Food Ingredients That Inhibit Cholesterol Absorption.

The absorption of cholesterol by the small intestine is a major route for the net entry of cholesterol into the body and can therefore affect the plasma low density lipoprotein-cholesterol (LDL-C) concentration. These studies used ezetimibe, a potent inhibitor of cholesterol absorption, to delineate the biochemical and molecular changes in intrahepatic metabolism and biliary lipid secretion when there is a major reduction in chylomicron cholesterol delivery to the liver. In female LDL receptor (LDLR)-deficient (LDLR-/-) mice fed a basal diet containing ezetimibe (0-10 mg/day/kg body weight), cholesterol absorption was reduced up to 91%, fecal neutral sterol excretion was increased up to 4.7-fold, and plasma total cholesterol concentrations decreased by up to 18%. Blocking cholesterol absorption prevented the accumulation of very low density lipoproteins and LDL in the circulation of LDLR-/- mice fed a lipid-rich diet. In female LDLR+/+ mice fed the lipid-rich diet with ezetimibe, the relative mRNA level for the LDLR in the liver was 2-fold greater than in matching mice given the lipid-rich diet alone. We conclude that in the mouse the reduction in plasma LDL-C levels induced by blocking cholesterol absorption reflects both a diminished rate of LDL-C production and a modest increase in hepatic LDLR expression.

Results of previous studies have shown that ezetimibe (10 mg/day) reduces LDL cholesterol in patients with mild hypercholesterolemia on a normal-cholesterol diet (dietary intake of 200-500 mg/day) by 16-22%. However, the LDL cholesterol-lowering effect of ezetimibe in subjects with an extremely low dietary cholesterol intake (vegetarians) has not been studied. We conducted a randomized, double-blind, placebo-controlled, two-phase crossover study in 18 healthy pure vegetarians to assess the effect of ezetimibe (10 mg/day) on plasma lipids, cholesterol absorption, and its synthesis. Treatment periods lasted 2 weeks each, with an intervening 2 week washout period. Fractional cholesterol absorption was determined using the continuous dual stable isotope feeding method. Mean dietary cholesterol intake in the pure vegetarians was extremely low and averaged 29.4 +/- 16.8 and 31.4 +/- 14.4 mg/day during the placebo and ezetimibe administration phases, respectively. Fractional cholesterol absorption during the placebo phase was 48.2 +/- 8.2% and was decreased by 58% during ezetimibe treatment to 20.2 +/- 6.2% (P < 0.001). This change in intestinal cholesterol absorption was followed by a significant reduction in LDL cholesterol of 17.3%. In individuals with extremely low dietary cholesterol intake, treatment with ezetimibe (10 mg/day) leads to a significant reduction of cholesterol absorption and a clinically relevant decrease of plasma LDL cholesterol, comparable to that of subjects with a normal dietary cholesterol intake. Thus, the lipid-lowering effect of ezetimibe is mediated mainly through a reduction of the absorption of endogenous (biliary) cholesterol.

To learn more about how the step of cholesterol uptake into the brush border membrane (BBM) of enterocytes influences overall cholesterol absorption, we measured cholesterol absorption 4 and 24 h after administration of an intragastric bolus of radioactive cholesterol in mice with scavenger receptor class B, type 1 (SR-BI) and/or cluster determinant 36 (CD36) deleted. The cholesterol absorption efficiency is unaltered by deletion of either one or both of the receptors. In vitro determinations of the cholesterol uptake specific activity of the BBM from the mice reveal that the scavenger receptors facilitate cholesterol uptake into the proximal BBM. It follows that cholesterol uptake into the BBM is not normally rate-limiting for the cholesterol absorption process in vivo; a subsequent step, such as NPC1L1-mediated transfer from the BBM into the interior of the enterocyte, is rate-limiting. The absorption of dietary cholesterol after 4 h in mice lacking SR-BI and/or CD36 and fed a high-fat/high-cholesterol diet is delayed to more distal regions of the small intestine. This effect probably arises because ATP binding cassette half transporters G5 and G8-mediated back flux of cholesterol from the BBM to the lumen of the small intestine limits absorption and causes the local cholesterol uptake facilitated by SR-BI and CD36 to become rate-limiting under this dietary condition.

The hypothesis tested in this study was that cholesterol esterification by ACAT2 would increase cholesterol absorption efficiency by providing cholesteryl ester (CE) for incorporation into chylomicrons. The assumption was that absorption would be proportional to Acat2 gene dosage. Male ACAT2⁺/⁺, ACAT2⁺/⁻, and ACAT2⁻/⁻ mice were fed a diet containing 20% of energy as palm oil with 0.2% (w/w) cholesterol. Cholesterol absorption efficiency was measured by fecal dual-isotope and thoracic lymph duct cannulation (TLDC) methods using [³H]sitosterol and [¹⁴C]cholesterol tracers. Excellent agreement among individual mice was found for cholesterol absorption measured by both techniques. Cholesterol absorption efficiency in ACAT2⁻/⁻ mice was 16% compared with 46-47% in ACAT2⁺/⁺ and ACAT2⁺/⁻ mice. Chylomicrons from ACAT2⁺/⁺ and ACAT2⁺/⁻ mice carried ∼80% of total sterol mass as CE, whereas ACAT2⁻/⁻ chylomicrons carried >90% of sterol mass in the unesterified form. The total percentage of chylomicron mass as CE was reduced from 12% in the presence of ACAT2 to ∼1% in ACAT2⁻/⁻ mice. Altogether, the data demonstrate that ACAT2 increases cholesterol absorption efficiency by providing CE for chylomicron transport, but one copy of the Acat2 gene, providing ∼50% of ACAT2 mRNA and enzyme activity, was as effective as two copies in promoting cholesterol absorption.

Toward Individualized Cholesterol-Lowering Treatment in End-Stage Renal Disease

Inhibition of intestinal absorption of cholesterol by surface-modified nanostructured aluminosilicate compounds

Compensatory phospholipid digestion is required for cholesterol absorption in pancreatic phospholipase A2-deficient mice

Niemann-Pick C1-Like 1 (NPC1L1) mediates intestinal cholesterol absorption. NPC1L1 knockout (L1-KO) mice were recently shown to be resistant to high-fat diet (HFD)-induced obesity in one study, which was contrary to several other studies. Careful comparison of dietary compositions in these studies implies a potential role of dietary cholesterol in regulating weight gain. To examine this potential, wild-type (WT) and L1-KO mice were fed one of three sets of diets for various durations: (1) a HFD without added cholesterol for 5 weeks; (2) a high-carbohydrate diet with or without added cholesterol for 5 weeks; or (3) a synthetic HFD with or without added cholesterol for 18 weeks. We found that L1-KO mice were protected against diet-induced weight gain only on a diet without added cholesterol but not on a diet containing 0.16% or 0.2% (w/w) cholesterol, an amount similar to a typical Western diet, regardless of the major energy source of the diet. Food intake and intestinal fat absorption were similar between the two genotypes. Intestinal cholesterol absorption was blocked, and fecal cholesterol excretion increased in L1-KO mice. Under all diets, L1-KO mice were protected from hepatosteatosis. In conclusion, increasing dietary cholesterol restores diet-induced weight gain in mice deficient in NPC1L1-dependent cholesterol absorption.

Type 2 diabetes has been associated with high synthesis and low absorption of cholesterol independent of weight, indicating that insulin resistance may be a link between glucose and cholesterol metabolism. Therefore, we investigated the relationship of serum cholesterol precursors, reflecting cholesterol synthesis, and serum plant sterols and cholestanol, reflecting cholesterol absorption efficiency, with insulin sensitivity measured with the hyperinsulinemic euglycemic clamp in 72 healthy normoglycemic men. Men in the most insulin-resistant tertile had higher serum cholesterol precursor ratios (P < 0.05), whereas no significant differences in serum absorption sterols were observed. In bivariate analysis, cholesterol synthesis markers correlated with fasting insulin (r = 0.36-0.46, P < 0.01) and the rates of insulin-stimulated whole-body glucose uptake (WBGU; r = -0.37-0.40, P < 0.01). Also, cholesterol absorption markers correlated with fasting insulin and WBGU (P < 0.05). Fasting insulin correlated with desmosterol (r = 0.286, P = 0.015) and lathosterol (r = 0.248, P = 0.037) even when the rates of WBGU and body mass index (BMI) were controlled for. We conclude that insulin resistance is linked to high cholesterol synthesis and decreased cholesterol absorption. Because fasting insulin correlated with cholesterol synthesis independent of the rates of BMI and WBGU, it is possible that regulation of cholesterol synthesis by hyperinsulinemia may be a link between insulin resistance and cholesterol metabolism.

Subjects with increased cholesterol absorption might benefit more from statin therapy combined with a cholesterol absorption inhibitor. We assessed whether baseline cholesterol absorption markers were associated with response to ezetimibe/simvastatin therapy, in terms of LDL-cholesterol (LDL-C) lowering and cholesterol absorption inhibition, in patients with familial hypercholesterolemia (FH). In a posthoc analysis of the two-year ENHANCE trial, we assessed baseline cholesterol-adjusted campesterol (campesterol/TC) and sitosterol/TC ratios in 591 FH patients. Associations with LDL-C changes and changes in cholesterol absorption markers were evaluated by multiple regression analysis. No association was observed between baseline markers of cholesterol absorption and the extent of LDL-C response to ezetimibe/simvastatin therapy (beta = 0.020, P = 0.587 for campesterol/TC and beta<0.001, P = 0.992 for sitosterol/TC). Ezetimibe/simvastatin treatment reduced campesterol levels by 68% and sitosterol levels by 62%; reductions were most pronounced in subjects with the highest cholesterol absorption markers at baseline, the so-called high absorbers (P < 0.001). Baseline cholesterol absorption status does not determine LDL-C lowering response to ezetimibe/simvastatin therapy in FH, despite more pronounced cholesterol absorption inhibition in high absorbers. Hence, these data do not support the use of baseline absorption markers as a tool to determine optimal cholesterol lowering strategy in FH patients. However, due to the exploratory nature of any posthoc analysis, these results warrant further prospective evaluation in different populations.

We previously reported that liver-specific overexpression of ABCG5/G8 in mice is not atheroprotective, suggesting that increased biliary cholesterol secretion must be coupled with decreased intestinal cholesterol absorption to increase net sterol loss from the body and reduce atherosclerosis. To evaluate this hypothesis, we fed low density lipoprotein receptor-knockout (LDLr-KO) control and ABCG5/G8-transgenic (ABCG5/G8-Tg)xLDLr-KO mice, which overexpress ABCG5/G8 only in liver, a Western diet containing ezetimibe to reduce intestinal cholesterol absorption. On this dietary regimen, liver-specific ABCG5/G8 overexpression increased hepatobiliary cholesterol concentration and secretion rates (1.5-fold and 1.9-fold, respectively), resulting in 1.6-fold increased fecal cholesterol excretion, decreased hepatic cholesterol, and increased (4.4-fold) de novo hepatic cholesterol synthesis versus LDLr-KO mice. Plasma lipids decreased (total cholesterol, 32%; cholesteryl ester, 32%; free cholesterol, 30%), mostly as a result of reduced non-high density lipoprotein-cholesterol and apolipoprotein B (apoB; 36% and 25%, respectively). ApoB-containing lipoproteins were smaller and lipid-depleted in ABCG5/G8-TgxLDLr-KO mice. Kinetic studies revealed similar 125I-apoB intermediate density lipoprotein/LDL fractional catabolic rates, but apoB production rates were decreased 37% in ABCG5/G8-TgxLDLr-KO mice. Proximal aortic atherosclerosis decreased by 52% (male) and 59% (female) in ABCG5/G8-TgxLDLr-KO versus LDLr-KO mice fed the Western/ezetimibe diet. Thus, increased biliary secretion, resulting from hepatic ABCG5/G8 overexpression, reduces atherogenic risk in LDLr-KO mice fed a Western diet containing ezetimibe. These findings identify distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism and atherosclerosis.

The present study investigated the role of apolipoprotein E (apoE) phenotype on intestinal cholesterol absorption and cholesterol synthesis. Studies were carried out in eight subjects homozygous for the apoE4 and 12 subjects homozygous for the E2 allele (six normocholesterolemic volunteers and six patients with type III hyperlipoproteinemia). Cholesterol absorption did not differ between the three groups of subjects and averaged 38 +/- 2% (mean +/- SEM) in normolipemic E2/2, 37 +/- 4% in type III hyperlipemic E2/2, and 41 +/- 3% in E4/4 subjects, respectively. Dietary intake of fat and cholesterol had no influence on cholesterol absorption efficiency. A positive correlation between efficiency of cholesterol absorption and the ratio of campesterol to cholesterol in plasma, an indirect marker for cholesterol absorption, was observed after combining the results of the three groups (r = 0.504; P < 0.02). Bile acid and total cholesterol synthesis were also not affected by the different apoE alleles, but the well-known relationship between body weight and cholesterol synthesis was noticed (r = 0.574; P < 0.01). Thus, the present study provides evidence that the efficiency of intestinal absorption and synthesis of cholesterol in humans are not related to the apoE phenotype.

The profile of cholesterol metabolism does not interfere with the cholesterol-lowering efficacy of phytostanol esters

Food Components that Reduce Cholesterol Absorption

The importance of the cholesterol transporter Niemann-Pick C1 like protein 1 (NPC1L1)in the small intestine for efficient absorption of cholesterol is well documented and its physiological importance is clear (1Davis Jr., H.R. Basso F. Hoos L.M. Tetzloff G. Lally S.M. Altmann S.W. Cholesterol homeostasis by the intestine: lessons from Niemann-Pick C1 Like 1 (NPC1L1).Atheroscler. Suppl. 2008; 9: 77-81Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Similarly, the drug ezetimibe (Zetia™), which blocks function of this transporter, reduces plasma cholesterol, primarily LDL, by dramatically reducing intestinal absorption of dietary and biliary cholesterol (2Davis H.R. Veltri E.P. Zetia: inhibition of Niemann-Pick C1 Like 1 (NPC1L1) to reduce intestinal cholesterol absorption and treat hyperlipidemia.J. Atheroscler. Thromb. 2007; 14: 99-108Crossref PubMed Scopus (142) Google Scholar). There are controversies about the exact mechanism by which NPC1L1 functions: whether the protein is on the apical cell membrane or intracellular vesicles or both (3Davies J.P. Scott C. Oishi K. Liapis A. Ioannou Y.A. Inactivation of NPC1L1 causes multiple lipid transport defects and protects against diet-induced hypercholesterolemia.J. Biol. Chem. 2005; 280: 12710-12720Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar–4Petersen N.H. Faergeman N.J. Yu L. Wustner D. Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells.J. Lipid Res. 2008; 49: 2023-2037Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 5Ge L. Wang J. Qi W. Miao H.H. Cao J. Qu Y.X. Li B.L. Song B.L. The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1.Cell Metab. 2008; 7: 508-519Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), and whether ezetimibe directly blocks NPC1L1 or interrupts its association with other proteins in the sterol absorption and transport pathway (6Altmann S.W. Davis H.R. Yao X. Laverty M. Compton D.S. Zhu L.J. Crona J.H. Caplen M.A. Hoos L.M. Tetzloff G. et al.The identification of intestinal scavenger receptor class B, type I (SR-BI) by expression cloning and its role in cholesterol absorption.Biochim. Biophys. Acta. 2002; 1580: 77-93Crossref PubMed Scopus (140) Google Scholar–7Garcia-Calvo M. Lisnock J. Bull H.G. Hawes B.E. Burnett D.A. Braun M.P. Crona J.H. Davis Jr, H.R. Dean D.C. Detmers P.A. et al.The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1).Proc. Natl. Acad. Sci. USA. 2005; 102: 8132-8137Crossref PubMed Scopus (650) Google Scholar, 8Ge L. Qi W. Wang L.J. Miao H.H. Qu Y.X. Li B.L. Song B.L. Flotillins play an essential role in Niemann-Pick C1-like 1-mediated cholesterol uptake.Proc. Natl. Acad. Sci. USA. 2011; 108: 551-556Crossref PubMed Scopus (17) Google Scholar). Nevertheless, it is now accepted that this protein is the key player in sterol absorption, although other transporters may play ancillary roles (9Nguyen D.V. Drover V.A. Knopfel M. Dhanasekaran P. Hauser H. Phillips M.C. Influence of class B scavenger receptors on cholesterol flux across the brush border membrane and intestinal absorption.J. Lipid Res. 2009; 50: 2235-2244Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 10Adams M.R. Konaniah E. Cash J.G. Hui D.Y. Use of NBD-cholesterol to identify a minor but NPC1L1-independent cholesterol absorption pathway in mouse intestine.Am. J. Physiol. Gastrointest. Liver Physiol. 2011; 300: G164-G169Crossref PubMed Scopus (12) Google Scholar). NPC1L1 is also abundant in human liver, although its physiological role with regard to hepatic cholesterol and lipoprotein metabolism is less clear. This lack of information reflects, in large part, species differences: mice, the most widely used animal model for NPC1L1 studies as well as cholesterol metabolism, do not express NPC1L1 in liver. Transgenic mice with liver-specific overexpression (20 times) of human NPC1L1 were found to have reduced biliary cholesterol secretion (11Temel R.E. Tang W. Ma Y. Rudel L.L. Willingham M.C. Ioannou Y.A. Davies J.P. Nilsson L.M. Yu L. Hepatic Niemann-Pick C1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe.J. Clin. Invest. 2007; 117: 1968-1978Crossref PubMed Scopus (301) Google Scholar). In this issue of the Journal of Lipid Research, Kurano et al. (12Kurano M. Hara M. Tsuneyama K. Okamoto K. Iso-O N. Matsushima T. Koike K. Tsukamoto K. Modulation of lipid metabolism with the over-expression of NPC1L1 in mice liver.J. Lipid Res. 2012; 53: 2275-2285Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) bring new light to the topic of hepatic NPC1L1 and also raise some intriguing questions regarding specific hepatic cholesterol pools and their differential metabolic utilization for specific lipoproteins. In these studies, Kurano et al. (12Kurano M. Hara M. Tsuneyama K. Okamoto K. Iso-O N. Matsushima T. Koike K. Tsukamoto K. Modulation of lipid metabolism with the over-expression of NPC1L1 in mice liver.J. Lipid Res. 2012; 53: 2275-2285Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) used adenoviral gene transfer to transiently express human NPC1L1 in mouse hepatocytes at levels approximately similar to those seen in human liver. Many of their data corroborate the findings of Temel et al. (11Temel R.E. Tang W. Ma Y. Rudel L.L. Willingham M.C. Ioannou Y.A. Davies J.P. Nilsson L.M. Yu L. Hepatic Niemann-Pick C1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe.J. Clin. Invest. 2007; 117: 1968-1978Crossref PubMed Scopus (301) Google Scholar) who analyzed transgenic mice with chronic hepatic expression of human NPC1L1. Both groups demonstrated a 5 to 10-fold decrease in biliary cholesterol that could be "corrected" by ezetimibe treatment (11Temel R.E. Tang W. Ma Y. Rudel L.L. Willingham M.C. Ioannou Y.A. Davies J.P. Nilsson L.M. Yu L. Hepatic Niemann-Pick C1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe.J. Clin. Invest. 2007; 117: 1968-1978Crossref PubMed Scopus (301) Google Scholar). This result shows one more parallel between enterocyte and hepatocyte function with regards to sterol and lipoprotein metabolism: sterol is recovered apically and secreted basolaterally as lipoproteins. Thus, teleologically, NPC1L1 functions to conserve cholesterol for the organism. Clinically, the data from these studies indicate, contrary to the general dogma, that ezetimibe reduces LDL not only by blocking cholesterol absorption in the intestine but also by increasing its hepatic biliary disposal and thereby increasing reverse cholesterol transport. A potential corollary to increased biliary cholesterol is that ezetimibe would increase the risk for gallstones. However, while low NPC1L1 expression was found in one cohort of gallstone disease patients (13Cui W. Jiang Z.Y. Cai Q. Zhang R.Y. Wu W.Z. Wang J.C. Fei J. Zhang S.D. Han T.Q. Decreased NPC1L1 expression in the liver from Chinese female gallstone patients.Lipids Health Dis. 2010; 9: 17-25Crossref PubMed Scopus (14) Google Scholar), ezetimibe has not been found to increase this risk in the SHARP trial (14Baigent C. Landray M.J. Reith C. Emberson J. Wheeler D.C. Tomson C. Wanner C. Krane V. Cass A. Craig J. et al.The effects of lowering LDL cholesterol with simvastatin plus ezetimibe in patients with chronic kidney disease (Study of Heart and Renal Protection): a randomized placebo-controlled trial.Lancet. 2011; 377: 2181-2192Abstract Full Text Full Text PDF PubMed Scopus (1886) Google Scholar) and there is some limited evidence that it may decrease risk due to the overriding increase in cholesterol excretion (15Wang H.H. Portincasa P. Mendez-Sanchez N. Uribe M. Wang D.Q. Effects of ezetimibe on the prevention and dissolution of cholesterol gallstones.Gastroenterology. 2008; 134: 2101-2110Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). As expected from decreased biliary output, Kurano et al. (12Kurano M. Hara M. Tsuneyama K. Okamoto K. Iso-O N. Matsushima T. Koike K. Tsukamoto K. Modulation of lipid metabolism with the over-expression of NPC1L1 in mice liver.J. Lipid Res. 2012; 53: 2275-2285Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) showed that intrahepatic cholesterol increased with NPC1L1 expression, while LDL receptor and nuclear SREBP2 decreased. In keeping with these changes, plasma LDL cholesterol and apoB levels increased while remnant clearance rate decreased. Curiously, hepatic NPC1L1 expression caused a decrease in VLDL triglyceride content although VLDL cholesterol was not changed. Kurano et al. (12Kurano M. Hara M. Tsuneyama K. Okamoto K. Iso-O N. Matsushima T. Koike K. Tsukamoto K. Modulation of lipid metabolism with the over-expression of NPC1L1 in mice liver.J. Lipid Res. 2012; 53: 2275-2285Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) present data suggesting a mechanism involving decreased MTTP and FASN expression due to decreased FoxO1 levels. Also curious, these effects were minimally changed by ezetimibe treatment. However, ezetimibe has been reported to increase MTTP expression in patients with nonalcoholic steatohepatitis and has been suggested as a possible therapy (16Yoneda M. Fujita K. Imajo K. Mawatari H. Kirikoshi H. Saito S. Nakajima A. Induction of microsomal triglyceride transfer protein expression is a candidate mechanism by which ezetimibe therapy might exert beneficial effects in patients with nonalcoholic steatohepatitis.J. Gastroenterol. 2011; 46: 415-416Crossref PubMed Scopus (8) Google Scholar). Another striking and unexpected result of hepatic NPC1L1 expression reported by both groups is the secretion of significant amounts of large apoE-rich HDL particles. Kurano et al. show that these particles are essentially devoid of apoA-I as well as apoB, and that their secretion is substantially reduced by ezetimibe treatment of the mice. They also present data showing increased apoE secretion by HepG2 cells when incubated with cholesterol-bile salt micelles and that this is also blocked by ezetimibe. The suggestion is that cholesterol recovered from bile is transported to a compartment that specifically utilizes apoE for HDL secretion. Contrary to some current models that showed the involvement of ABCA1 in HDL secretion (17Oram J.F. Vaughan A.M. ATP binding cassette cholesterol transporters and cardiovascular disease.Circ. Res. 2006; 99: 1031-1043Crossref PubMed Scopus (325) Google Scholar), ABCA1 is not necessarily part of this secretory pathway because its levels were not changed by expression of NPC1L1. Interestingly, the secretion of apoE-rich HDL appears to be NPC1L1 dependent because cholesterol accumulation by an alternative mechanism (lanosterol synthase overexpression) did not result in secretion of these particles. Also, there may be a requirement for apoE to mobilize this pool as NPC1L1 expression in apoE-knockout mice did not increase HDL levels. However, the latter mice have severe metabolic abnormalities that may have obscured the effect. The model generally described by Kurano et al. (12Kurano M. Hara M. Tsuneyama K. Okamoto K. Iso-O N. Matsushima T. Koike K. Tsukamoto K. Modulation of lipid metabolism with the over-expression of NPC1L1 in mice liver.J. Lipid Res. 2012; 53: 2275-2285Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) as well as others (11Temel R.E. Tang W. Ma Y. Rudel L.L. Willingham M.C. Ioannou Y.A. Davies J.P. Nilsson L.M. Yu L. Hepatic Niemann-Pick C1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe.J. Clin. Invest. 2007; 117: 1968-1978Crossref PubMed Scopus (301) Google Scholar) involves recovery of biliary cholesterol by hepatocytes expressing NPC1L1. However, it is also plausible that the presence of NPC1L1 in an endosomal recycling compartment associated with the apical membrane actually diverts bile-targeted cholesterol from being secreted and sequesters it into a unique pool. Such a mechanism would be consistent with current data as well as several previous studies that describe NPC1L1 as being a critical protein not only for cholesterol uptake but also for intracellular shuttling of vesicular cholesterol (3Davies J.P. Scott C. Oishi K. Liapis A. Ioannou Y.A. Inactivation of NPC1L1 causes multiple lipid transport defects and protects against diet-induced hypercholesterolemia.J. Biol. Chem. 2005; 280: 12710-12720Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar–4Petersen N.H. Faergeman N.J. Yu L. Wustner D. Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells.J. Lipid Res. 2008; 49: 2023-2037Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 5Ge L. Wang J. Qi W. Miao H.H. Cao J. Qu Y.X. Li B.L. Song B.L. The cholesterol absorption inhibitor ezetimibe acts by blocking the sterol-induced internalization of NPC1L1.Cell Metab. 2008; 7: 508-519Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 18Xie C. Li N. Chen Z.J. Li B.L. Song B.L. The small GTPase Cdc42 interacts with Niemann-Pick C1-like 1 (NPC1L1) and controls its movement from endocytic recycling compartment to plasma membrane in a cholesterol-dependent manner.J. Biol. Chem. 2011; 286: 35933-35942Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In either case, these results highlight the field's incomplete understanding of hepatic NPC1L1 function, as well as intracellular cholesterol traffic and metabolism, and highlight the need for additional detailed studies into how cholesterol is compartmentalized in cells, especially hepatocytes, and how these different pools are utilized. The clinical ramifications of the current and previous data from mice with hepatic NPC1L1 expression are not immediately apparent with regard to lipoprotein metabolism. Although ezetimibe lowered HDL somewhat in hamsters and apoE knockout mice (19van Heek M. Austin T.M. Farley C. Cook J.A. Tetzloff G.G. Davis H.R. Ezetimibe, a potent cholesterol absorption inhibitor, normalizes combined dyslipidemia in obese hyperinsulinemic hamsters.Diabetes. 2001; 50: 1330-1335Crossref PubMed Scopus (82) Google Scholar, 20Davis H.R. Compton D.S. Hoos L. Tetzloff G. Ezetimibe, a potent cholesterol absorption Inhibitor, inhibits the development of atherosclerosis in apoE knockout mice.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 2032-2038Crossref PubMed Scopus (199) Google Scholar), the drug has not been linked with significantly decreased HDL in humans as the current mouse data might suggest. Rather, clinical studies have shown the opposite in most cases, with reduced triglycerides, as well as LDL, upon Zetia™ treatment and essentially no change in HDL (14Baigent C. Landray M.J. Reith C. Emberson J. Wheeler D.C. Tomson C. Wanner C. Krane V. Cass A. Craig J. et al.The effects of lowering LDL cholesterol with simvastatin plus ezetimibe in patients with chronic kidney disease (Study of Heart and Renal Protection): a randomized placebo-controlled trial.Lancet. 2011; 377: 2181-2192Abstract Full Text Full Text PDF PubMed Scopus (1886) Google Scholar, 21Knopp R.H. Gitter H. Truitt T. Bays H. Manion C.V. Lipka L.J. LeBeaut A.P. Suresh R. Yang B. Veltri E.P. Effects of ezetimibe, a new cholesterol absorption inhibitor, on plasma lipids in patients with primary hypercholesterolemia.Eur. Heart J. 2003; 24: 729-741Crossref PubMed Scopus (370) Google Scholar, 22Leiter L.A. Betteridge D.J. Farnier M. Guyton J.R. Lin J. Shah A. Johnson-Levonas A.O. Brudi P. Lipid-altering efficacy and safety profile of combination therapy with ezetimibe/statin vs. statin monotherapy in patients with and without diabetes: an analysis of pooled data from 27 clinical trials.Diabetes Obes. Metab. 2011; 13: 615-628Crossref PubMed Scopus (73) Google Scholar). However, it is important to note that the mice in the current study were analyzed while on a chow diet with ∼10% of calories from fat, and some effects might be obscured in clinical studies that typically involve subjects with hyperlipidemia and/or metabolic disease who are consuming diets with higher quantities of fat. Further, mice lack CETP, which also profoundly affects lipoprotein metabolism in humans and most mammalian models other than mice and rats (23Jiang X-C. Beyer T.P. Li Z. Liu J. Quan W. Schmidt R.J. Zhang Y. Bensch W.R. Eacho P.I. Cao G. Enlargement of high density lipoprotein in mice via LXR activation requires apolipoprotein E and is abolished by cholesteryl ester transfer protein expression.J. Biol. Chem. 2003; 278: 49072-49078Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). An additional possibility is that transient expression of NPC1L1 has very different effects than chronic expression because compensatory mechanisms may take longer than 5 days to normalize metabolism. Nonetheless, these new studies reveal that hepatic NPC1L1 has important functions in the liver, and perhaps elsewhere, that remain poorly understood and merit further investigation. Cellular cholesterol levels can affect, directly or indirectly, several pathways related to fat and glucose metabolism and energy storage (24Bhonagiri P. Pattar G.R. Habegger K.M. McCarthy A.M. Tackett L. Elmendorf J.S. Evidence coupling increased hexosamine biosynthesis pathway activity to membrane cholesterol toxicity and cortical filamentous actin derangement contributing to cellular insulin resistance.Endocrinology. 2011; 152: 3373-3384Crossref PubMed Scopus (20) Google Scholar, 25Lee A.K. Yeung-Yam-Wah V. Tse F.W. Tse A. Cholesterol elevation impairs glucose-stimulated Ca(2+) signaling in mouse pancreatic β-cells.Endocrinology. 2011; 152: 3351-3361Crossref PubMed Scopus (25) Google Scholar), as well as lipid and lipoprotein metabolism. Thus, NPC1L1 and ezetimibe may affect the size and/or utilization of particular cholesterol pools that could have important clinical ramifications, especially in the context of metabolic disease characterized by obesity and insulin resistance. Lack of NPC1L1 reduces fatty liver disease in mice and in ezetimibe-treated humans (26Jia L. Ma Y. Rong S. Betters J.L. Xie P. Chung S. Wang N. Tang W. Yu L. Niemann-Pick C1-Like 1 deletion in mice prevents high-fat diet-induced fatty liver by reducing lipogenesis.J. Lipid Res. 2010; 51: 3135-3144Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 27Chan D.C. Watts G.F. Gan S.K. Ooi E.M. Barrett P.H. Effect of ezetimibe on hepatic fat, inflammatory markers, and apolipoprotein B-100 kinetics in insulin-resistant obese subjects on a weight loss diet.Diabetes Care. 2010; 33: 1134-1139Crossref PubMed Scopus (140) Google Scholar). Nomura et al. (28Nomura M. Ishii H. Kawakami A. Yoshida M. Inhibition of hepatic Neiman-Pick C1-Like 1 improves hepatic insulin resistance.Am. J. Physiol. Endocrinol. 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Importantly, improved insulin sensitivity has also been reported in several small clinical trials with ezetimibe (30Tamaki N. Ueno H. Morinaga Y. Shiiya T. Nakazato M. Ezetimibe ameliorates atherosclerotic and inflammatory markers, atherogenic lipid profiles, insulin sensitivity, and liver dysfunction in Japanese patients with hypercholesterolemia.J. Atheroscler. Thromb. 2012; 19: 532-538Crossref PubMed Scopus (32) Google Scholar, 31Hiramitsu S. Ishiguro Y. Matsuyama H. Yamada K. Kato K. Noba M. Uemura A. Yoshida S. Matsubara Y. Kani A. et al.The effects of ezetimibe on surrogate markers of cholesterol absorption and synthesis in Japanese patients with dyslipidemia.J. Atheroscler. Thromb. 2010; 17: 106-114Crossref PubMed Scopus (39) Google Scholar). The use of mice that demonstrate hepatic NPC1L1 expression will allow the distinction to be made between hepatic and intestinal contributions and will provide a more appropriate animal model for studies of the effects of ezetimibe and other cholesterol modulating therapies on the prevalent metabolic diseases of hepatosteatosis, gallstone disease, and insulin resistance.