Cholesterol 7-Hydroxylase Deficiency in Mice on an APOE*3-Leiden Background Impairs Very-Low-Density Lipoprotein Production
http://www.100md.com
《动脉硬化血栓血管生物学》
From TNO Prevention and Health (S.M.P., M.G., K.S., P.C.N.R., H.M.G.P.), Gaubius Laboratory, Leiden, The Netherlands; and the Department of General Internal Medicine (P.C.N.R.), Leiden University Medical Center, Leiden, The Netherlands.
ABSTRACT
Objective— Cholesterol 7-hydroxylase (cyp7a1) catalyzes the rate-limiting step in conversion of cholesterol to bile acids. To study the relationship between bile acid biosynthesis and triglyceride metabolism, we cross-bred mice lacking cyp7a1 on a hyperlipidemic APOE*3-Leiden background.
Methods and Results— Female mice received a chow or lipogenic diet. On both diets, fecal bile acid excretion was 70% decreased concomitantly with a 2-fold increased neutral sterol output. The differences in bile acid biosynthesis did not change plasma cholesterol levels. However, plasma triglyceride levels decreased by 41% and 38% in the cyp7a1-/-. APOE*3-Leiden mice as compared with APOE*3-Leiden mice on chow and lipogenic diet, respectively. Mechanistic studies showed that very-low-density lipoprotein (VLDL)–apolipoprotein B and VLDL–triglyceride production rates were reduced in cyp7a1-/-. APOE*3-Leiden mice as compared with APOE*3-Leiden mice (-34% and -35%, respectively). Cyp7a1 deficiency also increased the hepatic cholesteryl ester and triglyceride content (2.8-fold and 2.5-fold, respectively). In addition, hepatic anti-oxidative vitamin content, which can influence VLDL-production, was lower. Hepatic mRNA analysis showed decreased expression of genes involved in lipogenesis including srebf1.
Conclusions— Cyp7a1 deficiency in APOE*3-Leiden mice decreases the VLDL particle production rate, as a consequence of a strongly reduced bile acid biosynthesis, leading to a decrease in plasma triglycerides. These data underscore the close relationship between bile acid biosynthesis and triglyceride levels.
Key Words: bile acid biosynthesis ? cholesterol 7-hydroxylase ? mouse ? SREBP-1 ? triglycerides
Introduction
The liver plays a central role in the regulation and maintenance of the whole-body sterol balance. The conversion of cholesterol into bile acids and their subsequent fecal excretion is quantitatively the most important way for elimination of cholesterol from the body. In the intestine, bile acids are important as physiological detergents that facilitate the absorption of lipid-soluble vitamins and dietary fat. The classical or neutral route in bile acid biosynthesis is initiated by 7-hydroxylation of cholesterol catalyzed by the major rate-limiting enzyme cholesterol 7-hydroxylase (cyp7a1), which is located in the smooth endoplasmic reticulum.1,2 The rate of bile acid biosynthesis is under control of different molecular mechanisms in which nuclear receptors are involved. In most species, except for humans, the liver X receptor (LXR) is involved in the feed-forward regulation of cyp7a1 by cholesterol, whereas the feedback regulation by bile acids is mediated via the farnesoid X-receptor (FXR).3
Several studies have shown coordinate regulation between cholesterol 7-hydroxylase and the assembly and secretion of apolipoprotein B-containing lipoproteins in humans4,5 and in animal models.6,7 Patients treated with bile acid sequestrants, in which cyp7a1 expression is increased, have hypertriglyceridemia as a consequence of an induction in hepatic very-low-density lipoprotein (VLDL) secretion.8–10 In line with these studies, mice overexpressing cyp7a1 have increased VLDL production.6 These studies suggest a link between degradation of cholesterol into bile acids and hepatic VLDL production.
To further study the relationship between bile acid biosynthesis and triglyceride metabolism, we cross-bred mice lacking cyp7a111 on a hyperlipidemic APOE*3-Leiden background, thereby increasing the sensitivity of the effect of removal of cyp7a1 on lipoprotein and lipid metabolism. Because of the concomitant expression of APOE*3-Leiden and APOC1, these mice have a defective clearance of apolipoprotein B-containing lipoproteins and therefore show a more human-like lipoprotein profile.12
Our data show that female cyp7a1-/-.APOE*3-Leiden mice have a decreased VLDL production as a consequence of a strongly reduced bile acid biosynthesis, leading to reduced plasma triglyceride levels. These data reveal a major effect of physiological cyp7a1 expression on VLDL production and further underscore the link between bile acid biosynthesis and triglyceride levels.
Methods
Animals
Cyp7a1-/- mice11 were obtained from Jackson Laboratory (Bar Harbor, Me) and cross-bred on an APOE*3-Leiden background12 (line 2, >99% C57Bl/6). Specifically, male cyp7a1+/-.APOE*3-Leiden mice were crossed with female cyp7a1+/- mice to generate cyp7a1-/-.APOE*3-Leiden mice and APOE*3-Leiden littermate controls. Pregnant females were administered additional vitamins throughout the nursing period. All experiments were performed with 16- to 22-week-old female mice, which were maintained on a reversed 12-hour dark and 12-hour light cycle and allowed free access to food and water. Body weight of the mice and the consumption of diet and water were recorded weekly and did not differ between cyp7a1-/-.APOE*3-Leiden and APOE*3-Leiden mice during the whole experimental period. In some experiments (indicated in text or legends), mice were fed a sucrose-rich diet (Hope Farms, Woerden, The Netherlands)13 for 3 weeks. Institutional guidelines for animal care were observed in all experiments.
Analysis of Blood Parameters
After overnight fasting, blood samples were collected into paraoxonase-coated capillaries14 after tail incision. Plasma total cholesterol, triglycerides (corrected for plasma levels of free glycerol), and plasma ?-hydroxybutyrate (as a measure of in vivo ?-oxidation) were measured enzymatically (CHOD-PAP method, Roche, 236691, GPO-trinder, Sigma, 337-B, and Sigma Diagnostics, 310-A, respectively).
Hepatic RNA Isolation and Measurement of mRNA Levels by Real-Time Polymerase Chain Reaction
Total RNA was isolated from liver tissue as described previously.15 RNA was converted to single-stranded cDNA by a reverse-transcription procedure (Promega) according to the manufacturer’s protocol using random primers. cDNA levels were measured by real-time polymerase chain reaction (PCR) using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif), according to the manufacturer’s instructions. PCR mastermix from Eurogentec was used. Primers and probes were obtained from Biosource (Nivelles, Belgium). The probes were labeled with 3'-BHQ1 and 5'-FAM or 5'-TET. The mRNA levels were normalized to mRNA levels of different housekeeping genes (ie, cyclophilin, HPRT, and GAPDH). Primers and probes used for this study are summarized in Table 1. The level of mRNA expression for each gene of interest was calculated using the Ct values. Ct values are defined as the number of PCR cycles at which the fluorescent signal generated during the PCR reaches a fixed threshold. For each sample, the Ct for the target gene and for the housekeeping gene were determined to calculate Ct (Ct,target gene - Ct,housekeeping gene). Subsequently, Ct was determined by subtracting the Ct of the test group from the Ct of the control group, and the relative gene expression levels were calculated from 2-Ct, according to the manufacturer’s instructions (Applied Biosystems). In each case, we checked that the Ct method was valid by showing that Ct is constant over a range of cDNA solutions. For some signals (acyl-CoA synthase , steroyl-CoA desaturase , peroxisomal bifunctional enzyme , and thiolase ), we performed Northern blot analysis as described.15 Liver lipids were measured as described.15
TABLE 1. Primers and Probes for Real-Time PCR Analysis
VLDL–Triglyceride and Apolipoprotein B Production
After overnight fasting to prevent chylomicron production, mice were injected via the tail vein with 0.1 mL phosphate buffered saline (PBS) containing 100 μCi Tran35S-label (ICN), a mixture of 35S-labeled methionine and cysteine. After 30 minutes, they were treated with Triton WR-1339 (500 mg/kg intravenous), which virtually completely inhibits VLDL clearance by blocking LPL-mediated lipolysis. The accumulation of VLDL–triglyceride in plasma and the incorporation of radiolabeled amino acids into newly synthesized apolipoprotein B over 90 minutes were measured essentially as described.16
Fecal Excretion of Bile Acids and Neutral Sterols
APOE*3-Leiden and cyp7a1-/-.APOE*3-Leiden mice were housed at 3 mice per cage. Feces produced during 3 subsequent periods were separated from the wood shavings by sieving. Aliquots of lyophilized feces were used for determination of neutral and acidic sterol content by gas-liquid-chromatography procedures described.15
Lipolysis
Postheparin plasma from fasted mice (overnight) was collected from the tail vein at 20 minutes after intraperitoneal injection of heparin (1.0 U/g body weight). Postheparin plasma triacylglycerol hydrolase activity was determined in the presence or absence of 1 mol/L NaCl to estimate the lipoprotein lipase (LPL) activity, which was calculated as the portion of total triacylglycerol hydrolase activity inhibited by 1 mol/L NaCl.15
Western Blotting
Western blot analysis was performed as previously described.17 Liver homogenates (50 μg protein) were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and proteins were blotted onto nitrocellulose as described.17 Blots were developed with an anti-sterol regulatory element binding protein-1 (SREBP-1) primary antibody and horseradish peroxidase-conjugated secondary immunoglobulin. All antibodies were diluted in 20 mmol/L Tris (pH 7.4), 55 mmol/L NaCl, 0.1% (v/v) Tween-20, and 5% (wt/vol) bovine serum albumin and were used at a final concentration of 0.2 μg/mL. The Super Signal West Dura Extended Duration Substrate (Pierce, St Augustin, Germany) and the luminescent image workstation (Roche Diagnostics) were used for SREBP-1 visualization.
Diacylglycerol Acyltransferase Activity Assay
Hepatic diacylglycerol acyltransferase (DGAT) activity was determined by the esterification of oleate into triolein. The microsomal fraction of mouse liver (5 μg of protein diluted in 50 μL of 0.25 mol/L sucrose, 50 mmol/L Tris-HCl ) was added to 200 μmol/L 1,2-diacyl-sn-glycerol (Sigma) in acetone (final vehicle concentration 5% ), 62.5 μg BSA, and 25 μmol/L oleoyl-CoA (9 μCi/μmol) (NEN Life Science Products) in 100 mmol/L Tris-HCl, pH 7.5 (total volume 200 μL). After incubation for 5 minutes in a shaking water bath at 37°C, the reaction was terminated by adding 4 mL of chloroform/methanol (2:1 vol/vol); 20 μL of triolein (3000 dpm) (Amersham) in toluene was added to monitor the extraction efficiency. After phase separation by addition of 750 μL of dH2O, lipids were extracted, dried under nitrogen, dissolved in chloroform, and spotted onto a channeled 20x20-cm Silica gel TLC plate (LK5D 150 A; Whatman). The lipids were separated by running the TLC plate in chloroform/methanol (98:2 vol/vol, halfway), followed by chloroform/hexane (35:65 vol/vol). The radioactive triolein bands were visualized by autoradiography and scraped off for scintillation counting in 4 mL of Ultima Gold (Packard).
Vitamin Analysis
Liver samples were saponified during 30 minutes under reflux conditions using ethanolic KOH containing glycerol, Na2S, and 2% sodium ascorbate. After cooling to room temperature, vitamins were extracted by isopropylether. The extract was washed once using 5% KOH, followed by 2 washes with water. After dissolving the vitamins in isopropylether, the ether was dried under a gentle stream of nitrogen and the residue dissolved in heptane. Subsequently, the samples were analyzed for tocopherols (ie, vitamin E) and retinols (ie, vitamin A) using straight-phase HPLC with fluorescence detection.
Statistics
Data were analyzed statistically using Student unpaired t test, with the level of significance selected to be P<0.05. Values are expressed as means±SD.
Results
Effect of cyp7a1 Deficiency on Fecal Sterols and Plasma Lipid Levels
To evaluate the effect of cyp7a1 deficiency on regular and elevated plasma lipid levels, female APOE*3-Leiden and cyp7a1-/-.APOE*3-Leiden mice were fed a normal chow diet and a sucrose-rich diet, respectively. On chow diet, 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 (-73%) (Figure 1A). As a consequence, the fecal neutral sterol output was 2-fold increased in the cyp7a1-/-.APOE*3-Leiden mice (Figure 1B). The basal levels of fecal bile acid and neutral sterol output on sucrose-rich diet were significantly lower compared with chow diet (-37% and -57%, respectively). However, the relative effect on fecal bile acid and neutral sterol output was similar (Figure 1).
Figure 1. Cyp7a1 deficiency decreases fecal bile acid output concomitantly with increased fecal neutral sterol output in female APOE*3-Leiden mice on chow and sucrose-rich diet. Fecal bile acid (A) and neutral sterol (B) output in APOE*3-Leiden (E3L) and cyp7a-/-.APOE*3-Leiden mice on chow (white bars) or a semi-synthetic sucrose-rich diet (black bars) was determined as described in the Methods section. Data are means±SD of feces samples obtained from 2 groups of 3 mice per genotype during 3 time intervals. Significant differences between APOE*3-Leiden and cyp7a-/-.APOE*3-Leiden mice are indicated by an asterisk. *P<0.005; **P<0.001.
The differences in bile acid biosynthesis did not result in an effect on plasma cholesterol levels on an APOE*3-Leiden background as well as on a wild-type background on chow diet (Table 2). However, triglyceride levels were 41% and 29% decreased (mainly confined to the VLDL fraction) in the cyp7a1-/-.APOE*3-Leiden and cyp7a1-/- mice, respectively, as compared with their control littermates. On the sucrose-rich diet, basal cholesterol levels in the cyp7a1-/-.APOE*3-Leiden mice increased from 2.6±0.8 mmol/L to 4.4±1.1 mmol/L (P<0.05) and from 1.6±0.3 mmol/L to 2.6±0.6 mmol/L (P<0.05) in the cyp7a1-/- mice, whereas triglyceride levels did not change. The plasma lipid-inducing effects of the sucrose-rich diet in APOE*3-Leiden mice is lower as compared with our previous observations,12 which is probably related to continued back-crossing of the mice from the original CBA/J background to the C57Bl/6J background. On this diet, cyp7a1 deficiency on an APOE*3-Leiden and wild-type background had a similar decreasing effect on plasma triglyceride levels (38% and 38%, respectively) as on chow diet and had no effect on cholesterol levels (Table 2).
TABLE 2. Plasma Lipid Levels in Female Wild-Type, cyp7a1-/-, APOE*3-Leiden (E3L), and cyp7a1-/-.APOE*3-Leiden Mice on Chow and Sucrose-Rich Diet
Effect of cyp7a1 Deficiency on Plasma Lipolytic Activity, Ketone Bodies and VLDL Production
The decreasing effect of cyp7a1 deficiency on triglyceride levels could be caused by altered LPL activity, ?-oxidation of fatty acids, or VLDL production. To further investigate the mechanism behind the reduced plasma triglyceride levels in cyp7a1 deficiency, we measured postheparin LPL activity as a measure of peripheral lipolysis. It appeared that the triacylglycerol hydrolase activity of LPL did not differ between female APOE*3-Leiden and cyp7a1-/-.APOE*3-Leiden mice (fatty acid production rate 4.0±2.0 μmol/mL per hour versus 2.4±1.9 μmol/mL per hour, respectively; n=8 per group; P<0.05). Rather, a trend was observed toward decreased LPL levels in cyp7a1-/- mice. In addition, no changes were observed in plasma levels of ketone bodies (?-hydroxybutyrate) as markers of oxidation of fatty acids (1.79±0.53 mmol/L versus 1.79±0.33 mmol/L, respectively; n=8 per group), although the expression of various genes involved in hepatic ?-oxidation, eg, acyl-CoA oxidase (acox1), acyl-CoA synthase (acas1), peroxisomal bifunctional enzyme (ehhadh), and thiolase (acca1), were increased (Table 3). In contrast, hepatic VLDL–apolipoprotein B and VLDL–triglyceride production rates were reduced in the cyp7a1-/-.APOE*3-Leiden as compared with the APOE*3-Leiden mice (34% and 35%, respectively) (P<0.05) (Figure 2). The lipid composition of the nascent VLDL particles was not changed (see insert in Figure 2B), indicating that cyp7a1 deficiency results in a decreased hepatic VLDL particle production rate. Although the microsomal triglyceride transfer protein (mttp), apolipoprotein B (apob), and apolipoprotein B mRNA-editing complex-1 (apobec-1) play a critical role in the assembly and secretion of VLDL in the liver, the hepatic expression levels of these genes were not significantly different between cyp7a1-/-.APOE*3-Leiden and APOE*3-Leiden mice (Table 3).
TABLE 3. Hepatic mRNA Levels in Female cyp7a1-/-.APOE*3-Leiden and APOE*3-Leiden Mice
Figure 2. Decreased VLDL–triglyceride and VLDL–ApoB production in female cyp7a1-/-.APOE*3-Leiden on sucrose-rich diet. Tran35S-label (100 μCi) was injected into APOE*3-Leiden (E3L) (black circles) and cyp7a-/-.APOE*3-Leiden mice (white circles). After 30 minutes, Triton WR1339 (500 mg/kg body weight) was injected and plasma samples were taken at various time points. Plasma triglyceride levels were determined at indicated time points and corrected for the triglyceride level at the time of injection (A). Nascent VLDL was isolated by ultracentrifugation from pooled serum collected 120 minutes after Triton injection. 35S-labeled apolipoprotein B was precipitated from the VLDL fraction and radioactivity was subsequently measured (B). The values shown are means±SD of 8 mice (A), or means±SD of 4 VLDL samples of each group (B). Significant differences in triglyceride and apolipoprotein B production rates are indicated by an asterisk (*P<0.05). The insert in (B) represents the nascent VLDL lipid composition (values presented as % of total lipid). FC indicates free cholesterol; CE, cholesteryl esters; TG, triglycerides; PL, phospholipids. No significant differences were observed between cyp7a1-/-.E3L and E3L mice.
Effect of cyp7a1 Deficiency on Hepatic Lipogenesis, Lipid Content, and Vitamin Levels
To investigate the effect of the lack of cyp7a1 on hepatic triglyceride synthesis, mRNA and protein levels of the transcription factor SREBP-1 (srebf1), which regulates lipogenic genes, were measured. Also, the mRNA expression levels of enzymes involved in fatty acid synthesis, ie, fatty acid synthase (fasn), acyl-CoA carboxylase (acac) and steroyl-CoA desaturase (scd1) were measured. The srebf1 mRNA level was 31% lower in the cyp7a1-/-.APOE*3-Leiden mice, which was accompanied by a significant (P<0.05; n=4 per group) reduction in the amount of mature (100%±27% versus 35%±28%) and precursor (100%±47% versus 62%±54%) form of SREBP-1 and a lower expression of the lipogenesis genes acac and fasn (Table 3).
As a consequence of a decreased bile acid production and a reduction in VLDL secretion, hepatic free cholesterol, esterified cholesterol, and triglyceride levels were significantly increased (1.3-, 2.8-, and 2.5-fold, respectively) in the cyp7a1-/-.APOE*3-Leiden mice, whereas hepatic phospholipid levels were significantly decreased by -28% (Table 4). The increase in hepatic cholesterol levels was related to a 49% reduction in the expression of the low-density lipoprotein receptor (ldlr) (Table 3).
TABLE 4. Hepatic Lipid, Vitamin A, and Vitamin E Levels in Female cyp7a1-/-.APOE*3-Leiden and APOE*3-Leiden Mice
To investigate whether the changes in the hepatic content in phospholipids and triglycerides were caused by a change in the enzyme activity of DGAT, we performed a DGAT activity assay with microsomes isolated from the livers of the APOE*3-Leiden (n=6) and cyp7a1-/-.APOE*3-Leiden (n=7) mice fed the sucrose-rich diet. We found no significant difference between these groups with respect to the activity of this enzyme (10.0±1.7 nmol/min per mg protein versus 9.0±1.4 nmol/min per mg protein, respectively).
Anti-oxidative vitamins play a role in determining the hepatic content of reactive oxygen species, which can be involved in the degradation of apolipoprotein B. Therefore, we measured the amount of vitamin A and E in the liver. The data of Table 4 show that cyp7a1-/-.APOE*3-Leiden mice contain greatly reduced levels of the fat-soluble vitamins A and E in the liver as compared with their APOE*3-Leiden littermates.
Discussion
This study showed that the absence of cyp7a1 in female APOE*3-Leiden mice results in a decrease in plasma triglyceride levels as a consequence of a lower VLDL production.
Cyp7a1 knockout mice on an APOE*3-Leiden background as well as on a wild-type background of both sexes did not have different plasma cholesterol levels as compared with their control littermates. Although this is in agreement with a previous report,11 a hypercholesterolemic cyp7a1-/- colony has recently also been described.18 Similarly, conflicting effects on plasma triglyceride levels have been reported11,18 in mice lacking cyp7a1. No significant difference in plasma triglyceride levels was found in the cyp7a1-/- mice with normal cholesterol levels, whereas the females of the hypercholesterolemic cyp7a1-/- colony exhibited lower plasma triglyceride levels.18 The differences in phenotype may be because of sex, genetic background, diet, or other environmental factors. In our hands, cyp7a1 deficiency on a human-like lipoprotein profile and on a wild-type background consistently showed decreased plasma triglyceride levels in only the female mice similarly as previously described.18 The absence of an effect in male mice can possibly be explained by the lower level in VLDL–triglyceride production in males as compared with females as a result of the difference in hormone level.19
We have shown that the lower level of plasma triglycerides in the female cyp7a1-/-.APOE*3-Leiden mice is caused by inhibition of VLDL particle production. It has been known for many years that there is a link between bile acid metabolism, especially related to cyp7a1 expression, and hepatic VLDL production. Treatment with the bile acid sequestrant cholestyramine, which induces bile acid biosynthesis, leads to an increase in VLDL triglyceride and VLDL cholesterol production. This increase is seen in healthy individuals4 and in patients with various types of hyperlipidemia,9,20 and predominantly in patients with hypertriglyceridemia.8,9 In animals, this relation between bile acid biosynthesis and plasma triglyceride levels has also been detected. Disruption of the enzyme sterol 27-hydroxylase in mice leads to a 5-fold increase in cyp7a1 activity and a concomitant 2-fold increase in hepatic and plasma triglyceride levels.7 Furthermore, cyp7a1 transgenic mice have an increased production and secretion of apolipoprotein B-containing lipoproteins.6
We did not observe an effect of cyp7a1 deficiency on plasma cholesterol levels. Apparently, the reduced influx of cholesterol into plasma resulting from a reduced VLDL production and reduced intestinal neutral sterol cholesterol absorption is counterbalanced by a decreased hepatic cholesterol clearance. Indeed, the ldlr expression was reduced, probably as a result of downregulation, as induced by increased hepatic cholesterol levels.
The hepatic synthesis of VLDL particles requires many (in)dependent processes like: (1) the production of constituting lipids; (2) the synthesis and secretion of apolipoprotein B and apolipoprotein E; (3) the transfer of lipid by MTP to apolipoprotein B; and (4) association of the resulting precursor particle with bulk lipid to form mature VLDL.21 We did not find an effect of cyp7a1 deficiency on mttp mRNA levels. Because MTP protein and activity are tightly regulated by mttp mRNA levels,22 hepatic MTP activity is thus likely to be unaltered. In contrast, cyp7a1-overexpressing animals were reported to have increased mttp expression,6 which, however, may result from an unphysiologically high cyp7a1 expression.
Studies with hepatoma cells overexpressing cyp7a1 showed that as a result of increased mature SREBP-1, there was a coordinate induction of de novo lipogenesis and assembly and secretion of VLDL.23 We found a decrease in srebf1, mRNA, and SREBP-1 protein leading to suppression of genes involved in de novo lipogenesis, ie, fas and acc1, which may contribute to reduced VLDL secretion. Despite the decreased de novo lipogenesis, hepatic triglyceride levels were increased. These data indicate that a decreased de novo lipogenesis does not automatically lead to smaller triglyceride content in the liver, which is in line with the absence of an effect on hepatic triglyceride levels in srebf1-deficient mice.24 Alternatively, the decrease in lipogenesis can also be the consequence of the high level of triglycerides in the liver. In contrast, cyp7a1-/- mice on a wild-type background have no difference in hepatic triglyceride content and have decreased fatty acid synthesis.18 Differences between triglyceride and fatty acid metabolism in the cyp7a1-/- mice and cyp7a1-/-.APOE*3-Leiden mice may be explained by the presence of the APOE*3-Leiden and APOC1 gene.
Because the increased triglyceride content cannot be explained by the effect on de novo lipogenesis, we measured DGAT. DGAT is another important enzyme in determining the triglyceride content in the liver, producing triglycerides from diacylglycerides and acyl-CoAs. Because we found no alteration in the enzyme activity of DGAT, we can exclude the possibility that this enzyme is involved in the accumulation of triglycerides in the liver or the decrease in VLDL production. The increase in hepatic triglyceride content can also be a consequence of the extreme blockade in the output of fatty acids in the form of phospholipids via the bile, together with a decrease in VLDL production. Biliary bile acid excretion is an important determinant of biliary phospholipids and cholesterol excretion.25
Our data show that affecting bile acid metabolism has a major impact on hepatic fatty acid metabolism, not only on genes involved in de novo lipogenesis but also on genes involved in hepatic oxidation of fatty acids. The general effects of cyp7a1 deficiency in this animal model, except from the lack of effect on LPL, closely resemble the effects of treating mice with PPAR-agonists (ie, fibrates). It has been shown that fibrates decrease triglyceride levels, increase expression of genes involved in ?-oxidation, and reduce VLDL production and the expression of genes involved in fatty acid synthesis.26 We also reported that after treatment of mice with ciprofibrate, bile acid synthesis is suppressed via PPAR-mediated downregulation of cyp7a1 and sterol 27-hydroxylase.27 Therefore, we cannot exclude that PPAR plays a role in the effects observed in the cyp7a1-deficient mouse. In addition, bile acids have been shown to interfere with transactivation by PPAR, at least in part by impairing the recruitment of transcriptional co-activators.28 The decreased bile acid pool in the cyp7a1 knockout mice may explain the changes in PPAR-regulated genes involved in ?-oxidation eg, acox1, acac1, and ehhadh. The increase in acox1 has also been found by others.18 Despite the increase in expression levels of genes involved in ?-oxidation, which may have contributed to the differences in fatty acid and triglyceride metabolism in the cyp7a1-/-.APOE*3-Leiden, the concentration of plasma ketone bodies remained unaltered.
We showed that cyp7a1 deficiency affected the VLDL secretion rate. The nascent VLDL composition was not changed, although a trend was observed toward a higher cholesteryl ester/triglyceride ratio within the particle core. The constituting core lipids in VLDL (ie, cholesteryl esters and triglycerides) were available in sufficient amounts in the liver of the female cyp7a1-/-.APOE*3-Leiden mice, but it can be possible that deposition of these lipids in the liver is not present in the secretory route where proteins are located, which are involved in the packaging of lipids into VLDL particles. Moreover, we found that the amount of hepatic phospholipids was decreased. It has been reported that the hepatocellular phospholipid availability is a determinant for the regulation of VLDL assembly.29–32 Furthermore, intracellular proteolysis of apolipoprotein B, resulting in a diminished assembly of VLDL particles, may be increased in female cyp7a1-/-.APOE*3-Leiden mice. We and others11 showed that cyp7a1 deficiency in mice results in a low level of anti-oxidative vitamins caused by a reduction in the intestinal uptake.11 This consequently leads to an excess in intracellular reactive oxygen species. Recently, it has been shown that a novel pathway for destruction of newly synthesized apolipoprotein B is induced by intracellular reactive oxygen species and PI3-kinases.33,34 This may be a possible explanation for the difference in VLDL production in the knockout mice as compared with their littermates.
In conclusion, our data show that female cyp7a1-/-.APOE*3-Leiden mice have a decreased VLDL production as a consequence of a strongly reduced bile acid biosynthesis, leading to a decrease in plasma triglyceride levels. These data indicate a major impact of physiological cyp7a1 expression on VLDL production and further underscore the link between bile acid biosynthesis and triglyceride levels.
Acknowledgments
This work was supported by the Netherlands Heart Foundation (NHS grant 97.116), the Netherlands Organization for Scientific Research (NWO grant 980-10-024 and VIDI 917.36.351), the Marabou Foundation, Sweden, and by the Leiden University Medical Center (Gisela Thier fellowship to P. C. N. Rensen). We thank Elly de Wit for excellent technical assistance.
References
Russell DW, Setchell KD. Bile acid biosynthesis. Biochemistry. 1992; 31: 4737–4749.
Princen HMG, Post SM, Twisk J. Regulation of bile acid biosynthesis. Curr Pharmaceut Design. 1997; 3: 59–84.
Chiang JY. Bile acid regulation of gene expression: roles of nuclear hormone receptors. Endocr Rev. 2002; 23: 443–463.
Carrella M, Ericsson S, Del Piano C, Angelin B, Einarsson K. Effect of cholestyramine treatment on biliary lipid secretion rates in normolipidaemic men. J Intern Med. 1991; 229: 241–246.
Lipid Research Clinics Program. JAMA. 1984; 252: 2545–2548.
Miyake JH, Doung XD, Strauss W, Moore GL, Castellani LW, Curtiss LK, Taylor JM, Davis RA. Increased production of apolipoprotein B-containing lipoproteins in the absence of hyperlipidemia in transgenic mice expressing cholesterol 7alpha-hydroxylase. J Biol Chem. 2001; 276: 23304–23311.
Repa JJ, Lund EG, Horton JD, Leitersdorf E, Russell DW, Dietschy JM, Turley SD. Disruption of the sterol 27-hydroxylase gene in mice results in hepatomegaly and hypertriglyceridemia. Reversal by cholic acid feeding. J Biol Chem. 2000; 275: 39685–39692.
Beil U, Crouse JR, Einarsson K, Grundy SM. Effects of interruption of the enterohepatic circulation of bile acids on the transport of very low density-lipoprotein triglycerides. Metabolism. 1982; 31: 438–444.
Angelin B, Einarsson K, Hellstrom K, Leijd B. Bile acid kinetics in relation to endogenous triglyceride metabolism in various types of hyperlipoproteinemia. J Lipid Res. 1978; 19: 1004–1016.
Einarsson K, Ericsson S, Ewerth S, Reihner E, Rudling M, Stahlberg D, Angelin B. Bile acid sequestrants: mechanisms of action on bile acid and cholesterol metabolism. Eur J Clin Pharmacol. 1991; 40: S53–S58.
Schwarz M, Lund EG, Setchell KD, Kayden HJ, Zerwekh JE, Bjorkhem I, Herz J, Russell DW. Disruption of cholesterol 7alpha-hydroxylase gene in mice. II. Bile acid deficiency is overcome by induction of oxysterol 7alpha-hydroxylase. J Biol Chem. 1996; 271: 18024–18031.
van den Maagdenberg AM, Hofker MH, Krimpenfort PJ, de Bruijn, I, van Vlijmen B, van Der Boom H, Havekes LM, Frants RR. Transgenic mice carrying the apolipoprotein E3-Leiden gene exhibit hyperlipoproteinemia. J Biol Chem. 1993; 268: 10540–10545.
Post SM, Zoeteweij JP, Bos MH, de Wit EC, Havinga R, Kuipers F, Princen HM. Acyl-coenzyme A: cholesterol acyltransferase inhibitor, avasimibe, stimulates bile acid synthesis and cholesterol 7alpha-hydroxylase in cultured rat hepatocytes and in vivo in the rat. Hepatology. 1999; 30: 491–500.
Zambon A, Hashimoto SI, Brunzell JD. Analysis of techniques to obtain plasma for measurement of levels of free fatty acids. J Lipid Res. 1993; 34: 1021–1028.
Post SM, de Crom R, van Haperen R, van Tol A, Princen HM. Increased fecal bile acid excretion in transgenic mice with elevated expression of human phospholipid transfer protein. Arterioscler Thromb Vasc Biol. 2003; 23: 892–897.
Post SM, de Roos B, Vermeulen M, Afman L, Jong MC, Dahlmans VE, Havekes LM, Stellaard F, Katan MB, Princen HM. Cafestol increases serum cholesterol levels in apolipoprotein E*3-Leiden transgenic mice by suppression of bile acid synthesis. Arterioscler Thromb Vasc Biol. 2000; 20: 1551–1556.
Kleemann R, Gervois PP, Verschuren L, Staels B, Princen HM, Kooistra T. Fibrates down-regulate IL-1-stimulated C-reactive protein gene expression in hepatocytes by reducing nuclear p50-NFkappa B-C/EBP-beta complex formation. Blood. 2003; 101: 545–551.
Erickson SK, Lear SR, Deane S, Dubrac S, Huling SL, Nguyen L, Bollineni JS, Shefer S, Hyogo H, Cohen DE, Shneider B, Sehayek E, Ananthanarayanan M, Balasubramaniyan N, Suchy FJ, Batta AK, Salen G. Hypercholesterolemia and changes in lipid and bile acid metabolism in male and female cyp7A1-deficient mice. J Lipid Res. 2003; 44: 1001–1009.
van Vlijmen BJ, ’t Hof HB, Mol MJ, van Der Boom H, van der ZA, Frants RR, Hofker MH, Havekes LM. Modulation of very low density lipoprotein production and clearance contributes to age- and gender- dependent hyperlipoproteinemia in apolipoprotein E3-Leiden transgenic mice. J Clin Invest. 1996; 97: 1184–1192.
Einarsson K, Hellstrom K, Kallner M. Bile acid kinetics in relation to sex, serum lipids, body weights, and gallbladder disease in patients with various types of hyperlipoproteinemia. J Clin Invest. 1974; 54: 1301–1311.
Shelness GS, Sellers JA. Very-low-density lipoprotein assembly and secretion. Curr Opin Lipidol. 2001; 12: 151–157.
Raabe M, Flynn LM, Zlot CH, Wong JS, Veniant MM, Hamilton RL, Young SG. Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes. Proc Natl Acad Sci U S A. 1998; 95: 8686–8691.
Wang SL, Du EZ, Martin TD, Davis RA. Coordinate regulation of lipogenesis, the assembly and secretion of apolipoprotein B-containing lipoproteins by sterol response element binding protein 1. J Biol Chem. 1997; 272: 19351–19358.
Liang G, Yang J, Horton JD, Hammer RE, Goldstein JL, Brown MS. Diminished hepatic response to fasting/refeeding and liver X receptor agonists in mice with selective deficiency of sterol regulatory element-binding protein-1c. J Biol Chem. 2002; 277: 9520–9528.
Suchy FJ, Sippel CJ, Ananthanarayanan M. Bile acid transport across the hepatocyte canalicular membrane. FASEB J. 1997; 11: 199–205.
Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, Fruchart JC. Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation. 1998; 98: 2088–2093.
Post SM, Duez H, Gervois PP, Staels B, Kuipers F, Princen HM. Fibrates Suppress Bile Acid Synthesis via Peroxisome Proliferator-Activated Receptor-alpha-Mediated Downregulation of Cholesterol 7alpha-Hydroxylase and Sterol 27-Hydroxylase Expression. Arterioscler Thromb Vasc Biol. 2001; 21: 1840–1845.
Sinal CJ, Yoon M, Gonzalez FJ. Antagonism of the actions of peroxisome proliferator-activated receptor-alpha by bile acids. J Biol Chem. 2001; 276: 47154–47162.
Wiggins D, Gibbons GF. Origin of hepatic very-low-density lipoprotein triacylglycerol: the contribution of cellular phospholipid. Biochem J. 1996; 320: 673–679.
Yao ZM, Vance DE. The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes. J Biol Chem. 1988; 263: 2998–3004.
Verkade HJ, Fast DG, Rusinol AE, Scraba DG, Vance DE. Impaired biosynthesis of phosphatidylcholine causes a decrease in the number of very low density lipoprotein particles in the Golgi but not in the endoplasmic reticulum of rat liver. J Biol Chem. 1993; 268: 24990–24996.
Vermeulen PS, Lingrell S, Yao Z, Vance DE. Phosphatidylcholine biosynthesis is required for secretion of truncated apolipoprotein Bs from McArdle RH7777 cells only when a neutral lipid core is formed. J Lipid Res. 1997; 38: 447–458.
Fisher EA, Pan M, Chen X, Wu X, Wang H, Jamil H, Sparks JD, Williams KJ. The triple threat to nascent apolipoprotein B. Evidence for multiple, distinct degradative pathways. J Biol Chem. 2001; 276: 27855–27863.
Pan M, Cederbaum AI, Williams KJ, Fisher EA. Reactive oxygen species regulate basal and induced degradation of apolipoprotein B in rat hepatic cells. Circulation. 2002; 106 (suppl II): II-2.ABSTRACT.(Sabine M. Post; Martine G)
ABSTRACT
Objective— Cholesterol 7-hydroxylase (cyp7a1) catalyzes the rate-limiting step in conversion of cholesterol to bile acids. To study the relationship between bile acid biosynthesis and triglyceride metabolism, we cross-bred mice lacking cyp7a1 on a hyperlipidemic APOE*3-Leiden background.
Methods and Results— Female mice received a chow or lipogenic diet. On both diets, fecal bile acid excretion was 70% decreased concomitantly with a 2-fold increased neutral sterol output. The differences in bile acid biosynthesis did not change plasma cholesterol levels. However, plasma triglyceride levels decreased by 41% and 38% in the cyp7a1-/-. APOE*3-Leiden mice as compared with APOE*3-Leiden mice on chow and lipogenic diet, respectively. Mechanistic studies showed that very-low-density lipoprotein (VLDL)–apolipoprotein B and VLDL–triglyceride production rates were reduced in cyp7a1-/-. APOE*3-Leiden mice as compared with APOE*3-Leiden mice (-34% and -35%, respectively). Cyp7a1 deficiency also increased the hepatic cholesteryl ester and triglyceride content (2.8-fold and 2.5-fold, respectively). In addition, hepatic anti-oxidative vitamin content, which can influence VLDL-production, was lower. Hepatic mRNA analysis showed decreased expression of genes involved in lipogenesis including srebf1.
Conclusions— Cyp7a1 deficiency in APOE*3-Leiden mice decreases the VLDL particle production rate, as a consequence of a strongly reduced bile acid biosynthesis, leading to a decrease in plasma triglycerides. These data underscore the close relationship between bile acid biosynthesis and triglyceride levels.
Key Words: bile acid biosynthesis ? cholesterol 7-hydroxylase ? mouse ? SREBP-1 ? triglycerides
Introduction
The liver plays a central role in the regulation and maintenance of the whole-body sterol balance. The conversion of cholesterol into bile acids and their subsequent fecal excretion is quantitatively the most important way for elimination of cholesterol from the body. In the intestine, bile acids are important as physiological detergents that facilitate the absorption of lipid-soluble vitamins and dietary fat. The classical or neutral route in bile acid biosynthesis is initiated by 7-hydroxylation of cholesterol catalyzed by the major rate-limiting enzyme cholesterol 7-hydroxylase (cyp7a1), which is located in the smooth endoplasmic reticulum.1,2 The rate of bile acid biosynthesis is under control of different molecular mechanisms in which nuclear receptors are involved. In most species, except for humans, the liver X receptor (LXR) is involved in the feed-forward regulation of cyp7a1 by cholesterol, whereas the feedback regulation by bile acids is mediated via the farnesoid X-receptor (FXR).3
Several studies have shown coordinate regulation between cholesterol 7-hydroxylase and the assembly and secretion of apolipoprotein B-containing lipoproteins in humans4,5 and in animal models.6,7 Patients treated with bile acid sequestrants, in which cyp7a1 expression is increased, have hypertriglyceridemia as a consequence of an induction in hepatic very-low-density lipoprotein (VLDL) secretion.8–10 In line with these studies, mice overexpressing cyp7a1 have increased VLDL production.6 These studies suggest a link between degradation of cholesterol into bile acids and hepatic VLDL production.
To further study the relationship between bile acid biosynthesis and triglyceride metabolism, we cross-bred mice lacking cyp7a111 on a hyperlipidemic APOE*3-Leiden background, thereby increasing the sensitivity of the effect of removal of cyp7a1 on lipoprotein and lipid metabolism. Because of the concomitant expression of APOE*3-Leiden and APOC1, these mice have a defective clearance of apolipoprotein B-containing lipoproteins and therefore show a more human-like lipoprotein profile.12
Our data show that female cyp7a1-/-.APOE*3-Leiden mice have a decreased VLDL production as a consequence of a strongly reduced bile acid biosynthesis, leading to reduced plasma triglyceride levels. These data reveal a major effect of physiological cyp7a1 expression on VLDL production and further underscore the link between bile acid biosynthesis and triglyceride levels.
Methods
Animals
Cyp7a1-/- mice11 were obtained from Jackson Laboratory (Bar Harbor, Me) and cross-bred on an APOE*3-Leiden background12 (line 2, >99% C57Bl/6). Specifically, male cyp7a1+/-.APOE*3-Leiden mice were crossed with female cyp7a1+/- mice to generate cyp7a1-/-.APOE*3-Leiden mice and APOE*3-Leiden littermate controls. Pregnant females were administered additional vitamins throughout the nursing period. All experiments were performed with 16- to 22-week-old female mice, which were maintained on a reversed 12-hour dark and 12-hour light cycle and allowed free access to food and water. Body weight of the mice and the consumption of diet and water were recorded weekly and did not differ between cyp7a1-/-.APOE*3-Leiden and APOE*3-Leiden mice during the whole experimental period. In some experiments (indicated in text or legends), mice were fed a sucrose-rich diet (Hope Farms, Woerden, The Netherlands)13 for 3 weeks. Institutional guidelines for animal care were observed in all experiments.
Analysis of Blood Parameters
After overnight fasting, blood samples were collected into paraoxonase-coated capillaries14 after tail incision. Plasma total cholesterol, triglycerides (corrected for plasma levels of free glycerol), and plasma ?-hydroxybutyrate (as a measure of in vivo ?-oxidation) were measured enzymatically (CHOD-PAP method, Roche, 236691, GPO-trinder, Sigma, 337-B, and Sigma Diagnostics, 310-A, respectively).
Hepatic RNA Isolation and Measurement of mRNA Levels by Real-Time Polymerase Chain Reaction
Total RNA was isolated from liver tissue as described previously.15 RNA was converted to single-stranded cDNA by a reverse-transcription procedure (Promega) according to the manufacturer’s protocol using random primers. cDNA levels were measured by real-time polymerase chain reaction (PCR) using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif), according to the manufacturer’s instructions. PCR mastermix from Eurogentec was used. Primers and probes were obtained from Biosource (Nivelles, Belgium). The probes were labeled with 3'-BHQ1 and 5'-FAM or 5'-TET. The mRNA levels were normalized to mRNA levels of different housekeeping genes (ie, cyclophilin, HPRT, and GAPDH). Primers and probes used for this study are summarized in Table 1. The level of mRNA expression for each gene of interest was calculated using the Ct values. Ct values are defined as the number of PCR cycles at which the fluorescent signal generated during the PCR reaches a fixed threshold. For each sample, the Ct for the target gene and for the housekeeping gene were determined to calculate Ct (Ct,target gene - Ct,housekeeping gene). Subsequently, Ct was determined by subtracting the Ct of the test group from the Ct of the control group, and the relative gene expression levels were calculated from 2-Ct, according to the manufacturer’s instructions (Applied Biosystems). In each case, we checked that the Ct method was valid by showing that Ct is constant over a range of cDNA solutions. For some signals (acyl-CoA synthase , steroyl-CoA desaturase , peroxisomal bifunctional enzyme , and thiolase ), we performed Northern blot analysis as described.15 Liver lipids were measured as described.15
TABLE 1. Primers and Probes for Real-Time PCR Analysis
VLDL–Triglyceride and Apolipoprotein B Production
After overnight fasting to prevent chylomicron production, mice were injected via the tail vein with 0.1 mL phosphate buffered saline (PBS) containing 100 μCi Tran35S-label (ICN), a mixture of 35S-labeled methionine and cysteine. After 30 minutes, they were treated with Triton WR-1339 (500 mg/kg intravenous), which virtually completely inhibits VLDL clearance by blocking LPL-mediated lipolysis. The accumulation of VLDL–triglyceride in plasma and the incorporation of radiolabeled amino acids into newly synthesized apolipoprotein B over 90 minutes were measured essentially as described.16
Fecal Excretion of Bile Acids and Neutral Sterols
APOE*3-Leiden and cyp7a1-/-.APOE*3-Leiden mice were housed at 3 mice per cage. Feces produced during 3 subsequent periods were separated from the wood shavings by sieving. Aliquots of lyophilized feces were used for determination of neutral and acidic sterol content by gas-liquid-chromatography procedures described.15
Lipolysis
Postheparin plasma from fasted mice (overnight) was collected from the tail vein at 20 minutes after intraperitoneal injection of heparin (1.0 U/g body weight). Postheparin plasma triacylglycerol hydrolase activity was determined in the presence or absence of 1 mol/L NaCl to estimate the lipoprotein lipase (LPL) activity, which was calculated as the portion of total triacylglycerol hydrolase activity inhibited by 1 mol/L NaCl.15
Western Blotting
Western blot analysis was performed as previously described.17 Liver homogenates (50 μg protein) were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and proteins were blotted onto nitrocellulose as described.17 Blots were developed with an anti-sterol regulatory element binding protein-1 (SREBP-1) primary antibody and horseradish peroxidase-conjugated secondary immunoglobulin. All antibodies were diluted in 20 mmol/L Tris (pH 7.4), 55 mmol/L NaCl, 0.1% (v/v) Tween-20, and 5% (wt/vol) bovine serum albumin and were used at a final concentration of 0.2 μg/mL. The Super Signal West Dura Extended Duration Substrate (Pierce, St Augustin, Germany) and the luminescent image workstation (Roche Diagnostics) were used for SREBP-1 visualization.
Diacylglycerol Acyltransferase Activity Assay
Hepatic diacylglycerol acyltransferase (DGAT) activity was determined by the esterification of oleate into triolein. The microsomal fraction of mouse liver (5 μg of protein diluted in 50 μL of 0.25 mol/L sucrose, 50 mmol/L Tris-HCl ) was added to 200 μmol/L 1,2-diacyl-sn-glycerol (Sigma) in acetone (final vehicle concentration 5% ), 62.5 μg BSA, and 25 μmol/L oleoyl-CoA (9 μCi/μmol) (NEN Life Science Products) in 100 mmol/L Tris-HCl, pH 7.5 (total volume 200 μL). After incubation for 5 minutes in a shaking water bath at 37°C, the reaction was terminated by adding 4 mL of chloroform/methanol (2:1 vol/vol); 20 μL of triolein (3000 dpm) (Amersham) in toluene was added to monitor the extraction efficiency. After phase separation by addition of 750 μL of dH2O, lipids were extracted, dried under nitrogen, dissolved in chloroform, and spotted onto a channeled 20x20-cm Silica gel TLC plate (LK5D 150 A; Whatman). The lipids were separated by running the TLC plate in chloroform/methanol (98:2 vol/vol, halfway), followed by chloroform/hexane (35:65 vol/vol). The radioactive triolein bands were visualized by autoradiography and scraped off for scintillation counting in 4 mL of Ultima Gold (Packard).
Vitamin Analysis
Liver samples were saponified during 30 minutes under reflux conditions using ethanolic KOH containing glycerol, Na2S, and 2% sodium ascorbate. After cooling to room temperature, vitamins were extracted by isopropylether. The extract was washed once using 5% KOH, followed by 2 washes with water. After dissolving the vitamins in isopropylether, the ether was dried under a gentle stream of nitrogen and the residue dissolved in heptane. Subsequently, the samples were analyzed for tocopherols (ie, vitamin E) and retinols (ie, vitamin A) using straight-phase HPLC with fluorescence detection.
Statistics
Data were analyzed statistically using Student unpaired t test, with the level of significance selected to be P<0.05. Values are expressed as means±SD.
Results
Effect of cyp7a1 Deficiency on Fecal Sterols and Plasma Lipid Levels
To evaluate the effect of cyp7a1 deficiency on regular and elevated plasma lipid levels, female APOE*3-Leiden and cyp7a1-/-.APOE*3-Leiden mice were fed a normal chow diet and a sucrose-rich diet, respectively. On chow diet, 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 (-73%) (Figure 1A). As a consequence, the fecal neutral sterol output was 2-fold increased in the cyp7a1-/-.APOE*3-Leiden mice (Figure 1B). The basal levels of fecal bile acid and neutral sterol output on sucrose-rich diet were significantly lower compared with chow diet (-37% and -57%, respectively). However, the relative effect on fecal bile acid and neutral sterol output was similar (Figure 1).
Figure 1. Cyp7a1 deficiency decreases fecal bile acid output concomitantly with increased fecal neutral sterol output in female APOE*3-Leiden mice on chow and sucrose-rich diet. Fecal bile acid (A) and neutral sterol (B) output in APOE*3-Leiden (E3L) and cyp7a-/-.APOE*3-Leiden mice on chow (white bars) or a semi-synthetic sucrose-rich diet (black bars) was determined as described in the Methods section. Data are means±SD of feces samples obtained from 2 groups of 3 mice per genotype during 3 time intervals. Significant differences between APOE*3-Leiden and cyp7a-/-.APOE*3-Leiden mice are indicated by an asterisk. *P<0.005; **P<0.001.
The differences in bile acid biosynthesis did not result in an effect on plasma cholesterol levels on an APOE*3-Leiden background as well as on a wild-type background on chow diet (Table 2). However, triglyceride levels were 41% and 29% decreased (mainly confined to the VLDL fraction) in the cyp7a1-/-.APOE*3-Leiden and cyp7a1-/- mice, respectively, as compared with their control littermates. On the sucrose-rich diet, basal cholesterol levels in the cyp7a1-/-.APOE*3-Leiden mice increased from 2.6±0.8 mmol/L to 4.4±1.1 mmol/L (P<0.05) and from 1.6±0.3 mmol/L to 2.6±0.6 mmol/L (P<0.05) in the cyp7a1-/- mice, whereas triglyceride levels did not change. The plasma lipid-inducing effects of the sucrose-rich diet in APOE*3-Leiden mice is lower as compared with our previous observations,12 which is probably related to continued back-crossing of the mice from the original CBA/J background to the C57Bl/6J background. On this diet, cyp7a1 deficiency on an APOE*3-Leiden and wild-type background had a similar decreasing effect on plasma triglyceride levels (38% and 38%, respectively) as on chow diet and had no effect on cholesterol levels (Table 2).
TABLE 2. Plasma Lipid Levels in Female Wild-Type, cyp7a1-/-, APOE*3-Leiden (E3L), and cyp7a1-/-.APOE*3-Leiden Mice on Chow and Sucrose-Rich Diet
Effect of cyp7a1 Deficiency on Plasma Lipolytic Activity, Ketone Bodies and VLDL Production
The decreasing effect of cyp7a1 deficiency on triglyceride levels could be caused by altered LPL activity, ?-oxidation of fatty acids, or VLDL production. To further investigate the mechanism behind the reduced plasma triglyceride levels in cyp7a1 deficiency, we measured postheparin LPL activity as a measure of peripheral lipolysis. It appeared that the triacylglycerol hydrolase activity of LPL did not differ between female APOE*3-Leiden and cyp7a1-/-.APOE*3-Leiden mice (fatty acid production rate 4.0±2.0 μmol/mL per hour versus 2.4±1.9 μmol/mL per hour, respectively; n=8 per group; P<0.05). Rather, a trend was observed toward decreased LPL levels in cyp7a1-/- mice. In addition, no changes were observed in plasma levels of ketone bodies (?-hydroxybutyrate) as markers of oxidation of fatty acids (1.79±0.53 mmol/L versus 1.79±0.33 mmol/L, respectively; n=8 per group), although the expression of various genes involved in hepatic ?-oxidation, eg, acyl-CoA oxidase (acox1), acyl-CoA synthase (acas1), peroxisomal bifunctional enzyme (ehhadh), and thiolase (acca1), were increased (Table 3). In contrast, hepatic VLDL–apolipoprotein B and VLDL–triglyceride production rates were reduced in the cyp7a1-/-.APOE*3-Leiden as compared with the APOE*3-Leiden mice (34% and 35%, respectively) (P<0.05) (Figure 2). The lipid composition of the nascent VLDL particles was not changed (see insert in Figure 2B), indicating that cyp7a1 deficiency results in a decreased hepatic VLDL particle production rate. Although the microsomal triglyceride transfer protein (mttp), apolipoprotein B (apob), and apolipoprotein B mRNA-editing complex-1 (apobec-1) play a critical role in the assembly and secretion of VLDL in the liver, the hepatic expression levels of these genes were not significantly different between cyp7a1-/-.APOE*3-Leiden and APOE*3-Leiden mice (Table 3).
TABLE 3. Hepatic mRNA Levels in Female cyp7a1-/-.APOE*3-Leiden and APOE*3-Leiden Mice
Figure 2. Decreased VLDL–triglyceride and VLDL–ApoB production in female cyp7a1-/-.APOE*3-Leiden on sucrose-rich diet. Tran35S-label (100 μCi) was injected into APOE*3-Leiden (E3L) (black circles) and cyp7a-/-.APOE*3-Leiden mice (white circles). After 30 minutes, Triton WR1339 (500 mg/kg body weight) was injected and plasma samples were taken at various time points. Plasma triglyceride levels were determined at indicated time points and corrected for the triglyceride level at the time of injection (A). Nascent VLDL was isolated by ultracentrifugation from pooled serum collected 120 minutes after Triton injection. 35S-labeled apolipoprotein B was precipitated from the VLDL fraction and radioactivity was subsequently measured (B). The values shown are means±SD of 8 mice (A), or means±SD of 4 VLDL samples of each group (B). Significant differences in triglyceride and apolipoprotein B production rates are indicated by an asterisk (*P<0.05). The insert in (B) represents the nascent VLDL lipid composition (values presented as % of total lipid). FC indicates free cholesterol; CE, cholesteryl esters; TG, triglycerides; PL, phospholipids. No significant differences were observed between cyp7a1-/-.E3L and E3L mice.
Effect of cyp7a1 Deficiency on Hepatic Lipogenesis, Lipid Content, and Vitamin Levels
To investigate the effect of the lack of cyp7a1 on hepatic triglyceride synthesis, mRNA and protein levels of the transcription factor SREBP-1 (srebf1), which regulates lipogenic genes, were measured. Also, the mRNA expression levels of enzymes involved in fatty acid synthesis, ie, fatty acid synthase (fasn), acyl-CoA carboxylase (acac) and steroyl-CoA desaturase (scd1) were measured. The srebf1 mRNA level was 31% lower in the cyp7a1-/-.APOE*3-Leiden mice, which was accompanied by a significant (P<0.05; n=4 per group) reduction in the amount of mature (100%±27% versus 35%±28%) and precursor (100%±47% versus 62%±54%) form of SREBP-1 and a lower expression of the lipogenesis genes acac and fasn (Table 3).
As a consequence of a decreased bile acid production and a reduction in VLDL secretion, hepatic free cholesterol, esterified cholesterol, and triglyceride levels were significantly increased (1.3-, 2.8-, and 2.5-fold, respectively) in the cyp7a1-/-.APOE*3-Leiden mice, whereas hepatic phospholipid levels were significantly decreased by -28% (Table 4). The increase in hepatic cholesterol levels was related to a 49% reduction in the expression of the low-density lipoprotein receptor (ldlr) (Table 3).
TABLE 4. Hepatic Lipid, Vitamin A, and Vitamin E Levels in Female cyp7a1-/-.APOE*3-Leiden and APOE*3-Leiden Mice
To investigate whether the changes in the hepatic content in phospholipids and triglycerides were caused by a change in the enzyme activity of DGAT, we performed a DGAT activity assay with microsomes isolated from the livers of the APOE*3-Leiden (n=6) and cyp7a1-/-.APOE*3-Leiden (n=7) mice fed the sucrose-rich diet. We found no significant difference between these groups with respect to the activity of this enzyme (10.0±1.7 nmol/min per mg protein versus 9.0±1.4 nmol/min per mg protein, respectively).
Anti-oxidative vitamins play a role in determining the hepatic content of reactive oxygen species, which can be involved in the degradation of apolipoprotein B. Therefore, we measured the amount of vitamin A and E in the liver. The data of Table 4 show that cyp7a1-/-.APOE*3-Leiden mice contain greatly reduced levels of the fat-soluble vitamins A and E in the liver as compared with their APOE*3-Leiden littermates.
Discussion
This study showed that the absence of cyp7a1 in female APOE*3-Leiden mice results in a decrease in plasma triglyceride levels as a consequence of a lower VLDL production.
Cyp7a1 knockout mice on an APOE*3-Leiden background as well as on a wild-type background of both sexes did not have different plasma cholesterol levels as compared with their control littermates. Although this is in agreement with a previous report,11 a hypercholesterolemic cyp7a1-/- colony has recently also been described.18 Similarly, conflicting effects on plasma triglyceride levels have been reported11,18 in mice lacking cyp7a1. No significant difference in plasma triglyceride levels was found in the cyp7a1-/- mice with normal cholesterol levels, whereas the females of the hypercholesterolemic cyp7a1-/- colony exhibited lower plasma triglyceride levels.18 The differences in phenotype may be because of sex, genetic background, diet, or other environmental factors. In our hands, cyp7a1 deficiency on a human-like lipoprotein profile and on a wild-type background consistently showed decreased plasma triglyceride levels in only the female mice similarly as previously described.18 The absence of an effect in male mice can possibly be explained by the lower level in VLDL–triglyceride production in males as compared with females as a result of the difference in hormone level.19
We have shown that the lower level of plasma triglycerides in the female cyp7a1-/-.APOE*3-Leiden mice is caused by inhibition of VLDL particle production. It has been known for many years that there is a link between bile acid metabolism, especially related to cyp7a1 expression, and hepatic VLDL production. Treatment with the bile acid sequestrant cholestyramine, which induces bile acid biosynthesis, leads to an increase in VLDL triglyceride and VLDL cholesterol production. This increase is seen in healthy individuals4 and in patients with various types of hyperlipidemia,9,20 and predominantly in patients with hypertriglyceridemia.8,9 In animals, this relation between bile acid biosynthesis and plasma triglyceride levels has also been detected. Disruption of the enzyme sterol 27-hydroxylase in mice leads to a 5-fold increase in cyp7a1 activity and a concomitant 2-fold increase in hepatic and plasma triglyceride levels.7 Furthermore, cyp7a1 transgenic mice have an increased production and secretion of apolipoprotein B-containing lipoproteins.6
We did not observe an effect of cyp7a1 deficiency on plasma cholesterol levels. Apparently, the reduced influx of cholesterol into plasma resulting from a reduced VLDL production and reduced intestinal neutral sterol cholesterol absorption is counterbalanced by a decreased hepatic cholesterol clearance. Indeed, the ldlr expression was reduced, probably as a result of downregulation, as induced by increased hepatic cholesterol levels.
The hepatic synthesis of VLDL particles requires many (in)dependent processes like: (1) the production of constituting lipids; (2) the synthesis and secretion of apolipoprotein B and apolipoprotein E; (3) the transfer of lipid by MTP to apolipoprotein B; and (4) association of the resulting precursor particle with bulk lipid to form mature VLDL.21 We did not find an effect of cyp7a1 deficiency on mttp mRNA levels. Because MTP protein and activity are tightly regulated by mttp mRNA levels,22 hepatic MTP activity is thus likely to be unaltered. In contrast, cyp7a1-overexpressing animals were reported to have increased mttp expression,6 which, however, may result from an unphysiologically high cyp7a1 expression.
Studies with hepatoma cells overexpressing cyp7a1 showed that as a result of increased mature SREBP-1, there was a coordinate induction of de novo lipogenesis and assembly and secretion of VLDL.23 We found a decrease in srebf1, mRNA, and SREBP-1 protein leading to suppression of genes involved in de novo lipogenesis, ie, fas and acc1, which may contribute to reduced VLDL secretion. Despite the decreased de novo lipogenesis, hepatic triglyceride levels were increased. These data indicate that a decreased de novo lipogenesis does not automatically lead to smaller triglyceride content in the liver, which is in line with the absence of an effect on hepatic triglyceride levels in srebf1-deficient mice.24 Alternatively, the decrease in lipogenesis can also be the consequence of the high level of triglycerides in the liver. In contrast, cyp7a1-/- mice on a wild-type background have no difference in hepatic triglyceride content and have decreased fatty acid synthesis.18 Differences between triglyceride and fatty acid metabolism in the cyp7a1-/- mice and cyp7a1-/-.APOE*3-Leiden mice may be explained by the presence of the APOE*3-Leiden and APOC1 gene.
Because the increased triglyceride content cannot be explained by the effect on de novo lipogenesis, we measured DGAT. DGAT is another important enzyme in determining the triglyceride content in the liver, producing triglycerides from diacylglycerides and acyl-CoAs. Because we found no alteration in the enzyme activity of DGAT, we can exclude the possibility that this enzyme is involved in the accumulation of triglycerides in the liver or the decrease in VLDL production. The increase in hepatic triglyceride content can also be a consequence of the extreme blockade in the output of fatty acids in the form of phospholipids via the bile, together with a decrease in VLDL production. Biliary bile acid excretion is an important determinant of biliary phospholipids and cholesterol excretion.25
Our data show that affecting bile acid metabolism has a major impact on hepatic fatty acid metabolism, not only on genes involved in de novo lipogenesis but also on genes involved in hepatic oxidation of fatty acids. The general effects of cyp7a1 deficiency in this animal model, except from the lack of effect on LPL, closely resemble the effects of treating mice with PPAR-agonists (ie, fibrates). It has been shown that fibrates decrease triglyceride levels, increase expression of genes involved in ?-oxidation, and reduce VLDL production and the expression of genes involved in fatty acid synthesis.26 We also reported that after treatment of mice with ciprofibrate, bile acid synthesis is suppressed via PPAR-mediated downregulation of cyp7a1 and sterol 27-hydroxylase.27 Therefore, we cannot exclude that PPAR plays a role in the effects observed in the cyp7a1-deficient mouse. In addition, bile acids have been shown to interfere with transactivation by PPAR, at least in part by impairing the recruitment of transcriptional co-activators.28 The decreased bile acid pool in the cyp7a1 knockout mice may explain the changes in PPAR-regulated genes involved in ?-oxidation eg, acox1, acac1, and ehhadh. The increase in acox1 has also been found by others.18 Despite the increase in expression levels of genes involved in ?-oxidation, which may have contributed to the differences in fatty acid and triglyceride metabolism in the cyp7a1-/-.APOE*3-Leiden, the concentration of plasma ketone bodies remained unaltered.
We showed that cyp7a1 deficiency affected the VLDL secretion rate. The nascent VLDL composition was not changed, although a trend was observed toward a higher cholesteryl ester/triglyceride ratio within the particle core. The constituting core lipids in VLDL (ie, cholesteryl esters and triglycerides) were available in sufficient amounts in the liver of the female cyp7a1-/-.APOE*3-Leiden mice, but it can be possible that deposition of these lipids in the liver is not present in the secretory route where proteins are located, which are involved in the packaging of lipids into VLDL particles. Moreover, we found that the amount of hepatic phospholipids was decreased. It has been reported that the hepatocellular phospholipid availability is a determinant for the regulation of VLDL assembly.29–32 Furthermore, intracellular proteolysis of apolipoprotein B, resulting in a diminished assembly of VLDL particles, may be increased in female cyp7a1-/-.APOE*3-Leiden mice. We and others11 showed that cyp7a1 deficiency in mice results in a low level of anti-oxidative vitamins caused by a reduction in the intestinal uptake.11 This consequently leads to an excess in intracellular reactive oxygen species. Recently, it has been shown that a novel pathway for destruction of newly synthesized apolipoprotein B is induced by intracellular reactive oxygen species and PI3-kinases.33,34 This may be a possible explanation for the difference in VLDL production in the knockout mice as compared with their littermates.
In conclusion, our data show that female cyp7a1-/-.APOE*3-Leiden mice have a decreased VLDL production as a consequence of a strongly reduced bile acid biosynthesis, leading to a decrease in plasma triglyceride levels. These data indicate a major impact of physiological cyp7a1 expression on VLDL production and further underscore the link between bile acid biosynthesis and triglyceride levels.
Acknowledgments
This work was supported by the Netherlands Heart Foundation (NHS grant 97.116), the Netherlands Organization for Scientific Research (NWO grant 980-10-024 and VIDI 917.36.351), the Marabou Foundation, Sweden, and by the Leiden University Medical Center (Gisela Thier fellowship to P. C. N. Rensen). We thank Elly de Wit for excellent technical assistance.
References
Russell DW, Setchell KD. Bile acid biosynthesis. Biochemistry. 1992; 31: 4737–4749.
Princen HMG, Post SM, Twisk J. Regulation of bile acid biosynthesis. Curr Pharmaceut Design. 1997; 3: 59–84.
Chiang JY. Bile acid regulation of gene expression: roles of nuclear hormone receptors. Endocr Rev. 2002; 23: 443–463.
Carrella M, Ericsson S, Del Piano C, Angelin B, Einarsson K. Effect of cholestyramine treatment on biliary lipid secretion rates in normolipidaemic men. J Intern Med. 1991; 229: 241–246.
Lipid Research Clinics Program. JAMA. 1984; 252: 2545–2548.
Miyake JH, Doung XD, Strauss W, Moore GL, Castellani LW, Curtiss LK, Taylor JM, Davis RA. Increased production of apolipoprotein B-containing lipoproteins in the absence of hyperlipidemia in transgenic mice expressing cholesterol 7alpha-hydroxylase. J Biol Chem. 2001; 276: 23304–23311.
Repa JJ, Lund EG, Horton JD, Leitersdorf E, Russell DW, Dietschy JM, Turley SD. Disruption of the sterol 27-hydroxylase gene in mice results in hepatomegaly and hypertriglyceridemia. Reversal by cholic acid feeding. J Biol Chem. 2000; 275: 39685–39692.
Beil U, Crouse JR, Einarsson K, Grundy SM. Effects of interruption of the enterohepatic circulation of bile acids on the transport of very low density-lipoprotein triglycerides. Metabolism. 1982; 31: 438–444.
Angelin B, Einarsson K, Hellstrom K, Leijd B. Bile acid kinetics in relation to endogenous triglyceride metabolism in various types of hyperlipoproteinemia. J Lipid Res. 1978; 19: 1004–1016.
Einarsson K, Ericsson S, Ewerth S, Reihner E, Rudling M, Stahlberg D, Angelin B. Bile acid sequestrants: mechanisms of action on bile acid and cholesterol metabolism. Eur J Clin Pharmacol. 1991; 40: S53–S58.
Schwarz M, Lund EG, Setchell KD, Kayden HJ, Zerwekh JE, Bjorkhem I, Herz J, Russell DW. Disruption of cholesterol 7alpha-hydroxylase gene in mice. II. Bile acid deficiency is overcome by induction of oxysterol 7alpha-hydroxylase. J Biol Chem. 1996; 271: 18024–18031.
van den Maagdenberg AM, Hofker MH, Krimpenfort PJ, de Bruijn, I, van Vlijmen B, van Der Boom H, Havekes LM, Frants RR. Transgenic mice carrying the apolipoprotein E3-Leiden gene exhibit hyperlipoproteinemia. J Biol Chem. 1993; 268: 10540–10545.
Post SM, Zoeteweij JP, Bos MH, de Wit EC, Havinga R, Kuipers F, Princen HM. Acyl-coenzyme A: cholesterol acyltransferase inhibitor, avasimibe, stimulates bile acid synthesis and cholesterol 7alpha-hydroxylase in cultured rat hepatocytes and in vivo in the rat. Hepatology. 1999; 30: 491–500.
Zambon A, Hashimoto SI, Brunzell JD. Analysis of techniques to obtain plasma for measurement of levels of free fatty acids. J Lipid Res. 1993; 34: 1021–1028.
Post SM, de Crom R, van Haperen R, van Tol A, Princen HM. Increased fecal bile acid excretion in transgenic mice with elevated expression of human phospholipid transfer protein. Arterioscler Thromb Vasc Biol. 2003; 23: 892–897.
Post SM, de Roos B, Vermeulen M, Afman L, Jong MC, Dahlmans VE, Havekes LM, Stellaard F, Katan MB, Princen HM. Cafestol increases serum cholesterol levels in apolipoprotein E*3-Leiden transgenic mice by suppression of bile acid synthesis. Arterioscler Thromb Vasc Biol. 2000; 20: 1551–1556.
Kleemann R, Gervois PP, Verschuren L, Staels B, Princen HM, Kooistra T. Fibrates down-regulate IL-1-stimulated C-reactive protein gene expression in hepatocytes by reducing nuclear p50-NFkappa B-C/EBP-beta complex formation. Blood. 2003; 101: 545–551.
Erickson SK, Lear SR, Deane S, Dubrac S, Huling SL, Nguyen L, Bollineni JS, Shefer S, Hyogo H, Cohen DE, Shneider B, Sehayek E, Ananthanarayanan M, Balasubramaniyan N, Suchy FJ, Batta AK, Salen G. Hypercholesterolemia and changes in lipid and bile acid metabolism in male and female cyp7A1-deficient mice. J Lipid Res. 2003; 44: 1001–1009.
van Vlijmen BJ, ’t Hof HB, Mol MJ, van Der Boom H, van der ZA, Frants RR, Hofker MH, Havekes LM. Modulation of very low density lipoprotein production and clearance contributes to age- and gender- dependent hyperlipoproteinemia in apolipoprotein E3-Leiden transgenic mice. J Clin Invest. 1996; 97: 1184–1192.
Einarsson K, Hellstrom K, Kallner M. Bile acid kinetics in relation to sex, serum lipids, body weights, and gallbladder disease in patients with various types of hyperlipoproteinemia. J Clin Invest. 1974; 54: 1301–1311.
Shelness GS, Sellers JA. Very-low-density lipoprotein assembly and secretion. Curr Opin Lipidol. 2001; 12: 151–157.
Raabe M, Flynn LM, Zlot CH, Wong JS, Veniant MM, Hamilton RL, Young SG. Knockout of the abetalipoproteinemia gene in mice: reduced lipoprotein secretion in heterozygotes and embryonic lethality in homozygotes. Proc Natl Acad Sci U S A. 1998; 95: 8686–8691.
Wang SL, Du EZ, Martin TD, Davis RA. Coordinate regulation of lipogenesis, the assembly and secretion of apolipoprotein B-containing lipoproteins by sterol response element binding protein 1. J Biol Chem. 1997; 272: 19351–19358.
Liang G, Yang J, Horton JD, Hammer RE, Goldstein JL, Brown MS. Diminished hepatic response to fasting/refeeding and liver X receptor agonists in mice with selective deficiency of sterol regulatory element-binding protein-1c. J Biol Chem. 2002; 277: 9520–9528.
Suchy FJ, Sippel CJ, Ananthanarayanan M. Bile acid transport across the hepatocyte canalicular membrane. FASEB J. 1997; 11: 199–205.
Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, Fruchart JC. Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation. 1998; 98: 2088–2093.
Post SM, Duez H, Gervois PP, Staels B, Kuipers F, Princen HM. Fibrates Suppress Bile Acid Synthesis via Peroxisome Proliferator-Activated Receptor-alpha-Mediated Downregulation of Cholesterol 7alpha-Hydroxylase and Sterol 27-Hydroxylase Expression. Arterioscler Thromb Vasc Biol. 2001; 21: 1840–1845.
Sinal CJ, Yoon M, Gonzalez FJ. Antagonism of the actions of peroxisome proliferator-activated receptor-alpha by bile acids. J Biol Chem. 2001; 276: 47154–47162.
Wiggins D, Gibbons GF. Origin of hepatic very-low-density lipoprotein triacylglycerol: the contribution of cellular phospholipid. Biochem J. 1996; 320: 673–679.
Yao ZM, Vance DE. The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes. J Biol Chem. 1988; 263: 2998–3004.
Verkade HJ, Fast DG, Rusinol AE, Scraba DG, Vance DE. Impaired biosynthesis of phosphatidylcholine causes a decrease in the number of very low density lipoprotein particles in the Golgi but not in the endoplasmic reticulum of rat liver. J Biol Chem. 1993; 268: 24990–24996.
Vermeulen PS, Lingrell S, Yao Z, Vance DE. Phosphatidylcholine biosynthesis is required for secretion of truncated apolipoprotein Bs from McArdle RH7777 cells only when a neutral lipid core is formed. J Lipid Res. 1997; 38: 447–458.
Fisher EA, Pan M, Chen X, Wu X, Wang H, Jamil H, Sparks JD, Williams KJ. The triple threat to nascent apolipoprotein B. Evidence for multiple, distinct degradative pathways. J Biol Chem. 2001; 276: 27855–27863.
Pan M, Cederbaum AI, Williams KJ, Fisher EA. Reactive oxygen species regulate basal and induced degradation of apolipoprotein B in rat hepatic cells. Circulation. 2002; 106 (suppl II): II-2.ABSTRACT.(Sabine M. Post; Martine G)