JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M404027200 on August 25, 2004

J. Biol. Chem., Vol. 279, Issue 45, 47402-47410, November 5, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/45/47402    most recent
M404027200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jacobs, R. L.
Right arrow Articles by Vance, D. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jacobs, R. L.
Right arrow Articles by Vance, D. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Targeted Deletion of Hepatic CTP:phosphocholine Cytidylyltransferase {alpha} in Mice Decreases Plasma High Density and Very Low Density Lipoproteins*

René L. Jacobs{ddagger}§, Cecilia Devlin¶, Ira Tabas¶, and Dennis E. Vance{ddagger}||**

From the {ddagger}Canadian Institutes of Health Research Group on the Molecular and Cell Biology of Lipids and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada and the Departments of Medicine, Anatomy and Cell Biology, and Physiology and Cellular Biophysics, Columbia University, New York, New York 10032

Received for publication, April 12, 2004 , and in revised form, August 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
CTP:phosphocholine cytidylyltransferase (CT) is the key regulatory enzyme in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. Hepatic cells express both an {alpha} and a {beta}2 isoform of CT and can also synthesize phosphatidylcholine via the sequential methylation of phosphatidylethanolamine catalyzed by phosphatidylethanolamine N-methyltransferase. To ascertain the functional importance of CT{alpha}, we created a mouse in which the hepatic CT{alpha} gene was specifically inactivated by the Cre/LoxP procedure. In CT{alpha} knockout mice, hepatic CT activity (due to residual CT{beta}2 activity as well as activity in nonhepatic cells) was 15% of normal, whereas phosphatidylethanolamine N-methyltransferase activity was elevated 2-fold compared with controls. Lipid analyses of the liver indicated that female knockout mice had reduced phosphatidylcholine levels and accumulated triacylglycerols. The plasma phosphatidylcholine concentration was reduced in the CT{alpha} knockout (independent of gender), as were levels of high density lipoproteins (cholesterol and apoAI) and very low density lipoproteins (triacylglycerols and apoB100). Experiments in which mice were injected with Triton WR1339 indicated that apoB secretion was decreased in hepatic-specific CT{alpha} knockout mice compared with controls. These results suggest an important role for hepatic CT{alpha} in regulating both hepatic and systemic lipid and lipoprotein metabolism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphatidylcholine (PC)1 is vital for the structural integrity of mammalian membranes and is the primary phospholipid in bile, lung surfactant, and plasma lipoproteins. In all nucleated mammalian cells, PC is synthesized from choline via the Kennedy (CDP-choline) pathway (1), and the flux through this pathway is regulated by the activity of CTP:phosphocholine cytidylyltransferase (CT) (2–4). In the mouse, two genes encode CT, Pcyt1a and Pcyt1b (5). Hepatic cells have both a CT{alpha} isoform encoded by Pcyt1a and a CT{beta}2 isoform encoded by Pcyt1b. CT{alpha} is believed to be the predominant isoform in the liver (5). Liver cells are unique in that they can also synthesize PC via the sequential methylation of phosphatidylethanolamine catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT) (6). The PEMT pathway accounts for ~30% of hepatic PC biosynthesis, whereas the enzymes of the Kennedy pathway produce the remaining 70% (7–9).

In recent years, several studies have described specific roles for the PEMT and CDP-choline pathways in PC biosynthesis and lipoprotein metabolism. Experiments on Pemt-/- mice suggested that PEMT has been retained during evolution to provide PC when dietary choline is deficient, such as during starvation (10–12). Indeed, when PEMT knockout mice were fed a choline-deficient diet, they developed severe liver pathology and died within 4 days (11), thus highlighting the requirement for PEMT activity when biosynthesis of PC through the CDP-choline pathway is insufficient. The PEMT-deficient mice appeared normal on a standard chow diet, suggesting that the CDP-choline pathway can compensate for the loss of PEMT activity when choline is available. A specific role for PEMT in VLDL secretion has been demonstrated in vivo and in experiments with hepatocytes (13, 14). In male Pemt-/- mice fed a high fat/high cholesterol diet, plasma TG and PC levels were reduced by 50 and 20%, respectively, compared with those in Pemt+/+ mice. In contrast, the plasma content of these lipids was the same in female Pemt-/- and Pemt+/+ mice fed a high fat/high cholesterol diet (14). In hepatocytes from male Pemt-/-, compared with Pemt+/+ mice, apoB100 secretion was reduced by 60%, whereas TG secretion was reduced by 75% (13). This apparent reduction in VLDL secretion may explain why Pemt-/- mice fed the high fat/high cholesterol diet have a 5-fold higher level of hepatic TG than their Pemt+/+ counterparts (13). The above experiments demonstrate that PEMT can be limiting in VLDL secretion and highlight a possible gender difference in the relationship between PC metabolism and lipoprotein homeostasis.

Studies investigating the importance of the CDP-choline pathway in lipoprotein metabolism have primarily consisted of feeding choline-deficient (CD) diets to animals (15–17), or applying CD medium to hepatocytes or other cells in culture (18–20). It has been well established that hepatic and plasma PC concentrations are reduced in rats fed a CD diet. This reduction in dietary choline results in decreased plasma VLDL-TG and VLDL-apoB levels and TG accumulation in the liver (16). The presumption in these experiments was that the effects seen on lipid and lipoprotein metabolism were due to inhibition of PC biosynthesis via the CDP-choline pathway. Studies in hepatocytes initially seemed to agree with this conclusion, since VLDL secretion was impaired from rat hepatocytes incubated in medium deficient in methionine and choline and TG accumulated 6-fold in the cells (18). However, attributing these changes to impaired flux through the CDP-choline pathway was dubious for several reasons. First, supplementation of the medium with either choline or methionine returned cellular TG levels to normal and restored normal TG and apoB secretion (18). Second, the rate of PC synthesis from CDP-choline was not directly measured. However, these observations suggest that active PC biosynthesis from either PEMT or CDP-choline is required for normal VLDL secretion. Furthermore, Cui and Vance (21) observed that the specific radioactivity of 3H-labeled PC in CD hepatocytes was higher, not lower, than in choline-supplemented hepatocytes following incubation with [3H]choline. Moreover, Kulinski et al. (22) have shown that the rate of flux through the CDP-choline pathway is higher, not lower, in CD than in choline-supplemented murine hepatocytes and that VLDL secretion from murine hepatocytes is not impaired by choline deficiency. In light of these experiments, choline deficiency appears not to be an appropriate model in which to investigate the specific role of the CDP-choline pathway in PC biosynthesis or in lipid and lipoprotein metabolism.

To gain further insight for the role of the CDP-choline pathway in hepatic lipoprotein metabolism, we have generated mice in which the CT{alpha} gene was selectively disrupted in the liver by the Cre-Lox system (23, 24). Our hypothesis predicted that CT{alpha} is vital for normal secretion of lipoproteins from liver. Consequently, we predicted that mice deficient in hepatic CT{alpha} would have decreased circulating lipoproteins and fat accumulation in the liver. The results describe an important role for the hepatic CDP-choline pathway, and particularly CT{alpha}, in regulating both hepatic and systemic lipid and lipoprotein metabolism.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Anti-human CT{beta}2 and anti-human CT{alpha} rabbit polyclonal antibodies were generous gifts from Dr. S. Jackowski (St. Jude Children's Research Hospital, Memphis, TN). The sheep anti-human apoB antibody was purchased from Roche Applied Science, and the rabbit anti-human apoA1 and goat anti-human apoE antibodies were from Biodesigns (Kennebunk, ME). Both the donkey anti-sheep and the goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase were purchased from Pierce. The polyclonal antibody directed against the C-terminal dodecapeptide of rat PEMT2 was raised in rabbits in our laboratory (25). S-[methyl-3H]adenosylmethionine, [methyl-3H]choline, and [35S]Promix (methionine/cysteine) were purchased from Amersham Biosciences. Triton WR1339 was purchased from Sigma. All other chemicals and reagents were from standard commercial sources.

Generation and Identification of CT{alpha}flox Mice and CT{alpha}flox/AlbuminCre Mice—Homozgous CT{alpha}flox mice, generated previously (23), were identified by PCR of tail DNA (isolated by the DNesay Tissue Kit; Qiagen) using the following primers: CT382 (5'-TCTTTGCTTGCATCA-3') and CT3UL (5'-GAAGTAGGCACTGAACTTAGC-3'). A homozygous CT{alpha}flox mouse was crossed with a mouse expressing the Cre recombinase gene driven by the hepatic-specific albumin promoter (albuminCre) kindly provided by Dr. Mark A. Magnuson (Vanderbilt University). The resulting pups, which were heterozygous for both CT{alpha}flox and albuminCre, were then bred with homozygous CT{alpha}flox mice. Mice homozygous for CT{alpha}flox and heterozygous for albuminCre (i.e. mice having CT{alpha}-deficient livers) were identified (Fig. 1A) by PCR screening of tail DNA from the pups.



View larger version (40K):
[in this window]
[in a new window]
  		FIG. 1.
Cre-mediated disruption of the CT{alpha}flox gene in liver. A, PCR assay of tail DNA for Cre (left panel) and CT{alpha} (right panel) from homozygous CT{alpha}flox (lane 1), homozogous CT{alpha}flox + Cre (lane 2), and wild type (lane 3) mice. A reaction with no DNA template (negative control) was performed with all assays (lane 4). B, immunoblots of liver homogenates obtained from control and knockout mice; a CT{alpha}-specific antibody was used at 1:1500 dilution. C, homogenates from livers and hearts were assayed for CT activity in the presence of phosphatidylcholine/oleate vesicles. Values are expressed as means ± S.E. for 3–8 mice. The asterisks signify differences versus control, p < 0.05.

 
Mice—All procedures were approved by University of Alberta's Animal Welfare Committee and were in accordance with guidelines of the Canadian Council on Animal Care. Mice, housed 4–5 per cage, were exposed to a 12-h light/dark cycle beginning with light at 8:00 a.m. Adult male and female mice, 12–24 weeks old, were fed ad libitum a chow diet from LabDiet (PICO laboratory Rodent Diet 20) and had free access to water. For all experiments, mice were fasted overnight (16 h) prior to sacrifice.

Enzymatic Assays—Tissues were homogenized in a glass/Teflon homogenizer in 2 ml of buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride) followed by sonication for 30 s. Protein concentration was determined using the Coomassie Plus protein protocol (Bio-Rad), with bovine serum albumin as a standard. Total CT activity was measured in homogenates (50 µg of protein) from livers and hearts by monitoring the conversion of [3H]phosphocholine into CDP-choline (26). For measurement of PEMT activity, 45 µg of protein of liver homogenates were incubated with phosphatidylmonomethylethanolamine and S-adenosyl[methyl-3H]methionine, and the incorporation of radiolabel into PC was measured as described previously (27).

Immunoblotting of Hepatic CT{alpha}, PEMT, and CT{beta}2—Liver homogenates (20–50 µg of protein) were boiled in buffer containing 1% SDS, and proteins were separated on a 10% SDS-polyacrylamide gel. Proteins were transferred to nylon membranes and probed with anti-CT{alpha} (dilution 1:1500), anti-PEMT2 (1:5000), or anti-CT{beta}2 (1:1000) antibody. Immunoreactive bands were visualized by enhanced chemiluminescence according to the manufacturer's instructions (Amersham Biosciences). Quantification of the immunoreactive bands was performed using Image Gauge version 3.0 software by Fuji.

Analysis of ApoAI, ApoE, and ApoB48/B100 Levels in Plasma— Blood was collected from each mouse via cardiac puncture in the presence of trace amounts of 250 mM EDTA, and plasma was isolated by centrifugation. Plasma (2–4 µl) was resolved on a 10% (for apoAI and apoE) or 5% (for apoB48/100) SDS-polyacrylamide gel. Proteins were transferred to nylon membranes and probed with anti-apoAI (dilution 1:10,000), anti-apoE (dilution 1:2500), or anti-apoB (dilution 1:5000) antibody.

Determination of the Mass of Cholesterol, Cholesteryl Ester, and TG in Liver and Plasma—The amount of cholesterol, cholesteryl ester, and TG was determined in liver homogenates (0.5 mg of protein) or 50 µl of plasma. After digestion (2 h, 30 °C) of the phospholipids with phospholipase C, tridecanoin (20 ng) was added as an internal standard, and lipids were extracted. The mass of TG, cholesterol, and cholesteryl ester was determined by gas-liquid chromatography (28).

Plasma from individual animals was separated into lipoprotein fractions using high performance liquid chromatography with an Amersham Biosciences Superose 6 column attached to a Beckman Systems Gold or Nouveau Gold apparatus. In-line assays for total cholesterol (Sigma Infinity cholesterol reagent) and TG (Sigma Triglyceride GPO Trinder kit) were performed as previously described (14).

Determination of PE and PC Mass in Livers and Plasma—Phosphatidyldimethylethanolamine (0.01 mg) was added as an internal standard to an aliquot (0.5 mg of protein) of liver homogenate or 50 µl of plasma. Lipids were extracted from the samples by the method of Folch et al. (29), and phospholipids were separated and quantified by the HPLC method of Bergo et al. with minor modifications (30).

Determination of the Mass of Cholesterol, PC, and Bile Acids in Bile—Bile was collected directly from the gallbladders of anesthetized mice. Bile acids were separated and quantified by the HPLC method of Torchia et al. (31) using 3{alpha},12{alpha}-dihydroxy-23-nor-5{beta}-cholanic acid as an internal standard. Assay kits were used to determine the mass of biliary cholesterol (Wako) and PC (Sigma).

Analysis of ApoB Secretion in Vivo—Mice were fasted overnight and injected with 100 µl of phosphate-buffered saline containing 15% Triton WR1339 (v/v) and 250 µCi of [35S]Promix as previously described (14). After various times, the animals were sacrificed, blood was collected, and plasma was isolated. To 100 µl of plasma were added 450 µl of phosphate-buffered saline, 100 µl of buffer containing Tris-HCl (0.63 M, pH 7.4), NaCl (0.75 M), EDTA (25 mM), phenylmethylsulfonyl fluoride (5 mM) and Triton X-100 (5%, v/v), and 7.5 µl of anti-human apoB antibody. The mixture was incubated overnight at 4 °C with gentle shaking. Next, 45 mg of protein A-Sepharose was added, and the sample was mixed for 2 h at 4 °C. The apoB-protein A complex was pelleted by centrifugation of the sample for 2 min at 14,000 rpm in a microcentrifuge. The pellet was boiled in the presence of 100 µl of sample buffer. Following centrifugation, samples were electrophoresed on 5% SDS-polyacrylamide gels, after which the gels were soaked in Amplify solution, dried, and exposed to film. Quantification of the bands was performed using Image Gauge version 3.0 software by Fuji.

Histological Studies—Livers were quickly extirpated and fixed in 10% formalin, and sections were stained by a standard protocol with hematoxylin and eosin. Some formalin-fixed samples were frozen in liquid nitrogen before sectioning and were subsequently stained with Oil Red O.

Measurement of Plasma Aminotransferase Activities—Aspartate aminotransferase and alanine aminotransferase activities in plasma were measured using the INFINITY AST and ALT kits from ThermoTrace.

Statistical Analysis—Data are presented as means ± S.E. unless otherwise noted. There were 3–10 samples in each experimental group. Student's unpaired t test was performed to compare means. A p value of <0.05 was interpreted as a significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of the Liver-specific CT{alpha} Knockout Mice— Breeding of a homozygous CT{alpha}flox mouse with a mouse homozygous for CT{alpha}flox and heterozygous for Cre generated two possible genotypes: 1) homozygous CT{alpha}flox mice, which were used as controls in this study, and 2) homozygous CT{alpha}flox + heterozygous Cre mice, which lack hepatic CT{alpha} expression. DNA from tail clips were used to identify the genotype of the mice (Fig. 1A). In this figure, mice 1 and 2 were homozygous for CT{alpha}flox (right panel); however, only mouse 2 expressed Cre (left panel). Therefore, mouse 1 was a "control," whereas mouse 2 was a liver-specific CT{alpha} "knockout." Mouse 3 was homozygous for CT{alpha}wt (right panel) and did not have the Cre gene (left panel).

To determine whether disruption of the CT{alpha} gene resulted in decreased CT{alpha} protein expression, immunoblot analysis was performed on hepatic homogenates from control (CT{alpha}flox) and knockout (CT{alpha}flox + albuminCre) mice. As demonstrated in Fig. 1B, the presence of Cre reduced hepatic CT{alpha} expression by ~95%. The remaining CT{alpha} protein can be attributed to the presence of endothelial, Kupffer, and blood cells. Indeed, others have observed similar results in liver when using the albuminCre/LoxP technology for other hepatocyte-specific genes (32). Cardiac CT{alpha} protein expression was unchanged by the presence of the hepatic Cre gene (data not shown). CT activity was also measured in hepatic and cardiac homogenates (Fig. 1C). In livers of knockout mice, CT activity was reduced to 15% of control levels. The remaining CT activity can be attributed to the presence of CT{beta}2 in hepatocytes (Fig. 3B) as well as CT activity in nonhepatic cells. Cardiac CT activity was unaffected providing further evidence that CT{alpha} deletion is specific to the liver.



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 3.
Enhanced expression of hepatic PEMT and CT{beta}2 in CT{alpha}-deficient mice. A, liver homogenates were immunoblotted for PEMT, and PEMT activity was measured. Twenty µg of protein was used for immunoblotting PEMT. At least three mice were used for each genotype. Results from enzymatic assays are shown as means ± S.E. for 6–8 mice for each condition. The asterisks signify differences versus control, p < 0.05. B, immunoblots for CT{beta}2 from 40 µg of liver protein obtained from control and knockout mice. A CT{beta}2-specific antibody was used at 1:1500 dilution. Three livers were used from each genotype.

 
Histological Characterization of Livers and Assay of Aminotransferase Activities—The weights of knockout mice (21.6 ± 2.4 g) were comparable with those of their littermate controls (21.5 ± 3.6 g). Moreover, there was no difference in the weight of the liver as a percentage of body weight between genotypes (controls versus knockouts: 5.6 ± 1.2 versus 5.3 ± 0.7% body weight). Livers taken from female knockout mice appeared outwardly normal; however, histological examination with hemotoxylin staining revealed many large vacuoles (Fig. 2B). These vacuoles were present throughout the hepatic acinus and showed no particular pattern. Livers from male knockout mice were also indistinguishable in outward appearance; however, unlike the female mice, male knockouts had only mild vacuolization (Fig. 2D). Oil Red-O staining of liver slices confirmed the presence of neutral lipids in the vacuoles (data not shown).



View larger version (145K):
[in this window]
[in a new window]
  		FIG. 2.
Histology of livers from control and knockout mice. Mice (12–24 weeks old) were fasted overnight and sacrificed, and livers were removed. Sections of liver were fixed in 10% formalin, sliced, and stained with hemotoxylin/eosin. The hemotoxlin/eosin sections are shown at a magnification of x40. Livers from at least two mice for each condition were stained, and pictures are representative of all livers examined. The arrows indicate selected vacuoles.

 
Regardless of the degree of vacuolization, the CT{alpha}-deficient livers appeared to have fewer hepatocytes that were larger in size compared with control livers. We therefore counted the number of cells present on the slide at x 63 magnification. The livers taken from the knockout mice had 40% fewer hepatocytes per viewing area (control versus knockout: 105–130 versus 60–80 cells/viewing area). To confirm these results, we also measured the amount of DNA present/g of hepatic tissue. The CT{alpha}-deficient livers contained 30% less DNA/g of tissue compared with controls (0.78 ± 0.08 versus 1.17 ± 0.13 mg/g liver, p < 0.05). These results provide convincing evidence for the presence of fewer but larger hepatocytes in the knockout livers.

Because of the obvious differences between the livers of the CT{alpha} knock-out and control mice (Fig. 2, B and D) plasma aspartate aminotransferase and alanine aminotransferase activities were measured to assess the possibility of liver damage. The plasma activity of these enzymes was unaltered by disruption of the CT{alpha} gene in the liver, suggesting that the observed changes in plasma and hepatic lipids were due to loss of hepatic CT{alpha} activity rather than liver damage.

The Lipid Content of the Liver Is Altered in CT{alpha} Knockout Mice—The Kennedy (CDP-choline) pathway has been shown to produce ~70% of PC in the liver (7, 9). It was, therefore, expected that deletion of CT{alpha} would alter hepatic lipid concentrations. Table I shows data for hepatic PC, PE, cholesterol, cholesteryl esters, and TG. PC in the liver was decreased by 20% in female CT{alpha} knockout mice relative to controls. This decrease was not observed in male mice. Furthermore, hepatic TG was increased by 40% in the female knockout mice relative to control mice. Regardless of gender, no difference was observed in the levels of hepatic PE, cholesterol, or cholesteryl ester between control and knockout mice.


View this table:
[in this window]
[in a new window]
  		TABLE I
Concentration of PC, PE, cholesterol, cholesteryl ester, and TG in livers of hepatic CT{alpha} knockout and control mice Lipids were extracted from livers (0.5 mg of protein) and separated by high pressure liquid chromatography (for phospholipids) or by gas chromatography (for neutral lipids). The results are expressed as means ± S.E. from 6–10 mice.

 
Expression of Hepatic PEMT and CT{beta}2 Is Induced in the Liver-specific CT{alpha} Knockout Mice—To ascertain how the livers of the CT{alpha} knockout mice respond to decreased CT{alpha}, we investigated the other major pathway by which the liver can synthesize PC, the PEMT pathway. In the CT{alpha}-deficient livers, PEMT activity was increased irrespective of gender by 90% (Fig. 3A). Similarly, hepatic PEMT protein levels were 2-fold higher in the knockout mice than in control mice. Hepatic CT{beta}2 protein was also increased ~2-fold in the CT{alpha} KO mouse (Fig. 3B), consistent with previous data with CT{alpha}-deficient mice (23). These results suggest that normally CT{beta}2 represents only ~5% of total hepatic CT activity. This observation is consistent with another study that reports only minimal hepatic expression of CT{beta}2 (5).

Plasma Lipids Are Decreased in the Liver-specific CT{alpha} Knockout Mice—The plasma concentration of PC, the major phospholipid in mammalian plasma (33), was 60% lower in male knockout mice than in their control counterparts (Fig. 4). Moreover, the amount of plasma PC in female knockout mice was 50% less than in female controls. Interestingly, the large change in plasma PC was mirrored by changes in plasma cholesterol concentrations. In male knockout mice, total plasma cholesterol was 40% of control levels (Fig. 5), and in female knockout mice the plasma cholesterol level was half of that in control mice.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 4.
Plasma PC is decreased in the liver-specific CT{alpha} knockout mice. PC was extracted from 50 µl of plasma, separated from other lipids by HPLC, and quantified using phosphatidyldimethylethanolamine as an internal standard. Values are means ± S.E. for 6–10 mice for each condition. The asterisks signify differences versus control, p < 0.05.

 



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 5.
Plasma TG and cholesterol are decreased in the liver-specific CT{alpha} knockout mice. The amounts of total cholesterol (unesterified + esterified cholesterol) and TG were measured in 50 µl of plasma. After digestion (2 h, 30 °C) of the phospholipids with phospholipase C, tridecanoin (20 ng) was added as an internal standard, and lipids were extracted. The amounts of TG and total cholesterol were determined by gas-liquid chromatography. Values are expressed as means ± S.E. for 6–10 mice for each condition. The asterisks signify differences versus control, p < 0.05.

 
Whereas the extent of the changes in plasma PC and cholesterol was similar in male and female mice, modulation of plasma TG levels by hepatic CT{alpha} deficiency was different between genders. The plasma TG content in male mice was only modestly (20%, p < 0.05) reduced in knockout mice compared with controls (Fig. 5). However, the amount of plasma TG was strikingly lower (45%, p < 0.05) in female knockout mice than in littermate controls. These data are consistent with the larger accumulation of TG in the livers of female knockout mice than in their male counterparts and suggest a greater defect in lipoprotein secretion from the CT{alpha}-deficient livers of female, compared with male, mice.

Plasma was separated into lipoprotein fractions by HPLC. The content of TG (Fig. 6, A and C) in the VLDL fraction from both male and female knockout mice was lower than in control mice, in agreement with the quantitative data shown in Fig. 5. However, the decrease in plasma VLDL-TG in the male knockout mice compared with the control mice was larger than was predicted from the quantitative data of Fig. 5. The amount of cholesterol (Fig. 6, B and D) in the HDL fractions of both male and female knockout mice was lower than in their control littermates. The cholesterol content of the VLDL and low density lipoprotein fractions of both male (Fig. 6D) and female (Fig. 6B) knockout mice appeared to be lower than in control mice. However, the cholesterol level of these fractions was close to the limit of detection of these experiments.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 6.
Fractionation of plasma lipoproteins by high performance liquid chromatography. Plasma (25 µl) from female (A and B) and male (C and D) mice was separated into lipoprotein fractions on an Amersham Biosciences Superose 6 column with an in-line assay for either cholesterol (B and D) or triacylglycerol (A and C). Lipoproteins eluted in the following order: VLDL at 25 min, intermediate/low density lipoproteins (LDL) at 35 min, and HDL at 40–45 min. For both assays, 2–4 samples were measured in each group. Graphs are representative of all samples measured.

 
Bile Composition Is Unchanged in the Liver-specific CT{alpha} Knockout Mice—The concentration of PC in the gall bladders of male and female knockout mice (21.4 ± 3.2 nmol/liter) was similar to that in control mice (18.8 ± 3.5 nmol/liter). Cholesterol secretion into bile is closely associated with PC secretion. The amount of biliary cholesterol was also the same in the knockout (3.5 ± 0.8 nmol/liter) versus control (4.0 ± 1.6 nmol/liter) mice. Total bile acid concentration (control versus knockout: 213 ± 21.1 versus 246 ± 22.3 mM) and the levels of tauromuricholic acid (control versus knockout: 97.2 ± 7.4 versus 113.1 ± 9.9 mM) and taurocholic acid (control versus knockout: 115.4 ± 16.1 versus 134 ± 14.2 mM), the two major bile acids in mice, were not significantly affected by deletion of the hepatic CT{alpha} gene.

Plasma ApoB100 and ApoA1 Are Decreased in the Liver-specific CT{alpha} Knockout Mice—Since the plasma levels of HDL and VLDL were decreased in the knockout mice, we next determined whether or not the amounts of the corresponding plasma apolipoproteins were altered. To this end, plasma samples were immunoblotted for apoB48, apoB100, apoAI, and apoE. The plasma level of apoAI was ~60% lower in both male and female knockout mice than in their gender controls (Fig. 7). These results are consistent with the observation that the knockout mice contained lower levels of HDL-cholesterol than did the control mice (Fig. 6). Unlike apoAI, the amount of plasma apoE was unaltered by a deficiency of hepatic CT{alpha} irrespective of gender (Fig. 7). This result is not surprising, since apoE is only found in a small fraction (15%) of HDL particles (34). ApoB48 and apoB100 are prominently found in VLDL in mice. The level of plasma apoB100, but not apoB48, was markedly lower in both male and female knockout mice than in their control littermates (Fig. 7).



View larger version (58K):
[in this window]
[in a new window]
  		FIG. 7.
Separation and analysis of plasma apoAI, apoE, apoB100, and apoB48. Plasma proteins from fasted mice were fractionated by SDS-polyacrylamide gel electrophoresis, and immunoblotting was performed using antibodies raised against apoAI, apoE, and apoB (A). For each measurement, at least three mice were used from each test group. Intensities of band were quantitated using Image Gauge version 3.0 software by Fuji, and the results are presented in B.

 
To determine whether or not apoB secretion was decreased in the knockout mice, we performed in vivo labeling of proteins in the mice in the presence of Triton WR1339, a detergent that prevents the catabolism of TG in plasma VLDL by inhibition of lipoprotein lipase (35). Male mice were fasted overnight and then injected with 100 µl of 15% Triton WR1339 and 250 µCi of [35S]Promix. The animals were sacrificed at various times up to 3.5 h, and apoB100 and apoB48 were immunoprecipitated from plasma. The results from a time course are indicated in Fig. 8A, showing maximal incorporation for apoB100 at 2 h. For apoB48, the labeling continued to rise until 3.5 h. In subsequent experiments, the samples were taken at 1 h after injection of Triton WR1339 and radioactivity. As a means of controlling for injection volume, the labeling of plasma apoB was compared with the labeling of plasma albumin. Fig. 8B shows that the total amount of radioactivity was approximately equal in the plasma samples. The amount of labeled apoB100 recovered from the plasma was significantly lower in the liver-specific CT{alpha} knockout mice than in control mice (Fig. 8, B and C). Although the plasma apoB48 concentration was apparently unaltered by disruption of the CT{alpha} gene (Fig. 7), the amount of labeled apoB48 recovered in the plasma was significantly lower in the knockout mice than in the control mice (Fig. 8, B and C).



View larger version (47K):
[in this window]
[in a new window]
  		FIG. 8.
Secretion of hepatic apoB48 and apoB100 into plasma. Male mice were fasted overnight, after which the tail vein was injected with 100 µl of 15% Triton WR1339 (v/v) in phosphate-buffered saline containing 250 µCi of [35S]Promix. The animals were sacrificed after 0.5–3.5 h, and apoB was immunoprecipitated from 100 µl of plasma. Additionally, 5 µl of plasma was separated by SDS-PAGE for detection of labeled albumin. A, time course for the secretion of apoB100 and apoB48. The values are expressed relative to the amount of radioactivity in control sample after 30 min, which was set as 1. There was one mouse used for each time point. The genotypes were compared using a two-way analysis of variance test, and the curves were significantly different for both apoB100 (p = 0.035) and apoB48 (p = 0.048). B, fluorographs and total radioactivity recovered from 2 µl of plasma 1 h after the injection of detergent and radioactivity. C, quantitation of the apoB100, apoB48, and albumin bands from the fluorographs (B) from knockout and control mice using Image Gauge version 3.0 software. The results are expressed as means ± S.E. of three animals for apoB relative to albumin. The asterisks signify differences versus control, p < 0.05, Student's t test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Choline was classified as an essential nutrient by the American Food and Nutrition Board in 1998 (61). A major phenotype of choline deficiency is lipid accumulation in the liver (16), suggesting that choline deficiency inhibited PC synthesis and consequently impaired lipoprotein secretion from the liver. Early studies in hepatocytes showed that an active synthesis of PC is required for VLDL secretion (18). However, these studies were unable to ascertain a direct role for either the CDP-choline or PEMT pathway in this process. To address the role of PEMT, our laboratory generated mice in which the Pemt gene was disrupted. Hepatocytes isolated from Pemt-/- mice had a defect in lipoprotein (VLDL) secretion. PEMT-deficient hepatocytes secreted 70% less apoB100 and 50% less TG and PC associated with VLDL (13) than did wild-type controls. In vivo, Pemt-/- mice (fed a high fat/high cholesterol diet) also had lower than normal HDL (PC and cholesterol) levels. However, VLDL secretion, under the same dietary conditions, appeared to be dependent on gender. Male, but not female, Pemt-/- mice exhibited a defect in the secretion of VLDL (TG and apoB100) (14). The reason for the gender difference is unclear, but the combined data from the Pemt-/- mice provided a convincing argument for a vital role of PEMT in lipoprotein metabolism.

We now report the generation of a liver-specific CT{alpha} knockout mouse. Since CT catalyzes the rate-limiting step in the Kennedy pathway for PC synthesis (3, 36), this model allows us, for the first time, to investigate directly the impact of inhibiting the hepatic CDP-choline pathway on lipid and lipoprotein metabolism. The results of this study demonstrate that CT{alpha} is required for normal secretion of VLDL (apoB, PC, and TG) in mice fed a normal chow diet. In addition, plasma HDL (PC, cholesterol, and apoAI) was 50% lower in the knockout mice than in the control mice. These results suggest that hepatic PC supply from CT{alpha} is vital for homeostasis of both plasma VLDL and HDL.

How Does Hepatic PC Biosynthesis Regulate Plasma HDL?— Plasma levels of HDL-cholesterol are inversely correlated with the development of cardiovascular disease (37, 38). HDL is thought to inhibit atherosclerosis by stimulating transport of cholesterol from peripheral cells to the liver, where the cholesterol is converted to bile acids and/or excreted (39). The importance of the hepatic uptake of cholesterol in reverse cholesterol transport has been demonstrated using transgenic and knockout models of the scavenger receptor class B type 1 that mediates the selective uptake of cholesteryl esters and probably other lipids from HDL (40). Overexpression of scavenger receptor B1 in the livers of mice is accompanied by a decreased level of plasma HDL-cholesterol (41). Furthermore, SR-B1 gene deletion in mice results in a 90% reduction in cholesterol uptake by the liver and a 2-fold increase in the plasma level of cholesterol (42, 43). In rats, the hepatic uptake of PC from HDL is also a quantitatively important process, since nearly 40% of biliary PC originates from HDL (44). These observations might explain why the amount of biliary PC is not altered in mice deficient in hepatic CT{alpha}. We suggest that our data indicate that hepatic CT{alpha} deficiency might result in an increased uptake of HDL-PC with a corresponding reduction in the level of plasma HDL as observed in our liver-specific CT{alpha} knockout mice.

Alternatively, or in addition, livers deficient in CT{alpha} might compensate for impaired PC biosynthesis by limiting the amount of PC and cholesterol available for HDL formation. The formation of HDL from apoAI requires the cellular efflux of both cholesterol and PC (45). A change in the rate of hepatic cholesterol efflux has been previously shown to alter circulating HDL levels (46, 47). Overexpression of hepatic ATP-binding cassette binding protein A1 (required for the efflux of cholesterol and PC to apoAI) results in a 2-fold increase in plasma levels of both PC and cholesterol in HDL (47). Whether or not decreased hepatic PC biosynthesis reduces cholesterol efflux remains to be determined. No change was observed in apoAI secretion or nascent HDL formation in cultured hepatocytes deficient in methionine and choline, suggesting that PC biosynthesis is not required for hepatic cholesterol efflux (18). However, these results should be interpreted carefully, since an increased, rather than a decreased, flux through the Kennedy pathway has been observed in choline-deficient murine hepatocytes (22). Indeed, the generation of the PEMT knockout mice and the liver-specific CT{alpha} knockout mice allows a closer examination of the relationship between PC biosynthesis and cholesterol efflux to apoA1.

CT{alpha} and VLDL Secretion—The role of PC synthesis in VLDL secretion has been studied extensively in our laboratory (13, 14, 16–19, 21, 48, 49). When rats (16) and mice (22) are fed a CD diet, the level of plasma VLDL (apoB, PC, and TG) is reduced. Nevertheless, recent studies suggest that this reduction is not the result of either inhibition of the CDP-choline pathway or a defect in VLDL secretion from the liver (22).

As mentioned above, studies in Pemt-/- mice have strengthened the link between PE methylation and VLDL secretion. (We now provide evidence that hepatic CT{alpha} is required for normal VLDL secretion in mice.) Our data show that plasma TG, PC, and apoB100 are decreased in the knockout mice, compared with littermate controls, regardless of gender. It is interesting that the lower VLDL levels are independent of gender, since total hepatic PC is decreased only in female, not male, knockout mice. This observation further supports the idea that active PC biosynthesis is required for normal VLDL secretion. The PC level of Pemt-/- hepatocytes is also normal, but secretion of apoB100 and TG is inhibited (13). Impairment of apoB100 and apoB48 secretion was seen in male CT{alpha} knockout mice without a decrease in the level of PC in the liver. The decrease in apoB secretion may be partially explained by the reduction in the total number of hepatocytes in the CT{alpha}-deficient livers. However, preliminary results show that apoB100 secretion from hepatocytes isolated from knockout mice is reduced compared with that from control hepatocytes.2 It should be noted that induction of expression of PEMT and CT{beta}2, while probably important in maintaining hepatic PC levels, was insufficient to normalize hepatic VLDL secretion.

Other Roles for CT{alpha}—It is now clear that PC production via the CDP-choline pathway is vital for lipoprotein homeostasis in mice. However, regulation of CT activity has also been implicated in many other biological processes, including neurite growth (50), neural tube closure (51), and fetal development (52). It has also been suggested that dysregulation of hepatic PC biosynthesis disrupts the distribution of long chain polyunsaturated fatty acids in liver and plasma (53).

Furthermore, induction of PC biosynthesis is an essential step during cell division (54–56). In this process, CT{alpha} gene expression is stimulated by Sp1 during the S phase of the cell cycle (57). Consequently, the amount of CT is increased prior to mitosis, which might explain why CT{alpha}-deficient livers contain fewer hepatocytes than do control livers. It is possible that hepatocytes lacking CT{alpha} cannot produce enough PC for normal cell division. If this were the case, the liver-specific CT{alpha} knockout mouse model might provide in vivo evidence for the requirement of CT{alpha} in hepatic cell division. Thus, the results presented herein further support the hypothesis suggested by previous studies that PEMT activity cannot substitute for CT{alpha} in maintaining the cell cycle. First, overexpression of PEMT in Chinese hamster ovary-MT58 cells (these cells have a temperature-sensitive mutation in CT that prevents PC biosynthesis at 40 °C) did not rescue growth of MT58 cells at 40 °C, because insufficient PC was produced for cellular replication (58). Second, induction of hepatocyte proliferation after partial hepatectomy of rats is accompanied by a reduction in the amount of PEMT mRNA, protein, and activity (59, 60). This down-regulation of PE methylation coincided with maximal DNA synthesis and an elevation of CT activity, illuminating the importance of the CDP-choline pathway in rapidly dividing hepatocytes.

Conclusions—We have generated a liver-specific CT{alpha} knockout mouse to investigate the function of CT{alpha}-derived PC in the liver. The experiments presented in this study demonstrate that CT{alpha} plays an important role in regulating plasma levels of both HDL and VLDL. One surprising observation is that livers deficient in CT{alpha} have fewer hepatocytes, but of larger size, than do the control livers, suggesting a vital role for CT{alpha} in hepatic cell division. However, more in vivo data linking CT{alpha} and hepatic cell division are required. Our data clearly demonstrate that impairment in the hepatic CDP-choline pathway alters the metabolism of both hepatic and circulating lipids and lipoproteins.


   FOOTNOTES
 
* This work was supported by grants from the Canadian Institutes of Health Research and National Institutes of Health Grant H:54591. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of a Canadian Institutes of Health Research Postdoctoral Fellowship and an Alberta Heritage Foundation for Medical Research Postdoctoral Fellowship. Back

|| Holder of the Canada Research Chair in Molecular and Cell Biology of Lipids and Medical Scientist of the Alberta Heritage Foundation of Medical Research. Back

** To whom correspondence should be addressed. Tel.: 780-492-8286; Fax: 780-492-3383; E-mail: dennis.vance{at}ualberta.ca.

1 The abbreviations used are: PC, phosphatidylcholine; apo, apolipoprotein; CD, choline-deficient; CT, CTP:phosphocholine cytidylyltransferase; HDL, high density lipoprotein; HPLC, high performance liquid chromatography; PE, phosphatidylethanolamine; PEMT, phosphatidylethanolamine N-methyltransferase; TG, triacylglycerols; VLDL, very low density lipoprotein. Back

2 R. L. Jacobs and D. E. Vance, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Mark A. Magnuson for the albuminCre mouse, Dr. Suzanne Jackowski for antibodies to CT, and Dr. Jean Vance for helpful discussions. We thank Audric Moses, Sandra Ungarian, and Pricilla Gao for excellent technical assistance, Laura Hargraves and Jennifer Witmer for maintenance of the mouse colonies, and Dr. Mark Lee for analysis of histological data.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kennedy, E. P., and Weiss, S. B. (1956) J. Biol. Chem. 222, 193-214[Free Full Text]
  2. Vance, D. E. (1990) Biochem. Cell Biol. 68, 1151-1165[Medline] [Order article via Infotrieve]
  3. Johnson, J. E., Kalmar, G. B., Sohal, P. S., Walkey, C. J., Yamashita, S., and Cornell, R. B. (1992) Biochem. J. 285, 815-820
  4. Lykidis, A., Jackson, P., and Jackowski, S. (2001) Biochemistry 40, 494-503[CrossRef][Medline] [Order article via Infotrieve]
  5. Karim, M., Jackson, P., and Jackowski, S. (2003) Biochim. Biophys. Acta 1633, 1-12[Medline] [Order article via Infotrieve]
  6. Vance, D. E., and Ridgway, N. D. (1988) Prog. Lipid Res. 27, 61-79[CrossRef][Medline] [Order article via Infotrieve]
  7. Sundler, R., and Akesson, B. (1975) Biochem. J. 146, 309-315[Medline] [Order article via Infotrieve]
  8. Reo, N. V., Adinehzadeh, M., and Foy, B. D. (2002) Biochim. Biophys. Acta 1580, 171-188[Medline] [Order article via Infotrieve]
  9. DeLong, C. J., Shen, Y. J., Thomas, M. J., and Cui, Z. (1999) J. Biol. Chem. 274, 29683-29688[Abstract/Free Full Text]
  10. Walkey, C. J., Donohue, L. R., Bronson, R., Agellon, L. B., and Vance, D. E. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12880-12885[Abstract/Free Full Text]
  11. Walkey, C. J., Yu, L., Agellon, L. B., and Vance, D. E. (1998) J. Biol. Chem. 273, 27043-27046[Abstract/Free Full Text]
  12. Waite, K. A., Cabilio, N. R., and Vance, D. E. (2002) J. Nutr. 132, 68-71[Abstract/Free Full Text]
  13. Noga, A. A., Zhao, Y., and Vance, D. E. (2002) J. Biol. Chem. 277, 42358-42365[Abstract/Free Full Text]
  14. Noga, A. A., and Vance, D. E. (2003) J. Biol. Chem. 278, 21851-21859[Abstract/Free Full Text]
  15. Best, C. H., and Huntsman, M. E. (1932) J. Physiol. (Lond.) 75, 405-412
  16. Yao, Z. M., and Vance, D. E. (1990) Biochem. Cell Biol. 68, 552-558[Medline] [Order article via Infotrieve]
  17. Yao, Z. M., Jamil, H., and Vance, D. E. (1990) J. Biol. Chem. 265, 4326-4331[Abstract/Free Full Text]
  18. Yao, Z. M., and Vance, D. E. (1988) J. Biol. Chem. 263, 2998-3004[Abstract/Free Full Text]
  19. Yao, Z. M., and Vance, D. E. (1989) J. Biol. Chem. 264, 11373-11380[Abstract/Free Full Text]
  20. Vermeulen, P. S., Lingrell, S., Yao, Z., and Vance, D. E. (1997) J. Lipid Res. 38, 447-458[Abstract]
  21. Cui, Z., and Vance, D. E. (1996) J. Biol. Chem. 271, 2839-2843[Abstract/Free Full Text]
  22. Kulinski, A., Vance, D. E., and Vance, J. E. (2004) J. Biol. Chem., M312676200
  23. Zhang, D., Tang, W., Yao, P. M., Yang, C., Xie, B., Jackowski, S., and Tabas, I. (2000) J. Biol. Chem. 275, 35368-35376[Abstract/Free Full Text]
  24. Postic, C., Shiota, M., Niswender, K. D., Jetton, T. L., Chen, Y., Moates, J. M., Shelton, K. D., Lindner, J., Cherrington, A. D., and Magnuson, M. A. (1999) J. Biol. Chem. 274, 305-315[Abstract/Free Full Text]
  25. Cui, Z., Vance, J., Chen, M., Voelker, D., and Vance, D. (1993) J. Biol. Chem. 268, 16655-16663[Abstract/Free Full Text]
  26. Schneider, W. J., and Vance, D. E. (1978) Eur. J. Biochem. 85, 181-187[Medline] [Order article via Infotrieve]
  27. Ridgway, N. D., and Vance, D. E. (1992) Methods Enzymol. 209, 366-374[Medline] [Order article via Infotrieve]
  28. Myher, J. J., Kuksis, A., and Pind, S. (1989) Lipids 24, 408-418[Medline] [Order article via Infotrieve]
  29. Folch, J., Lees, M., and Stanley, G. H. S. (1957) J. Biol. Chem. 226, 497-509[Free Full Text]
  30. Bergo, M. O., Gavino, B. J., Steenbergen, R., Sturbois, B., Parlow, A. F., Sanan, D. A., Skarnes, W. C., Vance, J. E., and Young, S. G. (2002) J. Biol. Chem. 277, 47701-47708[Abstract/Free Full Text]
  31. Torchia, E. C., Labonte, E. D., and Agellon, L. B. (2001) Anal. Biochem. 298, 293-298[CrossRef][Medline] [Order article via Infotrieve]
  32. Postic, C., and Magnuson, M. A. (2000) Genesis 26, 149-150[CrossRef][Medline] [Order article via Infotrieve]
  33. Vance, D. E. (2002) in Biochemistry of Lipids, Lipoproteins and Membranes (Vance, D. E., and Vance, J. E., eds) 4th Ed., pp. 205-232, Elsevier, Amsterdam
  34. Fagan, A. M., Bu, G., Sun, Y., Daugherty, A., and Holtzman, D. M. (1996) J. Biol. Chem. 271, 30121-30125[Abstract/Free Full Text]
  35. Hayashi, H., Niinobe, S., Matsumoto, Y., and Suga, T. (1981) J. Biochem. (Tokyo) 89, 573-579[Abstract/Free Full Text]
  36. Vance, D. E., and Choy, P. C. (1979) Trends Biochem. Sci. 4, 145-148[CrossRef]
  37. Gordon, D. J., and Rifkind, B. M. (1989) N. Engl. J. Med. 321, 1311-1316[Medline] [Order article via Infotrieve]
  38. Tabas, I. (2002) in Biochemistry of Lipids, Lipoproteins and Membranes (Vance, D. E., and Vance, J. E., eds) pp. 573-600, Elsevier Science, Amsterdam
  39. Glomset, J. A. (1980) Adv. Intern. Med. 25, 91-116[Medline] [Order article via Infotrieve]
  40. Acton, S., Rigotti, A., Landschulz, K. T., Xu, S., Hobbs, H. H., and Krieger, M. (1996) Science 271, 518-520[Abstract]
  41. Wang, N., Arai, T., Ji, Y., Rinninger, F., and Tall, A. R. (1998) J. Biol. Chem. 273, 32920-32926[Abstract/Free Full Text]
  42. Van Eck, M., Twisk, J., Hoekstra, M., Van Rij, B. T., Van der Lans, C. A., Bos, I. S., Kruijt, J. K., Kuipers, F., and Van Berkel, T. J. (2003) J. Biol. Chem. 278, 23699-23705[Abstract/Free Full Text]
  43. Rigotti, A., Trigatti, B. L., Penman, M., Rayburn, H., Herz, J., and Krieger, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12610-12615[Abstract/Free Full Text]
  44. Portal, I., Clerc, T., Sbarra, V., Portugal, H., Pauli, A. M., Lafont, H., Tuchweber, B., Yousef, I., and Chanussot, F. (1993) Am. J. Physiol. 264, G1052-G1056
  45. Yokoyama, S. (2000) Biochim. Biophys. Acta 1529, 231-244[Medline] [Order article via Infotrieve]
  46. Wellington, C. L., Brunham, L. R., Zhou, S., Singaraja, R. R., Visscher, H., Gelfer, A., Ross, C., James, E., Liu, G., Huber, M. T., Yang, Y. Z., Parks, R. J., Groen, A., Fruchart-Najib, J., and Hayden, M. R. (2003) J. Lipid Res. 44, 1470-1480[Abstract/Free Full Text]
  47. Basso, F., Freeman, L., Knapper, C. L., Remaley, A., Stonik, J., Neufeld, E. B., Tansey, T., Amar, M. J., Fruchart-Najib, J., Duverger, N., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2003) J. Lipid Res. 44, 296-302[Abstract/Free Full Text]
  48. Jamil, H., Yao, Z. M., and Vance, D. E. (1990) J. Biol. Chem. 265, 4332-4339[Abstract/Free Full Text]
  49. Verkade, H. J., Fast, D. G., Rusinol, A. E., Scraba, D. G., and Vance, D. E. (1993) J. Biol. Chem. 268, 24990-24996[Abstract/Free Full Text]
  50. Carter, J. M., Waite, K. A., Campenot, R. B., Vance, J. E., and Vance, D. E. (2003) J. Biol. Chem. 278, 44988-44994[Abstract/Free Full Text]
  51. Fisher, M. C., Zeisel, S. H., Mar, M. H., and Sadler, T. W. (2002) FASEB J. 16, 619-621[Free Full Text]
  52. Cui, Z., Shen, Y. J., and Vance, D. E. (1997) Biochim. Biophys. Acta 1346, 10-16[Medline] [Order article via Infotrieve]
  53. Watkins, S. M., Zhu, X., and Zeisel, S. H. (2003) J. Nutr. 133, 3386-3391[Abstract/Free Full Text]
  54. Terce, F., Brun, H., and Vance, D. (1994) J. Lipid Res. 35, 2130-2142[Abstract]
  55. Jackowski, S. (1996) J. Biol. Chem. 271, 20219-20222[Free Full Text]
  56. Golfman, L. S., Bakovic, M., and Vance, D. E. (2001) J. Biol. Chem. 276, 43688-43692[Abstract/Free Full Text]
  57. Banchio, C., Schang, L. M., and Vance, D. E. (2003) J. Biol. Chem. 278, 32457-32464[Abstract/Free Full Text]
  58. Waite, K. A., and Vance, D. E. (2000) J. Biol. Chem. 275, 21197-21202[Abstract/Free Full Text]
  59. Houweling, M., Tijburg, L. B., Jamil, H., Vance, D. E., Nyathi, C. B., Vaartjes, W. J., and van Golde, L. M. (1991) Biochem. J. 278, 347-351
  60. Houweling, M., Cui, Z., Tessitore, L., and Vance, D. E. (1997) Biochim. Biophys. Acta 1346, 1-9[Medline] [Order article via Infotrieve]
  61. Food and Nutrition Board, Institute of Medicine (1998) Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin and Choline, National Academy Press, Washington, D. C.

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles: