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Physiol. Genomics 30: 192-204, 2007. First published April 10, 2007; doi:10.1152/physiolgenomics.00198.2006
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Received 11 September 2006; accepted in final form 3 April 2007.
Physiological Genomics 30:192-204 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Genome-wide analysis of PPAR{alpha} activation in murine small intestine

Meike Bünger1,2, Heleen M. van den Bosch1,2, Jolanda van der Meijde1, Sander Kersten1,2, Guido J. E. J. Hooiveld1,2 and Michael Müller1,2

1 Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University
2 Nutrigenomics Consortium, Wageningen Centre for Food Sciences, Wageningen, the Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The peroxisome proliferator-activated receptor alpha (PPAR{alpha}) is a fatty acid-activated transcription factor that governs a variety of biological processes. Little is known about the role of PPAR{alpha} in the small intestine. Since this organ is frequently exposed to high levels of PPAR{alpha} ligands via the diet, we set out to characterize the function of PPAR{alpha} in small intestine using functional genomics experiments and bioinformatics tools. PPAR{alpha} was expressed at high levels in both human and murine small intestine. Detailed analyses showed that PPAR{alpha} was expressed most highly in villus cells of proximal jejunum. Microarray analyses of total tissue samples revealed, that in addition to genes involved in fatty acid and triacylglycerol metabolism, transcription factors and enzymes connected to sterol and bile acid metabolism, including FXR and SREBP1, were specifically induced. In contrast, genes involved in cell cycle and differentiation, apoptosis, and host defense were repressed by PPAR{alpha} activation. Additional analyses showed that intestinal PPAR{alpha}-dependent gene regulation occurred in villus cells. Functional implications of array results were corroborated by morphometric data. The repression of genes involved in proliferation and apoptosis was accompanied by a 22% increase in villus height and a 34% increase in villus area of wild-type animals treated with WY14643. This is the first report providing a comprehensive overview of processes under control of PPAR{alpha} in the small intestine. We show that PPAR{alpha} is an important transcriptional regulator in small intestine, which may be of importance for the development of novel foods and therapies for obesity and inflammatory bowel diseases.

microarray; peroxisome proliferator-activated receptor alpha; gene expression; crypt-villus axis; lipid absorption; proximal-distal axis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR)-{alpha} is a ligand-activated transcription factor with diverse functions and is activated by a variety of synthetic compounds, including the lipid lowering fibrate drugs (35, 37). High-affinity natural ligands include eicosanoids, unsaturated as well as long-chain fatty acids, and their activated derivatives (acyl-CoA esters) (19, 25, 31, 32). In analogy with other nuclear receptors, PPAR{alpha} forms obligate heterodimers with the retinoid X receptor and stimulates gene expression by binding to peroxisome proliferator response elements (PPRE) located in the regulatory domain of genes (37). PPAR{alpha} is expressed in a variety of tissues including the small intestine (6, 16); however, its function has been almost exclusively studied in liver. In liver PPAR{alpha} is critical for the coordinate transcriptional activation of genes involved in lipid catabolism, including cellular fatty acid uptake and activation, mitochondrial ß-oxidation, peroxisomal fatty acid oxidation, ketone body synthesis, fatty acid elongation and desaturation, and apolipoprotein synthesis (35, 37). In addition, PPAR{alpha} is an important regulator of the hepatic acute phase response. While the function of PPAR{alpha} in liver is well studied, little is known about PPAR{alpha} and PPAR{alpha} target genes in nonhepatic tissues. This is especially true with respect to the role of PPAR{alpha} in the small intestine, which has only been addressed in few studies (39, 40). Knowledge of the regulatory and physiological function of PPAR{alpha} in the small intestine is of particular interest, since the average Western diet contains a high amount of triacylglycerols (5) that are hydrolyzed to monoacylglycerol and free fatty acids before entering the enterocyte (46). Consequently the small intestine is frequently exposed to high levels of PPAR{alpha} ligands.

Therefore we set out to determine the role of PPAR{alpha} in the small intestine. We first analyzed in detail the expression of PPAR{alpha} throughout the small intestine and then evaluated the outcome of specific PPAR{alpha} activation on small intestinal gene expression using microarrays and bioinformatics tools. This allowed the genome-wide identification of intestinal PPAR{alpha} target genes and corresponding processes. We conclude that PPAR{alpha} plays an important role in the regulation of intestinal function by governing diverse processes ranging from numerous metabolic pathways to the control of apoptosis and cell cycle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.
Pure bred wild-type (129S1/SvImJ) and PPAR{alpha}-null (129S4/SvJae) mice (34) were purchased from Jackson Laboratories (Bar Harbor, ME) and bred at the animal facility of Wageningen University.

Mice were housed in a light- and temperature-controlled facility and had free access to water and standard laboratory chow (RMH-B; Hope Farms, Woerden, the Netherlands). All animal studies were approved by the Local Committee for Care and Use of Laboratory Animals.

Experimental design and tissue handling.
Four independent studies were performed. In all studies 4- to 5-mo old male wild-type and PPAR{alpha}-null mice were used. Study A: Mice were fed chow or chow supplemented with 0.1% WY14643 (Chemsyn, Lenexa, KS) for 5 days (n = 6 mice per group). On the sixth day, mice were anaesthetized with a mixture of isoflurane (1.5%), nitrous oxide (70%), and oxygen (30%). Small intestines were excised and flushed with ice-cold PBS, and all subsequent tissue handling was performed on ice. Remaining fat and pancreatic tissue was carefully removed, and RNA was isolated from the complete full-length small intestine for microarray analysis. Study B: The above described experiment was repeated (n = 3 mice per group), except that after removal the small intestine was divided into 10 equal parts to study gene expression along the proximal-distal axis. Study C: Study A was repeated (n = 3–4 mice per group), except that after removal, the small intestine was inverted on a 0.75-mm-diameter rod, washed in ice-cold PBS, and divided into segments of 1 cm. Before continuing with the cell isolation protocol, we pooled segments of all animals within each experimental group. Fractions enriched in crypt or villus cells were isolated as described by Flint et al. (18). This isolation protocol was repeated 1 week later for the control group. Cell fractions were used for RNA isolation. Study D: The feeding experiment was repeated as described, except that in addition to WY14643 mice were fed chow supplemented with fenofibrate (0.1% wt/wt; Sigma, St. Louis, MO) for 5 days (n = 5 mice per group). RNA was isolated from the complete full-length small intestine for quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis.

RNA isolation and quality control.
Total RNA was isolated from small intestinal samples using TRIzol reagent (Invitrogen, Breda, the Netherlands) according to the manufacturer's instructions. RNA was treated with DNase and purified using the SV total RNA isolation system (Promega, Leiden, the Netherlands). Concentrations and purity of RNA samples were determined on a NanoDrop ND-1000 spectrophotometer (Isogen, Maarssen, the Netherlands). RNA integrity was checked on an Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam, the Netherlands) with 6000 Nano Chips according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits and displayed no chromosomal peaks or RNA degradation products. Total RNA from human tissues (FirstChoice Human Total RNA Survey Panel) was obtained from Ambion (Austin, TX). Each tissue pool comprises RNA from three or four donors.

Affymetrix GeneChip oligoarray hybridization and scanning.
For microarray analyses, we used RNA isolated from the full-length small intestine. RNA was hybridized on an Affymetrix GeneChip Mouse Genome 430A array. This array detects 22,626 transcripts that represent ~13,700 known genes. For each experimental group, three biological replicated were hybridized; thus in total 12 arrays were used. Detailed methods for the labeling and subsequent hybridizations to the arrays are described in the eukaryotic section of the GeneChip Expression Analysis Technical Manual, Revision 3, from Affymetrix (Santa Clara, CA). Array data have been submitted to the Gene Expression Omnibus, accession number GSE5475.

Analyses and functional interpretation of microarray data.
Scans of the Affymetrix arrays were processed using packages from the Bioconductor project (20). Expression levels of probe sets were summarized using the GC content-corrected robust multichip average algorithm (GCRMA) (62), where after differentially expressed probe sets were identified using Limma (51). P values were corrected for multiple testing using a false discovery rate (FDR) method (55). Probe sets that satisfied the criterion of FDR <5% (q value <0.05) were considered to be significantly regulated. Of these, probe sets that were also >1.5-fold changed in wild-type mice upon WY14643 treatment but were not changed in treated PPAR{alpha}-knockout mice were designated PPAR{alpha} regulated. Three complementary methods were applied to relate changes in gene expression to functional changes. One method is based on overrepresentation of Gene Ontology (GO) terms (33). Another approach, gene set enrichment analysis (GSEA), takes into account the broader context in which gene products function, namely in physically interacting networks, such as biochemical, metabolic, or signal transduction routes (57). Both applied methods have the advantage that it is unbiased, because no gene selection step is used, and a score is computed based on all genes in a GO term or gene set. In addition, biological interaction networks among PPAR{alpha} regulated genes were identified using Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, Redwood City, CA). Detailed descriptions of the applied methods are available in the supplemental text (supplemental_1).1

qRT-PCR.
Single-stranded complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the reverse-transcription system from Promega (Leiden, the Netherlands) according to the supplier's protocol. qRT-PCR was performed on a MyIQ thermal cycler (Bio-Rad, Veenendaal, the Netherlands) using Platinum Taq DNA polymerase (Invitrogen) and SYBR green (Molecular Probes, Leiden, the Netherlands). Most of the primer sequences were obtained from the PrimerBank at Harvard University (59). Primer sequences are listed in Table S1 of the supplemental data (supplemental_3). Samples were analyzed in duplicate and standardized to either cyclophilin or 18S expression. Expression levels in isolated villus cells were standardized to villin.

Histology.
For histology studies a fifth, independent experiment was performed, exactly as described in study A. After removal, intestines were divided into three equal parts, which are referred to as duodenum, jejunum, and ileum, respectively. Each section was prepared using a "Swiss roll" technique (38) to evaluate the entire longitudinal section on one slide. Tissues were fixed by immersion in 4% PBS-buffered formaldehyde, processed in an automatic tissue processor, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin. Sections were examined on a CKX41 microscope (Olympus, Zoeterwoude, the Netherlands) equipped with calibrated DP software, version 3.2 (Olympus). This software was used to measure villus height, crypt depth, and villus area of 50 villi per section for each animal. Statistical analysis between groups was performed with ANOVA, followed by the least significant difference post hoc test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PPAR{alpha} expression in human and murine small intestine.
To ascertain whether PPAR{alpha} may be functionally relevant in the small intestine, the expression of PPAR{alpha} was measured in 20 human tissues by qRT-PCR. In humans the highest expression levels of PPAR{alpha} were observed in kidney, followed by heart, small intestine, and liver (Fig. 1A). In mice, the expression of PPAR{alpha} was slightly higher in liver compared with small intestine of SV129 mice, whereas RXR{alpha} expression was comparable between both tissues (Fig. 1B). These data suggest that PPAR{alpha} may be functionally relevant in the small intestine.


Figure 1
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  		Fig. 1. Peroxisome proliferator-activated receptor (PPAR)-{alpha} is expressed at high levels in human and murine small intestine. A: expression levels of PPAR{alpha} in various human tissues. The top 10 out of the 20 tissues analyzed are presented, PPAR{alpha} expression in kidney was arbitrarily set to 100%. B: murine PPAR{alpha} (left) and RXR (right) expression levels in small intestine (SI) and liver. Data are presented as means ± SD, n = 5. The SI was arbitrarily set to 100%. For both human and mouse samples, expression levels were standardized to 18S.

 
Next, the expression levels of PPAR{alpha} and selected genes along the crypt-villus and proximal-distal axes of the small intestine were examined. As expected, mRNA levels of intestinal alkaline phosphatase (IAP) and villin, two markers for differentiated absorptive epithelial cells (17, 49) were maximal in fraction 1 (Fig. 2). Conversely, expression of pancreatic lipase-related protein-2 (PNLIPRP2), a marker for Paneth cells located at the base of villi (53), peaked in fractions 6–8. Importantly, expression of PPAR{alpha} declined from villus to crypt cell-enriched fractions, which was mimicked by fatty acid transport protein 4 (FATP4) and CD36/FAT, two proteins known to be highly expressed in enterocytes and involved in fatty acid uptake (9, 52). These data demonstrate that PPAR{alpha} is predominantly expressed in differentiated enterocytes and colocalizes with other genes involved in fatty acid metabolism.


Figure 2
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  		Fig. 2. PPAR{alpha} is predominantly expressed in differentiated enterocytes. qRT-PCR was used to determine relative expression levels of PPAR{alpha} and marker genes in fractions enriched in villus or crypt cells isolated from intestines from adult 129Sv mice. Cell fractions were isolated in 2 independent experiments, using 3 or 4 mice per isolation. mRNA levels were standardized to cyclophilin; fraction 8 was arbitrarily set to 1. Expression levels of intestinal alkaline phosphatase (IAP), villin, pancreatic lipase-related protein 2 (Pnliprp2), PPAR{alpha}, fatty acid transport protein 4 (FATP4, Slc27a4), and CD36/FAT. Data are presented as means ± SD.

 
A similar relationship was observed along the proximal-distal axis. PPAR{alpha} expression gradually increased from the duodenum throughout the distal jejunum and then decreased in ileum (Fig. 3). The same pattern of expression was observed for FATP4. Expression of liver-type fatty acid binding protein (L-FABP) peaked more proximally, whereas intestine-type fatty acid binding protein (I-FABP) expression was highest in the distal jejunum. As expected, the expression of IAP (Fig. 3A) and apical sodium-dependent bile salt transporter (ASBT) (Fig. 3B) was restricted either to the duodenum or terminal ileum, respectively (43, 61). Combined, these data demonstrate that PPAR{alpha} expression is highest in jejunal villus cells.


Figure 3
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  		Fig. 3. PPAR{alpha} is expressed at highest levels in jejunum. qRT-PCR was used to determine relative expression levels of PPAR{alpha} and marker genes in sections isolated along the proximal-distal axis of the SI from adult 129Sv mice (n = 3). The SI was divided into 10 equal parts; part 1 refers to the most proximal part (duodenum), part 10 refers to the most distal part (terminal ileum). mRNA levels were standardized to cyclophilin; part 10 was arbitrarily set to 1. Expression levels of IAP; apical sodium-dependent bile salt transporter (ASBT); liver-type fatty acid binding protein (L-FABP); intestine-type fatty acid binding protein (I-FABP); PPAR{alpha}; and FATP4, (Slc27a4). Data are presented as means ± SD. Comparable results were observed when 18S rRNA was used as reference.

 
Function of intestinal PPAR{alpha} as assessed by transcriptome analyses.
To study the function of PPAR{alpha} in the small intestine, wild-type and PPAR{alpha}-null mice were treated with the synthetic PPAR{alpha} agonist WY14643, followed by analyses of changes in global gene expression using Affymetrix MOE430A arrays. Results on the number of significantly regulated genes are summarized in Fig. 4. A complete list of regulated genes is available in supplemental_2. Additional qRT-PCR analyses were performed for selected genes, which confirmed the array results (Fig. 8 and supplemental_3). Under control conditions, expression levels of only 21 genes out of the ~13,700 genes analyzed were significantly different between wild-type and PPAR{alpha}-null mice [fold change (FC) >1,5; FDR <0.05]. Of these 21 genes, 16 genes were expressed at lower levels and five genes at higher levels in PPAR{alpha}-null mice (Fig. 5). Most of these genes are known to be involved in lipid metabolism, and several have been identified as direct PPAR{alpha} target genes in other tissues (37). In wild-type mice, activation of PPAR{alpha} resulted in differential expression of 1,138 genes, of which only two, BC018473 [hitchhiker, Entrez Gene (EG) ID: 193217] and Abcb1a (Mdr1a, EG ID: 18671) were also altered in the PPAR{alpha}-null mice upon WY14643 treatment (Fig. 4). Thus, in total 1,136 genes were PPAR{alpha}-dependently regulated in the small intestine; PPAR{alpha} activation resulted in increased mRNA levels of 567 genes, whereas 569 were repressed. To gain insight into the underlying biological phenomena affected by PPAR{alpha} activation, a scoring-based resampling method was applied to identify significantly overrepresented Gene Ontology (GO) classes (33). As input the >22,000 t-test P values from the probe set comparisons across the two diets in wild-type mice were used. Classes of genes that changed most significantly are listed in Table 1. With respect to the concept "biological process," terms on this top list were mainly descriptors for fatty acid and lipid metabolism. Other overrepresented GO classes included descriptors for immune system, cell proliferation and differentiation, and programmed cell death (Table 1), suggesting that PPAR{alpha} is involved in the regulation of these processes in the small intestine.


Figure 4
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  		Fig. 4. Identification of PPAR{alpha} target genes in SI. Affymetrix MOE430A arrays (n = 3 per group) were hybridized with RNA isolated from intestines from control and WY14643-treated wild-type and PPAR{alpha}-null mice. Groups were compared and genes that satisfied the criteria of fold change >1.5 and false discovery rat <0.05 were designated significantly changed genes. Numbers of genes are summarized in a Venn plot. PPAR{alpha}–/–control, PPAR{alpha}-null mice that received the control diet; PPAR{alpha}–/–WY14643, PPAR{alpha}-null mice that received the control diet supplemented with 0.1% WY14643 for 5 days; WTcontrol, wild-type mice that received the control diet; WTWY14643, wild-type mice that received the control diet supplemented with 0.1% WY14643 for 5 days.

 

Figure 5
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  		Fig. 5. Twenty-one genes are differentially expressed in PPAR{alpha}-null mice under basal conditions. Heat maps representing the expression levels of 21 genes that were differentially expressed in PPAR{alpha}-null mice compared with wild-type mice under control conditions. A: 5 genes significantly higher expressed in PPAR{alpha}-null mice. B: 16 genes significantly lower expressed in PPAR{alpha}-null mice. *Genes that were also regulated upon PPAR{alpha} activation. Signal intensities were log2 transformed and subjected to hierarchical clustering in Spotfire.

 

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  		Table 1. Gene Ontology classes overrepresented upon PPAR{alpha} activation

 
A parallel GSEA was used to focus on groups of genes that comprise specific biochemical, metabolic, or signal transduction routes (57). This method allows the identification of up- or downregulated processes (Table 2). However, due to overlap in the source databases, several functions are represented multiple times. The outcome of GSEA was similar to that of the GO-based analysis. Remarkable, almost all increased gene sets correspond to metabolic processes, including fatty acids catabolism, mitochondrial oxidative metabolism, and several pathways that feed intermediates into these processes. Other processes of interest that were upregulated include genes related to steroid and bile acid metabolism. The cellular responses represented by the downregulated gene sets were much more diverse and did not include metabolic pathways. Various pleiotropic signal transduction routes were suppressed, and the functional outcomes were summarized as acting on immune system, cell proliferation, migration and differentiation, and apoptosis. The functional outcomes of these transcriptome analyses are summarized in Fig. 6. The figure underscores the role of PPAR{alpha} as an important transcriptional regulator in small intestine, governing diverse processes ranging from apoptosis and cell cycle, to the immune response and numerous metabolic pathways.


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  		Table 2. Gene sets changed upon PPAR{alpha} activation

 

Figure 6
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  		Fig. 6. PPAR{alpha} regulates a variety of processes in SI. Summary of functional implications of PPAR{alpha} activation as assessed by analyses of predefined gene sets based on Gene Ontology, biochemical, metabolic, or signal transduction routes.

 
Many genes involved in fatty acid catabolism are known to be direct PPAR{alpha} target genes (37). However, the mechanisms by which PPAR{alpha} activation results in downregulation of numerous genes are less well understood. We therefore used IPA to search for biological interaction networks. As input all 1136 PPAR{alpha}-dependently regulated genes were used. Of these genes, 588 were eligible for network analysis. For the remaining 548 genes molecular interaction information was lacking. IPA computed 83 networks, of which five scored equally best, as judged by a statistical likelihood approach (8). The five networks with a score of 39 (P < 10–39) were combined to form a composite network representing the underlying biology of PPAR{alpha} activation in small intestine (Fig. 7). This complex comprised a network of 175 unique genes and their interactions. All genes were responsive to PPAR{alpha} activation, and every interaction between the genes was supported by published information. In addition, a right-tailed Fisher's exact test identified 23 canonical pathways significantly affected by PPAR{alpha} activation (P < 0.05) (data not shown). All of these were also identified with GSEA, and six canonical pathways linked to the composite network (Fig. 7). Moreover, we identified six nodes that were central in connecting many of the changed genes. As expected, PPAR{alpha} linked directly to most of the induced genes. The downregulated genes of the merged network all linked to MYC, caspase 3 (CASP3), major histocompatibility class (MHC) 2 transactivator (MHC2TA), epidermal growth factor receptor (EGFR), or lymphocyte-specific protein tyrosine kinase (LCK), which themselves were also PPAR{alpha}-dependently downregulated. No direct interaction between PPAR{alpha} and the other five central nodes could be identified. Since the maximum network size was limited to 35 genes, all genes presented in the composite network were regulated by PPAR{alpha}. Yet this does not imply that all genes connected to the central nodes were regulated. For example, IPA knowledge base linked PPAR{alpha} and MYC to 109 and 459 genes, respectively (data not shown). Of these, 35 (32%) respectively 60 (13%) genes were significantly regulated (FC >1.5, FDR <0.05) in the small intestine. Taken together, the generation of biological interaction networks identified five genes that may play a central role in mediating the pleiotropic suppressive effects of PPAR{alpha} activation.


Figure 7
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  		Fig. 7. Generation of biological interaction networks identifies central genes. Ingenuity Pathway Analysis was used to search for biological interaction networks (BINs). As input the 1,136 PPAR{alpha} dependently regulated genes were used, of which 588 were eligible for the analysis (focus genes). Five networks scored equally best; these were merged and are presented. Six major affected canonical pathways, indicated as CP, are linked to the merged network. *Genes that are detected 2 or more times on the array. Color coding: red, upregulated gene; green, downregulated gene. The intensity of the colors indicates the degree of up- or downregulation, respectively; a greater intensity represents a higher degree of regulation.

 
PPAR{alpha}-dependent gene regulation occurs in villus cells.
To confirm that PPAR{alpha}-dependent gene regulation occurred in the differentiated enterocytes, we isolated fractions enriched in villus cells from wild-type and PPAR{alpha}-null mice treated with WY14643. The expression for selected PPAR{alpha} target genes identified in the array analysis was then determined by qRT-PCR (Fig. 8, A–C). For comparison, microarray data and qRT-PCR results of total tissue are presented as well. Expression of "classical" PPAR{alpha} target genes, cytochrome P450, family 4, subfamily a, polypeptide 10 (CYP4A10); enoyl-coenzyme A, hydratase/3-hydroxyacyl coenzyme A dehydrogenase (EHHADH); 2–4-dienoyl-coenzyme A reductase 2, peroxisomal (DECR2); and angiopoietin-like 4 (ANGPTL4) (28, 29, 34, 37) was increased by WY14643 in total tissue and villus cells from wild-type but not PPAR{alpha} null mice (Fig. 8A). Similar expression patterns were observed for four putative intestinal PPAR{alpha} target genes aldo-keto reductase family 1, member B8 (AKR1B8); glutamate oxaloacetate transaminase 2, mitochondrial (GOT2); farnesoid X receptor; retinoid X receptor (FXR); and diacylglycerol O-acyltransferase 2 (DGAT2), which were specifically induced by WY14643 treatment in total tissue and isolated fractions (Fig. 8B). Expression of PPAR{alpha} itself was increased by WY14643 in total tissue and isolated fractions, indicating autoregulation of PPAR{alpha} gene expression. Besides PPAR{alpha} itself we also determined the expression levels of five other nodes identified by network analysis. Expression of CASP3, MHC2TA, EGFR, MYC, and LCK was PPAR{alpha}-dependently downregulated (Fig. 8C). Thus, for all genes analyzed we observed great similarity in regulation in total tissue between microarray and qRT-PCR, as well as between total tissue and isolated villus cells. This again demonstrates the robustness of our microarray analysis.


Figure 8
Figure 8
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  		Fig. 8. PPAR{alpha}-dependent gene regulation occurs in total tissue and villus cells. For selected genes the outcome of PPAR{alpha} activation was determined in wild-type and PPAR{alpha}-null mice. Independent sets of mice were analyzed. For each gene shown, left column: microarray data obtained from total tissue samples (n = 3 mice per group); middle column: qRT-PCR data from total tissue samples (n = 3 mice per group); right column: qRT-PCR data from fractions enriched in villus cells (n = 3–4 mice per group, fractions f1–f3 were pooled). A: effect of PPAR{alpha} activation on expression of "classical" PPAR{alpha} target genes in SI (28, 29, 34, 37): cytochrome P450, family 4, subfamily a, polypeptide 10 (CYP4A10); enoyl-coenzyme A, hydratase/3-hydroxyacyl coenzyme A dehydrogenase (EHHADH); 2-4-dienoyl-coenzyme A reductase 2, peroxisomal (DECR2); angiopoietin-like 4 (ANGPTL4). B: effect of PPAR{alpha} activation on expression of putative intestinal PPAR{alpha} target genes: aldo-keto reductase family 1, member B8 (AKR1B); glutamate oxaloacetate transaminase 2, mitochondrial (GOT2); farnesoid X receptor, retinoid X receptor (FXR); diacylglycerol O-acyltransferase 2 (DGAT2). C: effect of PPAR{alpha} activation on PPAR{alpha} expression itself and 5 central nodes identified by network analysis: caspase 3 (CASP3), major histocompatibility class (MHC) 2 transactivator (MHC2TA), epidermal growth factor receptor (EGFR), myelocytomatosis oncogene (MYC), and lymphocyte protein tyrosine kinase (LCK). Microarray data are presented as fold change compared with wild-type mice fed the control diet. qRT-PCR data were standardized to cyclophillin (total tissue) or villin (cell fractions); wild-type control was then arbitrarily set to 1. Data for total tissue samples are means ± SD. **Significant difference between the groups: for microarray data, gene satisfied the criterion of FDR <5% (q value < 0.05) and >1.5-fold changed in wild-type mice upon WY14643 treatment but was not changed in treated PPAR{alpha}-knockout mice; for total tissue qRT-PCR data P < 0.01.

 
PPAR{alpha} activation by fenofibrate.
To study the specificity of PPAR{alpha} activation, we compared the effects of WY14643 with the effects of fenofibrate another PPAR{alpha} agonist. qRT-PCR was used to analyze expression of a set of classical and putative PPAR{alpha} target genes as well as central nodes, which also were studied in isolated villus cells (Fig. 9). The effects of fenofibrate were comparable to those of WY14643, as both agonists induced the expression of PPAR{alpha} itself and (putative) PPAR{alpha} target genes and reduced the expression of the central nodes MHC2TA and CASP3. However, at the same concentration fenofibrate was less potent in activating or repressing genes compared with WY14643. Thus, we show that activation of PPAR{alpha} by two different ligands does not result in qualitative differences in expression of a specific set of genes.


Figure 9
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  		Fig. 9. Activation of PPAR{alpha} by 2 different ligands does not result in qualitative differences on expression of a set of genes. Wild-type (white bars) and PPAR{alpha}-null (black bars) mice were treated with WY14643 (0.1% wt/wt) or fenofibrate (0.1% wt/wt) for 5 days (n = 5 per group). For selected genes the outcome in expression was determined by qRT-PCR analysis. mRNA levels were standardized to cyclophilin; wild-type control was set to 1. Top row: "classical" PPAR{alpha} target genes; middle row: putative intestinal PPAR{alpha} target genes, bottom row: PPAR{alpha} and 2 other central nodes. Data are presented as means ± SD. a, b, c: different letters indicate a significant difference between the treatment groups (P < 0.05) according to ANOVA.

 
Morphometric changes of the villus of mice treated with WY14643.
One of the outcomes of the transcriptome analyses was that PPAR{alpha} activation suppressed cell proliferation and apoptosis and enhanced cell differentiation. We hypothesized that morphologically this should result in elongated villi. We therefore prepared paraffin sections of control and treated intestines from wild-type and PPAR{alpha}-null mice and studied gross intestinal morphology and height of the villi (Fig. 10, Table 3). Under basal conditions no differences were observed with respect to form, structure, and morphometric parameters between wild-type and PPAR{alpha}-null mice. In wild-type mice, treatment with WY14643 significantly increased the height of the villus, whereas no effect was observed in PPAR{alpha}-null mice. Crypt depth was not affected by WY14643 treatment in both groups. These data indicate that PPAR{alpha} activation specifically increases villus height but does not alter crypt depth. The morphometric assessments are in line with the functional outcomes of the transcriptome analyses.


Figure 10
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  		Fig. 10. Activation of PPAR{alpha} increases villus height. Histological staining was performed on jejunal sections from control and WY14643-treated wild-type and PPAR{alpha}-null mice. Two representative sections of different mice per treatment group are shown. I, II: wild-type mice fed the control diet; III, IV: wild-type mice fed the control diet supplemented with 0.1% WY14643 for 5 days; V, VI: PPAR{alpha}-null mice fed the control diet; VII, VII: PPAR{alpha}-null mice fed the control diet supplemented with 0.1% WY14643 for 5 days. Original magnification x88.

 

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  		Table 3. Villus height, crypt depth, and villus area upon PPAR{alpha} activation in the jejunum

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study we set out to determine the role of PPAR{alpha} in the small intestine using genomics tools. We find that PPAR{alpha} is very well expressed in the small intestine in both mouse and human. PPAR{alpha} expression is highest in villus cells and peaks in the proximal jejunum. Activation of PPAR{alpha} results in altered expression of a large set of genes involved in a variety of pathways, including intestinal lipid handling, cell cycle, differentiation, apoptosis, and host defense. These data suggest an important role for PPAR{alpha} in the regulation of gene expression in the small intestine.

Under control (fed) conditions we observed few changes in gene expression between wild-type and PPAR{alpha} null mice. Only 21 genes were significantly altered, most of which are involved in lipid metabolism. These observations are in accordance with numerous studies showing that the effect of PPAR{alpha} deletion becomes mainly noticeable under conditions of metabolic stress, and does not a priori imply that the physiological role of PPAR{alpha} is limited to lipid metabolism (14, 36, 41, 45).

Our finding that PPAR{alpha} is expressed highest in villus cells from the jejunum is supported by crude tissue distribution studies performed in rats (6, 16). Intestinal PPAR{alpha} expression thus coincides with the main anatomical location where long-chain fatty acids are digested, taken up, and secreted into the body, suggesting an important regulatory function of PPAR{alpha} in these processes. Indeed, activation of PPAR{alpha} resulted in the specific induction of genes involved in fatty acid uptake, binding, transport, and catabolism, as well as genes involved in triacylglycerol and glycerolipid metabolism, in the small intestine of wild-type but not PPAR{alpha}-null mice. Although PPAR{alpha} is known to regulate fatty acid metabolism in other organs (35, 37), the link between PPAR{alpha} and regulation of lipid handling in the intestine is new. Hence, at the level of the enterocyte, PPAR{alpha} serves as a fatty acid sensor that is part of a feed-forward mechanism in which fatty acids stimulate their own catabolism, storage, transfer through the enterocyte, and secretion as triacylglycerols. PPAR{alpha} thus tightly controls the intracellular levels of these potential toxic compounds.

Next to genes involved in fatty acid and triacylglycerol metabolism, genes coding for transcription factors and enzymes connected with steroid (sterol) and bile acid metabolism, including FXR, SHP, and SREBP1 are specifically induced. This demonstrates that cross talk between PPAR{alpha} and other lipid-regulated transcription factors also occurs in small intestine, in addition to liver (10, 26), and is functionally in line with the well-established roles of bile acids in absorption of dietary lipids (46). Moreover, since it was recently shown that intestinal FXR target genes are also involved in enteroprotection and inhibition of bacterial overgrowth (27), our data suggest that activation of PPAR{alpha} might also influence epithelial barrier function.

In addition to PPAR{alpha} itself, network analyses identified five central genes that connected many of the repressed genes. These central genes (MYC, EGFR, CASP3, LCK, and MHC2TA) themselves were also PPAR{alpha}-dependently downregulated. The MYC gene encodes a multifunctional nuclear phosphoprotein and plays a role in cell cycle progression, apoptosis, cellular transformation, and differentiation. The results of the microarray study show that MYC and related genes are repressed in a PPAR{alpha}-dependent manner, including cyclin D1 (CCND1), CASP3, nuclear factor-{kappa}B (NFkB), signal transducer and activator of transcription 1 (STAT1), and EGFR via platelet-derived growth factor receptor-ß (PDGFRB). Inasmuch as MYC is known to repress major histocompatibility complex, class I-B (HLA-B); PDGFRB; and N-myc downstream-regulated gene 1 (NDRG1) expression (21, 44, 60), the observed upregulation of these genes by PPAR{alpha} is likely mediated through repression of MYC. Our data for the first time connect PPAR{alpha} with regulation of cell proliferation, differentiation, and apoptosis in the small intestine. Our results indicate that an important consequence of intestinal PPAR{alpha} activation is blocking cells in transition to the G1-S checkpoint of the cell cycle, resulting in reduced proliferation and increased differentiation of cells (58). We speculate that the specific downregulation in villus cells of CASP3, a key enzyme in the apoptotic cascade (15), points to inhibition of apoptosis, as has been reported for other cell types (22, 42, 48, 63). Since cell shedding is strongly associated with apoptosis, this in turn may result in reduced shedding of cells from the villus tips (7, 47). Functional support of the gene expression data is provided by morphometric data showing a significant 22% increase in villus height, which is accompanied by a 34% increase in villus area. These findings were not observed in PPAR{alpha}-null mice treated with WY14643. Combined, our data demonstrate that PPAR{alpha} represses cell growth and apoptosis and stimulates cell differentiation, resulting in an increased number of mature absorptive enterocytes. We believe that this may be an important adaptation mechanism of the small intestine aimed at adjusting lipid absorptive capacity to increased dietary fats (i.e., hydrolyzed TAGs).

Our study connects PPAR{alpha} with the immune system of the small intestine. Activation of PPAR{alpha} suppresses complement activation, antigen presentation, and B-cell receptor signaling. It is known that PPAR{alpha}-null mice have abnormally prolonged hepatic responses to inflammatory stimuli (13). In vascular cells the expression of interleukin-6, vascular cell adhesion molecule, and cyclooxygenase-2 in response to cytokine activation can be inhibited by PPAR{alpha} ligands (11). In these cells PPAR{alpha} ligands may inhibit the functional expression of NF-{kappa}B, in part by augmenting the expression of inhibitor of NF-{kappa}B (I{kappa}B{alpha}) (12). However, inhibitory effects of PPAR{alpha} in the intestine as well as on the innate immune system have not been reported before. Although the lymphoid tissue of the gut is the primary system for host defense, it is known that intestinal epithelial cell expresses MHC2 molecules and can function as antigen-presenting cells, thus being capable of regulating mucosal T-cell responses (23, 24, 56). Moreover, there is evidence that some of the complement proteins, such as complement C3, are synthesized in enterocytes (1, 2). Network analyses showed that the repression of the positive "master" regulator MHC2TA by PPAR{alpha} is likely responsible for the suppression of MHC2 gene transcription, whereas suppressed B-cell receptor signaling is linked to decreased expression of LCK (54). Taken together, our data show that PPAR{alpha} influences the immune and inflammatory response in the intestine and support the possibility that enterocytes are involved in a local response to injury/inflammation at the epithelial surface. A repression of the inflammatory response in the intestine by PPAR{alpha} might be therapeutically valuable for patients with inflammatory bowel disease. Although several studies suggest a link between PPAR{gamma} and inflammatory bowel disease (3, 4, 30, 50), hardly anything is known about the effect of PPAR{alpha}.

In summary, by using a combination of functional genomics experiments and current bioinformatics tools, we are the first to identify the pathways and processes under control of PPAR{alpha} in the small intestine. Our data provide new insight into the role of PPAR{alpha} in the small intestine and may be of particular importance for the development of fortified foods and for prevention and therapies for treating obesity and inflammatory bowel diseases.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Dutch Ministry of Economic Affairs through the innovation-oriented research program on genomics IOP-IGE01016. Additional support was obtained from the Wageningen Centre for Food sciences.


   ACKNOWLEDGMENTS
 
The authors thank Rene Bakker and Bert Weijers for excellent assistance with animal experiments.


   FOOTNOTES
 
Address for reprint requests and other correspondence: M. Müller, Nutrition, Metabolism and Genomics Group, Div. of Human Nutrition, Wageningen Univ., PO Box 8129, NL-6700EV Wageningen, the Netherlands (e-mail: michael.muller{at}wur.nl)

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

1 The online version of this article contains supplemental material. Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
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 DISCUSSION
 GRANTS
 REFERENCES
 

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