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Role of plasma and liver cholesterol- and lipoprotein-metabolism determinants in LpX formation in the mouse
Biochimica et Biophysica Acta 1770 (2007) 979 – 988 www.elsevier.com/locate/bbagen
Role of plasma and liver cholesterol- and lipoprotein-metabolism determinants in LpX formation in the mouse Ignacio Bravo a , Ludwig Amigo a , David E. Cohen b , Flavio Nervi a , Attilio Rigotti a , Omar Francone c , Silvana Zanlungo a,⁎ a
Departamento de Gastroenterología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Marcoleta 367, Casilla 114-D, Santiago, Chile b Brigham and Women’s Hospital, Boston, MA 02115, USA c Pfizer Global Research and Development, Department of Cardiovascular and Metabolic Diseases, Groton, CT 06340, USA Received 30 October 2006; received in revised form 1 February 2007; accepted 21 February 2007 Available online 1 March 2007
Abstract Cholestasis is characterized by hypercholesterolemia and the appearance of an abnormal lipoprotein, lipoprotein X (LpX), in plasma. The mechanisms responsible for this cholestatic plasma lipid phenotype are not fully understood. We used ATP-binding cassette A1 (ABCA1)(−/−) and scavenger receptor class B type I (SR-BI)(−/−) mice to test the hypothesis that hepatic sinusoidal cholesterol transporters contribute to LpX formation and hypercholesterolemia during cholestasis. Bile-duct ligation (BDL) of both ABCA1(−/−) and SR-BI(−/−) mice, as well as their respective controls, induced a dramatic increase in plasma cholesterol and phospholipid concentrations. Plasma fractionation revealed the presence of LpX in plasma of cholestatic mice, irrespective of their genetic background. We observed that the presence of HDL before cholestasis, a decrease in the activity of LCAT, and an increase in VLDL synthesis were not required for hypercholesterolemia and lipoprotein modifications induced by obstructive cholestasis in mice. In addition, murine cholestasis resulted in increased hepatic cholesterol synthesis that may contribute to the higher plasma free cholesterol levels found during the early hours after BDL. Together these findings indicate that hypercholesterolemia and LpX formation associated with obstructive cholestasis are correlated with an increase in hepatic cholesterol synthesis and are independent of plasma HDL levels, LCAT activity, VLDL synthesis, and ABCA1 and SR-BI expression. © 2007 Elsevier B.V. All rights reserved. Keywords: Cholestasis; Hypercholesterolemia; Lipoprotein X
1. Introduction Cholestasis induces a dramatic increase in plasma total and free cholesterol and the appearance of an abnormal lipoprotein, lipoprotein X (LpX), in the plasma [1]. The exact mechanisms underlying these plasma lipid changes are not well understood. There is considerable evidence that during cholestasis there is a quantitative shift of lipid secretion from bile into the plasma compartment. First, the structure and composition of LpX are very similar to those of biliary secretory vesicles [2]. Second, Abbreviations: LpX, lipoprotein X; BDL, bile-duct ligation; ABCA1, ATPbinding cassette A1; cyp7A1, cholesterol 7α-hydroxylase; SR-BI, scavenger receptor class B type I; IDL, intermediate-density lipoprotein; MTTP, microsomal triglyceride transfer protein; pI–pC, polyinosinic/polycytidylic ribonucleic acid ⁎ Corresponding author. Tel.: +56 2 6863820; fax: 56 2 6397780. E-mail address: [email protected] (S. Zanlungo). 0304-4165/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2007.02.010
mice incapable of producing biliary vesicles – due to a disruption in the canalicular membrane phosphatidylcholine translocator, Mdr2 (Abcb4) – do not exhibit hypercholesterolemia or produce LpX particles during cholestasis [3]. These observations have led to the proposal that biliary vesicles, which are still formed during cholestasis and secreted into the canaliculus, are regurgitated from the biliary compartment to the plasma via a paracellular route [4]. However, not all the available data fit this model. For example, the paracellulartransport hypothesis is dependent on the tight junctions between hepatocytes during cholestasis being sufficiently damaged to allow a significant flux of lipid polymolecular complexes from bile into plasma [5–7]. However, this has not been observed at the beginning of cholestasis, whereas the changes in plasma lipids can be detected as early as 8 h after bile-duct ligation (BDL) in mice [8]. Also, the increase in plasma lipid levels during cholestasis is comparable to the efflux of lipids from the
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liver into the bile canaliculus in mice [3]. Therefore, it seems reasonable to propose that during lipid plasma accumulation in BDL, biliary lipids are reverse transcytosed through the hepatocyte and/or directly secreted from the liver cells into the plasma via a mechanism mediated by lipid transporters present in the hepatocyte sinusoidal membrane. Two plasma membrane proteins are good candidates for playing an important pathophysiological role in the plasma hypercholesterolemic phenotype observed during cholestasis. One is ATP-binding cassette A1 (ABCA1), a transmembrane lipid translocator involved in cellular cholesterol efflux. ABCA1 mediates cholesterol and phospholipid transport from cells into lipid-free apolipoproteins that interact with the plasma membrane [9,10]. ABCA1 is abundantly expressed in the liver [11], and several studies have found that hepatic ABCA1 is important to maintaining normal plasma levels of high-density lipoprotein (HDL) [12–14]. The second candidate for involvement in cholesterol cellular efflux is the scavenger receptor class B type I (SR-BI). SR-BI is involved in the bidirectional flow of lipids between cells and lipoproteins [15]. This receptor mediates HDL cholesterol uptake by the liver, is highly expressed in the liver sinusoidal membrane [16,17], and can mediate both selective cellular uptake of cholesterol esters from diverse lipoproteins [15] and nonesterified cholesterol efflux from cells [16,18,19]. Hyperlipidemia and LpX have also been described in other disease conditions related to lipoprotein metabolism, including those related to abnormal HDL such as deficiency of lecithin: cholesterol acyltransferase (LCAT) [20]. LCAT catalyzes freecholesterol esterification in HDL [21], and in LCAT deficiency the HDL fraction is dramatically decreased and LpX-like particles appear [22,23]. The same lipoprotein pattern has also been reported in other experimental models in which HDL metabolism was altered [24–26]. All these represent new models of LpX formation since its generation would be independent of cholestasis. Thus, questions arise as to how LpX is generated in noncholestatic conditions and whether the same mechanisms are involved in LpX formation with and without cholestasis. Abnormal remodeling of very-low-density lipoprotein (VLDL) could also be important in LpX formation. One study has demonstrated that in vitro lipolysis of VLDL generates LpX-like particles [27]. Since hepatocytes secrete lipids into the plasma compartment as VLDL particles [28], modification of these lipoproteins might also generate LpX. On the other hand, cholestatic hyperlipidemia has been associated with increased cholesterol synthesis in rats [29–33], which correlated with an increase in HMG-CoA reductase (HMG-CoA red) activity [34,35]. Hepatic newly synthesized cholesterol could contribute and might even be a key determinant of increased plasma cholesterol during obstructive cholestasis. In this paper we propose an important role for the liver in the lipid plasma changes that occur during cholestasis. We analyzed the relevance of ABCA1 and SR-BI expressions by inducing BDL in ABCA1(−/−) and SR-BI(−/−) mice models. We also analyzed LCAT activity and the role of VLDL and hepatic cholesterol synthesis during cholestasis in mice.
2. Materials and methods 2.1. Animals and diet The wild-type C57BL/6J murine strain was originally purchased from Jackson Laboratory (Bar Harbor, ME) and bred to generate our own colony. ABCA1-deficient mice (DBA/1-Abca1tm1Jdm) and LCAT transgenic mice (C57BL/6J) were obtained from Pfizer (Groton, CT) and bred for experiments in our animal facility. Genotypes of ABCA1 mice were identified using polymerase chain reaction (PCR)-based screening as described previously [36]. Mice with a targeted mutation in the sr-bi locus were obtained by gene disruption in a 129/Sv-derived embryonic stem-cell line as described previously [37] and also genotyped by PCR [37]. Liver-specific mttp-knockout mice were obtained after treating Mttpflox/floxMx1-Cre mice (donated by Dr. Steven Young, Gladstone Institute of Cardiovascular Disease, San Francisco, CA) with polyinosinic/polycytidylic ribonucleic acid (pI–pC; Sigma Chemical, St. Louis, MO) every other day for 6 days as described previously [38]. All mice had free access to water and a chow diet (< 0.02% cholesterol; Prolab RMH 3000, PMI Feeds, St. Louis, MO). Protocols were performed according to accepted criteria for the humane care of experimental animals, and were approved by the review board for animal studies of the Pontificia Universidad Católica de Chile.
2.2. Common bile duct ligation Two-month-old mice with different genetic backgrounds were subjected to BDL as described previously [3]. This procedure is a well-characterized model of liver damage induced by acute obstructive cholestasis. Animals were anesthetized by an intraperitoneal injection of Nembutal (50 mg/kg). The abdomen was opened by a midline incision, the common bile duct was ligated as close to the liver as possible, the gallbladder was ligated and removed, and then the abdomen was closed. Sham-operated animals were subjected to the same surgical procedure but without ligation of the common bile duct and removal of the gallbladder. Plasma and livers were collected 24 and 48 h later. Aliquots of liver tissue were frozen in liquid nitrogen for the subsequent preparation of protein and total RNA samples.
2.3. Plasma lipoprotein separation and plasma and hepatic biochemical analyses Plasma lipoprotein separation was performed by Superose 6 fast protein liquid chromatography (FPLC) gel filtration [37,39]. Plasma total, free, and lipoprotein cholesterol were measured as described previously [37,39]. The hepatic cholesterol content was determined by routine methods [40,41]. Plasma total phospholipids were measured as described previously [42].
2.4. LpX separation by density gradient ultracentrifugation At 1 day after BDL or the sham operation, plasma was centrifuged at 100,000 rpm for 1 h at 16 °C in a KBr gradient (density, 1.3 g/ml) as described previously [43]. After centrifugation, 1-ml fractions were collected from the gradient and their density and lipid contents were measured.
2.5. Immunoblotting analysis For ABCA1, SR-BI, Na+-taurocholate cotransporting protein, NTCP (Slc10a1), and multidrug resistance protein 3, MRP3 (ABCC3), immunoblotting, total membrane extracts from mouse liver were prepared [44] and separated (50 μg of protein/sample) by 8% SDS-PAGE. Samples were immunoblotted using anti-ABCA1 antiserum (donated by Dr. Mason Freeman, Harvard Medical School, Boston, MA), an anti-SR-BI antiserum (obtained from Dr. Monty Krieger, Massachusetts Institute of Technology, Cambridge, MA), and anti-Slc10a1 and anti-ABCC3 antisera (provided by Dr. Marco Arrese, Pontificia Universidad Católica, Santiago, Chile). Anti-ε-cop antibody (obtained from Dr. Monty Krieger) was used as a membrane protein-loading control. Antibody binding to protein samples was visualized using enhanced chemiluminescence and measured using a molecular imaging system (GS-525, BIO-RAD, Hercules, CA).
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2.6. Semiquantitative RT-PCR assay. Five micrograms of total liver RNA was reverse transcribed using random primer hexamers. Semiquantitative reverse-transcription PCR (RT-PCR) was performed for different genes using primers based on mouse and rat cDNA sequences available through GeneBank databases. The expressions of target genes cyp7A1, bsep (abcb11), hmg-CoA reductase, and mttp were analyzed by coamplification with β-actin or 18S internal standards (Ambion, Austin, TX) to quantify transcript abundance. PCR was performed in the presence of [α-32P] dCTP, and PCR products were resolved on a 2% agarose gel and transferred to a nylon filter. Radiolabeled bands were quantified using a molecular imaging system (GS-525, BIO-RAD), with the results normalized to the signal by from the radiolabeled β-actin or 18S PCR products.
2.7. LCAT-activity analysis The exogenous LCAT in plasma from different groups was assayed as described previously [45] by measuring the rate of synthesis of 3H-cholesteryl esters from unilamellar vesicles activated with apolipoprotein A-I.
2.8. Hepatic cholesterol synthesis in vivo The rate of cholesterol synthesis was measured after a 12-h fasting period. Each mouse received 50 mCi of [3H] water (Amersham Pharmacia Biotech, Piscataway, NJ) by intraperitoneal injection as described previously [39,46]. One hour later, animals were anesthetized and blood was obtained for the determination of the water-specific activity in plasma. Livers were removed and saponified, and digitonin-precipitable sterols were isolated as described previously [46]. Results are expressed as micromoles of [3H] water incorporated into digitonin-precipitable sterols per hour per gram of liver weight.
2.9. Statistics The two-tailed, unpaired Student’s t test was used to compare data sets. Differences were considered statistically significant at a probability value of p < 0.05.
3. Results 3.1. Hepatic expression of genes related to lipid metabolism and transport in BDL mice We first analyzed the hepatic expressions of various genes involved in cholesterol metabolism and transport in BDL mice. The hepatic expressions of cholesterol 7α-hydroxylase (cyp7a1), hmg-CoA red, microsomal triglyceride transfer protein (mttp), and bile salt export pump (abcb11) were analyzed by semiquantitative RT-PCR (Fig. 1). Hepatic mRNA levels of hmg-CoA red, mttp, and abcb11 were unchanged by BDL, whereas cyp7a1 mRNA levels decreased to 83% after 24 h of BDL and remained low at 48 h. ABCA1 and SR-BI were analyzed by immunoblotting as shown in Fig. 2. Hepatic ABCA1 protein levels decreased to 21% after 48 h of BDL and SR-BI protein levels decreased to 77% and 66% after 24 and 48 h of BDL, respectively. We also analyzed the hepatic expressions of the basolateral membrane transporters ABCC3 and Slc10a1 since it has been proposed that they play a major protective role against cholestasis [47]. ABCC3 protein expression did not differ between control and BDL mice, while Slc10a1 protein expression was dramatically reduced after 24 and 48 h of BDL (Fig. 2).
Fig. 1. Effect of BDL on hepatic expressions of cyp7A1, hmg-CoA red, mttp, and abcb11 in mice. RNA expressions were analyzed by semiquantitative RT-PCR in the presence of [α-32P]dCTP, with β-actin or 18S primers included in each sample as controls. The products were resolved in a 2% agarose gel, transferred to a nylon filter, and the radiolabeled signals were quantified. Results are representative of three independent experiments. (a) indicates a significant difference (p < 0.05) compared to sham-operated animals.
Together these results show that hepatic ABCA1, SR-BI, Slc10a1 and cyp7a1 levels are modulated early after the induction of obstructive cholestasis in mice. 3.2. Plasma lipoprotein cholesterol analysis in BDL wild-type and ABCA1(−/−) mice To assess whether ABCA1 expression is relevant to the hypercholesterolemic response observed during cholestasis, both wild-type (ABCA1(+/+)) and ABCA1(−/−) mice were subjected to BDL, and plasma was collected and analyzed after 24 and 48 h. As expected, in ABCA1(+/+) mice plasma total cholesterol and phospholipids were higher after 24 h of BDL, and increased further during the following 24 h (Fig. 3A). As described previously [3], the rise in plasma cholesterol during cholestasis was mainly due to the accumulation of free cholesterol (Fig. 3A). Plasma cholesterol and phospholipids levels were significantly lower in sham-operated ABCA1(−/−) mice than in controls, as has been described previously [48]. BDL in ABCA1(−/−) mice induced a significant rise in plasma total and free cholesterol and phospholipids at 24 and 48 h (Fig. 3A), similar to the values found in cholestatic ABCA1(+/+) mice. To analyze the cholesterol content in the different lipoproteins, plasma samples from sham-operated and ABCA1(+/+) and ABCA1(−/−) BDL mice were subjected to FPLC. Plasma from ABCA1(+/+) BDL mice showed increased cholesterol transport in large lipoproteins that eluted in the FPLC fractions corresponding to very low density (VLDL) and intermediatedensity/low-density lipoproteins (IDL/LDL) relative to control animals at 24 and 48 h (Fig. 3B), whereas the cholesterol content of the HDL fraction was dramatically reduced (Fig. 3B). LpX elutes in the void volume of the FPLC fractionation [3], and hence these results suggest that the dramatic increase in cholesterol transported in VLDL-sized lipoproteins observed in cholestatic mice was due to the presence of LpX in these FPLC fractions. This is supported by the increase in the plasma levels
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deficient mice occurred in an HDL peak that was larger and more heterogeneous than the SR-BI(+/+) HDL peak (Fig. 4B) [36]. SR-BI(+/+) and SR-BI(−/−) mice showed similar responses in their plasma levels of total and free cholesterol and phospholipids after BDL (Fig. 4A). Also, cholesterol lipoprotein profiles after BDL, including the appearance of the large VLDL-sized lipoproteins, were very similar in SR-BI(+/+) and SR-BI(−/−) mice (Fig. 4B). Together these results show that SR-BI expression is not critical to the hypercholesterolemic response present during cholestasis in mice. 3.4. LCAT activity in BDL mice
Fig. 2. Effect of BDL on hepatic expressions of ABCA1, SR-BI, ABCC3, and Slc10a1 in mice. Total membrane extracts were prepared, size fractionated by SDS-PAGE, immunoblotted with anti-ABCA1, anti-SR-BI, anti-ABCC3, and anti-Slc10a1 antibodies, and subjected to densitometric analysis. Anti-ε-cop and anti-prealbumin antibodies were used as protein-loading controls. (a) indicates a significant difference (p < 0.05) compared to sham-operated animals.
of free cholesterol and phospholipids, which are the two main lipid components of LpX (Fig. 3A). As expected, HDL levels were very low and the plasma cholesterol was mostly contained in VLDL particles in shamoperated ABCA1(−/−) mice [49] (Fig. 3B). Although we found some differences in the IDL/LDL-sized lipoprotein fractions between ABCA1(−/−) and ABCA1(+/+) BDL mice after 24 h, the cholesterol lipoprotein profiles were similar after 48 h, even though the profiles of sham-operated animals were very different. Together these results show that ABCA1 expression is not relevant to the hypercholesterolemic response determined by acute obstructive cholestasis. 3.3. Plasma lipoprotein cholesterol analysis in BDL wild-type and SR-BI(−/−) mice To analyze whether SR-BI expression is relevant to the hypercholesterolemic phenotype observed during cholestasis, both wild-type (SR-BI(+/+)) and SR-BI(−/−) mice were subjected to BDL. Plasma cholesterol and phospholipids levels were significantly higher in sham-operated SR-BI(−/−) mice than in controls, as described previously (Fig. 4A) [37]. As expected, most of the plasma cholesterol present in SR-BI-
Reduced LCAT activity has been linked to LpX appearance in humans and in animal models. Indeed, LpX particles are commonly seen in human LCAT-deficient plasma [22,23]. In addition, LCAT-deficient mice that are transgenic for SREBP1a form LpX-like particles in plasma [26]. To determine whether changes in LCAT activity were correlated with the hypercholesterolemic response and the appearance of LpX in cholestasis, LCAT activity was measured in mice after 24 and 48 h of BDL. LCAT activity was significantly reduced at both time points (Fig. 5). To further analyze the relevance of decreased LCAT activity to LpX formation during cholestasis, plasma cholesterol levels and lipoprotein profiles were analyzed in LCAT transgenic mice [45] subjected to BDL. No differences in the abnormal plasma lipid phenotype were observed between wild-type and LCAT transgenic mice subjected to BDL (data not shown). These results indicate that the decrease in LCAT activity observed in BDL mice is a secondary effect of cholestasis, with it not playing a major role in LpX formation during biliary obstruction. 3.5. Plasma lipoprotein cholesterol analysis in BDL wild-type and MTTP(−/−) mice MTTP is essential to the transfer of triglycerides into the lumen of the endoplasmic reticulum for VLDL assembly, and is required for the secretion of apolipoprotein B-100 from the liver [38]. To evaluate the possible contribution of hepatic VLDL production to LpX formation during cholestasis, we used the well-characterized mouse model deficient in the mttp gene harboring a “floxed” mttp allele, and then used Cre-mediated recombination to generate liver-specific Mttp-knockout mice after pI–pC treatment [38]. Plasma cholesterol accumulation was determined in wildtype and liver-specific MTTP(−/−) mice subjected to BDL. As shown in Fig. 6A, although the plasma cholesterol content was low in MTTP(−/−) mice before cholestasis, BDL induced similar changes in plasma concentrations of total and free cholesterol in MTTP(−/−) and wild-type mice. Also, the cholesterol content in density-fractionated LpX particles were very similar in the wild-type and MTTP(−/−) mice (Fig. 6B). These results indicate that LpX is not synthesized as a byproduct of the VLDL biosynthetic pathway (or at least that it
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Fig. 3. Plasma cholesterol and phospholipid levels during BDL in ABCA1(+/+) and ABCA1(−/−) mice. ABCA1(+/+) and ABCA1(−/−) mice were subjected to BDL for 24 or 48 h, or to a sham operation (48 h), and plasma samples were obtained. (A) Concentrations of total and free cholesterol and phospholipids in plasma. Data are mean ± SD values from five animals. (a), (b), (c), and (d) indicate a significant difference in the levels of total (a, b) and free (c, d) cholesterol after 24 and 48 h of BDL between ABCA1(+/+) (a, c) or ABCA1(−/−) (b, d) mice and their respective sham-operated controls. The levels of total and free cholesterol did not differ significantly between 24 and 48 h of BDL in either ABCA1(+/+) or ABCA1(−/−) mice, nor between ABCA1(+/+) and ABCA1(−/−) mice after 24 or 48 h of BDL. (e) and (f) indicate a significant difference in phospholipid levels after 24 and 48 h of BDL between ABCA1(+/+) (e) or ABCA1(−/−) (f) mice and their respective sham-operated controls. The phospholipid levels did not differ between 24 and 48 h of BDL in either ABCA1(+/+) or in ABCA1(−/−) mice, nor between ABCA1(+/+) and ABCA1 (−/−) mice after 24 or 48 h of BDL. (B) Plasma lipoprotein cholesterol profiles as analyzed by FPLC in plasma from ABCA1(+/+) (○) and ABCA1(−/−) (●) mice. The chromatograms are representative of several independent FPLC analyses for each experimental condition. The approximate elution positions of VLDL, IDL/LDL, and HDL are indicated.
does not derive from a VLDL-type particle) as a consequence of acute biliary obstruction. 3.6. Hepatic cholesterol content and synthesis in BDL mice Studies of hepatic cholesterol synthesis in cholestasis have been reported in BDL rats [29–35] and in pharmacologically induced intrahepatic cholestasis in mice [50]. As shown in Fig. 7, BDL increased the hepatic contents of total and free cholesterol by nearly 20% each at 24 h, and by 40% and 55% at 48 h, respectively. Despite these increases in liver cholesterol, in vivo hepatic synthesis of cholesterol was increased by 253% after 24 and 48 h of BDL. These results suggest that newly synthesized cholesterol contributes to the increase in hepatic cholesterol content, and also that this is a key contributor to the hypercholesterolemic response observed during obstructive cholestasis.
4. Discussion Cholestasis is characterized by hypercholesterolemia and the appearance of LpX in plasma. The mechanisms responsible for this cholestatic plasma lipid phenotype are not fully understood. Using Abcb4-deficient mice, Elferink et al. [3] have shown that the expression of the canalicular translocator for phophatidylcholine, the Abcb4 protein, is crucial to the generation of the cholestatic LpX. These results are in line with the hypothesis that LpX is derived from biliary vesicles that somehow appear in the plasma [3], increasing cholesterol levels in the bloodstream. However, how are these vesicles formed and how do they get into the plasma compartment? One possibility is that biliary vesicles secreted into the canaliculus are regurgitated from the biliary compartment to the plasma via a paracellular route [4]. However, this mechanism seems unlikely because it requires hepatocellular tight junctions to be sufficiently open to
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Fig. 4. Plasma cholesterol and phospholipid levels during BDL in SR-BI(+/+) and SR-BI(−/−) mice. SR-BI(+/+) and SR-BI(−/−) mice were subjected to BDL for 24 or 48 h, or to a sham operation (48 h), and plasma samples were obtained. (A) Concentrations of total and free cholesterol and phospholipids in plasma. Data are mean ± SD values from five animals. (a), (b), (c), and (d) indicate a significant difference in the levels of total (a, b) and free (c, d) cholesterol after 24 and 48 h of BDL between SR-BI(+/+) (a, c) or SR-BI(−/−) (b, d) mice and their respective sham-operated controls. The levels of total and free cholesterol did not differ significantly between 24 and 48 h of BDL in either SR-BI(+/+) or SR-BI(−/−) mice, nor between SR-BI(+/+) and SR-BI(−/−) mice after 24 h or 48 h of BDL. (e) and (f) indicate a significant difference in phospholipid levels after 24 and 48 h of BDL between SR-BI(+/+) (e) or SR-BI(−/−) (f) mice and their respective sham-operated controls. The phospholipid levels did not differ between 24 and 48 h of BDL in either SR-BI(+/+) or SR-BI(−/−) mice, nor between SR-BI(+/+) and SR-BI(−/−) mice after 24 h or 48 h of BDL. (B) Plasma lipoprotein cholesterol profiles as analyzed by FPLC in plasma from SR-BI(+/+) (○) and SR-BI(−/−) (●) mice. The chromatograms are representative of several independent FPLC analyses for each experimental condition. The approximate elution positions of VLDL, IDL/LDL, and HDL are indicated.
allow 70-nm vesicles to pass from the biliary into Disse’s space. At least at the early stages of cholestasis, this does not appear to be the case since hepatocyte tight junctions appear to be intact [7,51]. Furthermore, opening of the liver cell tight junctions to such an extent would lead to a complete loss of cell polarity, which has not been observed in several studies with cholestatic models [3,51,52]. Indeed, Abcb4 is still observed in the canalicular region, and also in the subapical region in pericanalicular vesicles during BDL in mice [3]. These observations suggest that LpX forms in a subapical compartment of the hepatocytes. Indeed, electron microscopy studies in BDL rats have shown LpX-like vesicles within the canaliculus and in the pericanalicular region in the presence of intact tight junctions [52]. With this background, we hypothesized that liver cells contribute actively to the hypercholesterolemic response and LpX formation in cholestasis. One possibility is that LpX formed in the subapical compartment is transcytosed toward the
sinusoidal membrane and released into the plasma. In addition, cholesterol derived from the intrahepatic metabolically active pool could contribute to plasma cholesterol and LpX by direct secretion through the sinusoidal membrane into Disse’s space. This latter hypothesis leads to the question of whether cholesterol gets into the plasma during cholestasis by a passive or a protein-mediated process. The second process seems quite plausible since the estimated sinusoidal lipid output during cholestatic conditions in mice is comparable to the biliary lipid secretion rates under normal conditions [3], a process that is mediated by several canalicular protein transporters [53]. Because ABCA1 and SR-BI are the most relevant cholesterol transporters localized to the sinusoidal membrane, we postulated that they are key regulators of the entry of cholesterol into the plasma compartment during cholestasis. However, our findings demonstrate that the sinusoidal cholesterol transporters ABCA1 and SR-BI are not relevant to the hypercholesterolemia found in cholestatic conditions in mice. These findings do not
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of HDL or because LpX is depleted of normal cofactors for LCAT activation. In fact, it has been demonstrated in vitro that isolated LpX, which does not contain apoA-I [2], the most potent LCAT activator [60], was not a suitable substrate for recombinant LCAT [2]. Furthermore, addition of ApoA-I to LpX resulted in LCAT-mediated cholesterol esterification [2]. Since we measured LCAT activity using an exogenous substrate, and our preliminary data indicate that LCAT plasma protein levels increased during cholestasis, we postulate that the enzyme is inhibited during cholestasis. Moreover, LCAT transgenic mice showed the typical hypercholesterolemic response during cholestasis, indicating that LCAT deficiency Fig. 5. Effect of BDL on plasma LCAT activity in mice. LCAT activity was measured as the rate of synthesis of 3H-cholesteryl esters (CE) from unilamellar vesicles incubated with 3H-cholesterol and activated with apolipoprotein A-I in control mice (48 h, sham operated) and in mice after 24 and 48 h of BDL. Data are mean ± SD values from three mice per group. (a) indicates a significant difference (p < 0.05) compared to sham-operated animals.
rule out the possibility that other sinusoidal lipid transport proteins (e.g., ABCG1) participate in delivering cholesterol to plasma during cholestasis. One interesting finding of this study is that the presence of HDL in plasma was not necessary for LpX formation. During BDL in mice, LpX formation and hypercholesterolemia were accompanied by a dramatic reduction in HDL levels. Moreover, humans with deficiency in LCAT – which is the main enzyme catalyzing the synthesis of cholesteryl esters in HDL [21] – show LpX-like particles in plasma [22,23]. Hypercholesterolemia and LpX-like particles are also observed in various animal models with altered HDL metabolism. For example, LpX was present in the plasma of double-knockout SR-BI(−/−) and apolipoprotein E(−/−) mice [25] and LCAT-deficient mice fed a high-fat diet [54]. These data suggested that HDL metabolism and LpX formation are related. However, the present study demonstrates that ABCA1-deficient mice, which present very low levels of plasma HDL, show an intact hypercholesterolemic response and hence presumably form LpX particles as do normal mice during BDL. Furthermore, we found a similar cholestasis-induced dyslipidemic phenotype in apolipoprotein A-I and PCTP gene-targeted murine strains, which also exhibit abnormal HDL metabolism in vivo [21,55] (data not shown). Previous data support a connection between LCAT deficiency and LpX formation [22,23]. Also, LCAT activity is decreased in cholestatic conditions (our results and [56,57]). The mechanism for the decrease in LCAT during cholestasis is unknown, but it could be related to the reduction in HDL levels, a decrease in the hepatic expression of the enzyme, or to inhibition of the enzyme activity. With regard to this latter mechanism, plasma bile acids are characteristically increased during cholestasis [47,58] and high concentrations of these amphipatic molecules reduce LCAT activity [59]. The decrease in LCAT activity may also be attributable to the inability of the enzyme to use LpX as a substrate, because the bilaminar structure of this abnormal lipoprotein is very different from that
Fig. 6. Plasma cholesterol levels during BDL in MTTP(+/+) and MTTP(−/−) mice. MTTP(+/+) and MTTP(−/−) mice were subjected to BDL for 24 h, or to a sham operation (24 h), and plasma samples were obtained. (A) Concentrations of total and free cholesterol in plasma. Data are mean ± SD values from three animals. (a), (b), (c), and (d) indicate a significant difference in the levels of total (a, b) and free (c, d) cholesterol between controls and after 24 h of BDL in MTTP(+/+) (a, c) and MTTP(−/−) (b, d) mice. (B) Plasma was loaded onto a KBr density gradient for the separation of lipoproteins by ultracentrifugation. The levels of total and free cholesterol were determined in the fraction where LpX was collected. (a), (b), (c), and (d) indicate a significant difference in the levels of total (a, b) and free (c, d) cholesterol between controls and after 24 h of BDL in MTTP(+/+) (a, c) and MTTP(−/−) (b, d) mice.
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Fig. 7. Effect of BDL on hepatic cholesterol content and synthesis in mice. (A) Hepatic contents of total and free cholesterol were determined after 24 and 48 h of BDL or the sham operation (48 h). (a) and (b) indicate that the contents of total (a) and free (b) cholesterol in mice after 24 and 48 h of BDL differed significantly from that in control mice (p < 0.05). (B) Hepatic cholesterol synthesis in vivo was determined after 24 and 48 h of BDL or the sham operation (48 h). Results are expressed as micromoles of [3H] water incorporated into digitonin-precipitable sterols per hour per gram of liver weight. (a) indicates a significant difference in cholesterol synthesis at 24 and 48 h of BDL compared to sham-operated animals.
was not a pathogenic contributor to LpX formation and that LCAT overexpression could not overcome the mechanism underlying the dyslipidemic phenotype associated with cholestasis. VLDL secretion represents the major mechanism of lipoprotein secretion from the liver into the plasma through the sinusoidal membrane, which makes it theoretically possible that LpX formation is related to VLDL secretion. However, we demonstrated that the accumulation of LpX, which elutes in the same fraction as a VLDL particle in an FPLC column, was not impaired by the lack of VLDL production in liver-specific MTTP(−/−) mice. BDL in mice induces changes in the hepatic expressions of various lipoprotein-metabolism-related genes. This study has shown for the first time that the expressions of hepatic ABCA1 and SR-BI are modulated during cholestasis in mice. We also found a significant decrease in hepatic expressions of cyp7a1 and Slc10a1, probably due to the induction of early protective mechanisms against bile-salt-mediated hepatotoxicity during cholestasis. The mechanism responsible for hepatic ABCA1 and SR-BI downregulation in cholestasis is not known. There is
recent evidence that the nuclear transcription factor PXR is involved in the regulation of the hepatic levels of ABCA1 and SR-BI. Sporstol et al. [61] demonstrated that the use of PXR agonists suppressed the activity of the promoter regions of human abca1 and sr-b1 genes in HepG2 cells and in primary rat hepatocyte cultures. Also, PXR may be activated during cholestasis by the accumulation of bile acids, which can act as PXR agonists. Interestingly, the increase in total plasma cholesterol during BDL-induced cholestasis is smaller in PXRdeficient mice than in wild-type controls, suggesting that a decrease in hepatic SR-BI and/or ABCA1 expression is indeed important to the mechanisms underlying the hyperlipidemia associated with BDL [58]. It is also possible that the nuclear factor FXR, which is also activated by the cholestasis-induced increase in hepatic bile acids, participate in hepatic SR-BI regulation due to obstructive cholestasis. In fact, FXR can suppress the hepatic levels of SR-BI mRNA in rats, mice, and humans by an indirect mechanism mediated by SHP and the activity of the transcription factor LRH-1 [62]. Another important finding of this study is the major impact of BDL-induced cholestasis on hepatic cholesterol metabolism in the mouse. The synthesis and content of hepatic cholesterol were both increased during murine cholestasis. The increase in hepatic cholesterogenesis was not inversely correlated with the increased levels of hepatic cholesterol and the expected feedback regulation between these two metabolic parameters. Similar results have been previously reported by a number of studies, including cholestatic BDL rats [29–35] and druginduced intrahepatic cholestasis in mice [50]. The mechanism by which BDL regulates cholesterol synthesis is not known, but it might be related to changes in the intracellular distribution of cholesterol, presumably in the endoplasmic reticulum where cholesterol sensing occurs. However, we found no changes in hepatic mRNA levels of HMG-CoA red, which is the key regulatory enzyme in cholesterol synthesis. Further studies are needed to establish the molecular basis for the upregulation of cholesterol synthesis in cholestasis, for which there are several possibilities. First, cholesterol accumulation may have occurred simply as a consequence of the obstruction in biliary cholesterol secretion. Second, there may have been a contribution from impaired hepatic cholesterol efflux toward the plasma due to reduced ABCA1 expression and/or the increase in the de novo hepatic synthesis, with the increase in hepatic cholesterol content being primarily due to an increase in the hepatic content of free cholesterol. Future studies should attempt to localize this free cholesterol. In summary, our data rule out the participation of ABCA1 and SR-BI in a lipid efflux process from hepatocytes to plasma during cholestasis. We have also shown that the presence of HDL precholestasis, a decrease in LCAT activity, and an intact VLDL synthesis are not required for the appearance of hypercholesterolemia and lipoprotein modifications during cholestasis in mice. The more-striking contribution of these experiments is the finding that cholestasis results in an increase in hepatic cholesterol synthesis that could contribute to the increase in free cholesterol detected in plasma under cholestatic conditions.
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Acknowledgments The authors thank Dr. Steven Young for providing the Mttpflox/floxMx1-Cre mice. This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT Grants #1030415 and #7050126 to SZ, #1030416 to AR, and #1030744 to FN), Programa de Mejoramiento de la Calidad y la Equidad de la Educación Superior (MECESUP Grant #PUC0005 to IB), and Pontificia Universidad Católica Medicine Resident Grants #PG-12/03 and #PG-16/04 to IB. References
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Role of plasma and liver cholesterol- and lipoprotein-metabolism determinants in LpX formation in the mouse Ignacio Bravo a , Ludwig Amigo a , David E. Cohen b , Flavio Nervi a , Attilio Rigotti a , Omar Francone c , Silvana Zanlungo a,⁎ a
Departamento de Gastroenterología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Marcoleta 367, Casilla 114-D, Santiago, Chile b Brigham and Women’s Hospital, Boston, MA 02115, USA c Pfizer Global Research and Development, Department of Cardiovascular and Metabolic Diseases, Groton, CT 06340, USA Received 30 October 2006; received in revised form 1 February 2007; accepted 21 February 2007 Available online 1 March 2007
Abstract Cholestasis is characterized by hypercholesterolemia and the appearance of an abnormal lipoprotein, lipoprotein X (LpX), in plasma. The mechanisms responsible for this cholestatic plasma lipid phenotype are not fully understood. We used ATP-binding cassette A1 (ABCA1)(−/−) and scavenger receptor class B type I (SR-BI)(−/−) mice to test the hypothesis that hepatic sinusoidal cholesterol transporters contribute to LpX formation and hypercholesterolemia during cholestasis. Bile-duct ligation (BDL) of both ABCA1(−/−) and SR-BI(−/−) mice, as well as their respective controls, induced a dramatic increase in plasma cholesterol and phospholipid concentrations. Plasma fractionation revealed the presence of LpX in plasma of cholestatic mice, irrespective of their genetic background. We observed that the presence of HDL before cholestasis, a decrease in the activity of LCAT, and an increase in VLDL synthesis were not required for hypercholesterolemia and lipoprotein modifications induced by obstructive cholestasis in mice. In addition, murine cholestasis resulted in increased hepatic cholesterol synthesis that may contribute to the higher plasma free cholesterol levels found during the early hours after BDL. Together these findings indicate that hypercholesterolemia and LpX formation associated with obstructive cholestasis are correlated with an increase in hepatic cholesterol synthesis and are independent of plasma HDL levels, LCAT activity, VLDL synthesis, and ABCA1 and SR-BI expression. © 2007 Elsevier B.V. All rights reserved. Keywords: Cholestasis; Hypercholesterolemia; Lipoprotein X
1. Introduction Cholestasis induces a dramatic increase in plasma total and free cholesterol and the appearance of an abnormal lipoprotein, lipoprotein X (LpX), in the plasma [1]. The exact mechanisms underlying these plasma lipid changes are not well understood. There is considerable evidence that during cholestasis there is a quantitative shift of lipid secretion from bile into the plasma compartment. First, the structure and composition of LpX are very similar to those of biliary secretory vesicles [2]. Second, Abbreviations: LpX, lipoprotein X; BDL, bile-duct ligation; ABCA1, ATPbinding cassette A1; cyp7A1, cholesterol 7α-hydroxylase; SR-BI, scavenger receptor class B type I; IDL, intermediate-density lipoprotein; MTTP, microsomal triglyceride transfer protein; pI–pC, polyinosinic/polycytidylic ribonucleic acid ⁎ Corresponding author. Tel.: +56 2 6863820; fax: 56 2 6397780. E-mail address: [email protected] (S. Zanlungo). 0304-4165/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2007.02.010
mice incapable of producing biliary vesicles – due to a disruption in the canalicular membrane phosphatidylcholine translocator, Mdr2 (Abcb4) – do not exhibit hypercholesterolemia or produce LpX particles during cholestasis [3]. These observations have led to the proposal that biliary vesicles, which are still formed during cholestasis and secreted into the canaliculus, are regurgitated from the biliary compartment to the plasma via a paracellular route [4]. However, not all the available data fit this model. For example, the paracellulartransport hypothesis is dependent on the tight junctions between hepatocytes during cholestasis being sufficiently damaged to allow a significant flux of lipid polymolecular complexes from bile into plasma [5–7]. However, this has not been observed at the beginning of cholestasis, whereas the changes in plasma lipids can be detected as early as 8 h after bile-duct ligation (BDL) in mice [8]. Also, the increase in plasma lipid levels during cholestasis is comparable to the efflux of lipids from the
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liver into the bile canaliculus in mice [3]. Therefore, it seems reasonable to propose that during lipid plasma accumulation in BDL, biliary lipids are reverse transcytosed through the hepatocyte and/or directly secreted from the liver cells into the plasma via a mechanism mediated by lipid transporters present in the hepatocyte sinusoidal membrane. Two plasma membrane proteins are good candidates for playing an important pathophysiological role in the plasma hypercholesterolemic phenotype observed during cholestasis. One is ATP-binding cassette A1 (ABCA1), a transmembrane lipid translocator involved in cellular cholesterol efflux. ABCA1 mediates cholesterol and phospholipid transport from cells into lipid-free apolipoproteins that interact with the plasma membrane [9,10]. ABCA1 is abundantly expressed in the liver [11], and several studies have found that hepatic ABCA1 is important to maintaining normal plasma levels of high-density lipoprotein (HDL) [12–14]. The second candidate for involvement in cholesterol cellular efflux is the scavenger receptor class B type I (SR-BI). SR-BI is involved in the bidirectional flow of lipids between cells and lipoproteins [15]. This receptor mediates HDL cholesterol uptake by the liver, is highly expressed in the liver sinusoidal membrane [16,17], and can mediate both selective cellular uptake of cholesterol esters from diverse lipoproteins [15] and nonesterified cholesterol efflux from cells [16,18,19]. Hyperlipidemia and LpX have also been described in other disease conditions related to lipoprotein metabolism, including those related to abnormal HDL such as deficiency of lecithin: cholesterol acyltransferase (LCAT) [20]. LCAT catalyzes freecholesterol esterification in HDL [21], and in LCAT deficiency the HDL fraction is dramatically decreased and LpX-like particles appear [22,23]. The same lipoprotein pattern has also been reported in other experimental models in which HDL metabolism was altered [24–26]. All these represent new models of LpX formation since its generation would be independent of cholestasis. Thus, questions arise as to how LpX is generated in noncholestatic conditions and whether the same mechanisms are involved in LpX formation with and without cholestasis. Abnormal remodeling of very-low-density lipoprotein (VLDL) could also be important in LpX formation. One study has demonstrated that in vitro lipolysis of VLDL generates LpX-like particles [27]. Since hepatocytes secrete lipids into the plasma compartment as VLDL particles [28], modification of these lipoproteins might also generate LpX. On the other hand, cholestatic hyperlipidemia has been associated with increased cholesterol synthesis in rats [29–33], which correlated with an increase in HMG-CoA reductase (HMG-CoA red) activity [34,35]. Hepatic newly synthesized cholesterol could contribute and might even be a key determinant of increased plasma cholesterol during obstructive cholestasis. In this paper we propose an important role for the liver in the lipid plasma changes that occur during cholestasis. We analyzed the relevance of ABCA1 and SR-BI expressions by inducing BDL in ABCA1(−/−) and SR-BI(−/−) mice models. We also analyzed LCAT activity and the role of VLDL and hepatic cholesterol synthesis during cholestasis in mice.
2. Materials and methods 2.1. Animals and diet The wild-type C57BL/6J murine strain was originally purchased from Jackson Laboratory (Bar Harbor, ME) and bred to generate our own colony. ABCA1-deficient mice (DBA/1-Abca1tm1Jdm) and LCAT transgenic mice (C57BL/6J) were obtained from Pfizer (Groton, CT) and bred for experiments in our animal facility. Genotypes of ABCA1 mice were identified using polymerase chain reaction (PCR)-based screening as described previously [36]. Mice with a targeted mutation in the sr-bi locus were obtained by gene disruption in a 129/Sv-derived embryonic stem-cell line as described previously [37] and also genotyped by PCR [37]. Liver-specific mttp-knockout mice were obtained after treating Mttpflox/floxMx1-Cre mice (donated by Dr. Steven Young, Gladstone Institute of Cardiovascular Disease, San Francisco, CA) with polyinosinic/polycytidylic ribonucleic acid (pI–pC; Sigma Chemical, St. Louis, MO) every other day for 6 days as described previously [38]. All mice had free access to water and a chow diet (< 0.02% cholesterol; Prolab RMH 3000, PMI Feeds, St. Louis, MO). Protocols were performed according to accepted criteria for the humane care of experimental animals, and were approved by the review board for animal studies of the Pontificia Universidad Católica de Chile.
2.2. Common bile duct ligation Two-month-old mice with different genetic backgrounds were subjected to BDL as described previously [3]. This procedure is a well-characterized model of liver damage induced by acute obstructive cholestasis. Animals were anesthetized by an intraperitoneal injection of Nembutal (50 mg/kg). The abdomen was opened by a midline incision, the common bile duct was ligated as close to the liver as possible, the gallbladder was ligated and removed, and then the abdomen was closed. Sham-operated animals were subjected to the same surgical procedure but without ligation of the common bile duct and removal of the gallbladder. Plasma and livers were collected 24 and 48 h later. Aliquots of liver tissue were frozen in liquid nitrogen for the subsequent preparation of protein and total RNA samples.
2.3. Plasma lipoprotein separation and plasma and hepatic biochemical analyses Plasma lipoprotein separation was performed by Superose 6 fast protein liquid chromatography (FPLC) gel filtration [37,39]. Plasma total, free, and lipoprotein cholesterol were measured as described previously [37,39]. The hepatic cholesterol content was determined by routine methods [40,41]. Plasma total phospholipids were measured as described previously [42].
2.4. LpX separation by density gradient ultracentrifugation At 1 day after BDL or the sham operation, plasma was centrifuged at 100,000 rpm for 1 h at 16 °C in a KBr gradient (density, 1.3 g/ml) as described previously [43]. After centrifugation, 1-ml fractions were collected from the gradient and their density and lipid contents were measured.
2.5. Immunoblotting analysis For ABCA1, SR-BI, Na+-taurocholate cotransporting protein, NTCP (Slc10a1), and multidrug resistance protein 3, MRP3 (ABCC3), immunoblotting, total membrane extracts from mouse liver were prepared [44] and separated (50 μg of protein/sample) by 8% SDS-PAGE. Samples were immunoblotted using anti-ABCA1 antiserum (donated by Dr. Mason Freeman, Harvard Medical School, Boston, MA), an anti-SR-BI antiserum (obtained from Dr. Monty Krieger, Massachusetts Institute of Technology, Cambridge, MA), and anti-Slc10a1 and anti-ABCC3 antisera (provided by Dr. Marco Arrese, Pontificia Universidad Católica, Santiago, Chile). Anti-ε-cop antibody (obtained from Dr. Monty Krieger) was used as a membrane protein-loading control. Antibody binding to protein samples was visualized using enhanced chemiluminescence and measured using a molecular imaging system (GS-525, BIO-RAD, Hercules, CA).
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2.6. Semiquantitative RT-PCR assay. Five micrograms of total liver RNA was reverse transcribed using random primer hexamers. Semiquantitative reverse-transcription PCR (RT-PCR) was performed for different genes using primers based on mouse and rat cDNA sequences available through GeneBank databases. The expressions of target genes cyp7A1, bsep (abcb11), hmg-CoA reductase, and mttp were analyzed by coamplification with β-actin or 18S internal standards (Ambion, Austin, TX) to quantify transcript abundance. PCR was performed in the presence of [α-32P] dCTP, and PCR products were resolved on a 2% agarose gel and transferred to a nylon filter. Radiolabeled bands were quantified using a molecular imaging system (GS-525, BIO-RAD), with the results normalized to the signal by from the radiolabeled β-actin or 18S PCR products.
2.7. LCAT-activity analysis The exogenous LCAT in plasma from different groups was assayed as described previously [45] by measuring the rate of synthesis of 3H-cholesteryl esters from unilamellar vesicles activated with apolipoprotein A-I.
2.8. Hepatic cholesterol synthesis in vivo The rate of cholesterol synthesis was measured after a 12-h fasting period. Each mouse received 50 mCi of [3H] water (Amersham Pharmacia Biotech, Piscataway, NJ) by intraperitoneal injection as described previously [39,46]. One hour later, animals were anesthetized and blood was obtained for the determination of the water-specific activity in plasma. Livers were removed and saponified, and digitonin-precipitable sterols were isolated as described previously [46]. Results are expressed as micromoles of [3H] water incorporated into digitonin-precipitable sterols per hour per gram of liver weight.
2.9. Statistics The two-tailed, unpaired Student’s t test was used to compare data sets. Differences were considered statistically significant at a probability value of p < 0.05.
3. Results 3.1. Hepatic expression of genes related to lipid metabolism and transport in BDL mice We first analyzed the hepatic expressions of various genes involved in cholesterol metabolism and transport in BDL mice. The hepatic expressions of cholesterol 7α-hydroxylase (cyp7a1), hmg-CoA red, microsomal triglyceride transfer protein (mttp), and bile salt export pump (abcb11) were analyzed by semiquantitative RT-PCR (Fig. 1). Hepatic mRNA levels of hmg-CoA red, mttp, and abcb11 were unchanged by BDL, whereas cyp7a1 mRNA levels decreased to 83% after 24 h of BDL and remained low at 48 h. ABCA1 and SR-BI were analyzed by immunoblotting as shown in Fig. 2. Hepatic ABCA1 protein levels decreased to 21% after 48 h of BDL and SR-BI protein levels decreased to 77% and 66% after 24 and 48 h of BDL, respectively. We also analyzed the hepatic expressions of the basolateral membrane transporters ABCC3 and Slc10a1 since it has been proposed that they play a major protective role against cholestasis [47]. ABCC3 protein expression did not differ between control and BDL mice, while Slc10a1 protein expression was dramatically reduced after 24 and 48 h of BDL (Fig. 2).
Fig. 1. Effect of BDL on hepatic expressions of cyp7A1, hmg-CoA red, mttp, and abcb11 in mice. RNA expressions were analyzed by semiquantitative RT-PCR in the presence of [α-32P]dCTP, with β-actin or 18S primers included in each sample as controls. The products were resolved in a 2% agarose gel, transferred to a nylon filter, and the radiolabeled signals were quantified. Results are representative of three independent experiments. (a) indicates a significant difference (p < 0.05) compared to sham-operated animals.
Together these results show that hepatic ABCA1, SR-BI, Slc10a1 and cyp7a1 levels are modulated early after the induction of obstructive cholestasis in mice. 3.2. Plasma lipoprotein cholesterol analysis in BDL wild-type and ABCA1(−/−) mice To assess whether ABCA1 expression is relevant to the hypercholesterolemic response observed during cholestasis, both wild-type (ABCA1(+/+)) and ABCA1(−/−) mice were subjected to BDL, and plasma was collected and analyzed after 24 and 48 h. As expected, in ABCA1(+/+) mice plasma total cholesterol and phospholipids were higher after 24 h of BDL, and increased further during the following 24 h (Fig. 3A). As described previously [3], the rise in plasma cholesterol during cholestasis was mainly due to the accumulation of free cholesterol (Fig. 3A). Plasma cholesterol and phospholipids levels were significantly lower in sham-operated ABCA1(−/−) mice than in controls, as has been described previously [48]. BDL in ABCA1(−/−) mice induced a significant rise in plasma total and free cholesterol and phospholipids at 24 and 48 h (Fig. 3A), similar to the values found in cholestatic ABCA1(+/+) mice. To analyze the cholesterol content in the different lipoproteins, plasma samples from sham-operated and ABCA1(+/+) and ABCA1(−/−) BDL mice were subjected to FPLC. Plasma from ABCA1(+/+) BDL mice showed increased cholesterol transport in large lipoproteins that eluted in the FPLC fractions corresponding to very low density (VLDL) and intermediatedensity/low-density lipoproteins (IDL/LDL) relative to control animals at 24 and 48 h (Fig. 3B), whereas the cholesterol content of the HDL fraction was dramatically reduced (Fig. 3B). LpX elutes in the void volume of the FPLC fractionation [3], and hence these results suggest that the dramatic increase in cholesterol transported in VLDL-sized lipoproteins observed in cholestatic mice was due to the presence of LpX in these FPLC fractions. This is supported by the increase in the plasma levels
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deficient mice occurred in an HDL peak that was larger and more heterogeneous than the SR-BI(+/+) HDL peak (Fig. 4B) [36]. SR-BI(+/+) and SR-BI(−/−) mice showed similar responses in their plasma levels of total and free cholesterol and phospholipids after BDL (Fig. 4A). Also, cholesterol lipoprotein profiles after BDL, including the appearance of the large VLDL-sized lipoproteins, were very similar in SR-BI(+/+) and SR-BI(−/−) mice (Fig. 4B). Together these results show that SR-BI expression is not critical to the hypercholesterolemic response present during cholestasis in mice. 3.4. LCAT activity in BDL mice
Fig. 2. Effect of BDL on hepatic expressions of ABCA1, SR-BI, ABCC3, and Slc10a1 in mice. Total membrane extracts were prepared, size fractionated by SDS-PAGE, immunoblotted with anti-ABCA1, anti-SR-BI, anti-ABCC3, and anti-Slc10a1 antibodies, and subjected to densitometric analysis. Anti-ε-cop and anti-prealbumin antibodies were used as protein-loading controls. (a) indicates a significant difference (p < 0.05) compared to sham-operated animals.
of free cholesterol and phospholipids, which are the two main lipid components of LpX (Fig. 3A). As expected, HDL levels were very low and the plasma cholesterol was mostly contained in VLDL particles in shamoperated ABCA1(−/−) mice [49] (Fig. 3B). Although we found some differences in the IDL/LDL-sized lipoprotein fractions between ABCA1(−/−) and ABCA1(+/+) BDL mice after 24 h, the cholesterol lipoprotein profiles were similar after 48 h, even though the profiles of sham-operated animals were very different. Together these results show that ABCA1 expression is not relevant to the hypercholesterolemic response determined by acute obstructive cholestasis. 3.3. Plasma lipoprotein cholesterol analysis in BDL wild-type and SR-BI(−/−) mice To analyze whether SR-BI expression is relevant to the hypercholesterolemic phenotype observed during cholestasis, both wild-type (SR-BI(+/+)) and SR-BI(−/−) mice were subjected to BDL. Plasma cholesterol and phospholipids levels were significantly higher in sham-operated SR-BI(−/−) mice than in controls, as described previously (Fig. 4A) [37]. As expected, most of the plasma cholesterol present in SR-BI-
Reduced LCAT activity has been linked to LpX appearance in humans and in animal models. Indeed, LpX particles are commonly seen in human LCAT-deficient plasma [22,23]. In addition, LCAT-deficient mice that are transgenic for SREBP1a form LpX-like particles in plasma [26]. To determine whether changes in LCAT activity were correlated with the hypercholesterolemic response and the appearance of LpX in cholestasis, LCAT activity was measured in mice after 24 and 48 h of BDL. LCAT activity was significantly reduced at both time points (Fig. 5). To further analyze the relevance of decreased LCAT activity to LpX formation during cholestasis, plasma cholesterol levels and lipoprotein profiles were analyzed in LCAT transgenic mice [45] subjected to BDL. No differences in the abnormal plasma lipid phenotype were observed between wild-type and LCAT transgenic mice subjected to BDL (data not shown). These results indicate that the decrease in LCAT activity observed in BDL mice is a secondary effect of cholestasis, with it not playing a major role in LpX formation during biliary obstruction. 3.5. Plasma lipoprotein cholesterol analysis in BDL wild-type and MTTP(−/−) mice MTTP is essential to the transfer of triglycerides into the lumen of the endoplasmic reticulum for VLDL assembly, and is required for the secretion of apolipoprotein B-100 from the liver [38]. To evaluate the possible contribution of hepatic VLDL production to LpX formation during cholestasis, we used the well-characterized mouse model deficient in the mttp gene harboring a “floxed” mttp allele, and then used Cre-mediated recombination to generate liver-specific Mttp-knockout mice after pI–pC treatment [38]. Plasma cholesterol accumulation was determined in wildtype and liver-specific MTTP(−/−) mice subjected to BDL. As shown in Fig. 6A, although the plasma cholesterol content was low in MTTP(−/−) mice before cholestasis, BDL induced similar changes in plasma concentrations of total and free cholesterol in MTTP(−/−) and wild-type mice. Also, the cholesterol content in density-fractionated LpX particles were very similar in the wild-type and MTTP(−/−) mice (Fig. 6B). These results indicate that LpX is not synthesized as a byproduct of the VLDL biosynthetic pathway (or at least that it
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Fig. 3. Plasma cholesterol and phospholipid levels during BDL in ABCA1(+/+) and ABCA1(−/−) mice. ABCA1(+/+) and ABCA1(−/−) mice were subjected to BDL for 24 or 48 h, or to a sham operation (48 h), and plasma samples were obtained. (A) Concentrations of total and free cholesterol and phospholipids in plasma. Data are mean ± SD values from five animals. (a), (b), (c), and (d) indicate a significant difference in the levels of total (a, b) and free (c, d) cholesterol after 24 and 48 h of BDL between ABCA1(+/+) (a, c) or ABCA1(−/−) (b, d) mice and their respective sham-operated controls. The levels of total and free cholesterol did not differ significantly between 24 and 48 h of BDL in either ABCA1(+/+) or ABCA1(−/−) mice, nor between ABCA1(+/+) and ABCA1(−/−) mice after 24 or 48 h of BDL. (e) and (f) indicate a significant difference in phospholipid levels after 24 and 48 h of BDL between ABCA1(+/+) (e) or ABCA1(−/−) (f) mice and their respective sham-operated controls. The phospholipid levels did not differ between 24 and 48 h of BDL in either ABCA1(+/+) or in ABCA1(−/−) mice, nor between ABCA1(+/+) and ABCA1 (−/−) mice after 24 or 48 h of BDL. (B) Plasma lipoprotein cholesterol profiles as analyzed by FPLC in plasma from ABCA1(+/+) (○) and ABCA1(−/−) (●) mice. The chromatograms are representative of several independent FPLC analyses for each experimental condition. The approximate elution positions of VLDL, IDL/LDL, and HDL are indicated.
does not derive from a VLDL-type particle) as a consequence of acute biliary obstruction. 3.6. Hepatic cholesterol content and synthesis in BDL mice Studies of hepatic cholesterol synthesis in cholestasis have been reported in BDL rats [29–35] and in pharmacologically induced intrahepatic cholestasis in mice [50]. As shown in Fig. 7, BDL increased the hepatic contents of total and free cholesterol by nearly 20% each at 24 h, and by 40% and 55% at 48 h, respectively. Despite these increases in liver cholesterol, in vivo hepatic synthesis of cholesterol was increased by 253% after 24 and 48 h of BDL. These results suggest that newly synthesized cholesterol contributes to the increase in hepatic cholesterol content, and also that this is a key contributor to the hypercholesterolemic response observed during obstructive cholestasis.
4. Discussion Cholestasis is characterized by hypercholesterolemia and the appearance of LpX in plasma. The mechanisms responsible for this cholestatic plasma lipid phenotype are not fully understood. Using Abcb4-deficient mice, Elferink et al. [3] have shown that the expression of the canalicular translocator for phophatidylcholine, the Abcb4 protein, is crucial to the generation of the cholestatic LpX. These results are in line with the hypothesis that LpX is derived from biliary vesicles that somehow appear in the plasma [3], increasing cholesterol levels in the bloodstream. However, how are these vesicles formed and how do they get into the plasma compartment? One possibility is that biliary vesicles secreted into the canaliculus are regurgitated from the biliary compartment to the plasma via a paracellular route [4]. However, this mechanism seems unlikely because it requires hepatocellular tight junctions to be sufficiently open to
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Fig. 4. Plasma cholesterol and phospholipid levels during BDL in SR-BI(+/+) and SR-BI(−/−) mice. SR-BI(+/+) and SR-BI(−/−) mice were subjected to BDL for 24 or 48 h, or to a sham operation (48 h), and plasma samples were obtained. (A) Concentrations of total and free cholesterol and phospholipids in plasma. Data are mean ± SD values from five animals. (a), (b), (c), and (d) indicate a significant difference in the levels of total (a, b) and free (c, d) cholesterol after 24 and 48 h of BDL between SR-BI(+/+) (a, c) or SR-BI(−/−) (b, d) mice and their respective sham-operated controls. The levels of total and free cholesterol did not differ significantly between 24 and 48 h of BDL in either SR-BI(+/+) or SR-BI(−/−) mice, nor between SR-BI(+/+) and SR-BI(−/−) mice after 24 h or 48 h of BDL. (e) and (f) indicate a significant difference in phospholipid levels after 24 and 48 h of BDL between SR-BI(+/+) (e) or SR-BI(−/−) (f) mice and their respective sham-operated controls. The phospholipid levels did not differ between 24 and 48 h of BDL in either SR-BI(+/+) or SR-BI(−/−) mice, nor between SR-BI(+/+) and SR-BI(−/−) mice after 24 h or 48 h of BDL. (B) Plasma lipoprotein cholesterol profiles as analyzed by FPLC in plasma from SR-BI(+/+) (○) and SR-BI(−/−) (●) mice. The chromatograms are representative of several independent FPLC analyses for each experimental condition. The approximate elution positions of VLDL, IDL/LDL, and HDL are indicated.
allow 70-nm vesicles to pass from the biliary into Disse’s space. At least at the early stages of cholestasis, this does not appear to be the case since hepatocyte tight junctions appear to be intact [7,51]. Furthermore, opening of the liver cell tight junctions to such an extent would lead to a complete loss of cell polarity, which has not been observed in several studies with cholestatic models [3,51,52]. Indeed, Abcb4 is still observed in the canalicular region, and also in the subapical region in pericanalicular vesicles during BDL in mice [3]. These observations suggest that LpX forms in a subapical compartment of the hepatocytes. Indeed, electron microscopy studies in BDL rats have shown LpX-like vesicles within the canaliculus and in the pericanalicular region in the presence of intact tight junctions [52]. With this background, we hypothesized that liver cells contribute actively to the hypercholesterolemic response and LpX formation in cholestasis. One possibility is that LpX formed in the subapical compartment is transcytosed toward the
sinusoidal membrane and released into the plasma. In addition, cholesterol derived from the intrahepatic metabolically active pool could contribute to plasma cholesterol and LpX by direct secretion through the sinusoidal membrane into Disse’s space. This latter hypothesis leads to the question of whether cholesterol gets into the plasma during cholestasis by a passive or a protein-mediated process. The second process seems quite plausible since the estimated sinusoidal lipid output during cholestatic conditions in mice is comparable to the biliary lipid secretion rates under normal conditions [3], a process that is mediated by several canalicular protein transporters [53]. Because ABCA1 and SR-BI are the most relevant cholesterol transporters localized to the sinusoidal membrane, we postulated that they are key regulators of the entry of cholesterol into the plasma compartment during cholestasis. However, our findings demonstrate that the sinusoidal cholesterol transporters ABCA1 and SR-BI are not relevant to the hypercholesterolemia found in cholestatic conditions in mice. These findings do not
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of HDL or because LpX is depleted of normal cofactors for LCAT activation. In fact, it has been demonstrated in vitro that isolated LpX, which does not contain apoA-I [2], the most potent LCAT activator [60], was not a suitable substrate for recombinant LCAT [2]. Furthermore, addition of ApoA-I to LpX resulted in LCAT-mediated cholesterol esterification [2]. Since we measured LCAT activity using an exogenous substrate, and our preliminary data indicate that LCAT plasma protein levels increased during cholestasis, we postulate that the enzyme is inhibited during cholestasis. Moreover, LCAT transgenic mice showed the typical hypercholesterolemic response during cholestasis, indicating that LCAT deficiency Fig. 5. Effect of BDL on plasma LCAT activity in mice. LCAT activity was measured as the rate of synthesis of 3H-cholesteryl esters (CE) from unilamellar vesicles incubated with 3H-cholesterol and activated with apolipoprotein A-I in control mice (48 h, sham operated) and in mice after 24 and 48 h of BDL. Data are mean ± SD values from three mice per group. (a) indicates a significant difference (p < 0.05) compared to sham-operated animals.
rule out the possibility that other sinusoidal lipid transport proteins (e.g., ABCG1) participate in delivering cholesterol to plasma during cholestasis. One interesting finding of this study is that the presence of HDL in plasma was not necessary for LpX formation. During BDL in mice, LpX formation and hypercholesterolemia were accompanied by a dramatic reduction in HDL levels. Moreover, humans with deficiency in LCAT – which is the main enzyme catalyzing the synthesis of cholesteryl esters in HDL [21] – show LpX-like particles in plasma [22,23]. Hypercholesterolemia and LpX-like particles are also observed in various animal models with altered HDL metabolism. For example, LpX was present in the plasma of double-knockout SR-BI(−/−) and apolipoprotein E(−/−) mice [25] and LCAT-deficient mice fed a high-fat diet [54]. These data suggested that HDL metabolism and LpX formation are related. However, the present study demonstrates that ABCA1-deficient mice, which present very low levels of plasma HDL, show an intact hypercholesterolemic response and hence presumably form LpX particles as do normal mice during BDL. Furthermore, we found a similar cholestasis-induced dyslipidemic phenotype in apolipoprotein A-I and PCTP gene-targeted murine strains, which also exhibit abnormal HDL metabolism in vivo [21,55] (data not shown). Previous data support a connection between LCAT deficiency and LpX formation [22,23]. Also, LCAT activity is decreased in cholestatic conditions (our results and [56,57]). The mechanism for the decrease in LCAT during cholestasis is unknown, but it could be related to the reduction in HDL levels, a decrease in the hepatic expression of the enzyme, or to inhibition of the enzyme activity. With regard to this latter mechanism, plasma bile acids are characteristically increased during cholestasis [47,58] and high concentrations of these amphipatic molecules reduce LCAT activity [59]. The decrease in LCAT activity may also be attributable to the inability of the enzyme to use LpX as a substrate, because the bilaminar structure of this abnormal lipoprotein is very different from that
Fig. 6. Plasma cholesterol levels during BDL in MTTP(+/+) and MTTP(−/−) mice. MTTP(+/+) and MTTP(−/−) mice were subjected to BDL for 24 h, or to a sham operation (24 h), and plasma samples were obtained. (A) Concentrations of total and free cholesterol in plasma. Data are mean ± SD values from three animals. (a), (b), (c), and (d) indicate a significant difference in the levels of total (a, b) and free (c, d) cholesterol between controls and after 24 h of BDL in MTTP(+/+) (a, c) and MTTP(−/−) (b, d) mice. (B) Plasma was loaded onto a KBr density gradient for the separation of lipoproteins by ultracentrifugation. The levels of total and free cholesterol were determined in the fraction where LpX was collected. (a), (b), (c), and (d) indicate a significant difference in the levels of total (a, b) and free (c, d) cholesterol between controls and after 24 h of BDL in MTTP(+/+) (a, c) and MTTP(−/−) (b, d) mice.
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Fig. 7. Effect of BDL on hepatic cholesterol content and synthesis in mice. (A) Hepatic contents of total and free cholesterol were determined after 24 and 48 h of BDL or the sham operation (48 h). (a) and (b) indicate that the contents of total (a) and free (b) cholesterol in mice after 24 and 48 h of BDL differed significantly from that in control mice (p < 0.05). (B) Hepatic cholesterol synthesis in vivo was determined after 24 and 48 h of BDL or the sham operation (48 h). Results are expressed as micromoles of [3H] water incorporated into digitonin-precipitable sterols per hour per gram of liver weight. (a) indicates a significant difference in cholesterol synthesis at 24 and 48 h of BDL compared to sham-operated animals.
was not a pathogenic contributor to LpX formation and that LCAT overexpression could not overcome the mechanism underlying the dyslipidemic phenotype associated with cholestasis. VLDL secretion represents the major mechanism of lipoprotein secretion from the liver into the plasma through the sinusoidal membrane, which makes it theoretically possible that LpX formation is related to VLDL secretion. However, we demonstrated that the accumulation of LpX, which elutes in the same fraction as a VLDL particle in an FPLC column, was not impaired by the lack of VLDL production in liver-specific MTTP(−/−) mice. BDL in mice induces changes in the hepatic expressions of various lipoprotein-metabolism-related genes. This study has shown for the first time that the expressions of hepatic ABCA1 and SR-BI are modulated during cholestasis in mice. We also found a significant decrease in hepatic expressions of cyp7a1 and Slc10a1, probably due to the induction of early protective mechanisms against bile-salt-mediated hepatotoxicity during cholestasis. The mechanism responsible for hepatic ABCA1 and SR-BI downregulation in cholestasis is not known. There is
recent evidence that the nuclear transcription factor PXR is involved in the regulation of the hepatic levels of ABCA1 and SR-BI. Sporstol et al. [61] demonstrated that the use of PXR agonists suppressed the activity of the promoter regions of human abca1 and sr-b1 genes in HepG2 cells and in primary rat hepatocyte cultures. Also, PXR may be activated during cholestasis by the accumulation of bile acids, which can act as PXR agonists. Interestingly, the increase in total plasma cholesterol during BDL-induced cholestasis is smaller in PXRdeficient mice than in wild-type controls, suggesting that a decrease in hepatic SR-BI and/or ABCA1 expression is indeed important to the mechanisms underlying the hyperlipidemia associated with BDL [58]. It is also possible that the nuclear factor FXR, which is also activated by the cholestasis-induced increase in hepatic bile acids, participate in hepatic SR-BI regulation due to obstructive cholestasis. In fact, FXR can suppress the hepatic levels of SR-BI mRNA in rats, mice, and humans by an indirect mechanism mediated by SHP and the activity of the transcription factor LRH-1 [62]. Another important finding of this study is the major impact of BDL-induced cholestasis on hepatic cholesterol metabolism in the mouse. The synthesis and content of hepatic cholesterol were both increased during murine cholestasis. The increase in hepatic cholesterogenesis was not inversely correlated with the increased levels of hepatic cholesterol and the expected feedback regulation between these two metabolic parameters. Similar results have been previously reported by a number of studies, including cholestatic BDL rats [29–35] and druginduced intrahepatic cholestasis in mice [50]. The mechanism by which BDL regulates cholesterol synthesis is not known, but it might be related to changes in the intracellular distribution of cholesterol, presumably in the endoplasmic reticulum where cholesterol sensing occurs. However, we found no changes in hepatic mRNA levels of HMG-CoA red, which is the key regulatory enzyme in cholesterol synthesis. Further studies are needed to establish the molecular basis for the upregulation of cholesterol synthesis in cholestasis, for which there are several possibilities. First, cholesterol accumulation may have occurred simply as a consequence of the obstruction in biliary cholesterol secretion. Second, there may have been a contribution from impaired hepatic cholesterol efflux toward the plasma due to reduced ABCA1 expression and/or the increase in the de novo hepatic synthesis, with the increase in hepatic cholesterol content being primarily due to an increase in the hepatic content of free cholesterol. Future studies should attempt to localize this free cholesterol. In summary, our data rule out the participation of ABCA1 and SR-BI in a lipid efflux process from hepatocytes to plasma during cholestasis. We have also shown that the presence of HDL precholestasis, a decrease in LCAT activity, and an intact VLDL synthesis are not required for the appearance of hypercholesterolemia and lipoprotein modifications during cholestasis in mice. The more-striking contribution of these experiments is the finding that cholestasis results in an increase in hepatic cholesterol synthesis that could contribute to the increase in free cholesterol detected in plasma under cholestatic conditions.
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Acknowledgments The authors thank Dr. Steven Young for providing the Mttpflox/floxMx1-Cre mice. This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT Grants #1030415 and #7050126 to SZ, #1030416 to AR, and #1030744 to FN), Programa de Mejoramiento de la Calidad y la Equidad de la Educación Superior (MECESUP Grant #PUC0005 to IB), and Pontificia Universidad Católica Medicine Resident Grants #PG-12/03 and #PG-16/04 to IB. References
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