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Originally published In Press as doi:10.1074/jbc.M312676200 on March 15, 2004

J. Biol. Chem., Vol. 279, Issue 23, 23916-23924, June 4, 2004
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A Choline-deficient Diet in Mice Inhibits neither the CDP-choline Pathway for Phosphatidylcholine Synthesis in Hepatocytes nor Apolipoprotein B Secretion*

Agnes Kulinski{ddagger}§, Dennis E. Vance{ddagger}||, and Jean E. Vance{ddagger}§**

From the {ddagger}Canadian Institutes of Health Research Group on the Molecular and Cell Biology of Lipids and the Departments of §Medicine and Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

Received for publication, November 19, 2003 , and in revised form, February 20, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Phosphatidylcholine is a major component of very low density lipoproteins (VLDLs) secreted by the liver. Hepatic phosphatidylcholine is synthesized from choline via the CDP-choline pathway and from the phosphatidylethanolamine N-methyltransferase pathway. Elimination of the methyltransferase in male mice reduces hepatic VLDL secretion. Our objective was to determine whether inhibition of the CDP-choline pathway for phosphatidylcholine synthesis (by restricting the supply of choline) also impaired VLDL secretion. In mice fed a choline-deficient (CD), compared with a choline-supplemented, diet for 21 days, the amounts of plasma apolipoproteins (apo) B100 and B48 were reduced and the liver triacylglycerol content was increased. Hepatocytes were isolated from male mice that had been fed the CD diet for 3 or 21 days, and the cells were incubated with or without choline. The secretion of apoB100 and B48 from CD hepatocytes was not reduced, and triacylglycerol secretion was only modestly decreased, compared with that from cells supplemented with choline. Remarkably, in light of widely held assumptions, the rate of phosphatidylcholine synthesis from the CDP-choline pathway was not decreased in CD hepatocytes. Rather, there was a trend toward increased phosphatidylcholine synthesis that might be explained by enhanced CTP:phosphocholine cytidylyltransferase activity. Although the concentration of phosphocholine in CD hepatocytes was reduced, the size of the phosphocholine pool remained well above the K for the cytidylyltransferase. Moreover, the amount and m activity of the cytidylyltransferase and methyltransferase were increased. The reduction in plasma apoB in mice deprived of dietary choline cannot, therefore, be attributed to decreased apoB secretion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In 1998, choline was classified as an essential nutrient for humans and a minimum dietary intake was recommended (53). Choline is ubiquitously present in animals and plants and is essential for survival and normal growth of cultured cells (1–4). Choline also appears to be an essential nutrient for humans (5, 6). Upon consumption of a choline-deficient (CD)1 diet for 3 weeks, humans develop incipient liver dysfunction, particularly when adequate levels of methionine and folate are also lacking (5). In addition, choline has been proposed to play an important function in brain development (7, 8), probably because in cholinergic neurons choline is a precursor of the neurotransmitter acetylcholine (6, 9). Choline is also a precursor of the abundant membrane phospholipids phosphatidylcholine (PC), sphingomyelin, and choline plasmalogens in eukaryotic cells. In all nucleated eukaryotic cells, PC is synthesized from choline via the CDP-choline pathway (10). In the liver, an additional PC biosynthetic pathway, the phosphatidylethanolamine N-methyltransferase (PEMT) pathway (11, 12), which utilizes S-adenosylmethionine, generates ~30% of hepatic PC (13–15).

Choline deprivation has been widely reported to be an effective tool for specifically inhibiting the CDP-choline pathway for PC synthesis in rat liver (Ref. 16; reviewed in Ref. 6), rat primary hepatocytes (17), hepatoma cells (18, 19), and fibroblasts (20). The concentration of PC in livers (21–24) and plasma (24, 25) of rats is decreased upon feeding a CD diet. Deprivation of dietary choline in rats (16, 24–29) also decreases the plasma content of triacylglycerol (TG) and induces TG accumulation in the liver. The amounts of apolipoprotein (apo) B100 and apoB48 in plasma very low density lipoproteins (VLDLs) are reduced by 63 and 89%, respectively, in rats fed a CD, compared with a choline-supplemented (CS), diet for 3 days (24). Although choline deficiency reduces plasma TG in both male and female rats, the response is more pronounced in males (26). These observations led to the widely held view that the development of fatty livers in animals fed a CD diet is the result of an impaired hepatic secretion of TG into plasma lipoproteins, primarily VLDLs, because of inhibition of PC synthesis from choline (24, 25, 27, 28, 30, 31).

To determine whether the reduction in plasma TG induced by choline deficiency in rats was the result of impaired lipoprotein secretion, rather than increased removal of TGs from plasma, Yao and Vance (17, 32) examined VLDL secretion by CD and CS primary rat hepatocytes. The hepatocytes were isolated from rats fed a CD diet for 3 days and subsequently incubated in medium containing or lacking choline and methionine. In general agreement with previous studies on choline deficiency, when the culture medium lacked both choline and methionine, the TG content of the hepatocytes was ~6-fold higher than when the hepatocytes were cultured in medium containing either choline or methionine. Moreover, the mass of TG, PC, apoB100, and apoB48 secreted into VLDLs from choline- and methionine-deficient hepatocytes was markedly less than from hepatocytes cultured in the presence of either choline or methionine. Addition of choline to the choline- and methionine-deficient medium increased the rate of incorporation of radiolabeled [3H]oleate into PC by ~3-fold and restored a normal level of cellular PC. These observations demonstrated that normal hepatic secretion of VLDLs requires active PC synthesis from CDP-choline or from the methylation pathway. However, in these studies the choline deficiency was induced in a background of methionine insufficiency. Therefore, one could not clearly establish whether or not a deficiency of choline alone (i.e. inhibition of the CDP-choline pathway) reduced VLDL secretion. Nor was the rate of PC synthesis from the CDP-choline pathway measured in the choline- and methionine-deficient rat hepatocytes.

More definitive studies using PEMT-deficient mice have, however, recently established a specific requirement of the PEMT pathway of PC synthesis for VLDL secretion. In male Pemt–/– mice fed a high fat/high cholesterol diet, the plasma content of TG was ~50% lower (33), and the rate of secretion of apoB100 and TG from Pemt–/– hepatocytes was 50–70% lower, than from Pemt+/+ hepatocytes (33). In addition, the hepatic TG content was ~5-fold higher in male Pemt–/– mice than in male Pemt+/+ mice fed a high fat diet (34). Moreover, expression of PEMT activity in McArdle 7777 rat hepatoma cells (that normally lack PEMT activity) enhanced the secretion of TG and apoB100 into VLDLs (34). In combination, these experiments demonstrated that PEMT activity can regulate VLDL secretion.

In light of these observations, we designed experiments to determine whether or not inhibition of PC synthesis via the CDP-choline pathway, by deprivation of choline, impairs hepatic VLDL secretion in mice. The experiments reveal that VLDL secretion by hepatocytes isolated from mice fed a CD diet for up to 21 days was not inhibited compared with that from mice fed a CS diet. Moreover, choline deficiency in murine hepatocytes did not reduce, but in contrast appeared to increase, the rate of PC synthesis from the CDP-choline pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The CD diet (ICN catalog no. 901387) was obtained from ICN (Costa Mesa, CA) and contained 20% fat as lard. The CS diet consisted of the CD diet to which was added 0.4% (w/w) choline chloride. Hanks' solution, collagenase, penicillin, streptomycin, and fetal bovine serum were obtained from Invitrogen (Burlington, Ontario, Canada). The CD medium was made in our laboratory according to the Invitrogen formula for Dulbecco's modified Eagle's medium, except that choline was omitted. CS medium consisted of CD medium to which was added 28 µM choline chloride. Choline chloride, phenylmethylsulfonyl fluoride, protein A-Sepharose CL-4B, phosphocholine standard for thin layer chromatography, tridecanoin standard for gas chromatography, phospholipase C from Clostridium welchii, and insulin were purchased from Sigma. Complete Protease Inhibitor Mixture tablets were from Roche (Laval, Quebec, Canada). [3H]Glycerol, [methyl-3H]choline, S-adenosyl-[3H]methionine, [35S]methionine, and protein molecular weight markers were from Amersham Biosciences (Baie d'Urfe, Quebec, Canada). All chemicals used for SDS-polyacrylamide gel electrophoresis were from Bio-Rad (Mississauga, Ontario, Canada). Goat anti-human apolipoprotein B polyclonal antibodies were purchased from Chemicon (San Diego, CA). Mouse anti-goat IgG linked to horseradish peroxidase was obtained from Pierce. The rabbit anti-rat PEMT-2 polyclonal antibodies were generated and characterized in our laboratory (35). The rabbit anti-human CT polyclonal antibody was generously provided by Dr. S. Jackowski (St. Jude Children's Research Institute, Memphis, TN). The rabbit polyclonal anti-rat albumin antibody used for immunoblotting was raised in our laboratory, whereas the rabbit polyclonal anti-human albumin antibody used for immunoprecipitations was purchased from Sigma. The rabbit polyclonal anti-bovine protein-disulfide isomerase antibodies and the rabbit anti-canine calnexin antibodies were purchased from StressGen (Vancouver, British Columbia, Canada). Mouse anti-rabbit IgG linked to horseradish peroxidase was from Pierce. The chemiluminescent reagent used for immunoblotting was from Amersham Biosciences. Standard PC for thin layer chromatography and the phosphatidylmonomethylethanolamine substrate for the PEMT assay were purchased from Avanti Polar Lipids (Alabaster, AL). The choline detection kit (Phospholipids B) was from Wako Chemicals GmbH (Neuss, Germany). BIOCOAT collagen-coated cell culture dishes (60 mm) and thin layer chromatography plates (Silica gel G, 0.25 mm thickness) were from VWR (Mississauga, Ontario, Canada). All other chemicals and reagents were from Fisher Scientific or Sigma.

Feeding of the Mice; Isolation and Culture of Murine Primary Hepatocytes—All mice were adult male Pemt+/+ mice with a mixed background of 129/J and C57bl/6, and were from a colony maintained by homozygous breeding at the University of Alberta (34, 36). The mice were placed in wire-bottomed cages with no bedding and were fed the semipurified CD or CS diet and water ad libitum for 3 or 21 days. For experiments in which plasma lipids and lipoproteins were analyzed, the mice were fasted for 16 h prior to removal of plasma. For isolation of hepatocytes, all mice were fed the CD diet for 3 or 21 days, as indicated. The mice were anesthetized by intraperitoneal injection of Somnotol (0.022 ml/50 g of body weight). A midline incision was made, and Hanks' EGTA solution containing 1 mg/ml insulin was perfused through the portal vein until the liver was clear of blood. The upper and lower vena cava were tied, and the perfusion was continued with Hanks' collagenase solution (100 units/ml) containing 1 mg/ml insulin until the liver became soft (~3 min). The liver was removed, cut into pieces, transferred to Hanks' collagenase solution, and mixed until all clumps of tissue were dissipated. The hepatocytes were washed three times in CD medium, then suspended in CD medium containing 10% delipidated and dialyzed fetal bovine serum (37). Cells were plated on collagen-coated 60-mm dishes (2 x 106 cells/dish). Cell viability (typically >90%) was estimated by trypan blue exclusion. After hepatocytes had attached to the dish (2–3 h), the medium was removed and the cells were washed. Fresh medium without serum ± 28 µM choline was added, and the cells were incubated overnight. The hepatocytes were designated as CD or CS hepatocytes, accordingly.

Determination of TG Mass in Livers, Plasma, and Hepatocytes— Livers (typically ~1 g) were homogenized in a Polytron (model Ultra-Turrax-T8) in 5 ml of homogenization buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride). For measurement of the TG content of hepatocytes, cells from one 60-mm culture dish were scraped into 1 ml of water and the lipids were extracted (38). The amount of TG was determined in the liver homogenates (2 mg of protein), 100 µl of plasma, the hepatocyte lysate (from one 60-mm dish), or 2 ml of culture medium. After digestion of the phospholipids with phospholipase C, tridecanoin (20 ng) was added as an internal standard and lipids were extracted. The mass of TG was determined by gas-liquid chromatography.

Determination of PC Mass in Livers and Plasma—Lipids from an aliquot (2 mg of protein) of liver homogenate or 100 µl of plasma were extracted by the method of Folch et al. (38) and separated by thin layer chromatography in the solvent system chloroform/methanol/acetic acid/formic acid/water (70:30:12:4:1). Bands corresponding to authentic PC were visualized by exposure to iodine vapor and scraped from the plate, and the mass of PC was measured by phosphorus assay (39).

Analysis of ApoB Content of Hepatocytes, Culture Medium, and Plasma—Hepatocytes were harvested from one culture dish and scraped into 750 µl of phosphate-buffered saline. Next, 250 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) was added. The cellular extracts were centrifuged in a microcentrifuge at 14,000 rpm for 10 min, after which the supernatant (1 ml) was incubated overnight at 4 °C with anti-human apoB antibody (7.5 µl). Protein A-Sepharose (45 mg) was added, and the sample was mixed end-over-end for 2 h at 4 °C. The apoB-protein A complexes were pelleted by centrifugation of the sample for 2 min at 14,000 rpm in a microcentrifuge. For immunoprecipitation of secreted apoB, culture medium was centrifuged for 2 min at 1,000 x g to remove cell debris prior to immunoprecipitation of apoB, as described above. For analysis of plasma apoB, proteins in 30 µl of plasma were dissolved in sample buffer containing 62.5 mM Tris, 2% SDS, and 10% glycerol. The proteins were separated by electrophoresis on 5% polyacrylamide gels in the presence of 0.4% SDS, then transferred to polyvinylidene difluoride membranes. Proteins were immunoblotted with goat anti-human apoB antibody and subsequently with anti-goat IgG linked to horseradish peroxidase. Immunoreactive proteins were detected using chemiluminescence. The amount of apoB was quantitated by densitometric scanning of the blots.

Measurement of the Rate of ApoB Secretion—Hepatocytes from male mice fed the CD diet for 3 or 21 days were incubated in CD or CS media without serum for 16 h. Next, the cells were cultured in methionine-free medium containing or lacking 28 µM choline for 1 h, after which medium containing 100 µCi/dish [35S]methionine (Amersham Biosciences) was added for 1 or 2 h. Medium was collected, and apoB and albumin were immunoprecipitated with goat anti-human apoB antibodies followed by rabbit anti-human albumin antibodies. ApoB and albumin were resolved by electrophoresis on 5 and 10% polyacrylamide gels, respectively. Proteins were fixed on the gels, and the signal was amplified in Amplify solution (Amersham Biosciences). The gels were dried, and apoB and albumin were visualized by autoradiography. The intensity of the bands was quantitated by densitometry.

Density Gradient Separation of Lipoproteins in Culture Medium— Plasma samples (100 µl) were collected from mice fed the CD or CS diet for 21 days and then combined with 1.3 ml of phosphate-buffered saline, mixed with 0.7 g of KBr, and placed in a 5.0-ml Quick-seal tube. The sample was overlaid with 3.5 ml of 0.9% NaCl. The samples were centrifuged at 416,000 x g for 1 h in a VTi 90.0 rotor (40). Ten fractions, 0.5 ml each, with densities ranging from 1.006 g/ml (fraction 10) to 1.21 g/ml (fraction 1), were collected from the bottom of the tube. The density of each fraction was determined as the weight of 1.0 ml. ApoB was immunoprecipitated, as described above, from each fraction and analyzed by electrophoresis on 5% polyacrylamide gels containing 0.4% SDS. The proteins were transferred to polyvinylidene difluoride membranes and immunoblotted, as described above, with anti-apoB antibody. The amount of apoB was quantitated by densitometric scanning of the blots.

Metabolic Labeling of PC and TG—CD and CS hepatocytes were washed twice with CD or CS medium, respectively, after which CD or CS medium containing 5 µCi/ml [3H]glycerol was added for 4 h. Cells were harvested, lipids were extracted, and PC and TG were separated by thin layer chromatography. The bands corresponding to authentic PC and TG were visualized and scraped, and radioactivity was determined.

Determination of the Mass of Phosphocholine—To measure the mass of phosphocholine, six dishes of CD or CS hepatocytes were combined and lipids were extracted (38). The upper, aqueous phase was collected, and solvents were evaporated under a stream of air. The residue was dissolved in 300 µl of water; 100 µl of this sample was used for quantitation of phosphocholine using the choline detection kit (Phospholipids B from Wako Chemicals, Neuss, Germany), according to instructions from the manufacturer using a standard curve of phosphocholine. Control experiments, using thin layer chromatography, established that 90% of the choline-containing metabolites in the aqueous sample consisted of phosphocholine. Therefore, the amount of phosphocholine was calculated as 90% of the total choline-containing compounds in this mixture.

Measurement of the Rate of PC Synthesis from the CDP-choline Pathway—Hepatocytes that had been incubated with CD or CS medium overnight were washed twice with CD medium followed by the addition of CD medium containing 5 µCi/ml [methyl-3H]choline for 1 h. Cells were subsequently washed and incubated with fresh unlabeled CD medium for 0, 0.5, or 1.0 h. Hepatocytes were harvested, and lipids were extracted (38). Aliquots (100 µl) of the lower phase from the lipid extraction were applied to thin layer chromatography plates for separation of the lipids in the solvent system chloroform/methanol/acetic acid/formic acid/water (70:30:12:4:1). The band corresponding to PC was scraped from the plate, and the incorporation of radioactivity was determined.

For measurement of radioactivity in phosphocholine, 100 µl of the upper, aqueous phase from the lipid extraction was separated by thin layer chromatography in the solvent system methanol/0.6% NaCl/NH4OH (10:10:0.9). The band corresponding to authentic phosphocholine was scraped from the plate, and radioactivity was determined.

Because the dpm lost from [3H]phosphocholine during the 1 h chase period were approximately equal to the dpm gained in PC over the same time period, the rate of PC synthesis was calculated from the dpm incorporated into PC during the first 60 min of the chase period (41). The specific radioactivity of phosphocholine (dpm/nmol) was calculated as dpm in phosphocholine/mg protein divided by the mass of phosphocholine/mg of protein. The average specific radioactivity of phosphocholine over the 60-min chase period was used in calculating the rate of PC synthesis. The rate of PC synthesis is given as nanomoles of PC synthesized/min/mg of protein, and was calculated as dpm incorporated into PC over the 60-min chase period divided by the average specific radioactivity of phosphocholine over the same time period.

Measurement of Activities of PC Biosynthetic Enzymes—For measurement of CTP:phosphocholine cytidylyltransferase activity, a liver homogenate was centrifuged for 5 min at 7,000 x g to pellet unbroken cells, after which the supernatant was centrifuged at 470,000 x g for 1 h. The membrane pellet was used for measurement of CT activity, as previously described (42) by monitoring the conversion of [3H]phosphocholine into CDP-choline.

For measurement of PEMT activity, aliquots (50 µg of protein) of liver homogenates were incubated with phosphatidylmonomethylethanolamine and S-adenosyl [3H]methionine, and the incorporation of radiolabel into PC was measured (43).

Immunoblotting of CT, PEMT-2, Albumin, Calnexin, and Proteindisulfide Isomerase—For immunoblotting of CT, liver membrane proteins were separated by electrophoresis on 10% polyacrylamide gels containing 0.1% SDS, then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 10% skimmed milk, then incubated overnight with anti-CT antibodies (1:1,000 dilution) that recognized both {alpha} and {beta} isoforms of CT, followed by incubation with anti-rabbit IgG linked to horseradish peroxidase (dilution 1:2,500). Immunoreactive bands were visualized by enhanced chemiluminescence. The relative amounts of CT protein were determined by densitometric scanning of the blots. The same procedure was used for immunoblotting calnexin, except that the anti-calnexin antibody was used at a dilution of 1:5,000.

For immunoblotting of PEMT, proteins from liver homogenates were separated by electrophoresis on 12% polyacrylamide gels containing 0.1% SDS, then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 10% skimmed milk, then incubated overnight with anti-PEMT antibody (1:1,000 dilution) followed by incubation with anti-rabbit IgG linked to horseradish peroxidase (dilution 1:2,500). Immunoreactive bands were visualized by enhanced chemiluminescence. The anti-PEMT antibody recognizes only the isoform of PEMT that resides on mitochondria-associated membranes (i.e. PEMT-2), not the PEMT isoform that resides on the endoplasmic reticulum (35). The same procedure was used for immunoblotting of protein-disulfide isomerase and albumin except that the anti-protein-disulfide isomerase and anti-albumin antibodies were used at dilutions of 1:4,000 and 1:5,000, respectively.

Other Methods—The protein content of samples was determined using the BCA protein assay kit (Pierce) with bovine serum albumin as standard.

Statistical Analysis—Student's t test was performed. Differences were considered significant at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Plasma Content of ApoB100 and ApoB48 Is Reduced by a Choline-deficient Diet—As a mechanism for specifically attenuating the CDP-choline pathway for PC biosynthesis, we fed mice a CD diet because choline is a required precursor of PC from this biosynthetic route (10). Our goal was to evaluate the specific requirement of PC derived from the CDP-choline pathway for VLDL secretion and to put these observations in the context of the previously demonstrated requirement for the PEMT pathway of PC synthesis for VLDL secretion. For 3 and 21 days, therefore, we fed male mice a diet that contained 20% fat, as used in the previous studies on Pemt–/– mice (33, 44).

Choline chloride (0.4%, w/w) was either included in (CS) or excluded from (CD) the diet. In addition, we used the same strain of mice (Pemt+/+) as in the previous study (33, 44).

Plasma was collected from mice fed the CD and CS diets for 3 and 21 days, and the amounts of apoB48 and apoB100 were assessed by immunoblotting. Fig. 1A shows that the amount of plasma apoB100 was reduced by 46% (p < 0.008) in mice fed the CD diet compared with the CS diet for 21 days, but was not reduced after 3 days. Plasma apoB48 was similarly reduced, by 50% (p < 0.02), after 21 days but was not reduced after 3 days (Fig. 1B). Plasma from mice fed the CS and CD diets for 21 days was separated on the basis of density by ultracentrifugation on KBr gradients, and apoB48 and apoB100 were analyzed by immunoblotting (Fig. 2). Consistent with the data shown in Fig. 1, the amounts of apoB100 (Fig. 2A) and apoB48 (Fig. 2B) were reduced in plasma from CD, compared with CS, mice. ApoB48 and B100 were both present primarily in fractions with densities corresponding to low density lipoproteins and VLDLs. These data indicate that the number of VLDL particles was reduced in the plasma of mice fed the CD diet for 21 days, compared with the CS diet.



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  		FIG. 1.
A choline-deficient diet decreases the plasma content of apoB. Plasma samples (30 µl) were collected from mice fed the CS or CD diet for 3 days (CS-3, CD-3) and 21 days (CS-21, CD-21). ApoB100 (panel A) and apoB48 (panel B) were analyzed by immunoblotting, and the immunoblots were quantitated by densitometric scanning. Data are shown as a percentage of the amount of apoB in CS plasma and are averages ± S.E. from seven CS-3 mice, seven CS-21 mice, three CD-3 mice, and five CD-21 mice. *, p < 0.008; **, p < 0.02.

 



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  		FIG. 2.
Density distribution of plasma apoB-containing lipoproteins. Plasma (100 µl) was isolated from mice fed the CD or CS diet for 21 days. Lipoproteins in plasma were separated on the basis of density by ultracentrifugation on a KBr gradient (40). Ten fractions with densities ranging from 1.21 g/ml (fraction 1) to 1.006 g/ml (fraction 10), were collected. ApoB was immunoprecipitated and analyzed by polyacrylamide gel electrophoresis and immunoblotting. The amounts of apoB100 (panel A) and apoB48 (panel B) in each fraction were determined by densitometric scanning of the blots. Open circles, CD; closed circles, CS. Data are averages ± S.E. of three mice fed each diet.

 
The TG content of plasma from CS and CD mice was also determined. All mice were fasted overnight so that plasma TG was present primarily in VLDLs. The amount of TG in the plasma of CD mice was slightly less (by 17%, p < 0.02) than in CS mice after 3 days, but was not significantly reduced after 21 days (Fig. 3A). The amount of PC in plasma was also slightly reduced (by 18%, p < 0.002) after 3 days and by 22% after 21 days (p < 0.01) (Fig. 3B). We performed parallel experiments in rats, using the same dietary regimen, and found that plasma TG and apoB were reduced (data not shown) to a greater extent than in mice, in general agreement with other studies (24).



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  		FIG. 3.
Plasma concentration of TG and PC. Plasma samples (100 µl) were collected from mice fed the CS or CD diet for 3 days (CS-3, CD-3) and 21 days (CS-21, CD-21). Panel A, the amount of TG was measured by gas-liquid chromatography. Panel B, PC was isolated by thin layer chromatography and quantitated by phosphorus analysis. For both panels, filled bars = CS; open bars = CD. Data are averages ± S.E. of seven CS-3 mice, seven CS-21 mice, three CD-3 mice, and five CD-21 mice. *, p < 0.02; **, p < 0.002; ***, p < 0.01.

 
We hypothesized that the lower levels of apoB and TG in plasma from CD, compared with CS, mice were a consequence of reduced hepatic secretion of VLDLs from CD, compared with CS, mice. One indicator of impaired VLDL secretion is an accumulation of TG in the liver. After 21 days, the mass of TG in the livers of mice fed the CD diet was 3.7-fold higher than in mice fed the CS diet (Fig. 4A). The PC content of the livers of mice fed the CD diet was not significantly different from that of mice fed the CS diet for 21 days, but was 30% lower after 3 days (Fig. 4B). Thus, the accumulation of TG in the livers of mice fed the CD diet for 21 days, and the reduction in apoB100 and apoB48 in the plasma of these mice (Figs. 1 and 2), are consistent with the hypothesis that choline deficiency inhibits hepatic VLDL secretion, although other explanations are also possible.



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  		FIG. 4.
TG and PC content of livers from mice fed the CD or CS diet. Livers from mice fed the CS or CD diet for 3 days (CS-3 and CD-3, respectively) and 21 days (CS-21 and CD-21, respectively) were collected and homogenized. The amount of TG was measured by gas-liquid chromatography (panel A). PC was isolated by thin layer chromatography and quantitated by phosphorus analysis (panel B). For both panels, filled bars = CS; open bars = CD. Data are averages ± S.E. of four CS-3 mice, four CS-21 mice, four CD-3 mice, and four CD-21 mice. *, p < 0.006; **, p < 0.0006.

 
Secretion of TG from Hepatocytes Is Only Modestly Reduced by Choline Deficiency—Because the level of apoB in plasma reflects a balance between the rate of secretion of apoB into plasma and the rate of removal of apoB from plasma, we next determined whether or not VLDL secretion from hepatocytes was inhibited by choline deficiency. For these studies, hepatocytes were isolated from mice that had been fed the CD diet for 3 or 21 days. The hepatocytes were subsequently incubated in CS or CD medium. In all the studies, the hepatocytes are accordingly designated as CS or CD hepatocytes, respectively.

ApoB-containing lipoproteins that have been secreted by cultured primary rat hepatocytes are not rapidly internalized (45). Consequently, the accumulation of VLDLs in the culture medium of hepatocytes reflects the secretion of apoB and TG in the absence of particle re-uptake. We first determined whether choline deficiency induced an accumulation of TG in the hepatocytes. Fig. 5A shows that, in hepatocytes from mice fed the CD diet for 21 days, there was a 2.1-fold accumulation of TG compared with that in CS hepatocytes, but that TG did not accumulate in hepatocytes of mice fed the CD diet for 3 days. This observation is consistent with the finding that the TG content of livers of mice fed the CD diet for 21 days, but not for 3 days, was higher than that of mice fed the CS diet (Fig. 4A). The mass of TG secreted during a 16-h time period from CD hepatocytes derived from mice fed the CD diet for 3 days was not significantly different from that of CS hepatocytes. However, the amount of TG in the medium of CD hepatocytes isolated from mice that had been fed the CD diet for 21 days was 30% less than from CS hepatocytes (Fig. 5B). In a control experiment, we demonstrated that the mass of TG secreted by CS hepatocytes was the same whether the hepatocytes had been derived from mice fed the CD or CS diet for 21 days (data not shown). These data show that choline deficiency, even over an extended period of time (21 days), only modestly decreases the amount of TG secreted by murine hepatocytes.



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  		FIG. 5.
TG content of cells and medium from CD and CS hepatocytes. Hepatocytes were isolated from livers of mice that had been fed the CD diet for 3 days or 21 days. The hepatocytes were incubated in CS medium (CS hepatocytes) or CD medium (CD hepatocytes) for 16 h. Lipids were extracted from cellular lysates (panel A) and the culture medium (panel B), and the TG content (µg/mg of cell protein) was determined by gas-liquid chromatography. Filled bars, CS hepatocytes isolated from mice fed the CD diet for 3 days (CS-3) and 21 days (CS-21); open bars, CD hepatocytes isolated from mice fed the CD diet for 3 days (CD-3) and 21 days (CD-21). For panel A, data are averages ± S.E. of three independent experiments (*, p < 0.04). For panel B, data are averages ± S.E. of triplicate analyses of an experiment that was repeated twice with similar results (**, p < 0.05).

 
Secretion of ApoB from Hepatocytes Is Not Reduced by Choline Deficiency—Immunoblotting experiments indicated that the amount of apoB100 secreted into the medium of CS and CD hepatocytes during a 16-h incubation was not significantly different (Fig. 6, A and B). Nor was the amount of apoB48 secreted by CD hepatocytes different from that secreted by CS hepatocytes (Fig. 6, A and B). The amount of a typical secretory protein, albumin, secreted into the medium was also unaffected by choline deficiency (Fig. 6A). In addition, the secretion of apoB from CS and CD hepatocytes was assessed in radiolabeling experiments using [35S]methionine. Fig. 7 shows that, in CD hepatocytes from mice fed the CD diet for 3 or 21 days, the amount of 35S-labeled apoB100, apoB48, and albumin secreted during a 1- or 2-h period was the same as from CS hepatocytes. Thus, even when the mice had been deprived of dietary choline for 21 days, the secretion of apoB from CD hepatocytes was not significantly impaired.



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  		FIG. 6.
Immunoblotting of apoB48 and apoB100 secreted by CS and CD hepatocytes. Mice were fed the CD diet for 3 or 21 days. Hepatocytes were isolated from the livers and incubated in CS or CD medium (CS or CD hepatocytes, respectively) for 16 h. Panel A, secreted apoB and albumin were immunoprecipitated from the culture medium and analyzed by polyacrylamide gel electrophoresis and immunoblotting. Panel B, the amounts of apoB100 and apoB48 were quantitated by densitometric scanning of the gels. Data are given as a percentage of the amount of apoB in the medium from CS hepatocytes and are averages ± S.E. of three independent experiments. Filled bars, CS hepatocytes from mice fed the CS diet for 3 days (CS-3) or 21 days (CS-21); open bars, CD hepatocytes from mice fed the CD diet for 3 days (CD-3) or 21 days (CD-21).

 



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  		FIG. 7.
Secretion of [35S]methionine-labeled apoB from CS and CD hepatocytes. Mice were fed the CD diet for 3 or 21 days. Hepatocytes were isolated from the livers and incubated in CS or CD medium (CS or CD hepatocytes, respectively) for 16 h, then cultured in methionine-free medium ± 28 µM choline for 1 h. For radiolabeling of the proteins, the cells were incubated in the same media containing 100 µCi/dish [35S]methionine for 1 and 2 h. ApoB and albumin were immunoprecipitated from the medium and analyzed by polyacrylamide gel electrophoresis and radiography. The intensities of the bands corresponding to apoB100, apoB48, and albumin were quantitated by densitometry. Shown are apoB100 and apoB48 secreted by CS and CD hepatocytes derived from mice fed the CD diet for 3 days (panel A) and 21 days (panel C), and albumin secreted by CS and CD hepatocytes from mice fed the CD diet for 3 days (panel B) and 21 days (panel D). Data are averages ± S.E. of triplicate analyses. Filled symbols, CS; open symbols, CD; circles, apoB100; squares, apoB48; triangles, albumin.

 
PC Synthesis from the CDP-choline Pathway—In light of the above observations that apoB secretion from CD hepatocytes was not diminished, we questioned whether our regimen for inducing choline deficiency had, indeed, inhibited PC synthesis via the CDP-choline pathway. We had based our experimental protocol on the generally accepted dogma that choline deficiency in rodents inhibits hepatic PC synthesis from the CDP-choline pathway (6, 16–20). We first confirmed by direct analysis of choline, using the Phospholipids B kit (Wako Chemicals), that the purchased CD diet contained undetectable amounts of choline (data not shown). Nevertheless, upon extensive searching of the literature, we were unable to find any valid measure of the extent to which choline deficiency reduces the rate of hepatic PC synthesis by this pathway.

The rate of PC synthesis could not be measured directly by monitoring the incorporation of radiolabeled choline into PC because choline deficiency would be expected to reduce the pool size, and therefore increase the specific radioactivity, of phosphocholine (a precursor of PC) in hepatocytes (18, 22, 46). Initially, therefore, we compared the incorporation of [3H]glycerol into PC in CS and CD hepatocytes as an indication of PC synthesis from the CDP-choline and PEMT pathways combined. Fig. 8A shows that, after 4 h, the incorporation of [3H]-glycerol into PC was 33% higher in CD, than in CS, hepatocytes from mice fed the CD diet for 3 days, but was slightly lower (by 15%) in CD hepatocytes than in CS hepatocytes from mice fed the CD diet for 21 days. Parallel experiments revealed that the incorporation of [3H]oleate into PC was not significantly different between CS and CD hepatocytes (data not shown). These data indicate that the amount of PC synthesized from the two biosynthetic pathways combined was similar in CD and CS hepatocytes. We also measured the incorporation of [3H]glycerol into TG in CS and CD hepatocytes (Fig. 8B). In hepatocytes from mice fed the CD diet for 21 days, the incorporation of radiolabel into TG after 4 h was 39% (p < 0.01) higher in CD hepatocytes than in CS hepatocytes, but was the same in CD and CS hepatocytes from mice fed the CD diet for 3 days.



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  		FIG. 8.
Incorporation of [3H]glycerol into PC and TG of CD and CS hepatocytes. Mice were fed the CD diet for 3 or 21 days, after which hepatocytes were isolated and cultured for 16 h in CS or CD medium. The CS and CD hepatocytes were incubated for 4 h with CS or CD medium, respectively, containing 5 µCi/ml [3H]glycerol. Lipids were extracted from cellular lysates, and PC (panel A) and TG (panel B) were isolated by thin layer chromatography. Data are averages ± S.E. of triplicate analyses from an experiment that was repeated twice with similar results. *, p < 0.00006; **, p < 0.009; ***, p < 0.01. Filled bars, CS hepatocytes from mice fed the CD diet for 3 days (CS-3) or 21 days (CS-21); open bars, CD hepatocytes from mice fed the CD diet for 3 days (CD-3) or 21 days (CD-21).

 
The rate of PC synthesis from the CDP-choline pathway was next measured directly by performing pulse-chase experiments with [3H]choline in CD and CS hepatocytes. The hepatocytes were radiolabeled with [3H]choline for 1 h, after which the radiolabel was removed and the incorporation of [3H]choline into phosphocholine and PC was monitored over a 1-h chase period. The radiolabel lost from phosphocholine was quantitatively gained in PC over the 60-min chase period in all experiments (data not shown). We also measured the pool size (mass) of phosphocholine in the CS and CD hepatocytes. Although there was considerable variation in the concentration of phosphocholine among hepatocyte preparations, within each experiment the phosphocholine content of CD hepatocytes was consistently lower than in CS hepatocytes (Fig. 9A). We, therefore, were able to calculate the rate of PC synthesis (nanomoles of PC synthesized/min/mg of protein) as the dpm incorporated into PC during the first 60 min of the chase period divided by the average specific radioactivity (dpm/nmol) of phosphocholine over the same time period (41). Fig. 9B shows that, contrary to our initial expectations, the rate of PC synthesis was not reduced in CD, compared with CS, hepatocytes isolated from mice fed the CD diet for either 3 or 21 days. Instead, the data showed a distinct trend toward a higher rate of PC synthesis from the CDP-choline pathway in CD, compared with CS, hepatocytes.



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  		FIG. 9.
The rate of PC synthesis is not reduced in CD hepatocytes. Panel A, the concentration of phosphocholine (P-Chol) was determined in CD and CS hepatocytes derived from mice fed the CD diet for 3 days (CD-3, CS-3) or 21 days (CD-21, CS-21). Data are averages ± S.E. of three independent experiments (*, p < 0.04). Panel B, the rate of PC synthesis from [3H]choline was determined in pulse-chase experiments in CD and CS hepatocytes isolated from livers of mice fed the CD diet for 3 days (CD-3, CS-3) and 21 days (CD-21, CS-21). Hepatocytes were incubated for 1 h with [3H]choline, after which the medium was removed and replaced with fresh medium. After chase periods of 0, 30, and 60 min, PC and phosphocholine were isolated and the incorporation of radioactivity was measured. The rate of PC synthesis (nanomoles of PC synthesized/min/mg of protein) was calculated from the dpm in PC/mg of protein divided by the average specific radioactivity of phosphocholine (dpm/nmol) between 0 and 60 min of the chase period. Data are averages ± S.E. of three independent experiments (**, p < 0.05).

 
Under most metabolic conditions, CTP:phosphocholine cytidylyltransferase (CT) catalyzes the rate-limiting reaction of the CDP-choline pathway for PC synthesis (47). The amount of membrane-associated CT is a measure of the capacity for PC synthesis by this pathway. We, therefore, performed immunoblotting experiments and found that the apparent increase in the rate of PC synthesis from choline (Fig. 9B) was accompanied by an increase in the amount of membrane-associated CT in the liver (Fig. 10A and B). The membrane proteins were also immunoblotted with anti-calnexin antibodies as a control to confirm that choline deficiency did not alter the expression of another microsomal membrane protein (Fig. 10A). In agreement with the immunoblotting data (Fig. 10, A and B), membrane-associated CT activity in the livers was also increased by the CD diet (Fig. 10C). Similar increases in membrane-associated CT activity and protein have been observed in livers of rats fed a CD diet (48, 49) and in CD hepatoma cells (18).



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  		FIG. 10.
Membrane-associated CT protein and activity are increased by choline deficiency. Livers from mice fed the CD and CS diets for 3 and 21 days were homogenized, and membranes were isolated by centrifugation. Panel A, membrane-associated proteins were separated by electrophoresis on 10% polyacrylamide gels containing 0.1% SDS and immunoblotted with rabbit anti-rat CT polyclonal antibody and anti-canine calnexin polyclonal antibody. The experiment was repeated twice (a total of six livers for each condition) with similar results. Panel B, the amount of immunoreactive CT protein was quantitated by densitometric scanning of the blots from the experiments described in panel A. Data are averages ± S.E. from six livers. *, p < 0.008; **, p < 0.024. Panel C, membrane-associated CT activity (nanomoles/min/mg of protein) was determined. Data are averages ± S.E. of four livers in which assays were performed in duplicate. ***, p < 0.021.

 
Immunoblotting of the membrane proteins with an anti-PEMT2 antibody also revealed that the amount of PEMT2 protein in livers of mice fed the CD diet for 3 and 21 days was higher than in CS livers (Fig. 11A). It is noteworthy that the antibody used for these immunoblotting studies recognizes only the PEMT2 isoform of PEMT that is located on mitochondria-associated membranes, and does not recognize the PEMT isoform that is present in the bulk of the endoplasmic reticulum (35). As a control, the expression of protein-disulfide isomerase, a protein of the endoplasmic reticulum, was found to be unaltered by the CD diet (Fig. 11A). Moreover, PEMT activity was higher in CD, than in CS, liver (Fig. 11B). This increase in PEMT expression is consistent with previous studies, in which PEMT expression was increased in livers of rats that had been deprived of dietary choline (49, 50).



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  		FIG. 11.
PEMT is induced by choline deficiency. Mice were fed a CD or CS diet for 3 and 21 days. Livers were removed and homogenized. Proteins in the homogenate were separated by electrophoresis on 12.5% polyacrylamide gels containing 0.1% SDS, and the amounts of PEMT-2 (PEMT) and protein-disulfide isomerase (PDI) were assessed by immunoblotting (panel A). The experiment was performed in three CS and three CD livers (3 days) and four CS and four CD livers (21 days) with similar results. Panel B, PEMT enzymatic activity was measured in liver homogenates. Data are averages ± S.E. of three mice fed the CD or CS diet for 3 days (CD-3 and CS-3, respectively) and five mice fed the CD or CS diet for 21 days (CD-21 and CS-21, respectively). *, p < 0.042; **, p < 0.015. Filled bars, CS; open bars, CD.

 
In combination, these findings demonstrate that elimination of choline from the diet of mice and from the culture medium of murine hepatocytes does not reduce but, in contrast, tends to increase the rate of PC synthesis from the CDP-choline pathway. We attribute this increase, at least in part, to an increase in membrane-associated CT activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study was designed to determine whether inhibition of the CDP-choline pathway for PC synthesis, by deprivation of murine hepatocytes of exogenous choline, inhibited the secretion of apoB-containing lipoproteins. Recent experiments from our laboratories have demonstrated that elimination of the methylation pathway for PC synthesis in male mice reduces the secretion of apoB100 and TG from hepatocytes by 50 to 70% (34). We, therefore, wished to ascertain whether inhibition of PC production from the CDP-choline pathway would also impair VLDL secretion. The experiments show that choline deprivation of hepatocytes isolated from male mice does not inhibit the secretion of apoB100 or apoB48 and only slightly reduces the secretion of TG. Surprisingly, in light of the widely held belief that limiting the supply of choline (a precursor of PC) inhibits the CDP-choline pathway for PC synthesis in hepatocytes (6, 16–20, 50), we found that, even when mice were fed a CD diet for 21 days, the rate of PC synthesis from this pathway in hepatocytes was not diminished but, instead, appeared to be enhanced.

Very Low Density Lipoprotein Secretion—Our observation that the secretion of VLDLs from murine hepatocytes was not inhibited by choline deficiency is contrary to previous assumptions (16, 24–29). However, our findings are not inconsistent with the studies of Yao and Vance (17, 32), in which the secretion of VLDLs from rat hepatocytes that had been cultured in medium deficient in both choline and methionine was reduced. In those studies, when either choline (100 µM) or methionine (200 µM) was given to these hepatocytes, the secretion of VLDLs was normal, indicating that choline deficiency alone did not inhibit VLDL secretion provided that adequate amounts of methionine were present. The authors concluded that the active synthesis of PC from either the CDP-choline pathway or the methylation pathway was required for VLDL secretion.

Several other studies on choline deficiency have been performed in rats (16, 24–29). The general conclusion from these studies was that feeding rats a CD diet decreases the plasma content of apoB100, apoB48, and TG, and causes a concomitant increase in the TG content of the liver. The results of the present study in mice are consistent with these reports. The magnitude of the reduction in the plasma content of TG and apoB in CD mice was, however, smaller than that reported in the studies with CD rats, suggesting that mice are more resistant than rats to choline deficiency. In light of the lack of inhibition of apoB secretion from CD hepatocytes, we attribute the decreased amount of apoB in plasma from mice fed the CD diet for 21 days to an increased removal of VLDL from the plasma in CD, compared with CS, mice. Possible mechanisms by which the removal of VLDLs might be increased are a stimulation of lipolysis of VLDL particles in the plasma, or an increased expression of low density lipoprotein receptors that would result in an increased uptake of apoB-containing lipoproteins from the plasma. The 3.7-fold accumulation of TG in the livers of mice fed the CD diet for 21 days, and the corresponding 2.1-fold increase in the TG content of hepatocytes isolated from these livers, remain to be explained.

Choline Deficiency and PC Synthesis—Exogenous choline that enters hepatocytes is rapidly phosphorylated to phosphocholine that is subsequently converted into CDP-choline by the action of CT in the rate-limiting step of this biosynthetic pathway (47). In the final step of the pathway, CDP-choline is converted into PC. Based on previous reports in the literature, we had anticipated that reduction in the exogenous supply of choline would markedly decrease the pool size of phosphocholine in the hepatocytes/liver, and thereby decrease the rate of PC synthesis from this pathway (18, 22, 46). Surprisingly, however, our data showed that the rate of PC synthesis from the CDP-choline pathway was not decreased by choline deficiency, but instead appeared to be increased. To our knowledge, in none of the previous studies on choline deficiency was the rate of PC synthesis from choline directly compared in CD and CS hepatocytes.

As we anticipated, however, the concentration of phosphocholine was reduced in CD, compared with CS, hepatocytes, although the reduction was relatively small. This observation provides a likely explanation for why the rate of PC synthesis via the CDP-choline pathway was not reduced by choline deficiency. The Km of CT for phosphocholine in rat liver is ~0.18 mM (51). As shown in Fig. 9A, the concentration of phosphocholine measured in all experiments in both CS and CD hepatocytes was severalfold higher than 0.2 mM. Although the concentration of phosphocholine varied among experiments, within each experiment the phosphocholine concentration was consistently lower in CD hepatocytes than in CS hepatocytes. However, in the CD hepatocytes used in our studies, the pool of phosphocholine was not reduced to a level that would have become rate-limiting for this biosynthetic pathway. Instead, the amount of CT protein and activity associated with membranes was increased in CD, compared with CS, livers, thereby allowing an increased flux of phosphocholine through the pathway and accounting for the apparent increase in the rate of PC synthesis.

As an additional regulatory mechanism for maintaining PC levels in the hepatocytes in the face of a reduced supply of exogenous choline, the amounts of PEMT protein and activity were also increased by the CD diet. Similar increases in PEMT expression have previously been noted in livers of rats fed a CD diet (49, 50). The importance of the PEMT pathway for PC synthesis in the liver was recently highlighted by experiments performed in Pemt–/– mice. When Pemt–/– mice were fed a CD diet for 3 days, the PC content of the liver decreased by 50% and liver failure occurred, whereas, in Pemt–/– mice fed a CS diet, the PC content and liver function were normal (52). We speculate that, as compensation for the reduced exogenous supply of choline, PC generated from the methylation pathway might be available as a source of choline/phosphocholine that could be released by the action of phospholipases C or D on PEMT-derived PC. In addition, an increased expression of PEMT in the CD livers might also be expected to increase the rate of VLDL secretion because previous experiments in our laboratory have shown that transfection of rat hepatoma cells (which normally lack PEMT) with a cDNA encoding PEMT increases VLDL secretion (34). Interestingly, however, studies in our laboratory have demonstrated that a 25-fold overexpression of CT activity in rat hepatoma cells does not increase VLDL secretion.2

Conclusion—The experiments presented herein demonstrate that VLDL secretion from murine hepatocytes is not compromised by deprivation of exogenous choline for an extended period. In addition, and contrary to commonly held assumptions, a deficiency of exogenous choline in murine hepatocytes does not decrease the rate of PC synthesis from the CDP-choline pathway. We propose that these observations can be explained by the pool size of phosphocholine not being reduced to a level that would limit the rate of the reaction catalyzed by CT, the rate-limiting step in this biosynthetic pathway. The present studies also emphasize that the two pathways of PC synthesis in the liver, the CDP-choline pathway and the PEMT pathway, are exquisitely regulated to ensure that PC homeostasis is maintained. Even though the CD diet did not markedly diminish hepatic PC levels or the rate of PC synthesis in hepatocytes, and only modestly reduced the level of phosphocholine, a signal indicating that the supply of choline was compromised must have been transmitted so that the activities of CT and PEMT activity were compensatorily increased to maintain normal levels of PC. These regulatory mechanisms probably exist so that at least one important function of hepatocytes, namely VLDL secretion, is not impaired during periods of dietary insufficiency of choline, such as might occur in animals in the wild during starvation.

More insight into the requirement of the CDP-choline pathway for hepatic lipoprotein secretion is likely to be revealed when mice are generated in which the genes encoding CT{alpha} and/or CT{beta} have been disrupted, perhaps specifically in the liver.


   FOOTNOTES
 
* This work was supported in part by a grant from the Heart and Stroke Foundation of Alberta, Northwest Territories and Nunavut. 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

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

** To whom correspondence should be addressed: 328 HMRC, University of Alberta, Edmonton, Alberta T6G 2S2, Canada. Tel.: 780-492-7250; Fax: 780-492-3383; E-mail: jean.vance{at}ualberta.ca.

1 The abbreviations used are: CD, choline-deficient; apo, apolipoprotein; CS, choline-supplemented; PC, phosphatidylcholine; CT, CTP: phosphocholine cytidylyltransferase; PEMT, phosphatidylethanolamine N-methyltransferase; TG, triacylglycerol; VLDL, very low density lipoprotein. Back

2 Y. Zhao and D. E. Vance, unpublished experiments. Back



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