 min from 100% of phase A to 100% of phase B, remaining at 100% of phase B until 35 min. From 35 to 37 min, the gradient was decreased from 100% of phase B to 100% of phase A. Detection was performed with an eight-electrode Coularray model 5600 system (Euroservice, Milan, Italy) with potentials set at –20, 0, 60, 120, 180, 240, 400, and 500 mV.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Body Weight and Food Intake

Both WT and KO mice gained weight steadily throughout the experiment, and no significant differences (P = 0.01) were observed in food consumption in animals following administration of quercetin (Table 1). Therefore, we attribute all transcriptomic and metabolite changes to dietary quercetin or to the functional deletion of hepatic POR.


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  		Table 1. Food consumption/exposure to quercetin in wild-type and POR-null mice

 
Quercetin Metabolism

HPLC analysis of plasma from WT mice fed the high dose of quercetin revealed two peaks corresponding to quercetin and isorhamnetin (3'-methyl-quercetin) (Fig. 1). At the 4-h time point, the concentration of quercetin metabolites was 22.3 ± 5.9 µM (Fig. 2A). All of the quercetin was glucuronidated and/or sulfated, and 27% was also methylated. At 24 h, the total quercetin concentration was 3.9 ± 0.98 µM, of which 40% was methylated (Fig. 2A). Even after 72 h, quercetin and isorhamnetin metabolites were detectable in murine plasma, showing the relatively long half-life of quercetin derivatives. A similar metabolite profile was observed in KO animals (Fig. 2A). There were also no significant differences in the plasma profile of quercetin between WT and KO mice fed the low-dose diet (Fig. 2B).


Figure 1
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  		Fig. 1. Representative HPLC chromatograms of mice plasma. A: wild-type (WT) mice 4 h high dose of quercetin. B: knockout (KO) mice 4 h high dose of quercetin. C: spiked plasma control with 3 µM of quercetin, isorhamnetin, and rhamnetin (internal standard).

 

Figure 2
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  		Fig. 2. Evolution of plasma concentration of quercetin metabolites in mice WT and KO receiving a high dose (A) or a low dose (B) of quercetin. Ingested amounts of quercetin were 1.4 mg and 7 mg per mouse for low dose and high dose, respectively.

 
Transcriptional Events Modulated by Quercetin

A thorough discussion of the murine transcriptomic response following the elimination of the hepatic POR will be reported elsewhere; however, global liver gene expression changes in this mouse model have previously been reported by Wang et al. (35). In the present manuscript, we have examined the transcriptional profiles of the liver, jejunum, ileum, and colon following the consumption of 7 mg quercetin in both WT and POR-null mice. Hierarchical clustering of the data clearly placed the various tissues in the expected biological context, i.e., the jejunum and ileum are most similar in global gene expression, followed by the colon and then the liver, thus establishing a high degree of confidence in the data set (Fig. 3). To put into perspective the influence of quercetin consumption vs. the elimination of liver POR on the comprehensive gene expression profile, we used PCA. By defining the magnitude of each gene's contribution to a tissue's comprehensive expression profile, we were able to reduce the original number of genes to only those that had a high impact on the separation between paired experimental conditions (i.e., effect of quercetin in WT vs. KO in a specific tissue). As illustrated in Fig. 4, PCA of the liver and jejunum transcriptional data sets indicated two distinct clusters. The experimental groups in the liver and jejunum do not cluster based on whether animals received quercetin or not, but rather based on whether the POR gene is functionally active or not. The clearly dominant effect of the liver POR deletion over quercetin consumption was further reinforced by simply comparing the number of liver genes identified as differentially regulated (P < 0.001): 316 genes in wild-type animals (WT + quercetin vs. WT) and 210 genes in knockout animals (KO + quercetin vs. KO) compared with the 1,110 genes differentially regulated between mouse strains (KO vs. WT). PCA analysis of microarray data obtained from the colon and ileum did not indicate that the liver POR deletion had a dominant effect on the transcriptional profiles of these tissues (data not shown); rather, PCA analysis suggested that the deletion of POR and the consumption of quercetin contributed significantly to defining the global transcriptional profiles of these organs. This equivalent contribution meant that no distinct clusters could be clearly resolved in the ileum and colon.


Figure 3
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  		Fig. 3. Hierarchical clustering of all experimental samples placed the comprehensive gene expression profiles of the ileum and jejunum closest together, followed by the colon, with the liver being the furthest away. Twelve microarrays per organ, labeled 1–12: L, liver; C, colon; J, jejunum; I, ileum.

 

Figure 4
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  		Fig. 4. Principal component (PC) analysis plots for the liver (A) and jejunum (B).

 
Tissue Specific Responses to Quercetin

In all tissues examined, quercetin consumption in the WT animals produced more changes in the expression profiles than in KO mice (Fig. 5). When selecting biological pathways that were significantly regulated, it was quickly apparent that quercetin has the ability to modulate disparate molecular functions in a tissue-dependent manner.


Figure 5
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  		Fig. 5. Number of genes differentially regulated in the various organs of both WT and KO animals following quercetin consumption.

 
Colon. In the colon, the predominant molecular network modulated was the antigen presentation pathway. Consumption of quercetin in WT animals led to the upregulation (P < 5 x 10–4) of several genes corresponding to transmembrane receptors in this canonical pathway (Cd74, Hla-dqa1, Hla-dma, Hla-dqb1, Hla-drb1, between 2.5- and 3.5-fold). Furthermore, additional analysis revealed that the immune response, composed of 14 genes (ApoE, Bc1, Bgn, Ccl5, Col6a1, Col6a3, Dmpk, Igfbp7, Rgs5, and the five aforementioned genes) whose expression were all significantly increased, was the most highly regulated biological function in the colon (Fig. 6A). In contrast, the colonic epithelia of KO mice responded with a downregulation in expression between 1.4- and 1.6-fold for the aforementioned transmembrane receptors (Fig. 6B, P < 6 x 10–6). A subsequent analysis revealed that in the colons of KO mice, quercetin most significantly affected genes associated with the biological function immune response by downregulating such genes as Btg2, Cd74, H2-Aa, Hla-dma, Hla-dqb1, Hla-drb1, Igfbp4, Ighm, Igj, and Igk-C. Gene ontology (GO) clustering further confirmed this finding by identifying such molecular functions as major histocompatibility complex (MHC) class II receptor activity (GO identifier: 45012), antigen binding (GO identifier: 3823), and peptide antigen binding (GO identifier: 42605); however, a greater significance (P values differed by ~1010) was attributed to regulated molecular functions identified in WT animals vs. those in KO animals (data not shown).


Figure 6
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  		Fig. 6. Antigen peptide presentation pathway in the colon. The biological network demonstrates the complex and highly interactive nature between its component genes. Shaded boxes indicate genes that were differentially regulated by quercetin (A, upregulated; B, downregulated), and open boxes are those genes that were not identified as differentially regulated. Both the number of genes in this pathway and their regulation were dependent on hepatic cytochrome P-450 oxidoreductase (POR) functionality. A: wild-type animals, where all shaded boxes indicate upregulated genes. B: KO animals, where all shaded boxes indicate downregulated genes. For gene descriptions, see Supplemental Table S1. Network was created using Ingenuity Systems software (http://www.ingenuity.com).

 
Ileum. Pathway analysis did not identify strongly modulated biological functions in the ileum of either WT or KO animals following the consumption of quercetin. Indeed, the only canonical pathway identified as significantly modulated in WT mice was purine metabolism (P < 0.006), but these genes were only subtly regulated. Furthermore, these genes did not belong to a single molecular network. Additionally, both chemokine and estrogen receptor signaling were identified in WT animals as being significantly modulated by quercetin (P < 0.02 and P < 0.04, respectively); however, the genes associated with these pathways did not belong to a single network. Interestingly, no significant canonical pathways were identified in the ileum of KO animals. GO clustering revealed that the most significantly modulated molecular function was protein binding (WT, P < 6 x 10–24; KO, P < 3 x 10–12); however, no single biological network was identified. The results emphasize the regional specificity of the intestinal tract in response to its environment and the importance of the hepatic P450 system on the normal function of extrahepatic tissues.

Jejunum and liver. Data analysis suggested that the gene expression profiles of the jejunum and liver were coordinately regulated, as revealed by the identification of shared biological pathways and the PCA analysis. Although both tissues were highly susceptible to hepatic POR function, quercetin consumption led to quantifiable and significant changes in several important biological functions: amino acid metabolism, lipid metabolism, and glutathione metabolism.

Amino Acid Metabolism

In the livers of both WT and KO animals the alanine and aspartate metabolism canonical pathway (composed of Adssl1, Asl, Got1, and Gpt2) was upregulated by quercetin (P = 0.008 and P = 0.001 in WT and KO mice, respectively). Specific pathways of amino acid metabolism were regulated in each of the mouse strains. For example, in WT animals, arginine and proline metabolism (P = 0.01), glycine, serine and threonine metabolism (P = 0.002), and methionine metabolism (P = 0.02) were all significantly modulated. Genes associated with these canonical pathways were upregulated (Bhmt, Cth, Mat1a, Agxt, Gldc, Shmt1, Aldh2, Arg1, Asl, and Got1), with the exception of Amd1, P4ha1, and Alas1. In KO animals, only liver tyrosine metabolism (P = 0.05) was modulated in addition to the alanine and aspartate metabolism pathway through increases in Adhfe1 and Got1 and a decrease in Moxd1. In contrast, the only amino acid metabolism canonical pathway to be modulated in the jejunum occurred with WT animals. All genes (Acaa1, Aldh4a1, Ehhadh, and Tmlhe) associated with lysine degradation (P = 0.04) had a decreased expression following quercetin consumption.

Lipid Metabolism

Several canonical pathways related to lipid metabolism were found to be differentially regulated in both the jejunum and/or liver, including butanoate metabolism, fatty acid metabolism, and sterol biosynthesis. In WT animals, butanoate metabolism was more significantly regulated in the liver (P = 0.008) than the jejunum (P = 0.04); however, the analysis revealed that butanoate metabolism was increased in the liver and decreased in the jejunum. Interestingly, in KO animals, butanoate metabolism was only identified in the jejunum (P = 0.03), where all genes in this pathway were upregulated, suggesting that in the POR-null animals the predominant location for the transcriptional regulation of butanoate metabolism shifts from the liver to the jejunum. Pathway analysis for changes to the fatty acid metabolism pathway revealed similar findings. In WT animals, fatty acid metabolism is under the coordinate control of both the jejunum and liver; indeed, fatty acid metabolism was identified in both organs as significantly regulated (jejunum, P = 0.03; liver, P = 0.04). All genes (Acaa1, Aldh4a1, Cyp1a1, Cyp2b10, Cyp2j2, Cyp3a2, and Ehhadh) in the jejunum of WT animals were downregulated, in contrast with both the up- and downregulation of hepatic genes (Acox2, Aldh2, Cyp2b10, Cyp2c39, and Hadh2 were upregulated; Cyp51a1 and Cyp2a13 were downregulated). The effect of quercetin in KO animals was shifted predominantly to the liver (P = 0.01), where all genes were upregulated (Acad10, Acox2, Adhfe1, Cyp2b10, Cyp2c8, and Cyp2c39). No additional tissues were identified as key players in the regulation of this biological function in KO animals. Additionally, a tissue relationship between the liver and jejunum was also demonstrated for sterol biosynthesis, where quercetin affects the liver canonical pathway in WT animals (P = 0.002) through the downregulation of Fdft1, Fdps, Idi1, and Sqle and the jejunal canonical pathway in KO animals (P = 1 x 10–5) through the upregulation of epithelial Fdft1, Idi1, Lss, and Sqle. GO clustering agreed with the pathway analysis by identifying steroid and lipid metabolism as significant biological functions in the livers of WT mice but not in KO animals (data not shown). Furthermore, in the jejunum of both WT and KO animals, lipid-related biological processes were identified as significantly modulated.

Glutathione Metabolism

Finally, the principal tissue responding to quercetin for glutathione metabolism in WT animals is the jejunum (P < 1 x 10–4), where all nine genes associated with this canonical pathway were downregulated between 1.3- to 1.8-fold following quercetin consumption (Gpx2, Gstu7, Gsta2, Gsta4, Gstk1, Gstm3, Gstm5, Gstm6, and Gstt1). Regulation of this biological function was shifted to the liver in KO animals (P = 0.005), where Gstm3 and Gstt3 were upregulated and Gclc, Gsta3, and Gstp1 were downregulated.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The molecular mechanisms underlying the many biological roles of flavonoids continue to challenge the biological community. As with the great majority of nutrients, the influences of quercetin within a biological system are broad, subtle, and often seemingly unrelated. Studies, mostly in vitro, have found quercetin capable of modulating carcinogenic, plasma antioxidant, and cardiovascular biomarkers (17, 20, 23, 30, 38), in addition to acting as both signaling molecules (37) and regulators of gene expression (15); however, it remains unclear to what extent data derived from cultured cell models can be transposed to an in vivo context (14, 26). Of critical importance is an understanding of quercetin bioavailability and metabolism within a biological organism. The current hypothesis would suggest that both the liver and small intestine participate in the metabolism of quercetin (8, 14, 26, 33). KO animals are characterized by an ~90% reduction in bile acid synthesis and secretion (12). As the pharmokinetic data for quercetin bioavailability in the WT and KO animals were equivalent, this would suggest that a dramatic decrease in bile does not have an effect on quercetin absorption or its enterohepatic circulation. Recently, Chen et al. (8) examined the pharmokinetics of quercetin in Sprague-Dawley rats that were bile duct cannulated and concluded that quercetin undergoes little to no enterohepatic circulation, thereby minimizing the importance of bile on quercetin metabolism. Furthermore, Crespy et al. (9) demonstrated that quercetin can be secreted into the bile; however, its biliary secretion was very low compared with other flavonoids, again minimizing the importance of bile on quercetin metabolism. Findings from the present study would support this notion. Both rat and human intestinal and hepatic cell models are capable of differentially metabolizing quercetin (33), and intestinal metabolism of quercetin is extensive (8, 29). Taken together, the functional elimination of all hepatic cytochrome P-450 enzymes has little effect on the circulating quercetin metabolite profile.

Although hepatic P450s do not influence the metabolism of quercetin, they exert a profound influence on the biological response, even in extrahepatic tissues. As exemplified by the greater number of genes (2- to 3-fold) significantly regulated in all WT tissues examined vs. KO tissues, it is apparent that the hepatic detoxification system will influence an organism's ability to "interpret" nutritional molecules. We demonstrated that the ileum and colon respond uniquely to quercetin and that a molecular relationship promoting physiological homeostasis exists between the jejunum and liver. First, as previously demonstrated (3, 27, 28), gene expression in response to the intestinal environment appears to be region specific. Coupling these transcriptomic changes with functional bioinformatic analyses has suggested that the regional differences in gene expression may contribute to the site-specific biological functions exerted along the gastrointestinal tract. Quercetin consumption clearly produced many gene expression changes in the jejunum, ileum, and colon; however, pathway analysis and GO clustering identified biological functions that are region specific. It is important to note that the effects of quercetin in the ileum and colon were minimal, presumably because the bulk of quercetin was absorbed in the proximal regions of the small intestine (21). As enterohepatic circulation for quercetin is apparently low in this KO model, this would imply that the epithelium of the distal small and large intestines may be exposed to considerably less quercetin than the jejunum. Hence, only highly significant expression changes will be identified and produce a coherent functional picture. No specific biological pathways were modulated in the ileum and only a single, yet highly relevant, pathway was identified in the colon (antigen presentation). Interestingly, the great majority of studies examining quercetin in the intestine have focused on its role on cell proliferation, apoptosis, and carcinogenesis, while little work has focused on its role in immune function (11). This fact may stem from the use of common carcinomic cell culture models (e.g., Caco-2, HT-29, T84 cells, etc.) to study flavonoid metabolism. As these cell lines are highly prolific, resistant to exogenous factors, and have low or nonexistent P450 activities, it is possible that the influence of quercetin on immune function has, in large part, been overshadowed by other more predominant effects in these cell lines, again questioning the appropriateness of transferring in vitro results to in vivo. Our findings show that quercetin has the ability to modulate colonic immune function and that this modulation is hepatic P450 dependent, and so at least partly bile acid dependent, as demonstrated by the upregulation and downregulation of genes in WT and KO mice, respectively.

The most unexpected and exciting finding in this study was the molecular relationship identified between the jejunum and liver. Using a systems biology approach in which the transcriptional profiles of multiple organs were profiled in conjunction with circulating quercetin metabolites, we are able to demonstrate that the biological organism is highly adaptive and robust to exogenous insults (2). Quercetin is clearly able to modulate several canonical pathways, such as amino acid metabolism, lipid metabolism, and glutathione metabolism; however, the data would suggest that the metabolizing capacity of quercetin within the biological organism will have an effect on these molecular mechanisms. A WT animal will predominantly regulate amino acid and sterol metabolism in the liver, share the control of fatty acid metabolism between the liver and jejunum, and coordinate glutathione metabolism in the small intestine. In contrast, KO animals do not seemingly regulate amino acid metabolism in the small intestine, they share the control of fatty acid metabolism between the liver and jejunum, and they shift the regulation of sterol metabolism from the liver to the jejunum and that of glutathione metabolism from the jejunum to the liver.

The creation of comprehensive analytical platforms capable of assessing genes, proteins, and metabolites has provided the stepping stones required to unravel the robustness of biological organisms faced with a perturbation. Robustness can imply not only redundancy in enzyme function within a given cell but also between more complex biological networks within a cell type or between tissues (18, 32). Indeed, from an evolutionary perspective, the robustness inherent to a biological organism confers its ability to resist phenotypic changes by allowing a degree of metabolic flexibility (4, 32). Such a paradigm has been previously hypothesized for both glucose utilization and fatty acid synthesis in mice with a targeted disruption of muscle insulin receptor (IR) and liver sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP), respectively. More specifically, mice with a targeted deletion of muscle IR avoided insulin resistance by shifting insulin sensitization and glucose metabolism from the muscle to the adipose tissue (6), and despite mice with a targeted deletion of hepatic SCAP displaying a marked reduction in liver fatty acid synthesis, total body fatty acid synthesis was unchanged due to a compensatory mechanism in adipose tissue (16). In the present study, we have identified a between-tissue robustness that has preserved the ability of mice to metabolize quercetin and maintain the regulation of key biological functions by this bioactive nutrient. These findings demonstrate, via a systems biology perspective, the adaptive capabilities of a biological organism characterized by a tissue-specific molecular dysfunction.


   ACKNOWLEDGMENTS
 
We thank Inmaculada Sanchez-Martin for assistance with sample preparation.

Present address of D. M. Mutch: Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Williamson, Nutrient Bioavailability, Nestlé Research Center Vers-Chez-Les-Blanc, PO Box 44, CH-1000 Lausanne 26, Switzerland (e-mail: gary.williamson{at}rdls.nestle.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* D. M. Mutch and V. Crespy contributed equally to this work. Back

1 The Supplemental Material for this article (Supplemental Table S1) is available online at http://ajpgi.physiology.org/cgi/content/full/00565.2005/DC1. Back


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