J. Lipid Res.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1194/jlr.M600439-JLR200 on November 27, 2006

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M600439-JLR200v1
48/3/699    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Telford, D. E.
Right arrow Articles by Huff, M. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Telford, D. E.
Right arrow Articles by Huff, M. W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Journal of Lipid Research, Vol. 48, 699-708, March 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

The molecular mechanisms underlying the reduction of LDL apoB-100 by ezetimibe plus simvastatinboxs

Dawn E. Telford*, Brian G. Sutherland*, Jane Y. Edwards*, Joseph D. Andrews*, P. Hugh R. Barrett{dagger} and Murray W. Huff1,*

* Vascular Biology, Robarts Research Institute, The University of Western Ontario, Canada
{dagger} School of Medicine and Pharmacology, University of Western Australia, Perth, Australia

boxs The online version of this article (available at http://www.jlr.org) contains an additional table and three figures. Back

Published, JLR Papers in Press, November 27, 2006.

1 To whom correspondence should be addressed. e-mail: mhuff{at}uwo.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The combination of ezetimibe, an inhibitor of Niemann-Pick C1-like 1 protein (NPC1L1), and an HMG-CoA reductase inhibitor decreases cholesterol absorption and synthesis. In clinical trials, ezetimibe plus simvastatin produces greater LDL-cholesterol reductions than does monotherapy. The molecular mechanism for this enhanced efficacy has not been defined. Apolipoprotein B-100 (apoB-100) kinetics were determined in miniature pigs treated with ezetimibe (0.1 mg/kg/day), ezetimibe plus simvastatin (10 mg/kg/day), or placebo (n = 7/group). Ezetimibe decreased cholesterol absorption (–79%) and plasma phytosterols (–91%), which were not affected further by simvastatin. Ezetimibe increased plasma lathosterol (+65%), which was prevented by addition of simvastatin. The combination decreased total cholesterol (–35%) and LDL-cholesterol (–47%). VLDL apoB pool size decreased 26%, due to a 35% decrease in VLDL apoB production. LDL apoB pool size decreased 34% due to an 81% increase in the fractional catabolic rate, both of which were significantly greater than monotherapy. Combination treatment decreased hepatic microsomal cholesterol (–29%) and cholesteryl ester (–65%) and increased LDL receptor (LDLR) expression by 240%. The combination increased NPC1L1 expression in liver and intestine, consistent with increased SREBP2 expression. Ezetimibe plus simvastatin decreases VLDL and LDL apoB-100 concentrations through reduced VLDL production and upregulation of LDLR-mediated LDL clearance.

Supplementary key words cholesterol absorption • lipoproteins • apolipoprotein B kinetics • gene expression

Abbreviations: apo B, apolipoprotein B; CE, cholesteryl ester; FC, free cholesterol; FCR, fractional catabolic rate; IG, intragastric gavage; LDLR, LDL receptor; NPC1L1, Niemann-Pick C1-like 1 protein; NS, not significant; qRT-PCR, quantitative real-time PCR; TC, total cholesterol; TG, triglyceride


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increased concentrations of LDL-cholesterol represent a major risk factor for atherosclerosis (1, 2). Clinical trials demonstrate that reduction of plasma LDL-cholesterol by diet and/or drugs constitutes a primary strategy for the prevention and regression of coronary heart disease (1, 2). LDL concentrations are regulated primarily in the liver; it is the source of VLDL, the precursor of plasma LDL, and is responsible for the majority of LDL clearance via the LDL receptor (LDLR) (3). Apolipoprotein B-100 (apo B-100) kinetic studies in patients with primary hypercholesterolemia and combined hyperlipidemia demonstrate that LDL-cholesterol concentrations are determined by the production and catabolism of LDL particles (4, 5). HMG-CoA reductase inhibitors (statins) decrease cholesterol synthesis primarily in the liver (3), resulting in reduced hepatocyte cholesterol concentrations and, ultimately, upregulation of the LDLR and enhanced clearance of LDL (as reviewed in Ref. 6). In some patients, statins decrease rates of VLDL production (7).

The liver also clears cholesterol absorbed from the small intestine, via uptake of chylomicron remnants (3, 8). In humans, approximately one third of cholesterol entering the small intestine is of dietary origin, whereas two thirds is derived from bile (9). Inhibition of cholesterol absorption as a therapeutic target has gained considerable attention with the introduction of ezetimibe, the first of a new class of selective cholesterol absorption inhibitors (10–13).

Recent research (14–16) has revealed that ezetimibe specifically inhibits the transport of cholesterol and phytosterols across the brush border membrane of the intestinal enterocyte by blocking the transport function of its molecular target, Niemann-Pick C 1-like 1 protein (NPC1L1), (as reviewed in Ref. 17). Triglyceride and fat-soluble vitamin absorption are unaffected (11). Less cholesterol is transported in chylomicrons, implying a reduction in cholesterol reaching the liver via chylomicron remnants (14–17). Reduced hepatic cholesterol has been reported in ezetimibe-treated mice (14, 18) and dogs (12), and in mice, increases in hepatic LDLR expression were observed (18). However, it has not been determined how the molecular mechanisms regulating cholesterol homeostasis in the liver and intestine in response to ezetimibe alter the kinetics of apoB metabolism. Recent apoB-100 kinetic studies demonstrated that in men with primary hypercholesterolemia, ezetimibe decreased LDL-cholesterol 22%, primarily through enhanced fractional catabolic rates (FCRs) of VLDL, intermediate density lipoprotein (IDL), and LDL, suggesting upregulation of hepatic LDLR activity (19).

Inhibition of cholesterol absorption in mice by ezetimibe leads to increases in cholesterol synthesis in intestine and liver (14, 16, 18). Potentially, this would attenuate reductions in hepatic cholesterol, limiting the extent of LDLR expression and ultimately the magnitude of LDL-cholesterol lowering. Thus, combining ezetimibe and a statin would enhance LDL-cholesterol lowering, through complementary mechanisms of action (8, 20). The combination enhances LDL-cholesterol reductions in dogs (12), and in clinical trials, combination therapy decreases LDL-cholesterol to a significantly greater extent than does either agent alone (21, 22). However, the mechanism whereby this combination modulates the kinetics of apoB metabolism has not been investigated. Furthermore, the impact of ezetimibe plus a statin on the expression of genes important for the regulation of LDL metabolism in the liver and intestine has not been defined.

We examined the effect of ezetimibe alone or in combination with simvastatin on apoB-100 kinetics in miniature pigs, a large-animal model of lipoprotein metabolism. Ezetimibe plus simvastatin decreased VLDL and LDL apoB-100 concentrations; ezetimibe inhibited cholesterol absorption, and simvastatin blocked the increase in cholesterol synthesis observed with ezetimibe alone. This resulted in significant reductions of hepatic cholesterol and a synergistic increase in hepatic LDLR expression. Plasma apoB decreased significantly through a modest reduction in VLDL production and a greatly enhanced LDLR-mediated LDL clearance, both of which are attributed to the reduction in hepatic cholesterol.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and diets
Female pigs (Ja/Mar Farms; Port Dover, Ontario, Canada) weighing 25–30 kg were housed individually in metabolic cages and were fed a pig chow-based diet (600 g/day) containing 34% of calories from fat and 400 mg/day of cholesterol (0.1%, 0.2 mg/kcal). The diet mimics a standard Western-type human diet. The ratio of polyunsaturated to mono-unsaturated to saturated fat (1:1:1) was achieved by mixing lard, butter, and safflower oil (23). Pigs were studied as littermates, three at a time, and matched with respect to baseline total plasma cholesterol. Pigs were randomized to ezetimibe (0.1 mg/kg/day) monotherapy or ezetimibe plus simvastatin (10 mg/kg/day) or placebo (n = 7/group). Blood was obtained through surgically implanted jugular vein catheters in conscious, unrestrained animals (24). The pigs were fasted for 20–22 h prior to blood collection. The protocol was approved by the Animal Care Committee of the University of Western Ontario.

Administration of drugs
Ezetimibe and simvastatin were obtained from Merck-Frosst/Schering Canada, Montreal, Canada. Oral doses of powdered compound were placed in gelatine capsules and fed by hand. In a pilot dose-response study (n = 3 per group), daily doses of ezetimibe at 0.03 and 0.1 mg/kg/day decreased cholesterol absorption by 50% and 76% and reduced LDL-cholesterol by 22% and 30%, respectively, compared with control. In a previous study (24), we demonstrated in this model using the same protocol, that simvastatin at a dose of 10 mg/kg/day decreased LDL-cholesterol by 24%. This dose of simvastatin was chosen because it permitted comparison of the biochemical and kinetic parameters reported previously (24) with those obtained in the present study with ezetimibe alone or in combination with simvastatin. Therefore, ezetimibe (0.1 mg/kg/day) and simvastatin (10 mg/kg/day) were used for the apoB kinetic studies.

Analytical assays
Lipoprotein separations, plasma and lipoprotein lipid analyses, and isolation of apoB have been described previously (23–27). The concentration of apoB in plasma, VLDL, IDL, and LDL was determined by ELISA and confirmed by isopropanol precipitation (25). Lipoprotein cholesterol distributions were evaluated in serum samples (50 µl) from three pigs in each group that were resolved by size exclusion chromatography on a Superose 6 column (28). Plasma lathosterol and plant sterol concentrations were assayed by GC-MS as described previously (29).

Intestinal cholesterol absorption
The plasma dual isotope ratio method described by Turley, Herndon, and Dietschy (30) was modified for use in pigs. The required amounts of [1,2-3H]cholesterol (Amersham; Oakville, Canada) and [4-14C]cholesterol (Amersham) in toluene were evaporated to dryness under nitrogen in separate glass tubes and redissolved in absolute ethanol (1 µl per µCi of radioactivity). The [3H]cholesterol was added dropwise to undiluted 20% Intralipid (0.1 ml per µCi; Baxter Corporation, Toronto, Canada) while vortexing for 3 min. The [14C]cholesterol was added dropwise to safflower oil (0.2 ml per µCi) while vortexing for 3 min. Multiple 10 µl aliquots of each isotope were added directly to 10 ml of Ready Flow III (Beckmann Coulter, Inc.; Fullerton, CA) and counted in a liquid scintillation counter (Beckmann LS3801, Beckmann Coulter, Inc.) to confirm uniform distribution of the tracers in the Intralipid or safflower oil. Following a 22 h fast, pigs were anesthetized using preanesthetics described previously (24) at 9 AM. Each pig received 50 µCi of [14C]cholesterol by intragastric gavage (IG) and 50 µCi of [3H]cholesterol via the indwelling catheter (IV). Immediately upon recovery from the anesthetic (approximately 5 min), animals received their daily dose of placebo, ezetimibe, or ezetimibe plus simvastatin with the standard ration of food. The typical feeding/dosing regimen was continued for 5 days. Plasma samples were obtained every 24 h via the indwelling catheter just prior to feeding. One milliliter of each plasma sample was added directly to the scintillation cocktail and counted to confirm that the plasma decay curves of the two tracers were parallel. The percent cholesterol absorption was calculated from the 72 h plasma samples using the following expression: plasma cholesterol absorption = [percent of IG dose ([14C]cholesterol) per ml plasma] / [percent of IV dose ([3H]cholesterol) per ml plasma] x 100.

Kinetic studies
Pigs received a bolus of trideuterated leucine ([5,5,5-2H3]L-leucine, 3 mg/kg) via the indwelling catheter, following a 22 h fast (31). Sequential blood samples were obtained, and VLDL, IDL, LDL, and HDL were separated completely by ultracentrifugation. ApoB was obtained by isopropanol precipitation, delipidated, and hydrolyzed to amino acids (29). Leucine was derivatized to the oxazolinone, and isotopic enrichment was determined by GC-MS as described previously (29). Kinetic parameters of apoB-100 metabolism were analyzed using SAAM II (SAAM Institute; Seattle, WA) according to the model reported previously (31).

Tissue analyses
Approximately 22 h after the last dose of drug, pigs were euthanized, and samples of tissues were flash frozen and stored at –80°C. In liver and jejunum, the concentrations of triglyceride (TG), free cholesterol (FC), and cholesteryl ester (CE), and the incorporation of oleate into CE and TG were measured as previously described (27). Hepatic and jejunal microsomal lipids and HMG-CoA reductase activities were determined (27).

Tissue mRNA determination
RNA was isolated using Trizol reagent (Life Technologies; Mississauga, Ontario, Canada). Gene-specific mRNA quantitation for apoB, HMG-CoA reductase, LDLR, SREBP2, SREBP1c, NPC1L1, ABCG5, ABCG8, ABCA1, LXR{alpha}, MTP, and GAPDH was performed by quantitative real-time PCR (qRT-PCR) on an ABI Prism (model 7900 HT) Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions (31). Samples (20–75 ng) of reverse transcribed total RNA were assayed in triplicate in 20 µl reactions using a two-step qRT-PCR protocol and the standard curve method to estimate specific mRNA concentrations. Expression levels for each gene were normalized to GAPDH expression levels for each tissue type. For genes with known pig (Sus scrofa) sequences (APOB, HMGCR, LDLR, SREBP2, MTP, LXR{alpha}, ABCA1, and GAPDH), primer and probe sets were designed using Primer Express 2.0 software (Applied Biosystems) (31). For genes with unknown pig sequences (NPC1L1, ABCG5, ABCG8, and SREBP1c), PCR primers were designed against the mRNA sequence of the human homolog using PrimerQuest software (Integrated DNA technologies). The default settings were used, except that the desired PCR range was set to between 250 and 500 base pairs. PCR primers were tested with pig cDNA prepared using Superscript II reverse transcriptase (Invitrogen) and pig liver or intestine total RNA as per the manufacturer's instructions. PCR products of expected sizes were purified using the QIAquick PCR purification kit (Qiagen), and sequenced on a 3730 DNA analyzer (Applied Biosystems). A minimum of 85% homology of the PCR product sequence to the human sequence was used for verification. Pig-specific primer and probe sets were then designed using Primer Express 2.0. All primers were obtained from Sigma-Genosys, and the fluorogenic probes, labeled with 6-carboxyfluorescein, were obtained from Applied Biosystems. Primers and probes for qRT-PCR analyses are listed in Table 1 .


View this table:
[in this window]
[in a new window]

  		TABLE 1. Probes and primer sets used in quantitative real-time PCR in the livers and intestines of control, ezetimibe-, and ezetimibe plus simvastatin-treated miniature pigs

 
Statistical analyses
Differences between control, ezetimibe-, and ezetimibe plus simvastatin-treated animals were compared for statistical significance by paired t-test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ezetimibe monotherapy reduced plasma total cholesterol (TC) 22% (P < 0.007) and LDL-cholesterol 31% (P < 0.006) (Fig. 1 ). Ezetimibe plus simvastatin produced significantly greater reductions in both total (–35%, P < 0.045) and LDL-cholesterol (–47%, P < 0.012), compared with ezetimibe alone (Fig. 1A). Other lipids were unchanged. Ezetimibe decreased VLDL apoB 20% (P < 0.024) and LDL apoB 20% (P < 0.001) (Fig. 1B). The combination decreased VLDL apoB 26% [not significant (NS) compared with ezetimibe alone], whereas the 34% reduction in LDL apoB (P < 0.023) was significantly greater than ezetimibe monotherapy.


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 1. Plasma lipid, lipoprotein (A), and apolipoprotein (B) concentrations in control, ezetimibe-, and ezetimibe plus simvastatin-treated miniature pigs. Lipid values are the mean ± SEM of three determinations per animal, and apolipoprotein B (apoB) values are the means ± SEM of all samples obtained during the kinetic study from seven animals per group. Bars denoted by different letters are significantly different (P < 0.05).

 
Intestinal cholesterol absorption in control pigs was 48%, similar to mean values reported for humans (32). Ezetimibe inhibited absorption 79% (P < 0.001) (Fig. 2A ) and decreased plasma phytosterol concentrations 91% (P < 0.000001) (Fig. 2B), demonstrating its efficacy in pigs (14, 33). Ezetimibe plus simvastatin had no additional effect on these parameters. Plasma lathosterol levels, a marker of cholesterol synthesis, were increased 65% (P < 0.044) by ezetimibe, reflecting the increase in cholesterol synthesis induced by ezetimibe. Lathosterol concentrations decreased to control levels with the addition of simvastatin, reflecting inhibition of hepatic cholesterol synthesis. The ratios of plasma campesterol, sitosterol, and lathosterol to the concentration of plasma cholesterol are shown in Fig. 2C. The ratios showed a pattern of response similar to those of the absolute concentrations of each sterol to each of the treatments.


Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 2. Fractional cholesterol absorption rates (A), plasma lathosterol, campesterol, and ß-sitosterol (B) and the ratios of plasma lathosterol, campesterol, and ß-sitosterol to cholesterol (C) in control, ezetimibe-, and ezetimibe plus simvastatin-treated miniature pigs. Control values for lathosterol (2.5 µmol/l), campesterol (73.8 µmol/l), and ß-sitosterol (14.0 µmol/l) were set at 100%. Values represent the mean ± SEM from seven animals per group. Bars denoted by different letters are significantly different (P < 0.05).

 
Trideuterated leucine was injected as a bolus into fasted animals. ApoB kinetic parameters were determined from simultaneous analysis of all isotope enrichment data as described previously (31). A fit of VLDL and LDL apoB enrichment curves for one set of animals is shown in supplementary Figure I (available in the online data supplement). Ezetimibe significantly decreased VLDL apoB pool size 20% (P < 0.024); this decrease was attributed to a 27% decrease in VLDL apoB production (NS) (Table 2 ). Ezetimibe plus simvastatin decreased the VLDL apoB pool size 26% (P < 0.018), due entirely to a 35% decrease in VLDL apoB production (P < 0.017). Neither parameter was significantly different from ezetimibe alone. VLDL FCR, the amount of VLDL apoB converted to LDL apoB, and the amount cleared directly from plasma were not different. IDL apoB kinetic parameters were unchanged (data not shown).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Metabolic parameters of VLDL and LDL apoB metabolism in control, ezetimibe-, and ezetimibe plus simvastatin-treated miniature pigs

 
Ezetimibe monotherapy significantly decreased the LDL apoB pool size 20% (P < 0.001, Table 2) due to a 42% (P < 0.013) increase in the LDL apoB FCR, as total LDL apoB production was unaffected. The combination decreased the LDL apoB pool size 34% (P < 0.001) due entirely to an 81% (P < 0.002) increase in the LDL apoB FCR. Compared with ezetimibe alone, the combination enhanced the decrease in pool size 70% (P < 0.003) and increased the FCR 2-fold (P < 0.05). LDL apoB direct synthesis was unaffected. Total apoB production into plasma (VLDL plus LDL direct synthesis) decreased 19% with ezetimibe (NS) and 35% with combination treatment (P < 0.019).

VLDL and LDL were analyzed for lipid and protein content (see supplementary Table I). Ezetimibe produced small decreases in the percent of cholesterol in VLDL (NS), whereas the percent of TG increased 4%, (P < 0.036). These changes were greater with combination therapy. Ezetimibe decreased the LDL percentage of FC 5% (P < 0.014) and increased the percentage of TG and protein 44% (P < 0.023) and 11% (P < 0.022), respectively. These changes were greater with combination therapy. Other lipid percentages were unaffected. The TG/CE ratio (wt/wt) in LDL increased significantly.

Size exclusion chromatography demonstrated ezetimibe-induced reductions in serum VLDL and LDL-cholesterol, which were enhanced by the addition of simvastatin (Fig. 3 ). Peak elution volumes of cholesterol within each lipoprotein fraction and the ratio of surface to core lipids (see supplementary Table I) were unaffected by either treatment, indicating unchanged lipoprotein sizes.


Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 3. Lipoprotein cholesterol profiles in serum from control, ezetimibe-, and ezetimibe plus simvastatin-treated pigs. Serum (50 µl) was resolved by size exclusion chromatography using a Superose 6 column. Total cholesterol (TC) concentrations were determined in fraction numbers 11 to 45, with each fraction having a total volume of 500 µl. The plotted values represent the means of three pigs per group, (control, black circles; ezetimibe, shaded circles; ezetimibe plus simvastatin, open circles).

 
Ezetimibe decreased liver TC, FC, and CE 19% (P < 0.018), 15% (P < 0.048), and 36% (P < 0.022), respectively (Fig. 4A ). The combination significantly reduced hepatic TC, FC, and CE (–17%, P < 0.031; –12%, P < 0.046, and –39%, P < 0.031, respectively), but reductions were not different from ezetimibe alone. Compared with control, liver TGs were decreased by 21% (P < 0.042) and 19% (P < 0.047) by ezetimibe and the combination, respectively. Ezetimibe decreased hepatic microsomal TC (–25%, P < 0.010), FC (–21%, P < 0.050), and CE (–47%, P < 0.019) (Fig. 4B). The combination also reduced hepatic microsomal TC (–29%, P < 0.003), FC (–24%, P < 0.033), and CE (–65%, P < 0.009). Previously, we reported that in pigs treated with simvastatin monotherapy (10 mg/kg/day), liver microsomal FC, CE, and TG were unaffected (24). Ezetimibe decreased oleate incorporation into liver CE (–8%, NS) and TG (–14%, NS). The combination produced significant reductions in oleate incorporation into both liver CE (–18%, P < 0.013) and TG (–22%, P < 0.04) compared with control (Fig. 4C). In the jejunum, lipid concentrations (see supplementary Figure II) and oleate incorporation into CE and TG (Fig. 4C) were unaffected by either treatment.


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 4. Tissue lipids, lipid synthesis, and HMG-CoA reductase activities in control, ezetimibe-, and ezetimibe plus simvastatin-treated miniature pigs. TC, free cholesterol (FC), and triglyceride (TG) concentrations were quantitated in whole liver (A) and liver microsomes (B). Oleate incorporation into cholesteryl ester (CE) and TG were determined in homogenates (C). HMG-CoA reductase activity was measured in microsomes (D). Values represent the mean ± SEM from seven animals per group. Bars denoted by different letters are significantly different (P < 0.05).

 
Relative mRNA expression of genes involved in hepatic cholesterol metabolism was determined in liver and jejunum. Liver LDLR mRNA increased 1.7-fold (P < 0.030) with ezetimibe, and increased further to 3.4-fold (P < 0.032 vs. control; P < 0.048 vs. ezetimibe) with the combination (Fig. 5A ). Similar increases were observed in the jejunum (Fig. 5B). Previously, we reported that in pigs treated with simvastatin monotherapy (10 mg/kg/day), liver and jejunum LDLR mRNA both increased 1.3-fold (24). Liver mRNA for HMG-CoA reductase increased 4.1-fold (P < 0.006) with ezetimibe, and increased further to 13-fold (P < 0.050 vs. control) with the combination (Fig. 5A). HMG-CoA reductase activity increased 2.3-fold (P < 0.002) in hepatic microsomes of ezetimibe-treated pigs and increased 9.6-fold (P < 0.006) in microsomes from the combination group (Fig. 4D). Similar step-wise increases in mRNA and activity of HMG-CoA reductase were observed in the jejunum (Figs. 4D and 5B).


Figure 5
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 5. Tissue mRNA concentrations in control, ezetimibe-, and ezetimibe plus simvastatin-treated miniature pigs. Results are the ratio of the gene of interest relative to GAPDH, and values are expressed relative to control and were set at 100% in liver (A) and in intestine (B). Values represent the mean ± SEM of data from seven animals per group. Bars denoted by different letters are significantly different (P < 0.05).

 
NPC1L1 expression increased in jejunum (1.3-fold, P < 0.044) and liver (2.8-fold, P < 0.049) with ezetimibe (Fig. 5). The combination increased NPC1L1 expression in jejunum (1.4-fold, NS) and liver (3.1-fold, P < 0.047), relative to controls; however, values were not different from ezetimibe alone. Although the expression of NPC1L1 in jejunum relative to liver was 9:1 (data not shown), the enhanced expression of NPC1L1 in liver reflected the degree of tissue cholesterol depletion (Fig. 4).

Ezetimibe increased SREBP2 mRNA in liver 2.0-fold (P < 0.002) and in jejunum 1.5-fold. The combination increased SREBP2 mRNA 2.9-fold (P < 0.006) in liver and 2.7-fold (P < 0.034) in the intestine, compared with control. SREBP1c mRNA was significantly decreased by combination treatment in the liver. All other genes examined, including APOB (Fig. 5), MTP, LXR{alpha}, ABCG5, ABCG8, and ABCA1 (see supplementary Figure III) were unaffected.

Figure 6A summarizes the major apoB-100 kinetic parameters observed following ezetimibe and ezetimibe plus simvastatin treatment. VLDL apoB pool size decreased, primarily because of the reduced appearance of newly synthesized VLDL apoB in plasma. LDL apoB concentrations decreased, primarily because of an increased FCR. For comparison, Fig. 6B summarizes the percent change from control (no treatment) in apoB kinetic parameters obtained in the present study compared with parameters determined in a previous study using simvastatin monotherapy (10 mg/kg/day) (24). Although the same animal model and the same protocol were used in both studies, the former study used iodinated tracers, whereas the present study used deuterated leucine to trace apoB. Although this may have resulted in some quantitative differences in calculated parameters, qualitative comparisons of kinetic parameters are possible. Ezetimibe, simvastatin, and the combination all decreased VLDL apoB pool size because of decreased production. Addition of simvastatin to ezetimibe did not appear to enhance these reductions. Ezetimibe alone significantly reduced LDL pool size, owing entirely to an increased FCR. In contrast, simvastatin monotherapy decreased LDL apoB through decreased production, inasmuch as the increases in FCR (+7%) and hepatic LDLR mRNA (+26%) were modest. The addition of simvastatin to ezetimibe enhanced the reduction in LDL apoB pool size, because of a substantial synergistic increase in LDL apoB FCR.


Figure 6
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 6. A: Effects of ezetimibe and ezetimibe plus simvastatin treatment compared with control on apoB metabolism in miniature pigs. Circled numbers represent apoB pool sizes (mg/kg); numbers next to arrows represent mean transport rates (production rates, mg/kg/h), and numbers in italics next to arrows represent fractional catabolic rates (pools/h). Conversion values of apoB to LDL represent apoB from VLDL plus intermediate density lipoprotein. Values denoted by different letters are significantly different (P < 0.05). B: Comparison of the percent change from control (no treatment) for the same parameters of apoB metabolism shown in A in pigs treated with ezetimibe, simvastatin alone (24), or ezetimibe plus simvastatin.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Inhibition of intestinal cholesterol absorption by ezetimibe has the potential to decrease cholesterol transport to the liver via chylomicron remnants. Reduced hepatic cholesterol would enhance expression of the LDLR, resulting in decreased plasma LDL-cholesterol. However, hepatic cholesterol depletion is accompanied by a compensatory increase in hepatic cholesterol synthesis, making the addition of an HMG-CoA reductase inhibitor an ideal combination for LDL-cholesterol reduction through complementary mechanisms of action. This study demonstrates for the first time in a large-animal model of lipoprotein metabolism the molecular mechanisms underlying the reductions in both VLDL and LDL apoB. The combination of ezetimibe plus simvastatin decreased VLDL and LDL apoB-100 concentrations through reduced VLDL production and enhanced LDLR-mediated LDL clearance. Ezetimibe inhibited cholesterol absorption, and simvastatin blocked the increase in cholesterol synthesis observed with ezetimibe alone, resulting in a significant reduction in hepatic cholesterol and a marked increase in hepatic LDLR expression. Furthermore, we show that expression of NPC1L1 in both intestine and liver is increased with ezetimibe and combination treatment, consistent with regulation by intra-cellular cholesterol.

Previously, we documented reduced VLDL apoB secretion in pigs treated with atorvastatin (24, 25), simvastatin (24), or the oxidosqualene:lanosterol cyclase inhibitor RO-0717625 (31). Reduced VLDL apoB secretion has been linked to decreased availability of cholesterol for hepatic lipoprotein assembly (7, 24, 34). Decreased liver TC and CE in pigs treated with ezetimibe and ezetimibe plus simvastatin is consistent with this concept. VLDL secretion can also be influenced by hepatic TG concentrations, which were reduced by ezetimibe monotherapy. The addition of simvastatin produced no further decrease in liver cholesterol or TG concentrations, consistent with our previous pig studies in which simvastatin monotherapy had no effect on hepatic lipid concentrations (24). The ezetimibe-induced reduction in hepatic TG has also been observed in mice (18) and hamsters (35), and given that ezetimibe does not affect fat absorption (11), decreased hepatic TG is probably secondary to diminished delivery and accumulation of hepatic cholesterol. The contribution of decreased hepatic TG to the diminished VLDL apoB secretion is difficult to determine. Previous studies in this model have demonstrated quantitatively similar reductions in VLDL apoB production, which were correlated with diminished hepatic cholesterol concentrations, yet hepatic TG synthesis and concentrations were unchanged (24, 25, 27, 31). In hypercholesterolemic patients treated with ezetimibe, Tremblay et al. (19) reported increased VLDL apoB production despite a decrease in VLDL apoB concentrations and increased VLDL apoB FCR. The reason for the increased production is unknown but may reflect the patient type studied or the model used. The efficacy of ezetimibe for LDL-cholesterol reduction in patients with homozygous familial hypercholesterolemia (36), and in LDLR–/– mice (18), also supports a role for ezetimibe in reducing hepatic VLDL production.

Ezetimibe significantly decreased LDL apoB concentrations entirely as a result of enhanced clearance of LDL apoB from plasma, consistent with the significant increase in hepatic LDLR mRNA. This effect was significantly potentiated by simvastatin, producing additive reductions in plasma LDL apoB and synergistic increases in hepatic LDLR mRNA. Enhanced LDL apoB clearance was observed previously in pigs treated with atorvastatin (10 mg/kg/day) (15%) (24), but was unaffected in pigs treated with a lower dose of atorvastatin (3 mg/kg/day) (25) or simvastatin (10 mg/kg/day) (24). In contrast, inhibition of the apical sodium-dependent bile acid transporter decreased LDL apoB as a result of significant increases in LDL apoB FCR, 18% in monotherapy and 45% in combination with atorvastatin (27, 37), demonstrating that combining drugs with complementary mechanisms of action can amplify LDL apoB clearance.

Our finding that ezetimibe monotherapy decreases liver cholesterol has been reported in ezetimibe-treated wild-type and LDLR–/– mice fed a lipid-rich diet (18). Consistent with the present study, these authors reported a 2-fold increase in hepatic LDLR mRNA in wild-type mice; however, LDL apoB kinetics were not reported. In ezetimibe-treated LDLR–/– mice, it was concluded that ezetimibe decreased LDL-cholesterol primarily through reduced VLDL apoB production, although the latter was not measured directly (18). Our results with ezetimibe monotherapy are consistent with those of Tremblay et al. (19), where decreased LDL-cholesterol in patients with primary hypercholesterolemia treated with ezetimibe was due to enhanced LDL apoB clearance. We now demonstrate that this is due to increased hepatic LDLR expression consequent to diminished hepatic cholesterol. Furthermore, addition of simvastatin enhanced the effect of ezetimibe monotherapy by further increasing LDLR mRNA and LDL apoB clearance 2-fold, thus providing the molecular mechanism for the enhanced decrease in LDL-cholesterol observed in patients treated with ezetimibe plus a statin.

As anticipated, ezetimibe increased hepatic and intestinal HMG-CoA reductase expression and activity (14, 18), consistent with the increased plasma lathosterol levels, a marker of cholesterol synthesis. Increased plasma lathosterol has been reported in patients treated with ezetimibe (13). Elevated hepatic HMG-CoA reductase expression and cholesterol synthesis has been observed in ezetimibe-treated mice (16). This compensatory increase in cholesterol synthesis probably attenuates the increase in LDLR expression and reductions in plasma LDL-cholesterol. We observed that addition of simvastatin greatly increased the expression and activity of HMG-CoA reductase in liver and intestine. This anticipated effect of statins is the result of a positive feedback regulatory mechanism, mediated by enhanced activity of the transcription factor SREBP-2. The increased HMG-CoA reductase activity reflects statin removal during microsomal preparations (24). Enhanced ex vivo HMG-CoA reductase expression and activity have been reported previously in pigs treated with simvastatin or atorvastatin, and the extent of increase in ex vivo reductase activity was proportional to the extent of cholesterol synthesis inhibition in vivo (24). In the present study, addition of simvastatin was sufficient to inhibit the ezetimibe-induced increase in cholesterol synthesis as plasma lathosterol concentrations decreased to control levels with combination therapy.

A wide range of inter-individual variability in the LDL-cholesterol response to ezetimibe has been observed (38), and this almost certainly encompasses attenuation of the LDL-cholesterol-lowering effect of ezetimibe over time in some patients. Although the molecular mechanisms are unclear, it is possible that individuals who might experience ezetimibe tachyphylaxis have relatively greater compensatory upregulation of hepatic cholesterol synthesis than do patients whose LDL-cholesterol response remains stable with time. Patients who show attenuation of the LDL-cholesterol response to ezetimibe may thus be ideal candidates for combination therapy with statins, although more investigation would be required to substantiate this concept.

LDLR and HMG-CoA reductase expression is primarily regulated by SREBP2. SREBP2 mRNA increased significantly in both the liver and intestine of pigs treated with ezetimibe and increased even further with the combination. This provides a mechanism for the increased expression of both of these sterol-responsive genes. The expression of SREBP1c was decreased in liver, and although this provides a potential explanation for the decrease in hepatic TGs with both treatments, the mechanism is unclear, perhaps reflecting diminished hepatic cholesterol levels.

NPC1L1 expression was increased in both the jejunum and liver by ezetimibe and was increased further by the combination. These increases reflected cholesterol depletion in both tissues, consistent with transcriptional regulation by sterols via sterol-regulated elements within the NPC1L1 promoter (16). This suggests that its expression is regulated similarly to that of other genes coding for proteins involved in cholesterol metabolism, including LDLR and HMG-CoA reductase (39). This upregulation may have increased the tissue content of NPC1L1 transporters; however, there appeared to be sufficient ezetimibe for inhibition of NPC1L1-mediated sterol absorption, as evidenced by the approximately 80% reduction in cholesterol absorption and 91% decrease in plasma phytosterols following treatment. In contrast to mice and rats, human liver also expresses NPC1L1 (16, 40). Although expression of NPC1L1 in pig intestine is 9-fold higher than in liver, increased expression was observed in both tissues. The physiological significance of NPC1L1 in hepatocytes remains to be elucidated. The apical location of NPC1L1 in hepatoma cells predicts a canalicular distribution of NPC1L1 in vivo, which has been confirmed in monkey liver (41). Both enterocytes and hepatocytes are polarized cells with their apical surface exposed to micelles containing cholesterol. NPC1L1 mediates intestinal cholesterol absorption from micelles in the intestinal lumen, and it is tempting to speculate that it may also promote cholesterol reuptake from micelles in the canalicular bile. Furthermore, in species that express hepatic NPC1L1, including humans and pigs, the pharmacological efficacy of ezetimibe may be partially attributed to the blocking of this proposed canalicular reuptake mechanism (41).

In conclusion, inhibition of NPC1L1 by ezetimibe and inhibition of cholesterol synthesis by simvastatin in the pig results in significant reductions in LDL apoB-100 concentrations relative to monotherapy. Ezetimibe inhibited cholesterol absorption and simvastatin blocked the increase in cholesterol synthesis observed with ezetimibe alone, resulting in a significant reduction in hepatic cholesterol and a marked synergistic increase in hepatic LDLR expression. Plasma apoB was significantly decreased by a modest reduction in VLDL apoB production and a greatly enhanced LDLR-mediated LDL apoB clearance, both of which are attributed to the reduction in hepatic cholesterol. Therefore, inhibition of NPC1L1 by ezetimibe enhances LDL lowering compared with statins alone, through a distinct, yet complementary mechanism of action. These studies support the findings of recent clinical trials in which enhanced reductions of LDL-cholesterol achieved by combining ezetimibe with statin therapy greatly facilitated the ability of patients to reach new, more-stringent LDL-cholesterol targets.


   ACKNOWLEDGMENTS
 
This work was supported by Grants T-5603 and PRG-5967 from the Heart and Stroke Foundation of Ontario (M.W.H.), National Institutes of Health Grant NIBIB #P41 EB-001975 (P.H.R.B.), and Merck-Frosst/Schering Canada (M.W.H.). The authors thank Kevin Dwyer, Kim Thomaes, and Shannon Bull for technical assistance, and acknowledge Dr. Stewart Whitman and Mirela Hasu, University of Ottawa Heart Institute, for size exclusion chromatography.

Manuscript received October 3, 2006 and in revised form November 21, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
  1. Grundy, S. M., J. I. Cleeman, C. N. Merz, H. B. J. Brewer, L. T. Clark, D. B. Hunninghake, R. C. Pasternak, S. C. J. Smith, and N. J. Stone. 2004. Implications of recent clinical trials for the National Cholesterol Education Program Adult Treatment Panel III guidelines. Circulation. 110: 227–239.

  2. Genest, J., J. Frohlich, G. Fodor, and R. McPherson. 2003. Recommendations for the management of dyslipidemia and the prevention of cardiovascular disease: summary of the 2003 update. CMAJ. 169: 921–924.[Free Full Text]

  3. Dietschy, J. M., S. D. Turley, and D. K. Spady. 1993. Role of the liver in the maintenance of cholesterol and low-density lipoprotein homeostasis in different animal species, including humans. J. Lipid Res. 34: 1637–1659.[Medline]

  4. Kesaniemi, Y. A., W. F. Beltz, and S. M. Grundy. 1985. Comparisons of metabolism of apolipoprotein B in normal subjects, obese subjects and patients with coronary heart disease. J. Clin. Invest. 76: 586–595.[Medline]

  5. Parhofer, K. G., and P. H. Barrett. 2006. What we have learned about VLDL and LDL metabolism from human kinetic studies. J. Lipid Res. 47: 1620–1630.[Abstract/Free Full Text]

  6. Ginsberg, H. N. 2006. Efficacy and mechanisms of action of statins in the treatment of diabetic dyslipidemia. J. Clin. Endocrinol. Metab. 91: 383–392.[Abstract/Free Full Text]

  7. Huff, M. W., and J. R. Burnett. 1997. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and hepatic apolipoprotein B secretion. Curr. Opin. Lipidol. 8: 138–145.[CrossRef][Medline]

  8. Turley, S. D., and J. M. Dietschy. 2003. Sterol absorption by the small intestine. Curr. Opin. Lipidol. 14: 233–240.[CrossRef][Medline]

  9. Grundy, S. M., and A. L. Metzger. 1972. A physiological method for estimation of hepatic secretion of biliary lipids in man. Gastroenterology. 62: 1200–1217.[Medline]

  10. van Heek, M., C. Farley, D. S. Compton, L. Hoos, K. B. Alton, E. J. Sybertz, and H. R. Davis, Jr. 2000. Comparison of the activity and disposition of the novel cholesterol absorption inhibitor, SCH58235, and its glucuronide, SCH60663. Br. J. Pharmacol. 129: 1748–1754.[CrossRef][Medline]

  11. van Heek, M., C. Farley, D. S. Compton, L. Hoos, and H. R. Davis. 2001. Ezetimibe selectively inhibits intestinal cholesterol absorption in rodents in the presence and absence of exocrine pancreatic function. Br. J. Pharmacol. 134: 409–417.[CrossRef][Medline]

  12. Davis, H. R., Jr., K. K. Pula, K. B. Alton, R. E. Burrier, and R. W. Watkins. 2001. The synergistic hypocholesterolemic activity of the potent cholesterol absorption inhibitor, ezetimibe, in combination with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in dogs. Metabolism. 50: 1234–1241.[CrossRef][Medline]

  13. Sudop, T., D. Lutjohann, A. Kodal, M. Igel, D. L. Tribble, S. Shah, and K. von Bergmann. 2002. Inhibition of intestinal cholesterol absorption by ezetimibe in humans. Circulation. 106: 1943–1948.

  14. Davis, H. R. J., L. J. Zhu, L. M. Hoos, G. Tetzloff, M. Maguire, J. Liu, X. Yao, S. P. Iyer, M. H. Lam, E. G. Lund, et al. 2004. Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis. J. Biol. Chem. 279: 33586–33592.[Abstract/Free Full Text]

  15. Garcia-Calvo, M., J. Lisnock, H. G. Bull, B. E. Hawes, D. A. Burnett, M. P. Braun, J. H. Crona, H. R. J. Davis, D. C. Dean, P. A. Detmers, et al. 2005. The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1). Proc. Natl. Acad. Sci. USA. 102: 8132–8137.[Abstract/Free Full Text]

  16. Altmann, S. W., H. R. J. Davis, L. J. Zhu, X. Yao, L. M. Hoos, G. Tetzloff, S. P. Iyer, M. Maguire, A. Golovko, M. Zeng, et al. 2004. Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption. Science. 303: 1201–1204.[Abstract/Free Full Text]

  17. Huff, M. W., R. L. Pollex, and R. A. Hegele. 2006. NPC1L1: evolution from pharmacological target to physiological sterol transporter. Arterioscler. Thromb. Vasc. Biol. 26: 2433–2438.[Abstract/Free Full Text]

  18. Repa, J. J., S. D. Turley, G. Quan, and J. M. Dietschy. 2005. Delineation of molecular changes in intrahepatic cholesterol metabolism resulting from diminished cholesterol absorption. J. Lipid Res. 46: 779–789.[Abstract/Free Full Text]

  19. Tremblay, A. J., B. Lamarche, J. S. Cohn, J. C. Hogue, and P. Couture. 2006. Effect of ezetimibe on the in vivo kinetics of apoB-48 and apoB-100 in men with primary hypercholesterolemia. Arterioscler. Thromb. Vasc. Biol. 26: 1101–1106.[Abstract/Free Full Text]

  20. Ballantyne, C. M. 2005. Rationale for targeting multiple lipid pathways for optimal cardiovascular risk reduction. Am. J. Cardiol. 96: 14K–19K.[CrossRef][Medline]

  21. Ballantyne, C. M., J. Houri, A. Notarbartolo, L. Melani, L. J. Lipka, R. Suresh, S. Sun, A. P. Lebeaut, P. T. Sager, and E. P. Veltri. 2003. Effect of ezetimibe coadministered with atorvastatin in 628 patients with primary hypercholesterolemia: a prospective, randomized, double-blind trial. Circulation. 107: 2409–2415.

  22. Bays, H. E., L. Ose, N. Fraser, D. L. Tribble, K. Quinto, R. Reyes, A. O. Johnson-Levonas, A. Sapre, and S. R. Donahue. 2004. A multicenter, randomized, double-blind, placebo-controlled, factorial design study to evaluate the lipid-altering efficacy and safety profile of the ezetimibe/simvastatin tablet compared with ezetimibe and simvastatin monotherapy in patients with primary hypercholesterolemia. Clin. Ther. 26: 1758–1773.[CrossRef][Medline]

  23. Burnett, J. R., L. J. Wilcox, D. E. Telford, S. J. Kleinstiver, P. H. R. Barrett, and M. W. Huff. 1998. Inhibition of cholesterol esterification by DuP 128 decreases hepatic apolipoprotein B secreted in vivo: effect of dietary fat and cholesterol. Biochim. Biophys. Acta. 1393: 63–79.[Medline]

  24. Burnett, J. R., L. J. Wilcox, D. E. Telford, S. J. Kleinstiver, P. H. R. Barrett, R. S. Newton, and M. W. Huff. 1999. The magnitude of decrease in hepatic very low density lipoprotein apolipoprotein B secretion is determined by the extent of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibition in miniature pigs. Endocrinology. 140: 5293–5302.[Abstract/Free Full Text]

  25. Burnett, J. R., L. J. Wilcox, D. E. Telford, S. J. Kleinstiver, P. H. R. Barrett, R. S. Newton, and M. W. Huff. 1997. Inhibition of HMG-CoA reductase by atorvastatin decreases both VLDL and LDL apolipoprotein B production in miniature pigs. Arterioscler. Thromb. Vasc. Biol. 17: 2589–2600.[Abstract/Free Full Text]

  26. Burnett, J. R., L. J. Wilcox, D. E. Telford, S. J. Kleinstiver, P. H. R. Barrett, R. S. Newton, and M. W. Huff. 1999. Inhibition of ACAT by avasimibe decreases both VLDL and LDL apolipoprotein B production in miniature pigs. J. Lipid Res. 40: 1317–1327.[Abstract/Free Full Text]

  27. Telford, D. E., J. Y. Edwards, S. M. Lipson, B. Sutherland, P. H. Barrett, J. R. Burnett, E. S. Krul, B. T. Keller, and M. W. Huff. 2003. Inhibition of both the apical sodium-dependent bile acid transporter and HMG-CoA reductase markedly enhances the clearance of LDL apoB. J. Lipid Res. 44: 943–952.[Abstract/Free Full Text]

  28. Daugherty, A., and D. L. Rateri. 1994. Presence of LDL receptor-related protein/alpha 2-macroglobulin receptors in macrophages of atherosclerotic lesions from cholesterol-fed New Zealand and heterozygous Watanabe heritable hyperlipidemic rabbits. Arterioscler. Thromb. 14: 2017–2024.[Abstract/Free Full Text]

  29. Chan, D. C., G. F. Watts, P. H. Barrett, L. J. Beilin, T. G. Redgrave, and T. A. Mori. 2002. Regulatory effects of HMG CoA reductase inhibitor and fish oils on apolipoprotein B-100 kinetics in insulin-resistant obese male subjects with dyslipidemia. Diabetes. 51: 2377–2386.[Abstract/Free Full Text]

  30. Turley, S. D., M. W. Herndon, and J. M. Dietschy. 1994. Reevaluation and application of the dual-isotope plasma ratio method for the measurement of intestinal cholesterol absorption in the hamster. J. Lipid Res. 35: 328–339.[Abstract]

  31. Telford, D. E., S. M. Lipson, P. H. Barrett, B. G. Sutherland, J. Y. Edwards, J. D. Aebi, H. Dehmlow, O. H. Morand, and M. W. Huff. 2005. A novel inhibitor of oxidosqualene:lanosterol cyclase inhibits very low-density lipoprotein apolipoprotein B100 (apoB100) production and enhances low-density lipoprotein apoB100 catabolism through marked reduction in hepatic cholesterol content. Arterioscler. Thromb. Vasc. Biol. 25: 2608–2614.[Abstract/Free Full Text]

  32. Bosner, M. S., L. G. Lange, W. F. Stenson, and R. E. Ostlund. 1999. Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry. J. Lipid Res. 40: 302–308.[Abstract/Free Full Text]

  33. Salen, G., K. von Bergmann, D. Lutjohann, P. Kwiterovich, J. Kane, S. B. Patel, T. Musliner, P. Stein, and B. Musser. 2004. Ezetimibe effectively reduces plasma plant sterols in patients with sitosterolemia. Circulation. 109: 966–971.

  34. Xie, C., L. A. Woollett, S. D. Turley, and J. M. Dietschy. 2002. Fatty acids differentially regulate hepatic cholesteryl ester formation and incorporation into lipoproteins in the liver of the mouse. J. Lipid Res. 43: 1508–1519.[Abstract/Free Full Text]

  35. van Heek, M., T. M. Austin, C. Farley, J. A. Cook, G. G. Tetzloff, and H. R. Davis. 2001. Ezetimibe, a potent cholesterol absorption inhibitor, normalizes combined dyslipidemia in obese hyperinsulinemic hamsters. Diabetes. 50: 1330–1335.[Abstract/Free Full Text]

  36. Gagne, C., D. Gaudet, and E. Bruckert. 2002. Efficacy and safety of ezetimibe coadministered with atorvastatin or simvastatin in patients with homozygous familial hypercholesterolemia. Circulation. 105: 2469–2475.

  37. Huff, M. W., D. E. Telford, J. Y. Edwards, J. R. Burnett, P. H. R. Barrett, S. R. Rapp, N. Napawan, and B. T. Keller. 2002. Inhibition of the apical sodium-dependent bile acid transporter reduces LDL cholesterol and apoB by enhanced plasma clearance of LDL apoB. Arterioscler. Thromb. Vasc. Biol. 22: 1884–1891.[Abstract/Free Full Text]

  38. Hegele, R. A., J. Guy, M. R. Ban, and J. Wang. 2005. NPC1L1 haplotype is associated with inter-individual variation in plasma low-density lipoprotein response to ezetimibe. Lipids Health Dis. 4: 16.[CrossRef][Medline]

  39. Davies, J. P., B. Levy, and Y. A. Ioannou. 2000. Evidence for a Niemann-pick C (NPC) gene family: identification and characterization of NPC1L1. Genomics. 65: 137–145.[CrossRef][Medline]

  40. Davies, J. P., C. Scott, K. Oishi, A. Liapis, and Y. A. Ioannou. 2005. Inactivation of NPC1L1 causes multiple lipid transport defects and protects against diet-induced hypercholesterolemia. J. Biol. Chem. 280: 12710–12720.[Abstract/Free Full Text]

  41. Yu, L., S. Bharadwaj, J. M. Brown, Y. Ma, W. Du, M. A. Davis, P. Michaely, P. Liu, M. C. Willingham, and L. L. Rudel. 2006. Cholesterol-regulated translocation of NPC1L1 to the cell surface facilitates free cholesterol uptake. J. Biol. Chem. 281: 6616–6624.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
M. A. Valasek, J. J. Repa, G. Quan, J. M. Dietschy, and S. D. Turley
Inhibiting intestinal NPC1L1 activity prevents diet-induced increase in biliary cholesterol in Golden Syrian hamsters
Am J Physiol Gastrointest Liver Physiol, October 1, 2008; 295(4): G813 - G822.
[Abstract] [Full Text] [PDF]

Home page
 NEJMHome page
D. Steinberg, E. J. Eichhorn, W. E. Connor, G. A. Diamond, S. Kaul, J. A. Blake, H. R. Davis Jr., N. J. Murgolo, M. P. Graziano, T. Kaye, et al.
Simvastatin with or without ezetimibe in familial hypercholesterolemia.
N. Engl. J. Med., July 31, 2008; 359(5): 529 - 530.
[Full Text] [PDF]

Home page
 J. Lipid Res.Home page
F. J. Field, K. Watt, and S. N. Mathur
Ezetimibe interferes with cholesterol trafficking from the plasma membrane to the endoplasmic reticulum in CaCo-2 cells
J. Lipid Res., August 1, 2007; 48(8): 1735 - 1745.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M600439-JLR200v1
48/3/699    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Telford, D. E.
Right arrow Articles by Huff, M. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Telford, D. E.
Right arrow Articles by Huff, M. W.
Social Bookmarking
Add to CiteULike   Add to Complore