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Originally published In Press as doi:10.1074/jbc.M504609200 on May 26, 2005

J. Biol. Chem., Vol. 280, Issue 30, 28103-28109, July 29, 2005
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Caveolin-1 Is Not Required for Murine Intestinal Cholesterol Transport*

Mark A. Valasek{ddagger}§, Jian Weng§, Philip W. Shaul||, Richard G. W. Anderson¶, and Joyce J. Repa{ddagger}**{ddagger}{ddagger}

From the Departments of {ddagger}Physiology, **Internal Medicine, ||Pediatrics and Cell Biology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390

Received for publication, April 27, 2005 , and in revised form, May 25, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Caveolin-1 (CAV1) is the structural protein of the filamentous coat that decorates the cytoplasmic surface of each caveola. Cell culture studies have implicated CAV1 in playing an important role in intracellular cholesterol trafficking. In addition, it has been reported that CAV1 forms a detergent-resistant protein complex with Annexin-2 in enterocytes that can be disrupted by the cholesterol absorption inhibitor ezetimibe, suggesting a possible role for CAV1 in cholesterol absorption. In this report, we have evaluated cholesterol homeostasis in Cav1 knock-out mice. Deletion of CAV1 does not result in either a compensatory increase of CAV2 or CAV3 in intestine. In addition, Cav1 knock-out mice display normal mRNA and protein levels of Annexin-2 or the putative cholesterol transport protein Niemann-Pick C1-like 1 (NPC1L1) in proximal intestinal mucosa. Fractional cholesterol absorption and fecal neutral sterol excretion are statistically similar in Cav1 knock-out mice and their wild-type littermates. Moreover, oral administration of ezetimibe is equally effective in decreasing cholesterol absorption in Cav1 null mice and wild-type controls. The mRNA expression levels of genes sensitive to intracellular cholesterol concentration (ATP-binding cassette transporters ABCA1 and ABCG5, hydroxymethylglutaryl-CoA synthase and the LDL receptor) are similarly altered in the proximal intestinal mucosa of Cav1 null and wild-type mice following ezetimibe treatment. These results demonstrate that CAV1 is not required for cholesterol absorption or ezetimibe sensitivity in the mouse.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ezetimibe is a member of the 2-azetidinone class of drugs that has recently been approved for clinical use in reducing plasma cholesterol levels (1). This drug significantly reduces the uptake of intestinal sterols by a mechanism that has not been fully elucidated. One protein that appears to be involved is NPC1L1 (2). Animals made deficient in NPC1L1 exhibit markedly reduced cholesterol absorption and are unresponsive to ezetimibe (2). Although the evidence appears strong that NPC1L1 plays a significant role in sterol absorption (3), a direct interaction between NPC1L1 and ezetimibe has yet to be demonstrated. The possibility exists, therefore, that other proteins are required for the inhibitory effects of this drug.

Another protein that has been suggested to be an ezetimibe target is caveolin-1 (CAV1)1 (4). In zebrafish and mice, CAV1 has been shown to tightly associate with annexin-2 to form a complex that is resistant both to high heat and to solubilization by a SDS detergent. Treatment of mice with ezetimibe can disrupt this complex, suggesting that it may be a molecular target for the drug. Additionally, CAV1 is an attractive candidate target protein because there is considerable evidence for its involvement in intracellular cholesterol transport. Originally discovered as an integral membrane component of the filamentous coat that decorates the cytoplasmic surface of each caveola (5), CAV1 is a cholesterol- (6) and fatty acid (7)-binding protein that is able to move between membrane compartments in the cell (8) as a soluble cytoplasmic intermediate (9, 10). Importantly, cells lacking CAV1 are defective in transporting cholesterol to caveolae (11, 12). CAV1 also has been linked to cholesterol efflux in animals that are susceptible to gallstones. When gallstone-susceptible C57L/J mice are placed on a lithogenic diet, liver CAV1 mRNA and protein appear to be upregulated (13). CAV1 expression may also be regulated by cholesterol depletion by chronic high density lipoprotein exposure in cultured cells (14).

An important test of protein function is the phenotype of animals that lack the protein. For example, the high density lipoprotein receptor SR-B1 is clearly involved in efflux and influx of cholesterol (15), yet animals lacking SR-B1 are not defective in intestinal absorption of cholesterol (16) and are fully sensitive to cholesterol absorption inhibition by ezetimibe (17). Likewise, if CAV1 is a target for ezetimibe, then entero-hepatic cholesterol transport should be markedly reduced in animals lacking CAV1, and they should be unresponsive to ezetimibe. Here we show that Cav1 null mice do not exhibit any defect either in cholesterol transport or in response to ezetimibe.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Studies
Cav1-deficient mice were generated by gene deletion of exon 2 as described previously (18). The resulting mouse strain was intercrossed with C57/Bl6 mice for 10 generations to establish a pure C57/Bl6 background. Mouse genotyping was performed by PCR using the primers F1, 5'-ttctgtgtgcaagcctttcc; R1, 5'-gtgtgcgcgtcatacacttg; and R2, 5'-ggggaggagtagaaggtggc to generate a product of 307 bp for the null allele and 260 bp for the wild-type allele (18). All studies were carried out with male mice 3 months of age. Although it has been reported that older Cav1 null mice are smaller than wild-type mice (19), the mice used in these studies showed no difference in body weight (WT, 26.65 ± 2.79; WT-EZ, 29.48 ± 3.2; Cav1 knock-out, 30.58 ± 1.57; Cav1 knock-out-EZ, 30.76 ± 1.59 g). Animals were housed individually in plastic colony cages containing wood shavings in a 22 °C room lit from 7 a.m. to 7 p.m.

Mice were fed ad libitum a cereal-based rodent diet (Teklad Diet number 7001, Madison, WI) that contains 0.02% (w/w) cholesterol and 4% total lipid. The mice were fed the powdered form of this diet, which in some experiments was supplemented with ezetimibe to provide 10 mg/day/kg of body weight (based on the consumption of 160 g of diet/day/kg of body weight). Ezetimibe was provided by Harry R. Davis, Jr. at the Schering-Plough Research Institute. Experiments were performed at the end of the dark cycle, and mice were in a fed-state at the time of study. Experiments were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center at Dallas.

Plasma Lipid Analyses
Mice were anesthetized and blood was sampled from the inferior vena cava into EDTA-containing microfuge tubes (final concentration of EDTA ~2 mM). Plasma was prepared by low speed centrifugation (5,000 g) for 10 min at 4 °C. For the lipoprotein profiles, equal volumes of plasma from individuals of each group were pooled then diluted 1:1 (v/v) with phosphate-buffered saline and filtered through a 0.22-µm syringe filter (Millipore). One-hundred microliters of diluted sample was injected onto a Superose 6 HR 10/30 gel filtration column for fractionation by fast protein liquid chromatography (Pharmacia {Delta}KTA) at 4 °C. Samples were eluted at a flow rate of 0.4 ml/min in buffer containing 0.05 M phosphate, pH 7.0, and 0.15 M NaCl. Fractions of 0.3 ml were collected for analysis.

Cholesterol concentrations were measured enzymatically using the Infinity Cholesterol Reagent (401-500P, Sigma). Plasma non-esterified fatty acid concentrations were measured enzymatically using a 1:2 ratio of color reagent A to color reagent B from NEFA-C kit (994-75409, Wako Chemicals, Neuss, Germany). Plasma total triglycerides were measured enzymatically with triglyceride (GPO-Trinder) reagents A and B (T2449 and F6428, Sigma). All kits provided appropriate controls to establish a standard curve for calculation.

Liver Lipid Analyses
Cholesterol—An aliquot of liver was saponified in 3% KOH (in ethanol) and extracted in petroleum ether (PE) with added standard (stigmastanol). An aliquot of the PE phase was dried down and resuspended in hexane prior to analysis by gas chromatography (20).

Triglyceride—An aliquot of liver (~250 mg) was extracted in 20 ml of chloroform:methanol (2:1, v/v) in the presence of [14C]triolein (American Radiolabeled Chemicals, Inc., St. Louis, MO). The entire extract was filtered and dried under air, and the residue was redissolved in 1 ml of hexane:methyl-t-butyl ether (100:1.5, v/v). This solution was run over a Sep-Pak Vac RC silica cartridge (500 mg, Waters Corporation, Milford, MA). Following elution of the cholesteryl esters, triacylglycerols were eluted with 12 ml of hexane:methyl-t-butyl ether (96:4, v/v). The eluate was brought to 100 ml with hexane, and 2-ml aliquots were dried under air and measured for radioactivity using a scintillation counter to calculate the percent recovery of the internal standard and triacylglycerol. Separate 2-ml aliquots were used for enzymatic determination of triacylglycerol quantity with Infinity triglycerides liquid stable reagent (ThermoTrace, Noble Park, Australia).

Cholesterol Balance Measurements
Absorption—Fractional cholesterol absorption was measured by a fecal dual isotope ratio method (21). Briefly, mice received a single intragastric dose of medium chain triglyceride oil containing [5,6-3H]sitostanol (2 µCi, American Radiolabeled Chemicals, Inc) and [14C]cholesterol (1 µCi, PerkinElmer Life Sciences). Stools were collected over the following 3 days. Samples of the dosing mixture and aliquots of stool were extracted, and the ratio of 14C:3H in each was determined to calculate percent cholesterol absorption (21).

Fecal Neutral Sterol and Acidic Sterol Excretion—Stools were collected from individually housed mice over 3 days. They were dried, weighed, and ground. An aliquot of this material was used to determine bile acid content by an enzymatic method (22). A second aliquot was saponified, solvent was extracted, and the amounts of cholesterol, coprostanol, epicoprostanol, and cholestanone was quantified by gas chromatography (23). The amounts measured were adjusted to reflect the daily excretion (based on feces collected over 3 days) per 100 g of body weight.

Preparation of Samples for RNA and Protein Measurements
Mice were anesthetized and exsanguinated via the descending vena cava. Small intestines were removed, flushed with ice-cold phosphate-buffered saline and cut into three sections of equal length (the proximal third denoted as duodenum). The sections were slit lengthwise, and the mucosae were gently scraped, frozen in liquid nitrogen, and stored at -85 °C. Whole livers were removed and snap-frozen in liquid nitrogen and then crushed to a fine powder with a Besselar Tissue pulverizer and stored at -85 °C, thus providing multiple homogenous aliquots for various assays. Total RNA was isolated from tissue samples using RNA STAT-60 (Tel-Test Inc.). Total protein was obtained from the organic phase remaining after RNA isolation by precipitating with isopropanol, consecutively washing with 0.3 M guanidine hydrochloride in 95% ethanol and ethanol, and then solubilizing the protein pellet in 1% SDS. RNA concentrations were determined by absorbance at 260 nm. Protein concentrations were determined using the BCA protein assay kit (Pierce).

Western Analysis
Total protein obtained from whole-cell lysates of duodenal mucosa (see above) was size-fractionated on 8% SDS-polyacrylamide gels (60 µg/lane), transferred electrophoretically to a polyvinylidene difluoride membrane, and incubated with one of the following antisera: CAV1, amino terminus "pAb1" (N20, Santa Cruz Biotechnology, 1:1000); CAV1, central "pAb2" (amino acids 1-97, BD Biosciences, 1:5000); CAV1, carboxyl terminus "pAb3" (produced in rabbits by injection of the peptide IFSNVRINLQKEI (24), 1:500); NPC1L1, kindly provided by Helen Hobbs and Jonathan Cohen (UT-Southwestern Medical Center), generated in rabbits by injection of Escherichia coli-expressed recombinant NPC1L1 protein fragments corresponding to amino acids 29-250 and 404-611 per GenBankTM accession number NP_997125 [GenBank] . This antiserum recognizes a ~200-kDa protein in intestine samples of wild-type but not NPC1L1-null mice,2 ANXA2 (Santa Cruz Biotechnology), and ERK1/2 (Upstate Group LLP, Charlottesville VA). Proteins were visualized by sequential treatment with specific antibodies, horseradish peroxidase-conjugated secondary antibodies, and an ECL kit (Amersham Biosciences). Signal intensity of bands on autoradiograms was measured by densitometry using a Molecular Dynamics Densitometer Model 300A.

RNA Measurement
Northern Analysis—Equal quantities of total RNA from the samples of each group were pooled, and poly(A)+ RNA was purified using oligo(dT)-cellulose columns (Pharmacia Biotech). mRNA (5 µg/lane) was size fractionated on a 1% formaldehyde-agarose gel and transferred to nylon membrane (Zetaprobe, Bio-Rad) for probing with 32P-labeled cDNAs. Probes for ANXA2 and NPC1L1 were generated by reverse transcription-PCR using RNA isolated from mouse duodenum as template and the following primers: ANXA2-F, 5'-gctctcagcgatacgtgc; ANXA2-R, 5'-gagcgaagtctctagaacg (yielding an ANXA2 product consisting of nucleotides 31-1167, GenBankTM NM_007585 [GenBank] ); NPC1L1-F, 5'-agacgagggttatcactagag; NPC1L1-R, 5'-atttataaataccttggccata (yielding full-length mouse NPC1L1 per GenBankTM XM_137497, a fragment generated by PstI digestion containing nucleotides 658-1446 was used as a probe). A second cDNA produced against the 3'-untranslated region of NPC1L1 gave identical results by Northern analysis and was generated using the same reverse primer and NPC1L1-F2, 5'-aatggagtaggagcttgtc (yielding a product containing nucleotides 4127-4572). HMG-CoA Syn cDNA was provided by Michael Brown and Joseph Goldstein (UTSW, (25)), ABCG5 cDNA was provided by Helen Hobbs (UTSW, (26)), and the CAV1 cDNA probe contained the full-length human CAV1 transcript (27).

Quantitiative Real Time PCR—Quantitiative real time (qRT)-PCR was performed using an Applied Biosystems Prism 7900HT sequence detection system as described (28). Briefly, total RNA was treated with DNase I (RNase-free, Roche Molecular Biochemicals), and reverse-transcribed with random hexamers using SuperScript II (Invitrogen) to generate cDNA. Primers for each gene were designed using Primer Express Software (PerkinElmer Life Sciences) and validated by analysis of template titration and dissociation curves. Primer sequences are available upon request. Each qRT-PCR contained (final volume of 10 µl) 25 ng of reverse-transcribed RNA, each primer at 150 nM, and 5 µl of 2X SYBR Green PCR Master Mix (Applied Biosystems), and each sample was analyzed in triplicate. Results were evaluated by the comparative CT method (User Bulletin No. 2, PerkinElmer Life Sciences) using cyclophilin as the invariant control gene. RNA levels are expressed relative to those obtained for the wild-type mice fed the basal diet and reflect the average ± S.E. for n = 5-6 animals/group.

Statistical Analysis of Data—Data are reported as the mean ± S.E. for the specified number of animals. GraphPad Prism software (GraphPad, San Diego, CA) was used to perform all statistical analyses. If unequal variance was indicated by Bartlett's test, log transformation was performed prior to statistical analysis. Two-way analysis of variance was used (factors: genotype, drug). In no instance was a significant interaction of factors observed; therefore, statistical differences for each factor are fully described in each figure legend: *, p < 0.05; **, p < 0.01; ***, p < 0.001.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To investigate the role of CAV1 in cholesterol homeostasis, we evaluated a number of metabolic parameters indicative of cholesterol absorption and trafficking in intact mice. In addition, we measured the RNA and protein levels of key transporters, enzymes, and trafficking proteins in intestine and liver to corroborate the metabolic changes observed in these mice. Cav1 null mice and their wild-type controls were fed a standard rodent diet containing 4% total lipid and 0.02% cholesterol for 15 days. Some mice received this diet supplemented with 10 mg of ezetimibe/kg of body weight. This dose of ezetimibe has previously been established as effective in mice and results in a greater than 90% decrease in fractional cholesterol absorption (29, 30). Stool samples were collected from days 7-10 to measure fecal sterol and bile acid excretion, and mice received an intragastric dose of labeled sterols on day 12 to measure cholesterol absorption by the fecal dual isotope method (20, 21). After completion of the 15-day dietary regimen, mice were anesthetized and exsanguinated, and tissue samples were obtained.

Western blot analyses were performed using whole-cell lysates prepared from the proximal third of the small intestine of individual mice (Fig. 1). The genotype of mice used in the study was confirmed by the absence of the 22-kDa CAV1 protein (5) in samples from the Cav1 knock-out mice. Importantly, there was no evidence of any immunoreactive CAV1 polypeptide fragment generated in these mice as assessed by a variety of polyclonal antisera that target the amino terminus (pAb1) through the carboxyl terminus (pAb3). This was necessary, as a truncated CAV1 transcript was still evident by Northern analysis in these samples (Fig. 2C). The absence of invaginated caveolae in vascular endothelial cells (data not shown) further confirm the deletion of this gene product. The inclusion of ezetimibe in the diet had no effect on the CAV1 protein levels measured in the wild-type mice.

NPC1L1 was detected as a large protein (between 150 and 200 kDa) by a novel polyclonal antibody. This protein size is in agreement with that described for rat and human NPC1L1 (31, 32). The specificity of the NPC1L1 antiserum was confirmed by immunoblotting of intestinal samples from wild-type and Npc1l1 knock-out mice, with no detected protein in the null animals.2 Protein quantification by densitometry revealed that neither the Cav1 genotype nor the ezetimibe treatment had a significant effect on NPC1L1 protein levels (WT control diet, 0.68 ± 0.05 density units relative to loading standard ERK1/2; WT EZ, 0.80 ± 0.20; CAV1-/-control, 0.56 ± 0.09; CAV1-/-EZ, 0.66 ± 0.15, n = 5-6 mice/group).

Blotting with an ANXA2 polyclonal antiserum revealed three protein bands, with the most prominent (~37 kDa) in agreement with previous reports (11). The larger molecular weight forms could be a result of ANXA2 ubiquitination as has been observed with extracts from intestinal mucosa (33). Densitometric measurement of the principle ANXA2 band indicated that neither the Cav1 genotype nor the ezetimibe treatment resulted in altered ANXA2 expression levels (WT control diet, 0.18 ± 0.07 density units relative to loading standard ERK1/2; WT EZ, 0.30 ± 0.10; CAV1-/-control: 0.24 ± 0.07; CAV1-/-EZ, 0.19 ± 0.09, n = 5-6 mice/group). In summary, the Western analyses shown in Fig. 1 conclusively demonstrate that the absence of CAV1 does not affect the protein levels of NPC1L1 and ANXA2 in the mouse intestine. In addition, treatment of mice with the cholesterol absorption inhibitor ezetimibe does not alter duodenal levels of NPC1L1 and ANXA2 proteins.



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  		FIG. 1.
NPC1L1 and annexin-2 protein levels are unaltered in Cav1 null mice and unaffected by ezetimibe feeding. Wild-type (+) and Cav1-/- (-) mice were fed a powdered basal diet with or without ezetimibe (to provide 10 mg/kg of body weight/day) for 15 days. Protein was isolated from mucosa of the proximal third of the mouse small intestine. 60 µg of total protein was loaded in each lane, and analyzed by Western blot analysis using polyclonal antisera specific for CAV1 (pAb1 targets the amino terminus, pAb2 detects the central region of the protein, and pAb3 detects carboxyl-terminal amino acids), NPC1L1, and annexin-2 (ANXA2). ERK1/2 protein levels were used to assess equal sample loading. Shown are the representative results for three (of six analyzed) mice/group.

 
Additional evaluation of NPC proteins, CAV isoforms, and gene products important for enterocyte cholesterol balance was performed by using qRT-PCR to measure RNA levels (Fig. 2, A and B). CAV1 RNA was not detected in the Cav1 knock-out mice by this method. CAV2 and CAV3 levels were unchanged by either genotype or drug treatment. The cycle numbers needed to amplify the CAV PCR products allow for a rough approximation of relative expression of these three isoforms, and suggest that CAV1 is most abundant (CT = 24.8) and CAV3 RNA (CT = 30.9) most sparse in the mouse intestine. This is in agreement with immunoblot analyses that suggest a similar relative expression of these CAV family members in the mouse intestine (9). RNA levels of the Niemann-Pick type C proteins NPC1 and NPC2, involved in the movement of endocytosed LDL-cholesterol from the lysosome/late endosome into the cytosol of cells, were unaffected by the absence of CAV1 or treatment with ezetimibe.

The duodenal mRNA levels for acyl-CoA cholesterol acyltransferase-2 (ACAT2), the principle cholesterol-esterifying enzyme of the intestine, and the microsomal triglyceride transfer protein, a protein essential for chylomicron assembly, were similar in all groups of mice (Fig. 2B). The scavenger receptor type BI did not exhibit a change in RNA levels by drug or Cav1 genotype. Several mRNA species whose expression is under the regulation of sterol-sensing transcription factors exhibited altered levels following ezetimibe administration. The liver X receptors, LXR{alpha} and LXR{beta}, both present in intestine and activated by cholesterol-derived oxysterols, regulate the expression of ABCA1 and ABCG5 (34). Sterol regulatory element-binding protein-2 (SREBP-2) undergoes proteolytic cleavage under low intracellular sterol levels to release its transcription factor to the nucleus to increase transcription of HMG-CoA Syn and the LDL-receptor (35). The observed gene expression changes (ABCA1, ABCG5, HMG-CoA Syn, and LDLR) strongly suggest that ezetimibe treatment results in diminished intracellular sterol levels in the enterocyte and agrees with similar observations made using an ezetimibe analog (36). Importantly, these gene changes were observed in both wild-type and Cav1 knock-out mice.



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  		FIG. 2.
Relative expression of various genes involved with cholesterol handling in the duodenum. Wild-type and Cav1-/- mice were fed a powdered basal diet with or without ezetimibe (EZ) (to provide 10 mg/kg of body weight/day) for 15 days. RNA was isolated from mucosa of the proximal third of the mouse small intestine (see B for legend). A and B, mRNA levels were determined for duodenum of individual mice by quantitative real time PCR using SYBR green chemistry with gene-specific primers and calculated by the comparative CT method using cyclophilin as the invariant control. The average cycle number at threshold is inset for the control group (wild-type mice, basal diet), note, cyclophilin CT = 20.3. Values represent the mean ± S.E. of data from 5-6 mice/group. Statistical analyses were performed by two-way analysis of variance with genotype and ezetimibe treatment as the two factors. No significant interaction of drug and genotype was observed for any mRNA species measured, and aside from CAV1 mRNA levels, no other RNA species exhibited different levels because of Cav1 deletion. The asterisks denote significant difference due to ezetimibe treatment. *, p < 0.05; ***, p < 0.0001. C, Northern analysis for selected mRNAs was also performed using 5 µg of poly(A)+ RNA/lane purified from a pooled sample obtained from 5-6 mice/treatment. MTP, microsomal triglyceride transfer protein; SR-BI, scavenger receptor class B type I.

 
Northern analysis was performed for selected genes (Fig. 2C) to detect potential alternative splice variants and to corroborate qRT-PCR results. A truncated CAV1 transcript of 2.5 kb was detected in the knock-out mouse intestine. This mRNA was not amplified by qRT-PCR, as one of the PCR primers was directed against exon 2, which was deleted in the targeting vector used to generate this knock-out mouse strain. However, as shown in Fig. 1, no detectable CAV1 protein is produced by this aberrant transcript. A single band of 5.2 kilobases was detected for NPC1L1 in intestine (and not in liver, data not shown), allowing it to be easily distinguished from related family members NPC1 (5.8 kb (37)) and NPC2 (1.3 kb (38)). Northern analyses of ABCG5 showed decreased expression upon treatment with ezetimibe, whereas HMG-CoA Syn was increased by drug treatment. These data are consistent with the results obtained by qRT-PCR.

Total plasma cholesterol concentrations were significantly greater in the Cav1 null mice, a trend previously observed in male mice of an independently generated strain of Cav1-deficient mice (Fig. 3A) (19). The majority of this elevated serum sterol is present in a more buoyant, LDL/IDL-particle (fractions 35-42, Fig. 3C). Ezetimibe treatment did not evoke a significant decrease in plasma cholesterol, although there appeared to be a trend toward lower values. A reduction in serum cholesterol levels following ezetimibe administration has, to date, only been observed in mouse strains that exhibit hyper-cholesterolemia, such as apoE or Ldlr knock-out mice (29, 30, 36). Hepatic total cholesterol levels did not differ by genotype or drug treatment (Fig. 3B). Fractional cholesterol absorption was dramatically reduced by ezetimibe treatment (Fig. 3D), and the values obtained for the C57/Bl6 wild-type mice are nearly identical to those previously reported in this mouse strain at this dose (29). There was no indication that CAV1 plays a critical role in cholesterol absorption as the Cav1 knock-out mice showed similar cholesterol absorption efficiency in the absence of drug and responded similarly to ezetimibe treatment. With this block in cholesterol absorption, fecal excretion of neutral sterols was increased nearly 3-fold in ezetimibe-treated mice (Fig. 3E). There was no change in fecal bile acid excretion between the wild-type and Cav1 null mice, and ezetimibe did not affect bile acid excretion (Fig. 3F). As bile acid excretion is generally a good indicator of bile acid synthesis, these findings suggest that although ezetimibe has dramatic effects on entero-hepatic cholesterol balance, under the conditions of this study bile acid metabolism is unaffected.



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  		FIG. 3.
Sterol metabolism in wild-type and caveolin-1 knock-out mice is similar, and ezetimibe works comparably to block cholesterol absorption. Male mice at 3 months of age were fed a basal rodent diet (Teklad 7001) with or without added ezetimibe (EZ) to provide 10 mg/kg body weight daily for 15 days. A, plasma cholesterol was greater in Cav1-/- mice than wild-type mice (p < 0.01). Ezetimibe treatment had no significant effect on plasma cholesterol. B, liver total cholesterol levels did not differ by genotype or ezetimibe treatment. C, plasmas from the 6 mice/group were pooled and fractionated by fast protein liquid chromatography using a Superose 6 column. The cholesterol content of each fraction was determined. D, cholesterol absorption efficiency was determined by the fecal dual-isotope method and found to be similar in wild-type and knock-out mice. Ezetimibe caused a significant decrease in absorption (p < 0.001), regardless of genotype. E, fecal neutral sterol excretion was greater in mice treated with ezetimibe (p < 0.001). F, fecal bile acid excretion was similar in all groups.

 
Plasma triglyceride levels were elevated in the knock-out mice, as reported previously (Fig. 4A) (19), but unaffected by drug treatment. Plasma non-esterified fatty acid concentrations were no different by genotype or ezetimibe administration. Liver mass (as percent of body weight) was modestly, but significantly, greater in Cav1 null mice, although this could not be attributed to an increase in hepatic triglyceride levels (no differences among groups, Fig. 4B) or cholesterol levels (Fig. 3B). Further evaluation of liver phenotype was performed by qRT-PCR measurement of RNA levels for critical genes (Fig. 5). CAV1 mRNA is less abundantly expressed in liver (CT = 27.8) than small intestine (CT = 24.8, Fig. 2A), in agreement with reports on relative protein levels in these two mouse tissues (9). CAV2 RNA levels were modestly, but significantly, reduced in the Cav1 null mice. This is consistent with previous reports that CAV2 protein levels are diminished upon the loss of CAV1 in other tissues (lung, adipose, heart) of the Cav1 knock-out mouse (18, 39). CAV3 mRNA was undetectable (CT > 35 cycles) in liver. These results suggest that the mild to absent hepatic phenotype in the Cav1 null mouse is not because of a compensatory increase of another caveolin family member. Among the other RNA species measured, only HMG-CoA Syn showed a significant change, an increase in ezetimibe-treated mice. The decreased delivery of cholesterol from intestine to liver in the ezetimibe-treated mouse is accompanied by a compensatory increase in hepatic cholesterol synthesis (30, 36). In the chow-fed mice, it appears that this newly synthesized sterol is sufficient to restore basal hepatic concentrations (Fig. 3B), and no further changes in sterol-sensitive gene expression (i.e. LXR target genes, ABCG5, angiopoietin-like protein 3, SREBP-1c, carbohydrate-responsive element binding protein) are observed in the liver.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Cholesterol homeostasis is maintained by a fine balance between cholesterol acquisition (synthesis and absorption) and cholesterol elimination (fecal excretion of cholesterol and bile acids) (40). A wide variety of agents have been identified that affect these processes to ultimately reduce serum LDL cholesterol levels with the goal of lowering the incidence of athero-genesis and coronary events. Statins clearly reduce cholesterol biosynthesis, decrease LDL cholesterol levels, and reduce the mortality and morbidity associated with coronary heart disease (41). Several additional agents have been identified that reduce cholesterol absorption, and the elucidation of the mechanisms of action for these absorption inhibitors has identified key proteins involved in the processes that move free cholesterol from the lumen of the intestine into the enterocyte, where it is esterified by ACAT2 and ultimately packaged into chylomicrons for delivery into the lymphatic circulation.



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  		FIG. 4.
Plasma and liver lipid profiles show similar patterns in wild-type and Cav1-/- given ezetimibe. Mice were fed a powdered basal diet with or without ezetimibe (EZ) (to provide 10 mg/kg of body weight/day) for 15 days. A, plasma triglyceride levels were significantly greater in Cav1 knock-out mice (p < 0.005), but no effect of ezetimibe was observed. Plasma non-esterified fatty acid levels were similar in all groups. B, liver mass was statistically greater in Cav1-/- mice than wild-type (p < 0.01). There was no significant effect of ezetimibe. Liver triglyceride concentration (expressed as mg/g wet liver weight) was not different among groups. Values represent the mean ± S.E. of data from 5-6 mice/group. Statistical analyses were performed by two-way analysis of variance.

 
Several ACAT inhibitors have been characterized and have demonstrated the critical role of this enzyme in cholesterol absorption in animal models (42). Furthermore, the deletion of Acat2 in mice results in decreased cholesterol absorption (43, 44). Agonists of the nuclear hormone receptors retinoid X receptor and LXR are potent cholesterol absorption inhibitors (45), and the search for the receptor target genes responsible for this effect revealed the ABC transporters ABCG5 and ABCG8 (26, 46), which reside on the apical membrane of enterocytes to efflux free cholesterol back into the lumen, thereby reducing cholesterol absorption efficiency. Overexpression of ABCG5/G8 results in decreased cholesterol absorption efficiency (47) and deletion of Abcg5/g8 is associated with decreased fecal excretion of sterols (48). Finally, ezetimibe, a potent cholesterol absorption inhibitor, has been found to act in the enterocyte by a mechanism involving NPC1L1 (2). Mice lacking Npc1l1 exhibit diminished cholesterol absorption efficiency (3, 31) and show no further reduction following ezetimibe treatment (2).



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  		FIG. 5.
Relative expression of various genes of hepatic lipid metabolism. Wild-type and Cav1-/- mice were fed a powdered basal diet with or without ezetimibe (EZ) (to provide 10 mg/kg of body weight/day) for 15 days. A and B, mRNA levels were determined for liver of individual mice by quantitative real-time PCR using SYBR green chemistry with gene-specific primers and calculated by the comparative CT method using cyclophilin as the invariant control. The average cycle number at threshold for the control group (wild-type mice, basal diet) is provided for each gene to allow for an approximate comparison of relative abundance of different RNAs (note, cyclophilin CT = 19.2). Values represent the mean ± S.E. of data from 5-6 mice/group. Statistical analyses were performed by two-way analysis of variance with genotype and ezetimibe treatment as the two factors. No significant interaction of drug and genotype was observed for any mRNA species measured. The only statistically significant differences observed were CAV2 mRNA reduction due to Cav1 deletion (*, p < 0.05) and elevated HMG-CoA Syn expression in mice treated with ezetimibe (*, p < 0.05). INSIG, insulin-induced gene; ChREBP, carbohydrate-responsive element-binding protein.

 
Additional proteins involved in cellular cholesterol trafficking in the intestine could also be important in cholesterol absorption. Caveolin-1 is such a candidate as it is expressed in the intestine, binds cholesterol, and is known to move among cellular compartments. It has also been reported that the behavior of caveolin-1 is altered in the intestine of zebrafish and mice after exposure to ezetimibe (4). A detergent-resistant complex of CAV1 and annexin-2 is disrupted by prior treatment of these animals with ezetimibe, strongly suggesting that CAV1 and/or annexin-2 could play a role in cholesterol absorption. The studies described in this report demonstrate that the absence of CAV1 does not affect the intestinal expression of NPC1L1, ACAT2, ABCG5/G8, or other previously identified proteins involved in cholesterol absorption. Nor does the deletion of CAV1 result in a compensatory change in CAV2 or CAV3. In response to ezetimibe, similar changes in expression were observed in Cav1 knock-out mice and wild-type controls. NPC1L1 and ANXA2 levels remained unchanged upon treatment of mice with ezetimibe, unlike SREBP target genes (i.e. HMG-CoA Syn, LDLR) or LXR target genes (i.e. ABCA1, ABCG5), which showed increased or decreased expression, respectively. This suggests that NPC1L1 and ANXA2 expression may be dependent upon other mechanisms than the SREBP or LXR sterol-sensing pathways. Interestingly, we found that treatment with ezetimibe completely eliminated cholesterol absorption (fractional absorption was approximately zero), suggesting that the ezetimibe-sensitive pathway could account for all, or nearly all, intestinal cholesterol absorption under these conditions. Finally, mice devoid of CAV1 show normal fractional cholesterol absorption and are fully sensitive to ezetimibe. These findings demonstrate that caveolin-1 is not required for intestinal cholesterol absorption in the mouse model.


   FOOTNOTES
 
* This work was supported by grants from the American Heart Association-Texas Affiliate (to J. J. R.), Grants GM07062 (to M. A. V.), HL20948 (to R. G. W. A.), GM52016 (to R. G. W. A.), and HL58888 (to P. W. S.) from the National Institutes of Health, The Perot Family Foundation (to R. G. W. A.), and the Cecil H. Green Distinguished Chair in Cellular and Molecular Biology (to R. G. W. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Depts. of Physiology and Internal Medicine, Touchstone Center for Diabetes Research, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8854. Tel.: 214-648-9431; Fax: 214-648-9191; E-mail: joyce.repa{at}utsouthwestern.edu.

1 The abbreviations used are: CAV1, caveolin-1; WT, wild type; HMG-CoA Syn, 3-hydroxy-3-methylglutaryl-coenzyme A synthase; qRT-PCR, quantitative real time PCR; ERK1/2, extracellular signal-regulated kinase 1/2; EZ, ezetimibe; ANXA2, annexin-2; LDL, low density lipoprotein; LDLR, LDL receptor; ACAT2, acyl-CoA cholesterol acyltransferase-2; LXR, liver X receptor; SREBP, sterol regulatory element-binding protein; ABC, ATP-binding cassette transporter. Back

2 H. Hobbs and J. Cohen, personal communication. Back


   ACKNOWLEDGMENTS
 
The caveolin-1 knock-out mice were generously provided by Timothy C. Thompson (Baylor College of Medicine, Houston TX). We thank Helen Hobbs and Jonathan Cohen for supplying the NPC1L1-specific polyclonal antiserum. cDNA probes used in Northern analyses were provided by Michael Brown, Joseph Goldstein, and Helen Hobbs. We thank Stephen Turley, Stephen Osterman, and Heather Waddell for their assistance with the cholesterol balance measurements. We thank John W. Thomas for critical proofreading of the manuscript.



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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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