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Am J Physiol Gastrointest Liver Physiol 294: G1171-G1180, 2008. First published March 20, 2008; doi:10.1152/ajpgi.00360.2007
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MUCOSAL BIOLOGY

A cholesterol-free, high-fat diet suppresses gene expression of cholesterol transporters in murine small intestine

Heleen M. de Vogel-van den Bosch,1,2 Nicole J. W. de Wit,1,2 Guido J. E. J. Hooiveld,1,2 Hanneke Vermeulen,1,2 Jelske N. van der Veen,3 Sander M. Houten,4 Folkert Kuipers,2,3 Michael Müller,1,2 and Roelof van der Meer1,2,5

1Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, Wageningen; 2Nutrigenomics Consortium, TI Food and Nutrition, Wageningen; 3Center for Liver, Digestive, and Metabolic Diseases, Laboratory of Pediatrics, University-Medical Center Groningen, Groningen; 4Department of Pediatrics/Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam; and 5NIZO Food Research, Ede, The Netherlands

Submitted 9 August 2007 ; accepted in final form 12 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transporters present in the epithelium of the small intestine determine the efficiency by which dietary and biliary cholesterol are taken up into the body and thus control whole-body cholesterol balance. Niemann-Pick C1 Like Protein 1 (Npc1l1) transports cholesterol into the enterocyte, whereas ATP-binding cassette transporters Abca1 and Abcg5/Abcg8 are presumed to be involved in cholesterol efflux from the enterocyte toward plasma HDL and back into the intestinal lumen, respectively. Abca1, Abcg5, and Abcg8 are well-established liver X receptor (LXR) target genes. We examined the effects of a high-fat diet on expression and function of cholesterol transporters in the small intestine in mice. Npc1l1, Abca1, Abcg5, and Abcg8 were all downregulated after 2, 4, and 8 wk on a cholesterol-free, high-fat diet. The high-fat diet did not affect biliary cholesterol secretion but diminished fractional cholesterol absorption from 61 to 42% (P < 0.05). In an acute experiment in which triacylglycerols of unsaturated fatty acids were given by gavage, we found that this downregulation occurs within a 6-h time frame. Studies in LXR{alpha}-null mice, confirmed by in vitro data, showed that fatty acid-induced downregulation of cholesterol transporters is LXR{alpha} independent and associated with a posttranslational increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity that reflects induction of cholesterol biosynthesis as well as with a doubling of neutral fecal sterol loss. This study highlights the induction of adaptive changes in small intestinal cholesterol metabolism during exposure to dietary fat.

cholesterol absorption; ABC transporters; Npc1l1; fatty acids

THE EPITHELIUM OF THE SMALL intestine acts as a gatekeeper that controls the efficiency by which dietary and biliary cholesterol enter the body and thus plays a critical role in the regulation of whole-body cholesterol balance (20, 39, 46). Intestinal cholesterol absorption has long been considered as a passive process; however, this view has changed considerably during the last years. Niemann-Pick C1 Like Protein 1 (Npc1l1) has been identified as a cholesterol absorption transporter localized at the apical membrane of the small intestine (2, 13). ATP-binding cassette transporters Abca1, Abcg5, and Abcg8 are presumed to facilitate cholesterol efflux from the enterocyte. Abca1 promotes cholesterol efflux at the basolateral membrane to Apo-AI for HDL formation (15, 27, 28, 36). In contrast, the Abcg5/g8 heterodimer is responsible for transport of cholesterol from the enterocyte back into the intestinal lumen, a process that limits overall absorption efficacy (47, 52). Mutations in Abcg5 and Abcg8 contribute to plant sterol accumulation in plasma that is seen in patients with sitosterolemia (5). Abca1, Abcg5, and Abcg8 are target genes of the nuclear receptor liver X receptor (LXR), a receptor that is known to be involved in control of cholesterol-related metabolic processes (34, 45). Oxysterols, which are derivatives of cholesterol, are natural ligands of LXRs. 27-Hydroxycholesterol, which is synthesized from cholesterol by cytochrome P-450, family 27, subfamily a (Cyp27a1) (23), is a common oxysterol in the small intestine (22).

During fat absorption from the small intestinal lumen, cholesterol is required for the formation of chylomicrons (1, 33, 50). Therefore, as fat intake increases and more chylomicrons have to be formed (18), more cholesterol is theoretically needed for this process. We hypothesized that on a cholesterol-free, high-fat diet cholesterol will be retained in the enterocyte to be available for chylomicron formation to allow for efficient transport of dietary fat out of the enterocyte. Such an effect could theoretically be mediated by a downregulation of cholesterol efflux transporters. To study this hypothesis, we performed a low- and high-fat diet intervention in mice. We found that in the small intestine gene expression levels of cholesterol efflux transporters were suppressed on the high-fat diet. Surprisingly, this coincided with a downregulated gene expression of the cholesterol absorption protein Npc1l1 and with diminished fractional cholesterol absorption. In additional studies performed to investigate the mechanism behind this downregulation, we found that these high-fat-induced changes in gene expression of intestinal cholesterol transporters were LXR{alpha} independent.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Palmitic acid, stearic acid, oleic acid, linoleic acid, docosahexaenoic acid, fatty acid-free BSA, 3-hydroxy-3-methylglutaryl (HMG)-CoA (HMG-CoA), and carboxy methylcellulose were obtained from Sigma-Aldrich (Zwijndrecht, the Netherlands). 27-Hydroxycholesterol was purchased from Research Plus (Manasquan, NJ). Radiolabeled [14C]HMG-CoA was obtained from Amersham Biosciences (Diegem, Belgium). Trilinolein, trilinolenin, tridocosahexaenoin, and trieicosapentaenoin, which are synthetic triacylglycerols with three identical acyl moieties, were from Nu-Chek-Prep (Elysian, MN), whereas triolein was from Fluka (Zwijndrecht, the Netherlands). Cholesterol-D7 and cholesterol-D5 were purchased from Cambridge Isotope Laboratories (Andover, MA). Intralipid was obtained from Fresenius Kabi (Den Bosch, The Netherlands).

Animals and Diets

Male C57BL/6J mice were purchased from Harlan (Horst, The Netherlands). Lxra+/+ and Lxra–/– mice, generated by Deltagen using standard gene-targeting methods, were bred at the animal facility of the University Medical Center Groningen, Groningen, The Netherlands (32). These mice have a mixed background of C57BL/6J and 129/OlaHsd strains. SV129 mice were originally from Charles River (Maastricht, The Netherlands) and were bred at the animal facility of Wageningen University, Wageningen, The Netherlands. All 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 experiments were approved by the Ethical Committee on animal testing of Wageningen University and were performed in accordance with the national law.

In a first study we investigated the effect of a cholesterol-free, high-fat diet intervention on intestinal gene expression of C57BL/6J mice. To adapt mice to the diets, all mice were fed the low-fat purified diet for 3 wk. Thereafter, the mice were divided into two groups and the 9-wk-old mice were fed a powdered, cholesterol-free, high- or low-fat purified diet (n = 6) for 2, 4, and 8 wk. Low-fat and high-fat diets have been based on Research Diets formulas: D12450B/D12451, with some adaptations with regard to fat (Research Diet Services, Wijk bij Duurstede, The Netherlands), to mimic the fatty acid composition of a human, Western-type diet. The compositions of the diets are given in Supplemental Table S1. It should be noticed that the energy density of all nutrients, except for fat and carbohydrate, was made identical in each diet. After 2, 4, and 8 wk, mice were anesthetized with a mixture of isoflurane (1.5%), nitrous oxide (70%), and oxygen (30%). Blood was collected by orbital puncture. Plasma was obtained by centrifugation at 203 g for 10 min and stored at –80°C. The small intestines were excised and the remaining fat and pancreatic tissue were carefully removed. The small intestines were divided in three equal parts (proximal, middle, and distal), cut open longitudinally, and washed with PBS. Small intestinal mucosa was scraped, snap frozen in liquid nitrogen, and stored at –80°C until RNA isolation.

In a second study, male C57BL/6J were subjected to a 2-wk intervention with a high- or low-fat diet, as was described for the first study (low-fat: n = 6, high-fat: n = 8). After 2 wk, mice received an intravenous dose of 0.3 mg (0.763 µmol) cholesterol-D7 dissolved in 20% Intralipid and an oral dose of 0.6 mg (1.535 µmol) cholesterol-D5 dissolved in medium-chain triglyceride oil. Blood spots were collected from the tail on filter paper before and 3 days after administration of labeled cholesterol for measurement of fractional cholesterol absorption. At 72 h after administration, mice were anesthetized by intraperitoneal injection with Hypnorm (fentanyl/fluanisone, 1 ml/kg) and diazepam (10 mg/kg), and the gallbladder was cannulated as described (42). Bile was collected for 15 min. Body temperature was stabilized by use of a humidified incubator. Fractional cholesterol absorption was measured with the dual-isotope method (40).

In a third study, 3-mo-old male SV129 mice were given an oral gavage with 400 µl of the synthetic triacylglycerols triolein (~396 mg), trilinolein (~349 mg), trilinolenin (~360 mg), trieicosapentaenoin (~370 mg), and tridocosahexaenoin (~370 mg). An oral gavage with carboxy methylcellulose was given as a control. At 2 wk before the start of the experiment, the mice were switched to a diet consisting of a modified AIN76A diet, in which corn oil was replaced by olive oil (Research Diet Services, Wijk bij Duurstede, The Netherlands). Four hours before the gavage, the mice were fasted. Six hours after the oral gavage the mice were anesthetized. The small intestines were isolated and flushed with ice-cold PBS, and the remaining fat and pancreatic tissue were carefully removed. The small intestines were snap frozen in liquid nitrogen and stored at –80°C until RNA isolation.

In a fourth study, 4- to 5-mo-old female LXR{alpha}-null mice (32) and corresponding LXR{alpha}+/+ littermates (wild-type) were fed the same high- and low-fat diets as in the first study for 2 wk. Prior to this diet intervention, the mice were fed for 2 wk the cholesterol-free, low-fat diet, to adapt to the purified diet. Mice were housed two in a cage. Per cage, feces were collected after 24 h, 48 h, and 2 wk of diet intervention. After 2 wk of diet intervention mice were anesthetized, blood was collected via a heart puncture, and plasma was stored at –80°C. Small intestines were processed the same way as in the first experiment.

Cell Culture

Mouse small intestinal epithelial (MSIE) cells were a generous gift from Robert Whitehead (Vanderbilt University, Nashville, TN) (49). Cells were grown in RPMI 1640 medium (Cambrex, Verviers, Belgium) supplemented with 5% fetal bovine serum, 100 U penicillin/ml, 100 µg streptomycin/ml (all from Cambrex), and 5 U/ml of murine IFN-{gamma} (Invitrogen, Breda, The Netherlands). The MSIE cells were derived from the transgenic temperature-sensitive simian virus 40 large-T antigen Immortimouse and, as such, were grown at the permissive temperature of 33°C. For all experiments, cells were maintained in serum-free medium that contained 11 g/l of fatty acid-free BSA and were supplemented with additives (5 µM 27-hydroxycholesterol, or 500 µM fatty acids). Before being added to the cells, fatty acids were bound to BSA by preincubation in the BSA-containing medium for 30 min at 37°C. A 50 mmol/l stock solution of each fatty acid was prepared by dissolving the fatty acid in 70 mmol/l KOH. 27-Hydroxycholesterol was dissolved in ethanol.

RNA Isolation

Total RNA was isolated by using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. For microarray hybridization the isolated RNA was further column purified (SV total RNA isolation system Promega, Leiden, The Netherlands). RNA concentration was measured on a NanoDrop ND-1000 UV-Vis spectrophotometer (Isogen, Maarssen, The Netherlands) and analyzed on a bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands) with 6000 Nano Chips according to the manufacturer's instructions.

Array Hybridization and Microarray Data Analysis

RNA of the proximal segment and the middle segment of the small intestine were pooled per treated group (n = 6) and hybridized to Affymetrix Mouse genome 430 2.0 arrays. Detailed methods for the labeling and subsequent hybridizations to the arrays are described in the eukaryotic section in the GeneChip Expression Analysis Technical Manual Rev. 3 from Affymetrix, which is available upon request (Santa Clara, CA). Arrays were scanned on a GeneChip Scanner 3000 (Affymetrix). Data analysis was performed in Microarray Analysis Suite version 5.0 (Affymetrix). Array data have been submitted to the Gene Expression Omnibus, accession number GSE8582.

Real-Time PCR Analysis

cDNA of the middle segment of the small intestine was synthesized from 1 µg of total RNA by using the reverse transcription system (Promega, Leiden, The Netherlands) following the supplier's protocol.

cDNA was PCR amplified with Platinum Taq DNA polymerase (all reagents were from Invitrogen). Most of the primer sequences were obtained from the PrimerBank of the Harvard University (48), or otherwise constructed with primer3 (http://primer3.sourceforge.net). Primers were tested for specificity by BLAST analysis. The sequences of primers used are available on request. PCR was carried out by using SYBRgreen on a MyIQ thermal cycler (Bio-Rad Laboratories, Veenendaal, The Netherlands) with the following thermal cycling conditions: 8 min at 94°C, followed by 45 cycles of 94°C for 15 s and 60°C for 1 min. All samples were performed in duplicate and normalized to cyclophilin A and 18S expression. Only the results of the cyclophilin A normalization are shown because they are representative for the results of the 18S normalization.

Analysis of Bile Composition and Cholesterol Absorption

Analytical procedure. Cholesterol was extracted from blood spots with 1 ml of 95% ethanol-acetone (1:1 vol/vol) for gas chromatography-mass spectrometric (GC-MS) analysis according to Neese et al. (20). Unesterified cholesterol from blood spots was subsequently derivatized using N,O-bis-(trimethyl)trifluoroacetamide with 1% trimethylchlorosilane at room temperature. Biliary lipids were extracted according to Bligh and Dyer (7). Biliary concentrations of cholesterol and phospholipids were determined as previously described (31). Bile salts were measured enzymatically.

GC-MS measurements of mass isotopomer distribution. Cholesterol trimethylsilylether derivatives were separated with a Trace MS plus GC-MS (Interscience, Breda, the Netherlands), using a 20 m x 0.18 mm (0.18-µm film thickness) DB17 ms column (J&W Scientific, Falson, CA). The oven temperature was programmed from 140 to 280°C at 20°/min. A splitless injection was applied. Ions monitored were m/z 458–465 corresponding to the m0–m8 mass isotopomers. The fractional isotopomer distribution measured (m0–m8) was corrected for the fractional distribution due to natural abundance of 13C and 2H by multiple linear regression as described by Lee et al. (21) to obtain excess fractional distribution of mass isotopomers (M0–M8) resulting from isotope dilution of administered labeled compounds. In this approach, M5 represented the fractional contribution of the orally administered label and M7 the fractional contribution of the intravenously administered label.

Fractional cholesterol absorption measurement. Fractional cholesterol absorption was calculated as described for the plasma dual-isotope ratio method (40). Blood spots obtained at 72 h after intravenous and oral administration were used for the calculation of fractional cholesterol absorption (8). Fractional cholesterol absorption was calculated as the ratio of the fraction orally administered cholesterol-D5 and the fraction intravenously administered cholesterol-D7 as measured in bloodspots obtained 72 h after administration, after being corrected for its orally and intravenously administered dose.

Hmgcr Activity Assay

Activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) was measured in the study with LXR{alpha}–/– and wild-type mice as described by Brown et al. (9) with some modifications. We used scrapings of the proximal part of the intestine, as the middle part was completely used to study gene expression. The scrapings were dissolved in Hmgcr assay buffer containing 100 mM KPi, 200 mM KCl, 5 mM EGTA, 5 mM EDTA, 10 mM DTT, and 10 mg/ml leupeptin (pH 7.1). The tissue was disrupted by use of an Ultra-turrax followed by sonication (twice at 8-W output, 40 J, at room temperature). The homogenate was centrifuged for 5 min at 1,000 g. Protein concentration was determined in the supernatant by the Bradford method. The homogenates were diluted to ~0.5 mg/ml. One hundred microliters of the resulting homogenate was preincubated for 10 min at 37°C with 60 µl of cofactor-mix containing 175 mM glucose-6-phosphate, 6.7 mM NADPH, 16.7 mM EDTA, and 0.7 unit glucose-6-phosphate dehydrogenase. The enzyme reactions were started with the addition of 1.8 nmol of [14C]HMG-CoA and 5.6 nmol of HMG-CoA in 40 µl of H2O. After a 30-min incubation period at 37°C, reactions were terminated by adding 50 µl of 1.2 N HCl. After 30 min, the product was extracted three times with 2 ml of ethyl acetate. The extracts were evaporated to dryness and analyzed by silica thin-layer chromatography using a solvent system toluene-acetone (1:1) dried with Na2SO4. The formed product was quantified by phosphorimaging (Fuji FLA-3000) with the aid of the Aida software package using samples with known amounts of [14C]mevalonate. For each mouse, the activity of Hmgcr was determined in duplicate.

Other Analytical Methods

Feces were lyophilized, weighed, and homogenized. Neutral sterols were analyzed according to Arca et al. (3). Total bile acids in feces were determined as described previously (17). Pooled plasma samples from all animals of one group were used for lipoprotein separation by fast protein liquid chromatography (FPLC) as described previously (42). Total cholesterol was measured by use of a commercially available kit (Roche Molecular Biochemicals, Mannheim, Germany).

Statistical Analysis

All data are reported as means ± SE. The differences between the mean values were tested for statistical significance by the two-tailed Student's t-test. P values <0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cholesterol Transporter Genes Are Downregulated in Small Intestines of Mice on a Cholesterol-Free, High-Fat Diet

In this study we first focused on the expression of genes involved in cholesterol transport in the small intestine of C57BL/6J wild-type mice fed purified, cholesterol-free, low- or high-fat diets. Microarray results of cholesterol transporters and related genes at weeks 2, 4, and 8 of the middle segment of the small intestine are shown in Table 1. For week 2, additional quantitative RT-PCR (qRT-PCR) analyses were performed, which accurately confirmed our array results (Table 1). Similar gene expression patterns as were found in the middle part of the small intestine were also seen in the proximal segments. However, since changes were most prominent in the middle segment, we focused on this part of the small intestine.


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  		Table 1. Microarray results of high-fat diet intervention in C57B1/6J mice

 
An expected upregulation of microsomal triglyceride transfer protein (Mttp), involved in chylomicron assembly, was observed because it is known that an elevated fat intake stimulates chylomicron formation (18, 25, 44). In line with the assumption that more chylomicron formation means an elevated requirement of cholesterol, we found a decrease in gene expression of cholesterol efflux transporters Abca1, Abcg5, and Abcg8. Surprisingly, the cholesterol absorption transporter Npc1l1 was also downregulated. Additionally, Cyp27a1 was downregulated, which suggests that the conversion of cholesterol into 27-hydroxycholesterol is reduced on a high-fat diet. Gene expression of Hmgcr, the key enzyme for cholesterol synthesis, was not changed. On the basis of the downregulation of Npc1l1, we hypothesized that on a cholesterol-free, high-fat diet, cholesterol absorption is diminished. Moreover, the cholesterol efflux at the apical and basolateral side via Abcg5, Abcg8, and Abca1 will be reduced, since cholesterol in the enterocyte is mainly required for chylomicron formation (Fig. 1).


Figure 1
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  		Fig. 1. Hypothesis for the high-fat (HF)-induced suppression of cholesterol efflux transporters. The enterocyte cartoon shows a schematic overview of the microarray results. White means not regulated, light gray means downregulated, and dark gray means upregulated. Shaded circle, chylomicron; open star, 27-hydroxycholesterol (27-OH); its concentration was not measured. On the basis of the microarray data we hypothesized that on a cholesterol-free, high-fat diet, cholesterol absorption is diminished and that cholesterol in the enterocyte is mainly used for chylomicron formation to transport fat out of the enterocyte. Therefore, less cholesterol is converted to the liver X receptor (LXR) ligand 27-hydroxycholesterol via Cyp27a1, leading to a possible downregulation of LXR target genes Abca1, Abcg5, and Abcg8.

 
A Cholesterol-Free, High-Fat Diet Decreases Fractional Cholesterol Absorption

Fractional cholesterol absorption was measured to check whether the reduced gene expression of the cholesterol absorption transporter Npc1l1 functionally leads to diminished cholesterol absorption. Figure 2 shows that a cholesterol-free, high-fat diet for 2 wk fractional cholesterol absorption from 61% to 42% compared with a cholesterol-free, low-fat diet in C57BL/6J wild-type mice, which is a reduction of 31%. Additionally, we determined biliary secretion rates of cholesterol and bile acids to check whether differences in cholesterol absorption could be due to altered bile formation induced on a high-fat diet. However, we found no differences in these parameters between a low- and high-fat diet-fed mice (Table 2). Since the mice were fed cholesterol-free diets, unchanged biliary cholesterol secretion implies that the absolute cholesterol absorption was reduced by 31% on a high-fat diet.


Figure 2
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  		Fig. 2. Fractional cholesterol absorption in high-fat- and low-fat (LF)-fed C57BL/6J mice. Cholesterol absorption in high-fat (solid bar)- and low-fat (open bar)-fed mice was measured by the plasma dual-isotope method (LF: n = 5, HF: n = 8). After 2 wk of treatment, mice received an intravenous injection of cholesterol-D7 and an oral dose of cholesterol-D5. Plasma samples obtained 72 h after administration were used for the calculation of fractional cholesterol absorption. Data are presented as means ± SE, n = 5 (LF), n = 8 (HF) *Significant differences by Student's t-test (P < 0.05).

 

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  		Table 2. Bile flow and biliary secretion rates in high-fat- and low-fat-fed C57BL/6J mice

 
Downregulation of Cholesterol Transporters Is an Acute Response to Dietary Triacylglycerols

To investigate whether the suppression of gene expression of the cholesterol transporters reflects an acute process, we gavaged SV129 mice with 400 µl of unsaturated fatty acids in the form of triacylglycerols. Six hours after gavage, the small intestine was harvested and gene expression was analyzed by qRT-PCR (Fig. 3). This experiment showed that cholesterol efflux transporters were already downregulated after 6 h exposure to all fatty acids studied without marked differences between the different fatty acids. Furthermore, a pronounced decrease in gene expression of Npc1l1 was found in this short-term study. In addition, Mttp was induced, with most pronounced induction by C18:1, whereas Cyp27a1 was equally downregulated by all acyl species. In contrast to the long-term diet intervention, Hmgcr expression was induced by C18:1 but not by C20:5 or C22:6 fatty acids. Additionally, this acute study in SV129 mice showed that downregulation of cholesterol efflux and absorption transporter gene expression by dietary fat is mouse strain independent.


Figure 3
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  		Fig. 3. Relative mRNA levels in small intestines of SV129 mice gavaged with triacylglycerols. Four- to 5-mo-old male SV129 mice were given synthetic triacylglycerols via oral gavage. mRNA expression was measured by quantitative RT-PCR (qRT-PCR), with the control set to 1. Significance was determined by an unpaired Student's t-test and for all treatments was calculated compared with the control. *P < 0.05. Data are presented as means ± SE, n = 4.

 
Downregulation of Abca1 Is Caused by Unsaturated Fatty Acids and Is Not Due to Shortage of the Endogenous LXR Ligand 27-Hydroxycholesterol

Because 27-hydroxycholesterol is the major natural LXR ligand present in the enterocytes (22), suppression of Cyp27a1 expression might indicate that a reduced expression of LXR target genes Abca1, Abcg5, and Abcg8 upon high-fat feeding is due to shortage of endogenous LXR ligand (Fig. 1). To evaluate this possibility, we incubated MSIE cells with fatty acids with and without additional 27-hydroxycholesterol (Fig. 4). Because Abcg5, Abcg8, and Npc1l1 were not expressed in this cell line, only Abca1 expression was used as readout. As expected, Abca1 was strongly upregulated by 27-hydroxycholesterol (5-fold). The unsaturated fatty acids oleic acid, linoleic acid, and docosahexaenoic acid all suppressed expression of Abca1. Even after induction of basal Abca1 expression with 27-hydroxycholesterol, these unsaturated fatty acids were able to downregulate expression to a similar extend (Fig. 4). The saturated species palmitic and stearic acids did not affect Abca1 expression. These data indicate that downregulation of the cholesterol efflux transporter Abca1 is caused by unsaturated fatty acids. This downregulation is in contrast to our hypothesis as depicted in Fig. 1, independent on the level of the LXR ligand 27-hydroxycholesterol.


Figure 4
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  		Fig. 4. Effect of 27-hydroxycholesterol and fatty acids on gene expression of Abca1 in mouse small intestinal epithelial (MSIE) cells. Confluent MSIE cells were incubated for 6 h with 500 µM fatty acids and/or 5 µM 27-hydroxycholesterol. mRNA expression was measured by qRT-PCR. The y-axes are in a log-10 scale. Significance was determined by an unpaired Student's t-test and for all treatments was calculated compared with the control. *P < 0.05. Data are presented as means ± SE, n = 3.

 
Downregulation of Cholesterol Efflux Transporters Is Lxr{alpha} Independent

To address whether the fat-induced downregulation of the ABC transporters is due to interference with LXR{alpha} signaling, we fed LXR{alpha}–/– and wild-type mice similar cholesterol-free, high-fat and low-fat diets for 2 wk (Supplemental Table S1). Gene expression was analyzed with qRT-PCR (Fig. 5). Abca1, Abcg5, and Abcg8 were similarly downregulated by the high-fat diet in the wild-type mice and the LXR{alpha}–/– mice. Also, Npc1l1, Hmgcr, Mttp, and Cyp27a1 showed the same differential gene expression in response to high-fat feeding in wild-type and LXR{alpha}–/– mice (Fig. 5). This demonstrates that downregulation of cholesterol efflux transporters in the small intestine by dietary fat is LXR{alpha} independent.


Figure 5
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  		Fig. 5. Relative mRNA levels in small intestines of wild-type and LXR{alpha}–/– mice fed a high-fat diet for 2 wk. Four- to 5-mo-old female wild-type (WT) and LXR{alpha}–/– (KO) mice were fed a low-fat or high-fat, cholesterol-free diet for 2 wk. mRNA expression was measured by qRT-PCR. Open bars represent the LF diet group; solid bars represent the HF diet group. WT LF values were set to 1. Significance was determined by an unpaired Student's t-test. *P < 0.05. Data are presented as means ± SE, n = 6 (WT), n = 4 (KO).

 
A Cholesterol-free, High-Fat Diet Increases Fecal Neutral Sterol Excretion and Small Intestinal Hmgcr Activity Without Affecting Plasma Cholesterol in Mice

Because changes in cholesterol absorption generally affects fecal sterol concentrations (11, 12, 24), we measured neutral sterols in feces of wild-type and LXR{alpha}–/– mice fed a low-fat and a high-fat diet for 24 h, 48 h, and 2 wk (Fig. 6). In addition, fecal bile acids were determined. Already on the first day, the high-fat diet increased the fecal excretion of neutral sterols in wild-type and LXR{alpha}–/– mice (Fig. 6A), corroborating the acute downregulation of Npc1l1, shown in Fig. 3. This indicates that the diminished cholesterol absorption found after 2 wk on a high-fat diet (Fig. 2) represents an acute response to dietary fat. Bile acid excretion, on the other hand, was elevated only after 2 wk on a high-fat diet (Fig. 6B).


Figure 6
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  		Fig. 6. Fecal neutral sterol and bile acid excretion in wild-type and LXR{alpha}–/– mice fed high- and low-fat diets. Four- to 5-mo-old female wild-type and LXR{alpha}–/– mice were fed a low-fat or high-fat for 2 wk. Feces were collected after 24 and 48 h and after 2 wk of diet intervention and analyzed for neutral sterol and bile acid concentrations. A: neutral sterol concentrations in feces after 24 h, 48 h, and 2 wk. B: bile acid concentrations in feces after 24 h, 48 h, and 2 wk. Open bars represent the LF diet group; solid bars represent the HF diet group. Neutral sterol and bile acid concentrations were normalized for body weight. Significance was determined by an unpaired Student's t-test. *P < 0.05. Data are presented as means ± SE, n = 3 (WT), n = 2 (KO).

 
Since no cholesterol was present in the diet and fractional cholesterol absorption was reduced, we speculated that small intestinal cholesterol synthesis must be increased on a high-fat diet because cholesterol is needed for chylomicron synthesis. This elevation was not evident at the gene expression level (Table 1, Fig. 5); however, it is known that Hmgcr activity is determined by posttranscriptional regulatory mechanisms (16). Therefore, we measured Hmgcr activity in the small intestine and found it indeed to be significantly elevated in mice fed a high-fat diet compared with mice fed a low-fat diet for 2 wk (Fig. 7).


Figure 7
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  		Fig. 7. Hmg-CoA reductase activity in intestinal scrapings of high-fat and low-fat fed wild-type and LXR{alpha}–/– mice. Four- to 5-mo-old female wild-type and LXR{alpha}–/– mice were fed a low-fat or high-fat for 2 wk. Hmg-CoA reductase activity was measured in scrapings of the proximal part of the small intestine. Open bars represent the LF diet group; solid bars represent the HF diet group. Significance was determined by an unpaired Student's t-test. *P < 0.05. Data are presented as means ± SE, n = 6 (WT), n = 4 (KO).

 
To determine whether the observed suppression of cholesterol transporter expression is reflected in plasma cholesterol levels, we measured cholesterol in plasma of FPLC fractions of pooled plasma of wild-type and LXR{alpha}–/– mice fed a low-fat or a high-fat diet for 2 wk (Fig. 8). Separation of plasma lipoproteins by FPLC revealed that the HDL cholesterol levels were higher in the wild-type than in the LXR{alpha}–/– mice (Fig. 8). No changes were found in the HDL-sized fractions in the high-fat-fed mice compared with the low-fat-fed mice in both wild-type and LXR{alpha}–/– backgrounds.


Figure 8
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  		Fig. 8. Fast protein liquid chromatography (FPLC) separation of plasma lipoproteins of high-fat- and low-fat-fed wild-type (A) and LXR{alpha}–/– (B) mice. Four- to 5-mo-old female wild-type and LXR{alpha}–/– mice were fed a low-fat or high-fat for 2 wk. Plasma from all individual mice per group was pooled and subjected to gel filtration using Superose 6 columns. Cholesterol content in each fraction was measured. HDL, high-density lipoprotein.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major finding of this study is that, in the murine small intestine, cholesterol efflux transporters Abca1, Abcg5, and Abcg8 and the cholesterol absorption transporter Npc1l1, as well as the fractional cholesterol absorption, were all suppressed by feeding a cholesterol-free, high-fat diet to mice. The downregulation appeared to be an acute response that is maintained during a chronic diet intervention and is, surprisingly, not LXR{alpha} dependent, as revealed by in vitro studies and diet intervention in LXR{alpha}-null mice.

We showed with an in vivo gavage study that downregulation of cholesterol efflux and absorption transporters by fatty acids is an acute process. The gavage was given after a fasting period of 4 h, after which the mouse stomach is nearly empty (4). This implies that in this acute experiment only the gavaged triacylglycerols influence small intestinal gene expression and that the downregulation of small intestinal cholesterol transporters is a fat-induced effect. In these studies we could only apply triacylglycerols of unsaturated fatty acids, since these are fluid at room temperature. However, saturated and unsaturated fatty acids were compared by an in vitro approach, in which only the unsaturated species showed a downregulation of Abca1. On the basis of this observation, we speculate that, also in vivo, only unsaturated fatty acids induce downregulation of cholesterol transporters, but this requires further investigation.

The diminished fractional cholesterol absorption in our study is in line with results reported by Satchithanandam et al. (35), who found that a high-fat sesame oil diet decreases the lymphatic cholesterol absorption in the small intestine of rats. In our study, the high-fat diet almost doubled fecal neutral sterol excretion (Fig. 6). However, on the basis of the biliary cholesterol secretion that was not affected by the high-fat diet (Table 2), we could calculate that the diminished cholesterol absorption should only lead to a 50% increase [i.e., (0.58/0.39 – 1)x100%] in cholesterol excretion. We suggest that this higher fecal neutral sterol excretion could be due to a direct cholesterol secretion by the intestinal epithelium, as recently shown by Van der Velde et al. (43). It appears that this novel pathway is stimulated by high-fat-diet feeding in mice. Studies in humans did not show increased neutral sterol levels in feces on a high-fat diet (10), which might suggest a different effect of a high-fat diet on intestinal cholesterol absorption and secretion between humans and rodents. Whether this is due to species-dependent differences in fat-induced regulation of Npc1l1 requires further investigation. Because reduced cholesterol absorption on a high-fat intake is conflicting with the higher need of cholesterol for chylomicron formation, we expected increased cholesterol synthesis in the enterocyte. However, gene expression data showed that the key enzyme for cholesterol synthesis, i.e., Hmgcr, was only upregulated in the acute fatty acid experiment and was not changed after 2, 4, and 8 wk of high-fat feeding. Therefore, we additionally measured Hmgcr activity, and it turned out to be increased in the cholesterol-free, high-fat-fed mice, implying elevated small intestinal cholesterol synthesis. This observation is in line with increased intestinal cholesterol synthesis in rat studies with corn oil by Stange et al. (37, 38). In conjunction with downregulation of cholesterol efflux transporters, this increased intestinal cholesterol synthesis probably provides sufficient cholesterol for chylomicron formation despite the overall reduction in cholesterol absorption.

Fecal neutral sterol levels were higher and fractional cholesterol absorption was lower on a cholesterol-free, high-fat diet, despite a strongly reduced expression of Abcg5 and Abcg8. As previously described, deficiency of Abcg5 and Abcg8 leads to no (30) or only mild (51, 53) decrease in fecal neutral sterol content. On the basis of these data, we conclude that the effect of downregulation of cholesterol uptake (Npc1l1) overrules the effect of downregulation of the presumed cholesterol efflux transporters (Abcg5 and Abcg8). In the last few years, Npc1l1 has emerged as an important key component of the small intestinal sterol uptake system (2, 13) and here we show that Npc1l1 likely plays a pivotal role in the control of cholesterol absorption during exposure to a high-fat diet. Future research is needed to investigate the mechanism of this reduced expression of Npc1l1 during a high-fat diet intervention.

In contrast to the acute elevation of neutral sterols, fecal bile acid secretion was only elevated after 2 wk of high-fat diet intervention. As a result of a high-fat diet more chylomicrons are formed, causing cholesterol accumulation in the liver (6). Synthesis of bile acids is one of the predominant mechanisms for the excretion of excess cholesterol from hepatocytes, implying that the observed increase in bile acid secretion, which reflects hepatic bile acid synthesis, might be a secondary effect of the high-fat diet. This hypothesis is in line with liver gene expression data that showed that Cyp7a1 was not changed in the acute experiment but was increased in the long-term high-fat diet intervention (data not shown).

It should be noted that our intervention studies were performed with cholesterol-free, purified low- and high-fat diets. The in vitro study showed that the downregulation of cholesterol transporter genes was not caused by a lack of cholesterol derivatives as even with addition of 27-hydroxycholesterol unsaturated fatty acids still could decrease expression of Abca1. To investigate whether the downregulation of the cholesterol efflux transporters is mediated by LXR we used LXR{alpha}–/– mice. Because in wild-type and knockout mice the same degree of downregulation was seen, we concluded that the downregulation is LXR{alpha} independent. However, it has to be noted that in these LXR{alpha}–/– mice LXRβ is still present and might be able to compensate the loss of LXR{alpha}. Although LXR double-knockout mice would be a preferable model to study LXR involvement, we believe that it is not very likely that LXRβ can completely compensate for the loss of LXR{alpha}. In case of a partial compensation we would have expected a diminished downregulation of the cholesterol transporters in the LXR{alpha}-null mice. So far, compensation of LXR{alpha} by LXRβ in the intestine has not been reported, but it is known that in liver LXRβ is not able to compensate for the loss of LXR{alpha} (29). Moreover, we showed in our in vitro experiment with a ligand for both LXR{alpha} and LXRβ that unsaturated fatty acids still could downregulate Abca1.

Duval et al. (14) implied that Npc1l1 is LXR dependently downregulated. However, in their study no LXR–/– mice were included to discern direct or indirect involvement of LXR. Our results indicate that dietary fat-induced downregulation of Npc1l1 and the cholesterol efflux transporters in the intestine is LXR{alpha} independent, which implies that another transcription factor is involved in this process. The study of Duval et al. furthermore suggests that Npc1l1 is not repressed by PPAR{alpha}. However, Valasek et al. (41) recently showed that Npc1l1 is PPAR{alpha} dependently downregulated by fenofibrate. Furthermore, from studies in our own laboratory with wild-type and PPAR{alpha}–/– mice we know that PPAR{alpha} activation with WY14,643 results in reduced levels of Npc1l1 (unpublished data). So, PPAR{alpha} might be involved in the fatty acid-dependent downregulation of Npc1l1 and, thereby, in the control of intestinal cholesterol absorption on a high-fat diet. On the other hand it is known that Abca1 is PPAR{alpha} dependently upregulated (19). In addition, Abcg5 and Abcg8 are not known to be regulated by PPAR{alpha}. This implies that PPAR{alpha} is not the common regulator in the fatty acid-induced downregulation of cholesterol transporters. This suggests a different mechanism for Npc1l1 and cholesterol efflux transporters. Another potential candidate regulator is PPAR{delta}, because it has been previously described that Npc1l1 is PPAR{delta} dependently downregulated in the murine intestine and CaCo2 cells (42).

In conclusion, our data show that on a cholesterol-free, high-fat diet, fractional cholesterol absorption is diminished. We propose that, possibly in an attempt to spare intracellular cholesterol for the chylomicron formation, cholesterol efflux via ABC transporter-related pathways is reduced. In addition, to compensate for reduced uptake of cholesterol in the enterocyte, cholesterol synthesis is induced. This work shows that the downregulation of cholesterol transporters is mediated by unsaturated fatty acids. Studies with LXR{alpha}-null mice indicate, surprisingly, that this downregulation is not dependent on the presence of LXR{alpha}. PPARs might be feasible candidates to regulate cholesterol transporters on a high-fat diet, but additional studies are required to pinpoint the mechanism by which unsaturated fatty acids downregulate cholesterol transporters in the small intestine.


   ACKNOWLEDGMENTS
 
The authors thank Denise Jonker-Termont, Rick Havinga, Renze Boverhof, and Annelies Stroeve for excellent technical assistance; Bert Weijers, René Bakker, and Juul Baller for skillful biotechnical assistance; Mechteld Grootte Bromhaar and Jenny Jansen for expert microarray hybridizations; and Bert Groen for stimulating discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. de Wit, Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen Univ., Bomenweg 2, NL-6703 HD, Wageningen, the Netherlands (e-mail: nicole.dewit{at}wur.nl)

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.


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