Hepatic Niemann-Pick C1–like 1 regulates biliary cholesterol concentration and is a target of ezetimibe (original) (raw)
NPC1L1 is expressed in the liver and localizes to the canalicular membrane in NPC1L1 transgenic mice. To test our hypotheses, 2 independent NPC1L1 transgenic (L1-Tg) mouse lines, L1-Tg20 and L1-Tg112, were created. These mice were fertile and appeared grossly normal. Immunoblot analysis of multiple tissues detected an approximately 175-kDa protein only in the livers of L1-Tg mice (Figure 1A). A protein of slightly different size was seen in the kidneys of L1-Tg mice, but was deemed not to be NPC1L1 because it was also observed in the kidneys of WT mice (data not shown). NPC1L1 is highly _N_-glycosylated (3, 10). The approximately 175-kDa protein represents the _N_-glycosylated NPC1L1 in L1-Tg livers, because _N_-glycosidase digestion caused the apparent size of the protein to decrease to about 145 kDa (Figure 1B), the predicted molecular weight of NPC1L1. The relative expression level of human NPC1L1 transgene in the liver of L1-Tg20 and L1-Tg112 mice was about 3.5- and 5.7-fold higher, respectively, than that seen in human HepG2 hepatocarcinoma cells, based on densitometry of human NPC1L1 immunoblots of the same amount of whole-tissue homogenates or cell lysates (Figure 1C). NPC1L1 protein was readily detectable from 50 μg of fresh human hepatocyte and human liver lysates (Figure 1D). Interestingly, a substantial interindividual variation in hepatic NPC1L1 protein levels was observed in the 7 human liver specimens collected. When the amount of NPC1L1 was normalized by a glycoprotein, the receptor-associated protein (RAP) (21), its level in the L1-Tg20 liver was about 86% of that in human liver specimen 1 (Figure 1D); however, when the NPC1L1 protein was normalized by the protein concentration used for immunoblotting, its level was about 20-fold higher in the L1-Tg20 liver versus human liver specimen 1. Slight differences in the size of NPC1L1 protein were observed among different tissues and cells (Figure 1D), which may represent differential glycosylation of NPC1L1 in these tissues and cells because _N_-glycosidase digestion caused the protein from mouse and human samples to shift to the similar position (Figure 1E). The extent of _N_-glycosylation of NPC1L1 may be less in human liver than in rodent liver. This may explain why NPC1L1 protein size was smaller in human liver tissue than in mouse livers (Figure 1D). The invisible shift of the strong lower band of NPC1L1 protein in the human liver tissue (Figure 1E) may suggest that a majority of NPC1L1 was slightly _N_-glycosylated in the human liver tissue collected. When a large protein is slightly glycosylated, deglycosylation will not cause a visible shift of the protein size. The smaller size of human versus rodent RAP was consistent with a previous report (22).
NPC1L1 is expressed in L1-Tg mouse and human liver tissues. (A) Tissues were collected from 3 male L1-Tg112 mice (3 months old), and equal amounts of tissue from each mouse were pooled and processed for preparation of membrane proteins as described previously (50). Membrane proteins from adrenal glands (25 μg) and other tissues (50 μg) were fractionated by 8% polyacrylamide gel in the presence of SDS and immunoblotted with a rabbit anti-human NPC1L1 (anti-hNPC1L1) antibody (L1Ab) (10) and a rabbit anti-rat RAP serum (21). RAP was used as a loading control. Lanes 1 and 2, liver; lane 3, jejunum; lane 4, kidney; lane 5, pancreas; lane 6, lung; lane 7, heart; lane 8, spleen; lane 9, muscle; lane 10, testis; lane 11, cerebrum; lane 12, cerebellum; lane 13, epididymal fat; lane 14, adrenal glands. Similar results were observed in L1-Tg20 mice. (B) Membrane proteins (50 μg) from L1-Tg112 mice were treated with or without Peptide:_N_-glycosidase F (PNGaseF; New England Biolabs) followed by immunoblotting using L1Ab. (C) Mouse liver homogenates and HepG2 cell lysates were immunoblotted with the 69B and RAP antibodies. (D) Mouse liver homogenates (2.5 μg) and cell lysates or human liver homogenates (50 μg) were immunoblotted with the preimmune serum from the rabbit from which the 69B antiserum was obtained. The same membrane was stripped and immunoblotted with 69B and RAP antibodies. (E) L1-Tg20 mouse liver homogenates (2.5 μg) and fresh human hepatocyte lysates or human liver homogenates (50 μg) were deglycosylated by Peptide:_N_-glycosidase F, followed by immunoblotting with preimmune serum and 69B antiserum.
Immunofluorescence studies showed that NPC1L1 colocalized with a canalicular P-glycoprotein, ABCB1 (Figure 2A), demonstrating its canalicular location in the L1-Tg liver. No NPC1L1 staining was seen in the WT liver. Additionally, NPC1L1 did not show colocalization with cholangiocytes lining bile ducts (Figure 2B), indicating that NPC1L1 expression is hepatocyte specific. NPC1L1 was also found to localize to the canalicular membrane in human liver by fluorescent immunohistocytochemistry (Figure 2C).
NPC1L1 localizes to the canalicular membrane in L1-Tg and human liver tissues. (A) Acetone-fixed frozen sections of liver samples from 3-month-old L1-Tg112 and WT male mice fed the 0.015% cholesterol diet were processed for fluorescence immunohistochemistry with Bsn4052 antibody to human NPC1L1 and mouse monoclonal C219 antibody to ABCB1 as described in Methods. Arrows denote canalicular staining in the section of L1-Tg liver. Original magnification, ×630. Scale bars: 10 μm. (B) Fluorescence immunohistochemistry of human NPC1L1 was conducted as described in Methods, and sections were examined immediately using an Axioplan 2 fluorescence microscope. Images were taken to show the triad region of liver. Green autofluorescence from elastins in the blood vessel wall was included to distinguish bile duct from blood vessels. Arrows denote canalicular pattern (rhodamine red) of human NPC1L1 staining by Bsn4052 in the L1-Tg liver. Green and red stain appears white. a, artery; b, bile duct; v, vein. Original magnification, ×630. (C) Acetone-fixed frozen sections of human liver specimens were processed for fluorescence immunohistochemistry using C219, Bsn4052, and Bsn4052 preimmune serum as described in Methods, and sections were examined using an Axioplan 2 fluorescence microscope. Arrows denote canalicular pattern of human NPC1L1 staining by Bsn4052 and its colocalization with ABCB1 stained by C219. Scale bars: 35 μm.
Biliary cholesterol is selectively reduced in L1-Tg mice. To test the hypothesis that hepatic NPC1L1 modulates biliary cholesterol excretion, lipid compositions of gallbladder bile were measured in L1-Tg mice (Figure 3A). When fed a 0.015% cholesterol diet, L1-Tg20 and L1-Tg112 mice showed 13-fold and 23-fold decreases, respectively, in gallbladder cholesterol concentrations compared with WT mice, while phospholipid (PL) and BA concentrations were largely maintained. Feeding a 0.2% versus 0.015% cholesterol diet resulted in an increase in gallbladder cholesterol concentrations for mice of all genotypes. However, the cholesterol concentration of L1-Tg20 and L1-Tg112 mice was still 5- and 12-fold lower, respectively, than that of WT mice. No significant differences in gallbladder PL or BA concentration were seen on the 0.2% cholesterol diet. Because bile can be concentrated in the gallbladder, potentially altering lipid concentrations, molar ratios of biliary lipids were calculated. The molar percentages of biliary cholesterol of L1-Tg20 and L1-Tg112 mice were 14- and 19-fold lower on the 0.015% cholesterol diet and 5- and 10-fold lower on the 0.2% cholesterol diet, respectively, versus WT mice. L1-Tg mice had slightly higher molar percentages of BA and unchanged molar ratios of PL on both diets compared with WT mice.
Hepatic expression of NPC1L1 results in a dramatic reduction in biliary cholesterol but not PL and BA. (A) Concentration of gallbladder cholesterol, PL, and BA in male mice was determined as described in Methods. Molar ratios of each lipid in total biliary lipids were calculated from lipid concentrations. Data are mean ± SEM of 4–6 samples. (B and C) Liver total mRNAs or membrane proteins were prepared as described previously (50) from mice in A, and equal amounts of mRNA or protein from each mouse within a group were pooled (n = 5 per group). Levels of selected mRNAs and proteins were determined by quantitative real-time PCR (B) and by immunoblotting (C), respectively, as described previously (10, 50). RAP was used as the loading control, and human NPC1L1 was immunoblotted with L1Ab for genotype validation. The experiment was repeated twice with different sets of animals, and similar results were obtained. P < 0.05 between * and # groups for each measurement (ANOVA).
Because lack of ABCG5 and/or ABCG8 disrupts biliary cholesterol secretion (11, 23–25), the possibility existed that NPC1L1 expression was indirectly changing biliary cholesterol content by decreasing hepatic levels of these proteins. Thus, hepatic mRNA and protein levels of ABCG5 and ABCG8 were determined in mice fed either the 0.015% or 0.2% cholesterol diet. On both diets, the mRNA levels of ABCG5 and ABCG8 were slightly higher in L1-Tg112 versus WT mice (Figure 3B). While the amounts of ABCG5 and ABCG8 proteins remained unchanged on the 0.015% cholesterol diet, ABCG5 protein appeared to be modestly increased in L1-Tg liver compared with WT liver on the 0.2% cholesterol diet (Figure 3C). Regardless of genotype, consumption of the 0.2% cholesterol diet resulted in the expected increase in hepatic ABCG5 and ABCG8 mRNAs and proteins compared with the 0.015% cholesterol diet because the 2 genes are targets of liver X receptor (LXR) (26). Thus, the observed reduction in biliary cholesterol of L1-Tg mice was not coupled to decreased hepatic expression of ABCG5 and ABCG8. Because ABCB4 and ABCB11 also alter biliary cholesterol secretion (27, 28), mRNA levels of ABCB4 and ABCB11 were measured, and no appreciable changes were found (Figure 3B).
Hepatic cholesterol content is minimally affected in L1-Tg mice. To probe the metabolic consequence of NPC1L1-mediated decrease in biliary cholesterol excretion, hepatic and plasma lipids were analyzed. No differences were detected among the 0.015% cholesterol–fed WT, L1-Tg20, and L1-Tg112 mice in the hepatic content of FC, triglyceride (TG), and PL while the cholesteryl ester (CE) content in L1-Tg20 mice was slightly but significantly higher than in WT mice (Table 1). When fed the 0.2% cholesterol diet, L1-Tg20 and L1-Tg112 mice had modestly but significantly higher amounts of hepatic FC and PL compared with WT mice (Table 1). No differences were found in the hepatic content of CE and TG among L1-Tg20, L1-Tg112, and WT mice fed the 0.2% cholesterol diet. Thus, the NPC1L1-mediated inhibition of biliary cholesterol excretion only resulted in modest accumulation of cholesterol in the liver. In addition, no differences were observed between WT and L1-Tg mice in body weights, liver weights, liver/body weight ratios, and plasma levels of aspartate aminotransferase, alanine aminotransferase, and bilirubin (Table 1), which indicates that L1-Tg mice grow normally and have normal liver function.
Hepatic and plasma parameters of WT and L1-Tg mice
The minimal changes in the hepatic cholesterol concentration of L1-Tg mice was consistent with the minor changes in the mRNA levels of LXR target genes and genes in the cholesterol biosynthetic pathway, including SREBP-1c, PL transfer protein, ABCG5/ABCG8 (Figure 3B), SREBP-2, HMG coenzyme A reductase, and HMG coenzyme A synthase (data not shown).
Plasma cholesterol is increased in L1-Tg mice. To probe the trafficking of NPC1L1-derived cholesterol, plasma lipids were measured. Interestingly, significant increases in plasma cholesterol concentrations were observed in L1-Tg mice (Table 1). When fed the 0.015% cholesterol diet, L1-Tg20 and L1-Tg112 mice showed a 43% increase in plasma total cholesterol (TC) and a 41% and 21% increase, respectively, in FC compared with WT mice. Feeding the 0.2% versus 0.015% cholesterol diet caused plasma TC and FC to increase for all 3 genotypes. However, L1-Tg20 and L1-Tg112 mice still displayed significantly higher plasma TC and FC concentrations than in WT mice. Plasma TG concentrations were similar for mice of all 3 genotypes on both diets.
Increased plasma cholesterol largely accumulates in apoE-rich HDL in L1-Tg mice. To further characterize the changes in plasma cholesterol concentration, plasma lipoprotein cholesterol and apolipoprotein distributions were determined for L1-Tg112 and WT mice (Figure 4, A and B). On both 0.015% and 0.2% cholesterol diets, L1-Tg112 compared with WT mice had increases in HDL-cholesterol. However, the apolipoprotein composition of L1-Tg112 and WT HDL was very similar (Figure 4B; fractions 29–32), with the major constituent being apoA-I and the minor constituents being apoA-II, apoA-IV, and apoCs. In addition to the increase in HDL-cholesterol, L1-Tg112 mice had a dramatic increase in cholesterol carried on lipoproteins with a size indicative of either small LDL or large HDL. Although minor amounts of apoB100, apoA-I, apoA-II, and apoCs were present, the main apolipoprotein on this lipoprotein species in both L1-Tg112 and WT mice was apoE (Figure 4B; fractions 24–26). Because the amount of apoE on the apolipoprotein distribution gel is not quantitative, plasma apoE levels were determined by immunoblotting and densitometry. Compared with WT mice, the plasma apoE protein level was significantly higher in L1-Tg mice (Figure 4C).
Increased plasma cholesterol is mainly carried on the apoE-rich HDL. (A) Plasma lipoprotein cholesterol distribution in male WT and L1-Tg112 mice was determined as described in Methods. (B) Apolipoprotein distribution of plasma lipoproteins from male WT and L1-Tg112 mice was determined as described in Methods. Data represent a qualitative, not a quantitative, analysis of apolipoproteins. (C) Quantification of plasma apoE levels by immunoblotting. Plasma was collected from 4 WT and 4 L1-Tg112 mice fed the 0.2% cholesterol diet for 21 days. After 1:200 dilution in saline, 14 μl of diluted samples was immunoblotted with a rabbit anti-rat apoE serum and quantified as described in Methods. Values are mean ± SEM of 4 samples. (D) FA composition of lipoprotein CE was determined as described in Methods in male WT and L1-Tg112 mice. Values are the sum of the percentages of saturated (16:0 and 18:0) and monounsaturated (16:1 and 18:1) FAs divided by the percentage of polyunsaturated (18:2, 20:4, 20:5, and 22:6) FAs. *P < 0.05 versus WT (Student’s t test).
To characterize the apoE-rich lipoprotein (ERL), we isolated LDL, ERL, and HDL from WT and L1-Tg112 mice by size exclusion chromatography and determined the percent chemical compositions of these particles as described previously (29). Similar to apolipoprotein compositions (Figure 4B), the percent chemical compositions were nearly identical within each lipoprotein class isolated from WT and L1-Tg112 mice (Table 2). However, percent chemical compositions of LDL, ERL, and HDL were dramatically different. ERL had percentages of FC and protein that were similar to those of LDL. In contrast, the percentages of TG and PL on ERL were like those of HDL. Thus, ERL appeared to have chemical compositions similar to both LDL and HDL. However, compared with LDL and HDL, ERL was unique in that it contained an intermediate amount of CE and had the lowest CE/FC ratio, indicating the enrichment of FC in this lipoprotein particle.
Chemical composition of plasma lipoproteins
It has been shown that in mice, saturated and monounsaturated fatty acids (FAs), which are predominantly used by acyl-CoA:cholesterol acyltransferase 2 (ACAT2) in liver, are the major FAs in LDL-CE, while polyunsaturated FAs, which are preferentially used by lecithin:cholesterol acyltransferase (LCAT) in plasma, represent the bulk of FAs in HDL-CE (30, 31). To further define ERL, the FA composition of CE in ERL was determined. The ratios of saturated plus monounsaturated FA to polyunsaturated FA for LDL-CE, ERL-CE, and HDL-CE were approximately 3, 0.8, and 0.6, respectively (Figure 4D). These results indicate that the FA composition of ERL-CE is similar to that found in HDL-CE, which is mainly LCAT derived. Thus, we designated these lipoprotein particles “apoE-rich HDL.”
Because large apoE-rich HDL particles are also observed in mice lacking scavenger receptor class B type I (SR-BI) (32, 33) and in transgenic mice overexpressing ABCA1 in the liver (34), hepatic expression of SR-BI and ABCA1 were determined. Regardless of genotype and cholesterol feeding, hepatic mRNA levels of SR-BI and ABCA1 were largely unaffected (data not shown). SR-BI protein levels also remained unchanged (Figure 5). Interestingly, ABCA1 protein levels were consistently higher in L1-Tg mice compared with WT mice fed either diet (Figure 5). When compared with the mice fed the 0.015% cholesterol diet, both WT and L1-Tg mice fed the 0.2% cholesterol diet had higher ABCA1 protein levels. To further explore the mechanism underlying cholesterol accumulation in the plasma of L1-Tg mice, the hepatic mRNA levels of other genes important in lipoprotein metabolism — including LDL receptor (LDLR), apoB, apoE, and proprotein convertase subtilisin/kexin type 9a (PCSK9) — were also quantified, and no changes were found (data not shown). The protein level of LDLR was also determined, and no changes were observed (Figure 5).
Hepatic ABCA1 protein is increased in L1-Tg mice. The immunoblotting filter used in Figure 2C was stripped and reblotted with the antibodies to ABCA1 (51), SR-BI (46), and LDLR (46). The immunoblot of human NPC1L1 and RAP shown in Figure 3C are reproduced here as a control. The experiment was repeated twice with similar results.
Ezetimibe restores biliary cholesterol excretion in L1-Tg mice. To test our hypothesis that ezetimibe inhibits hepatic NPC1L1 function, the effect of ezetimibe on biliary cholesterol excretion was determined in WT and L1-Tg20 mice treated with either vehicle or 10 mg ezetimibe/kg/d for 4 days. Bile was collected by cannulation of the common bile duct, and its lipid composition was measured (Figure 6A). Based upon molar ratio, 10-fold less cholesterol was found in the bile of vehicle-treated L1-Tg20 versus WT mice. However, the molar ratio of biliary cholesterol in ezetimibe-treated L1-Tg20 mice was similar to that of ezetimibe-treated WT mice. Results were similar for biliary cholesterol concentrations. Unlike cholesterol, biliary PL and BA concentrations and molar ratios were not different in WT and L1-Tg20 mice treated with either vehicle or ezetimibe. Furthermore, it was determined that ezetimibe increased fecal neutral sterol (cholesterol and its bacterial metabolites) excretion in both WT and L1-Tg20 mice (Figure 6B), thus demonstrating that the effect of ezetimibe on intestinal cholesterol absorption was maintained in L1-Tg mice.
Ezetimibe restores biliary and plasma cholesterol concentrations in L1-Tg mice. (A) Bile was collected by common bile duct cannulation from male WT and L1-Tg20 mice treated with ezetimibe (Ezet) as described in Methods. Biliary lipid concentrations were analyzed, and lipid molar ratios were calculated. Veh, vehicle. (B) Fecal neutral sterol excretion was measured in WT and L1-Tg20 mice treated with ezetimibe as described in Methods. (C) After being fed the 0.015% cholesterol diet for 18 days, male WT and L1-Tg112 mice were gavaged daily on days 18–24 with either 10 mg/kg ezetimibe or vehicle. Mice were maintained on the same diet during the treatment. After a 4-hour fast on day 25, mice were sacrificed, and plasma TC concentration was determined. Values are mean ± SEM of 4–5 (A and B) or 5–7 (C) samples. P < 0.05 among *, #, and † groups for each measurement (ANOVA).
Ezetimibe restores plasma cholesterol concentration in L1-Tg mice. To examine the effect of ezetimibe on plasma cholesterol metabolism, plasma TC was measured in mice treated with either vehicle or ezetimibe (10 mg/kg/d) for 7 days (Figure 6C). When treated with vehicle, L1-Tg112 mice maintained a significant 38% increase in plasma TC compared with WT mice. However, administration of ezetimibe to L1-Tg112 mice caused the TC concentration to significantly decrease by 26% compared with vehicle-treated L1-Tg112 mice. In addition, the TC concentration of ezetimibe-treated L1-Tg112 mice was not significantly different from that of either vehicle-treated or ezetimibe-treated WT mice.