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Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2724-2730
Published online before print September 28, 2006, doi: 10.1161/01.ATV.0000247260.42560.e1
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2724.)
© 2006 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Cholesterol 7{alpha}-Hydroxylase Deficiency in Mice on an APOE*3-Leiden Background Increases Hepatic ABCA1 mRNA Expression and HDL-Cholesterol

Sabine M. Post; Martine Groenendijk; Caroline C. van der Hoogt; Catherine Fievet; Gérald Luc; Menno Hoekstra; Hans M.G. Princen; Bart Staels; Patrick C.N. Rensen

From TNO-Quality of Life, Department of Biomedical Research, (S.M.P., M.G., C.C.H., H.M.G.P., P.C.N.R.) Gaubius Laboratory, Leiden, The Netherlands; Département d’Athérosclérose and INSERM U545 (C.F., G.L., B.S.), Institut Pasteur de Lille and Faculté de Pharmacie, Université de Lille II, Lille, France; Leiden University Medical Center (C.C.H., P.C.N.R.), Department of General Internal Medicine, Endocrinology, and Metabolic Diseases, Leiden, The Netherlands; and Leiden/Amsterdam Center for Drug Research (M.H.), Division of Biopharmaceutics, Leiden, The Netherlands.

Correspondence to Patrick C.N. Rensen, PhD, Leiden University Medical Center, Department of Endocrinology and Metabolic Diseases, C4-R81, Albinusdreef 2, PO Box 9600, 2300 RC Leiden, The Netherlands. E-mail P.C.N.Rensen{at}lumc.nl


*    Abstract
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 *Abstract
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Objective— High-density lipoprotein (HDL) plays a key role in protection against development of atherosclerosis by reducing inflammation, protecting against LDL oxidation, and promoting reverse cholesterol transport from peripheral tissues to the liver for secretion into bile. Cholesterol 7{alpha}-hydroxylase (Cyp7a1) catalyzes the rate-limiting step in the intrahepatic conversion of cholesterol to bile acids that may have a role in HDL metabolism. We investigated the effect of Cyp7a1 deficiency on HDL metabolism in APOE*3-Leiden transgenic mice.

Methods and Results— Reduced bile acid biosynthesis in Cyp7a1–/–.APOE*3-Leiden mice versus APOE*3-Leiden mice did not affect total plasma cholesterol levels, but the distribution of cholesterol over various lipoproteins was different. Cholesterol was decreased in apoB-containing lipoproteins (ie, VLDL and IDL/LDL), whereas cholesterol was increased in HDL. The activity of PLTP and LCAT, which play a role in HDL catabolism, were not changed, and neither was HDL clearance. However, the hepatic cholesterol content was 2-fold increased, which was accompanied by a 2-fold elevated expression of hepatic ABCA1 and increased rate of cholesterol efflux from the liver to HDL.

Conclusions— Strongly reduced bile acid synthesis in Cyp7a1–/–.APOE*3-Leiden mice leads to increased plasma HDL-cholesterol levels, as related to an increased hepatic expression of ABCA1.

High levels of plasma HDL-cholesterol are correlated with a low risk of cardiovascular disease. We show that strongly reduced bile acid formation in APOE*3-Leiden transgenic mice by cholesterol 7{alpha}-hydroxylase (Cyp7a1)-deficiency increases HDL-cholesterol. The clearance of HDL was not changed but hepatic ABCA1 mRNA expression was increased, suggesting an increased rate of cholesterol efflux from the liver to HDL.


Key Words: ATP binding cassette transporter A1 • bile acid synthesis • cholesterol 7{alpha}-hydroxylase • high-density lipoprotein • transgenic mice


*    Introduction
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 *Introduction
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The level of plasma HDL-cholesterol is inversely correlated with the risk of cardiovascular disease.1 Besides having antiinflammatory and antioxidative properties, HDL can remove cholesterol from the arterial wall via the ATP binding cassette transporter A1 (ABCA1) and subsequently deliver cholesterol to the liver2–4 via the HDL-receptor scavenger receptor class B type I (SR-BI). On internalization, the cholesterol can be converted into bile acids or secreted directly into the bile. This process is called reverse cholesterol transport. Various plasma enzymes are involved in modulation and maturation of HDL particles, including phospholipid transfer protein (PLTP), lecithin cholesterol acyl transferase (LCAT), and cholesteryl ester transfer protein (CETP). In addition, plasma HDL cholesterol levels are determined by several processes, including the generation of HDL precursors from hepatically and intestinally derived apoAI,5,6 the efflux of cholesterol to HDL particles from the peripheral tissues7 and the liver8 by ABCA1, and the selective clearance of HDL-cholesteryl esters via SR-BI.9

The conversion of cholesterol into bile acids and its subsequent fecal excretion is quantitatively the most important way for elimination of cholesterol from the body and represents the final step in reverse cholesterol transport.10 Cholesterol 7{alpha}-hydroxylase (Cyp7a1) is the major enzyme which catalyzes the rate-limiting step in the classical or neutral route in bile acid formation.11,12 In addition to their role in the solubilization of fat and cholesterol, bile acids are signaling molecules that regulate the expression of genes involved in their own synthesis and in cholesterol homeostasis as mediated via the nuclear hormone receptor farnesoid X receptor (FXR).13 Several studies point at a potential role for bile acids in HDL metabolism; eg, by modulating the expression of apoAI, the major protein constituent of HDL. In cultured cells, bile acids negatively regulate human apoAI gene expression by activation of FXR.14 Treatment of gallstone patients and cerebrotendinous xanthomatosis patients with chenodeoxycholic acid lowers serum HDL levels.15,16 In addition, cholestatic patients, who have impaired bile secretion, show lower serum apoAI levels.17

To gain further insight into the role of bile acids and/or bile acid biosynthesis in the regulation of HDL metabolism, we used Cyp7a1–/– mice, which have a low bile acid biosynthesis and small bile acid pool size. We bred these mice onto a hyperlipidemic APOE*3-Leiden background. Because of the concomitant expression of APOE*3-Leiden and APOC1, APOE*3-Leiden mice have an attenuated clearance of apoB-containing lipoproteins. Therefore, APOE*3-Leiden mice show, in contrast to wild-type mice, a human-like lipoprotein profile with substantial amounts of VLDL, IDL, and LDL.18 Because extensive lipid exchange occurs between VLDL and HDL metabolism in humans, including transfer of phospholipid surface remnants from VLDL to HDL, we reasoned that the sensitivity of the effect of removal of Cyp7a1 on HDL metabolism would be increased on this human-like background.

Our data show that the absence of Cyp7a1 in APOE*3-Leiden mice causes an increase in plasma HDL-cholesterol as related to an increased hepatic expression of ABCA1, which is involved in the basolateral efflux of cholesterol from the liver to the plasma compartment.


*    Methods
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 *Methods
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Please see an expanded version of the Methods section online, available at http://atvb.ahajournals.org.

Animals
Cyp7a1–/– mice19 were bred on a wild-type (C57Bl/6) or on a APOE*3-Leiden background (>99% C57Bl/6) to generate Cyp7a1–/–.APOE*3-Leiden mice. Female mice were used. Institutional guidelines for animal care were observed in all experiments.

Analysis of Blood Parameters
Plasma cholesterol and triglyceride (TG) concentrations were determined by enzymatic assays. Plasma HDL-cholesterol levels were measured after precipitation of apoB-containing lipoproteins. Plasma levels of apoAI, apoAII, and apoB were determined by an immunonephelemetric assay.20 Lipoproteins were separated using a Superose 6 HR 10/30 column.

PLTP and LCAT Activity
PLTP and LCAT activities were measured as previously detailed.21

Radiolabeling and Plasma Clearance of HDL
Human HDL was isolated from plasma,22 and labeled with [1{alpha},2{alpha}(n)-3H]cholesteryl oleate ([3H]CO).23 [3H]CO-labeled HDL was injected into the tail vein of 4 hour-fasted conscious mice and the disappearance of label from blood was followed.

Immunoblot Analysis of SR-BI
Liver samples were lysed, cell debris was removed, and the protein concentration was determined by Lowry et al.24 Proteins were separated on SDS-PAGE gels and transferred to nitrocellulose membrane. Immunolabeling was performed using rabbit polyclonal {alpha}SRBI (anti-BI495)25 and rabbit polyclonal {alpha}GAPDH, followed by goat–anti-rabbit IgG. Immunolabeling was detected by enhanced chemiluminescence and quantified by densitometric scanning.

Hepatic [3H]Cholesterol Secretion to Plasma HDL
80-nm-sized [3H]CO-labeled chylomicron-like TG-rich emulsion particles26,27,28 (0.5 mg of TG) were injected via the tail vein into 4 hour–fasted conscious mice. After rapid uptake by the liver (t1/2 {approx}5 minutes),29 the 3H-cholesterol was liberated and secreted back into the plasma in a time-dependent manner. At the indicated times after injection, blood samples were taken.30 Lipoproteins were separated using a Superose 6 column, and eluted fractions were assayed for 3H activity.

RNA Isolation and Measurement of mRNA Levels by Real-Time Polymerase Chain Reaction
Total RNA was isolated from liver tissue as described,31 and converted into single stranded cDNA. cDNA levels were measured by real-time polymerase chain reaction (PCR). Primers and probes are summarized in supplemental Table I. The level of mRNA expression was calculated using the Ct values.32 Northern blot analysis of apoAI and liver lipid analysis were performed as described.31

Statistics
Data were analyzed statistically using the nonparametric Mann–Whitney test. mRNA levels were analyzed statistically using Student unpaired t test.


*    Results
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Cyp7a1 Deficiency Increases Plasma HDL Cholesterol Levels in APOE*3-Leiden Mice
We have previously shown that the absence of Cyp7a1 in APOE*3-Leiden mice resulted in a decrease in bile acid biosynthesis as reflected by a reduced fecal bile acid excretion (–70%) compared with their APOE*3-Leiden mice littermates. As a consequence, the fecal neutral sterol output was 2-fold increased in the Cyp7a1–/–.APOE*3-Leiden mice.32 Cyp7a1 deficiency resulted in 41% decreased plasma TG levels, but did not affect the plasma cholesterol levels.32 We now demonstrate that, despite the absence of an effect on total plasma cholesterol, the distribution of the cholesterol over the various lipoproteins was changed. Knocking-out Cyp7a1 in APOE*3-Leiden mice resulted in a decreased cholesterol content in the apoB-containing lipoproteins (ie, VLDL and IDL/LDL; Figure 1) and a similarly decreased plasma apoB level (Table 1). Concomitantly, an {approx}50% increase in the amount of cholesterol in the HDL fraction was observed (Figure 1). In line with this observation, the plasma concentrations of apoAI and apoAII were increased, albeit that statistical significance was only reached for apoAII (Table 1). Cyp7a1 deficiency on a wild-type background did not substantially affect the plasma lipoprotein profile (supplemental Figure I).


Figure 1
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  		Figure 1. Decreased amount of cholesterol in apoB-containing lipoproteins (VLDL and IDL/LDL) and increased amount of cholesterol in HDL of Cyp7a1–/–.APOE*3-Leiden mice. Plasma was isolated from APOE*3-Leiden mice (black circles) and Cyp7a–/–.APOE*3-Leiden littermates (white circles) and pooled (n=10 per group). Lipoproteins were separated by gel filtration chromatography, and fractions were assayed for cholesterol.


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  		TABLE 1. Plasma Lipid and Apolipoprotein Levels in APOE*3-Leiden and Cyp7a1–/–.APOE*3-Leiden Mice

Cyp7a1 Deficiency Does Not Change PLTP and LCAT Activities in APOE*3-Leiden Mice
To investigate the mechanism(s) underlying the increased HDL concentration on Cyp7a1 deficiency in APOE*3-Leiden mice, we measured the activities of PLTP and LCAT, which are enzymes involved in modulating HDL particles. However, their activities did not differ between Cyp7a1–/–.APOE*3-Leiden and APOE*3-Leiden mice. In addition, the amount of apoAI in pre–ß-HDL was not different in Cyp7a1–/–.APOE*3-Leiden as compared with APOE*3-Leiden mice (supplemental Table II).

Cyp7a1 Deficiency Increases Hepatic Lipid Content and Affects Hepatic mRNA Levels in APOE*3-Leiden Mice
To further evaluate the effect of Cyp7a1-deficiency on lipid levels, we measured the liver lipid content. The absence of Cyp7a1 in APOE*3-Leiden mice resulted in an increased hepatic content of cholesteryl esters (2-fold) and TG (1.9-fold), whereas hepatic free cholesterol and phospholipids did not change (Table 2). Whereas Cyp7a1 deficiency on a wild-type background did result in a similarly increased hepatic content of TG (2.5-fold), no effects were observed on hepatic total cholesterol, cholesteryl esters, and phospholipids (not shown). In addition, hepatic mRNA levels of various genes involved in both lipoprotein metabolism and biliary lipid output were differentially expressed in the Cyp7a1–/–.APOE*3-Leiden mice (Table 3). In line with the increased cholesterol content in the liver, HmgcoA-reductase mRNA levels were lower (–55%) in the Cyp7a1–/–.APOE*3-Leiden mice as compared with their APOE*3-Leiden littermates. The expression of the canalicular transporter genes for bile acids (ie, Bsep) and sterols (ie, Abcg5 and Abcg8) were all significantly decreased in the absence of Cyp7a1 (–23%, –65%, and –21%, respectively). The expression of Ntcp, the bile acid transporter involved in the uptake of bile acids by the liver, and the expression of Apoa1 (in liver as well as in intestine) were not different between both groups. In contrast, the expression of Pltp and Sr-b1 were mildly but significantly decreased in Cyp7a1–/–.APOE*3-Leiden as compared with their APOE*3-Leiden littermates (–11% and –28%, respectively). However, of all genes examined, Cyp7a1 deficiency had the most impact on the hepatic expression of Abca1, which was as much as 98% increased (Table 3). In contrast, although Cyp7a1 deficiency on a wild-type background did reduce the hepatic expression of Pltp (–39%) and Bsep (–38%), no effects were observed on the expression of other genes, including HmgcoA-reductase and Abca1 (supplemental Table III).


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  		TABLE 2. Hepatic Lipid Levels in APOE*3-Leiden and Cyp7a1–/–.APOE*3-Leiden Mice


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  		TABLE 3. Hepatic and Intestinal mRNA Levels in APOE*3-Leiden and Cyp7a1–/–.APOE*3-Leiden Mice

Cyp7a1 Deficiency Does Not Affect Hepatic HDL Clearance in APOE*3-Leiden Mice
To evaluate whether a decreased expression of Sr-b1 may have resulted in delayed HDL clearance in Cyp7a1-deficient mice on an APOE*3-Leiden background, thereby increasing plasma HDL levels, we injected [3H]CO-labeled HDL into the tail vein of the mice and followed the disappearance of the radiolabel in the blood by taking blood samples in time. Because mice do not express CETP, the radiolabel does not redistribute from the administered HDL, and the disappearance of radiolabel from serum thus truly reflects the SR-BI–dependent selective clearance of HDL-cholesteryl esters.33 As shown in Figure 2A, the serum half-life of [3H]CO was {approx}80 minutes, but no differences were detected with respect to the clearance of HDL between the two groups. This observation is in agreement with immunoblot analysis, which revealed that hepatic SR-BI protein (as normalized for GAPDH protein) did not differ between Cyp7a1–/–.APOE*3-Leiden and APOE*3-Leiden mice (1.50±0.19 versus 1.45±0.17 arbitrary units, P=0.71) (Figure 2B).


Figure 2
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  		Figure 2. Unchanged serum decay of [3H]CO-labeled HDL in Cyp7a–/–.APOE*3-Leiden mice. A, [3H]CO-labeled HDL (10 µg protein {approx}0.6x106 dpm) was injected into the tail vein of APOE*3-Leiden mice (black circles) and Cyp7a–/–.APOE*3-Leiden littermates (white circles). At the indicated time points, blood samples were taken. Serum was counted for radioactivity and corrected for the total plasma volume. Data are expressed as means±SD of 6 mice per group. B, Immunoblot analysis of SR-BI and GAPDH in liver samples from APOE*3-Leiden or Cyp7a1–/–.APOE*3-Leiden mice. Numbers indicate pixel counts of the SR-BI and GADPH bands after densitometric scanning (arbitrary units), as well as their ratio (SR-BI/GAPDH). Data are shown for 5 mice per group.

Cyp7a1 Deficiency Increases Hepatic Cholesterol Secretion to Plasma HDL in APOE*3-Leiden Mice
To examine the effect of the 2-fold increased hepatic ABCA1 expression in Cyp7a1-deficient mice on an APOE*3-Leiden background on hepatic cholesterol flux to HDL, [3H]CO-labeled chylomicron-like emulsion particles were injected into the tail vein of the mice. Two minutes after injection, 3H-activity was still solely associated with the emulsion particles, as evidenced by elution of all radioactivity in plasma in the void volume after Superose 6 column chromatography (Figure 3A). However, 3H-activity did appear in the HDL fractions at 1 hour after injection, and reached a maximum at 8 hours after injection. In contrast, in vitro incubation of [3H]CO-labeled chylomicron-like emulsion particles with plasma from Cyp7a1–/–.APOE*3-Leiden mice for 8 hours at 37°C, at a ratio similar to the in vivo situation (0.5 mg emulsion-TG: 1 mL plasma), did not result in transfer of radiolabel to HDL (supplemental Figure II). Apparently, after rapid and specific internalization of [3H]CO-labeled emulsion particles by the liver,27,28 liberated [3H]cholesterol is secreted back into plasma as a constituent of HDL. In line with an increased Abca1 expression, Cyp7a1-deficiency increased the appearance of radioactivity in HDL with 37% (Figure 3B).


Figure 3
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  		Figure 3. Increased hepatic cholesterol efflux to HDL in Cyp7a–/–.APOE*3-Leiden mice. [3H]CO-labeled chylomicron-like emulsion particles (0.5 mg TG {approx} 3.106 dpm) were injected into the tail vein of APOE*3-Leiden mice (black circles) and Cyp7a–/–.APOE*3-Leiden littermates (white circles). Two minutes (A) and 8 hours (B) after injection, blood samples were collected, plasma was isolated, and lipoproteins were separated by gel filtration chromatography. 50 µL-fractions were collected and assayed for 3H-activity. Data are expressed as means±SEM of 10 mice per group. *P<0.05.


*    Discussion
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 up arrowMethods
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 *Discussion
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Although several studies have shown an effect of bile acids on factors that influence HDL metabolism,14–17 these studies have been inconsistent with respect to the effect of bile acids on HDL levels. In this study, we have investigated the effect of impaired bile acid assembly as caused by Cyp7a1 deficiency on HDL metabolism in APOE*3-Leiden mice with a human-like lipoprotein profile. We demonstrated that decreased bile acid biosynthesis leads to an increased hepatic cholesteryl ester content, increased hepatic expression of Abca1, and an increased hepatic cholesterol efflux to HDL, resulting in increased plasma HDL levels.

Cyp7a1 knockout mice on an APOE*3-Leiden background did not have different total plasma cholesterol levels as compared with their control littermates. Although a hypercholesterolemic Cyp7a1–/– colony has recently been described,34 our data are in agreement with previous observations from Schwarz et al.19 Despite the absence of an effect on total plasma cholesterol levels, the distribution of cholesterol over the different plasma lipoproteins was changed in the Cyp7a1–/–.APOE*3-Leiden mice as compared with their APOE*3-Leiden littermates. The amount of cholesterol present in the apoB-containing lipoproteins (ie, VLDL and IDL/LDL) was decreased, which we have recently demonstrated to be attributable to a 35% decreased VLDL particle production.32 Reciprocally, we found an {approx}50% increased presence of cholesterol in HDL. In wild-type mice, Cyp7a1 deficiency did not alter the hepatic cholesteryl ester content (consistent with previous observations by Schwarz et al35), hepatic Abca1 expression, or plasma HDL-cholesterol. These data are consistent with the hypothesis that elevated hepatic cholesterol, attributable to Cyp7a1-deficiency, may in turn induce hepatic Abca1 transcription ultimately contributing to increased plasma HDL-cholesterol.

The steady-state level of HDL in de circulation is determined by factors that determine the balance between HDL synthesis and clearance.9 Whereas hepatic and intestinal apoAI synthesis, hepatic ABCA1, and plasma LCAT and PLTP contribute to the formation of mature plasma HDL, hepatic SR-BI is crucially involved in the selective clearance of HDL-derived cholesterol from plasma.33 Because apoAI promotes the efflux of cholesterol and phospholipids via the lipid transporter ABCA1, we have examined the effect of Cyp7a1 deficiency in APOE*3-Leiden mice on the expression of Apoa1. However, Cyp7a1 deficiency did not lead to any effect on hepatic or intestinal Apoa1 expression, and the plasma apoAI levels were also not significantly affected.

Previous human studies36,37 as well as animal studies38,39 point at a link between bile acids and hepatic Apoa1 mRNA and plasma apoAI levels. For example, treatment of rats with the bile acid sequestrant cholestyramine increased hepatic mRNA of Apoa1 in addition to plasma apoAI levels.38 Cholestyramine treatment of humans also resulted in increased plasma apoAI levels,37 and an increased concentration of the LpAI subfraction of HDL,36 which contains only apoAI and no apoAII. Reciprocally, treatment of wild-type and APOA1 transgenic mice with the bile acid cholate decreases hepatic mRNA of murine Apoa1 and human APOA1, respectively, and concomitantly decreases plasma apoAI.39 This is in line with the observation that bile acids suppress the expression of the human Apoa1 gene via a negative response element for FXR.14 However, in our mouse model, in which the hepatic bile acid biosynthesis is severely impaired, the expression of Apoa1 is apparently not altered. It can be speculated that Cyp7a1 deficiency not only influences the production of FXR agonists, but also other bile acids that may, via FXR-independent pathways, influence metabolism (eg, of apoAI). Alternatively, it cannot be excluded that adaptive pathways exist that counterbalance the expected increase in Apoa1 expression in our mice.

In contrast to Apoa1, expression of other target genes of FXR were lower in the Cyp7a1–/–.APOE*3-Leiden mice as compared with their APOE*3-Leiden littermates. For example, the canalicular bile acid transporter gene Bsep, was lower in the Cyp7a1–/–.APOE*3-Leiden, which was also found in Cyp7a1–/– mice on a wild-type background (supplemental Table III) and by others,40 and in FXR-null mice.41 Other FXR responsive genes, such as those coding for the canalicular transporters ABCG5,42 and possibly also ABCG8,41,42 which are involved in the biliary excretion of sterols, also showed decreased expression on Cyp7a1 deficiency. We have to realize that the genes described in this study are not only regulated by FXR but that other metabolic changes in these mice such as the increased hepatic sterol content may interfere with the FXR mediated regulation, eg, by stimulating LXR responses. In addition, the FXR target gene Pltp, which is involved in the modulation of HDL particles, showed a slight but significant decrease, similarly to previous observations in mice with a decreased bile acid pool.40,41 This small change in Pltp expression level did not result in a significant effect on the activity of PLTP. Nevertheless, reduced PLTP expression has been shown to lower HDL-cholesterol levels in a gene-dose–dependent manner,43 and can thus not account for the increased HDL levels as induced by Cyp7a1 deficiency. We did observe a modest reduction in hepatic Sr-b1 mRNA expression, which however was not accompanied by differences in hepatic SR-BI protein levels or HDL-CE clearance from plasma. Therefore, we can conclude that the Cyp7a1-deficiency–related increase in HDL level in APOE*3-Leiden mice, as induced by the Cyp7a1 deficiency, is not caused by impaired maturation of HDL in the circulation by PLTP and/or LCAT, nor by increased SR-BI–mediated clearance of HDL-CE.

Finally, we considered the possibility that increased hepatic cholesterol secretion would lead to higher HDL-CE levels. The impaired bile acid biosynthesis on Cyp7a1 deficiency resulted in an increase in hepatic cholesterol content, which was also found in the females of the hypercholesterolemic Cyp7a1–/– colony.34 However, Cyp7a1–/– mice with normal plasma cholesterol levels exhibited no changes in liver cholesterol content and showed an increased hepatic de novo sterol synthesis.35 We also did not observe an effect of Cyp7a1 deficiency on a wild-type background on the liver cholesterol content, consistent with the absence of an effect of Cyp7a1 deficiency on the hepatic expression of HmgcoA-reductase and the cholesterol transporters Abca1, Abcg5, and Abcg8. The differences in phenotype may be attributed to sex, genetic background, diet, or other environmental factors. In our hands, Cyp7a1 deficiency on a human-like lipoprotein profile consistently showed increased hepatic cholesterol content which consequently leads to a marked reduction in the expression levels of HmgcoA-reductase as previously shown in FXR-null mice.40,41 We speculated that the higher hepatic cholesterol content can result in increased expression of the transporter ABCA1.44

Recent findings have demonstrated the crucial involvement of hepatic ABCA1 in determining plasma HDL levels by promoting nascent HDL production through enhancing cholesterol efflux from hepatocytes.8 Bone marrow transplantation studies45,46 have shown that ABCA1 expression in macrophages does not have a large contribution to determine plasma HDL levels, and that hepatic ABCA1 is the major contributor to plasma HDL-cholesterol levels. Indeed, the ABCA1 transporter is localized on the basolateral surface of polarized hepatocytes,47 and hepatic overexpression of ABCA1 in mice by adenoviral gene transfer increases HDL-cholesterol.8 In our study, we found that Cyp7a1 deficiency led to a 2-fold increased hepatic Abca1 expression, which was accompanied by an increased hepatic secretion of cholesterol to HDL. Although we were unable to determine hepatic ABCA1 protein, hepatic Abca1 mRNA expression has been consistently found to correlate with hepatic ABCA1 protein levels.8,48,49 Thus, under conditions of reduced bile acid synthesis, this process represents a way to get rid of excess hepatic cholesterol via basolateral secretion via ABCA1. Increased HDL levels are generally thought to be atheroprotective, although an increase in HDL may also result from accumulation of HDL that are dysfunctional in several steps of cholesterol transport, eg, as observed in LCAT transgenic mice.50 Whether or not the elevated HDL levels resulting from Cyp7a1 deficiency represent functional HDL will be subject of future studies.

Recently, Sahoo et al51 have shown in vitro that the ABCA1-mediated increased HDL particle formation was accompanied by a reduction in VLDL particle secretion by limiting the availability of cholesterol for VLDL assembly. This mechanism may contribute to the decreased VLDL particle production in Cyp7a1–/–.APOE*3-Leiden mice compared with APOE*3-Leiden mice as we previously reported,32 despite the fact the total hepatic cholesterol and triglyceride levels are increased in Cyp7a1–/–.APOE*3-Leiden mice.

In conclusion, reduced bile acid synthesis in Cyp7a1-deficient APOE*3-Leiden mice leads to increased plasma HDL-cholesterol levels, as related to increased hepatic Abca1 expression. Furthermore, these data underscore a major impact of hepatic ABCA1 on determination of HDL-cholesterol levels.


*    Acknowledgments
 
We thank Elly de Wit for excellent technical assistance.

Sources of Funding

This work was performed in the framework of the "Leiden Center for Cardiovascular Research LUMC-TNO", and supported by the Netherlands Heart Foundation (NHS grant 97.116 and ZON-MW/NHS grant 980-10-024), the Netherlands Organization for Scientific Research (ZON-MW/NHS grant 980-10-024 and NWO-VIDI grant 917.36.351 to P.C.N.R.), and the LUMC (Gisela Thier Fellowship to P.C.N.R.).

Disclosures

None.


*    Footnotes
 
S.M.P. and M.G. contributed equally to this study.

Original received March 29, 2006; final version accepted August 29, 2006.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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