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Originally published In Press as doi:10.1194/jlr.M700278-JLR200 on June 26, 2007

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Journal of Lipid Research, Vol. 48, 2058-2064, September 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

The phosphatidylethanolamine N-methyltransferase pathway is quantitatively not essential for biliary phosphatidylcholine secretion

Henkjan J. Verkade1,*, Rick Havinga*, David J. Shields2,{dagger}, Henk Wolters*, Vincent W. Bloks*, Folkert Kuipers*, Dennis E. Vance{dagger} and Luis B. Agellon{dagger}

* Pediatric Gastroenterology/Research Laboratory Pediatrics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
{dagger} Canadian Institutes of Health Research Group on Molecular and Cell Biology of Lipids and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada

Published, JLR Papers in Press, June 26, 2007.

2 Present address of D. J. Shields: Moores UCSD Cancer Center, 3855 Health Sciences Drive, #0803, La Jolla, CA 92093-0803. Back

1 To whom correspondence should be addressed. e-mail: h.j.verkade{at}med.umcg.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The phosphatidylethanolamine N-methyltransferase (PEMT) pathway of phosphatidylcholine (PC) biosynthesis is not essential for the highly specific acyl chain composition of biliary PC. We evaluated whether the PEMT pathway is quantitatively important for biliary PC secretion in mice under various experimental conditions. Biliary bile salt and PC secretion were determined in mice in which the gene encoding PEMT was inactivated (Pemt–/–) and in wild-type mice under basal conditions, during acute metabolic stress (intravenous infusion of the bile salt tauroursodeoxycholate), and during chronic metabolic stress (feeding a taurocholate-containing diet for 1 week). The activity of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of PC biosynthesis via the CDP-choline pathway, and the abundance of multi-drug-resistant protein 2 (Mdr2; encoded by the Abcb4 gene), the canalicular membrane flippase essential for biliary PC secretion, were determined. Under basal conditions, Pemt–/– and wild-type mice exhibited similar biliary secretion rates of bile salt and PC (~145 and ~28 nmol/min/100 g body weight, respectively). During acute or chronic bile salt administration, the biliary PC secretion rates increased similarly in Pemt–/– and control mice. Mdr2 mRNA and protein abundance did not differ between Pemt–/– and wild-type mice. The cytidylyltransferase activity in hepatic lysates was increased by 20% in Pemt–/– mice fed the basal (bile salt-free) diet (P < 0.05). We conclude that the biosynthesis of PC via the PEMT pathway is not quantitatively essential for biliary PC secretion under acute or chronic bile salt administration.

Supplementary key words biliary lipids • bile salts • phosphatidylcholine biosynthesis • liver • cholesterol


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The liver secretes large amounts of phosphatidylcholine (PC) into plasma, as a surface component of lipoproteins, and into bile. Previously, we estimated that the hepatic secretion of PC into lipoproteins was as quantitatively important as that into bile (1). Biliary PC secretion accounts for a large amount of PC expenditure, with an estimated daily amount equivalent to the entire PC content of the liver (2) or ~70% of hepatic phospholipid content (3). Bile PC is specific in its acyl chain composition, containing predominantly C16:0 fatty acid at the sn-1 position and C18:1 or C18:2 fatty acid at the sn-2 position (4, 5). The liver has two pathways available for PC biosynthesis: via the CDP-choline pathway (de novo from choline) or via the phosphatidylethanolamine N-methyltransferase (PEMT) pathway (three successive methylations of phosphatidylethanolamine catalyzed by a single enzyme, PEMT). Previously, we demonstrated in PEMT-deficient (Pemt–/–) mice that the acyl chain specificity of bile PC was independent of its biosynthetic origin (5). However, it is not known whether PEMT is quantitatively essential for biliary PC secretion.

It is unclear whether or not the amount of PC secreted into bile is influenced by the route or rate of hepatic PC biosynthesis. Robins and Armstrong (6) found that supplementation of the diet with choline increased the biliary PC secretion rate in rats. It was suggested that dietary choline increased hepatic PC synthesis, which then became available for biliary secretion. In contrast, however, LeBlanc et al. (7) did not observe an increased biliary PC secretion rate in rats fed a choline-supplemented diet. The availability of the Pemt–/– mouse strain uniquely allows us to address the contribution of hepatic PC biosynthesis to the amount of PC secreted into bile. The secretion of PC into bile is deleterious for the liver and is lethal upon the inhibition of PC biosynthesis via both the PEMT and CDP-choline pathways (through inactivation of the Pemt gene and withdrawal of dietary choline, respectively) (8). Prevention of biliary PC secretion, through inactivation of the PC-specific flippase multi-drug resistant protein 2 (Mdr2; encoded by the Abcb4 gene), enables Pemt–/– mice to survive the inhibition of de novo PC synthesis (8, 9). In this study, we addressed whether the PEMT pathway is of quantitative importance for biliary PC secretion, particularly under conditions of metabolic stress, such as acute or chronic bile salt administration. We compared biliary PC secretion rates in Pemt–/– and wild-type (control) mice under basal conditions, during acute metabolic stress [intravenous tauroursodeoxycholate (TUDC) infusion], and during chronic metabolic stress (feeding a taurocholate-supplemented diet).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and diets
Experiments involving the use of animals were performed according to protocols approved by the University of Alberta Health Sciences Animal Welfare Committee and in accordance with the guidelines established by the Canadian Council on Animal Care. Pemt–/– and Pemt+/+ (wild-type) mice were maintained by homozygous breeding, given free access to chow and water, and maintained in a temperature-controlled environment under a reverse 12 h light/dark cycle. The Pemt–/– and wild-type mouse colonies have a mixed genetic background of 129/Ola and C57BL/6J. The semisynthetic (control) diet (No. 901387; ICN Biomedicals, Montreal, Quebec, Canada) contained 0.4 wt% choline chloride and 0.05 wt% cholesterol. The major species of fatty acids in the control diet were C16:0 (24%), C18:0 (16%), C18:1 (39%), C18:2 (9%), and C20:4 (0.5%). In another experiment, Pemt–/– and wild-type mice were exposed to chronic metabolic stress in the form of feeding the same diet supplemented with sodium taurocholate (0.5 wt%; Sigma-Aldrich Canada, Ltd., Oakville, Ontario, Canada) for 1 week.

Experimental design
To study biliary secretion rates, the gallbladders of Pemt–/– and wild-type mice (fed the control or the chronic metabolic stress diet) were cannulated after distal ligation of the common bile duct under ketamine/xylazine and diazepam anesthesia. During the same procedure, the mice also received a jugular vein catheter. For 30 min, bile was collected in two 15 min fractions, after which the mice were infused with TUDC (43 mM TUDC in phosphate-buffered saline, pH 7.4) through the jugular vein. The TUDC dose administered to each mouse was increased stepwise: 150 nmol/min for 30 min, 300 nmol/min for 30 min, 450 nmol/min for 30 min, and 600 nmol/min for 60 min. The bile salt infusion in mice fed the basal diet was extended for another 60 min at a dose of 750 nmol/min. During the infusion period, the mice were kept under anesthesia and bile was collected in 15 min fractions, except for the 60 min infusion dosages, for which bile was collected in 10 min fractions. Body temperature was maintained by placing the mice in an incubator during the experiment. Bile flow was determined gravimetrically, assuming a density of 1 g/ml bile.

Separate Pemt–/– and wild-type mice were used to measure CTP-phosphocholine cytidylyltransferase (CT) activity and hepatic Mdr2 mRNA and protein contents. Mice fed the control or chronic metabolic stress diet were euthanized by cardiac puncture under anesthesia. Immediately thereafter, the liver was excised, divided for the different analyses, frozen in liquid nitrogen, and stored at –70°C until analysis.

Biochemical analyses
The total bile salt and cholesterol concentrations in bile were determined using commercial diagnostic kits based on 3{alpha}-hydroxysteroid dehydrogenase for total bile salts and cholesterol oxidase for unesterified cholesterol. PC concentration was determined by a choline oxidase assay for PC (Wako Chemical USA, Inc., Richmond, VA). The results from the inorganic phosphorous and choline concentrations in the bile samples, as determined by the method of Bartlett (10) and the choline oxidase-based assay, respectively, were similar.

For the determination of hepatic CT activity, total membranes were isolated by centrifugation of the liver homogenates at 600 g for 10 min to pellet unbroken cells and nuclei. The supernatant was centrifuged at 100,000 g for 1 h. The membrane pellet, containing membrane-associated CT, was resuspended in homogenization buffer (11), and the supernatant contained the soluble fraction. CT activity was measured in the homogenate, as well as in soluble and microsomal fractions, in the presence of PC/oleate vesicles, as described previously (11).

Amount of Mdr2 protein in hepatic plasma membranes
Plasma membranes were isolated as described previously (12). Liver tissue (~2 g) pooled from three or four wild-type or Pemt–/– mice fed the control diet (i.e., without taurocholate supplementation) or fed for 1 week the same diet supplemented with taurocholate were used. Protein concentrations were determined according to Lowry et al. (13). Relative enrichments of Na+/K+-ATPase as a marker enzyme for the plasma membrane fraction (i.e., the specific activity of this enzyme in the isolated plasma membrane preparation divided by its activity in the homogenate) were used to determine the degree of purification of the isolated membranes in the different experimental groups (12).

Plasma membranes equivalent to 7.5 µg of protein were electrophoresed through a 4–15% polyacrylamide gel at 100 V. The proteins were electrophoretically transferred onto a nitrocellulose filter (Amersham, Little Chalfont, UK) by tank blotting. Ponceau S staining was performed to check equal protein transfer. The filters were blocked for 1 h at 4°C in a solution of Tris-buffered saline with 0.1% Tween and 4% skim milk powder, pH 7.4. The blots were incubated with the primary antibody anti-MDR3 (kindly provided by Dr. J. Schepers, Amsterdam) cross-reacting with murine Mdr2 (14) at a 1:1,000 dilution overnight at room temperature and washed; immune complexes were then detected using horseradish peroxidase-conjugated goat anti-mouse IgG2b (Southern Biotechnology Associated, Birmingham, AL) by the ECL Western blotting kit (Amersham). Protein density was determined by scanning the blots using an Image Master VDS system (Pharmacia Biotech, Uppsala, Sweden). Blots were run after loading of equal protein amounts and after correction for plasma membrane enrichments (Na+K+-ATPase).

RNA isolation and measurement of mRNA levels by real-time PCR
Total RNA was isolated from ~30 mg of liver tissue by the TRIzol method (Invitrogen, Paisley, UK). RNA was converted to single-stranded cDNA by a reverse transcription procedure with Moloney murine leukemia virus-RT (Roche Diagnostics, Mannheim, Germany) according to the protocol of the manufacturer using random primers. cDNA levels were measured by real-time PCR using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA).

For the PCR amplification studies, an amount of cDNA corresponding to 30 ng of total RNA was amplified using the qPCR core kit (Eurogentec, Seraing, Belgium) essentially according to the protocol of the manufacturer and optimized for amplification of the particular gene using the appropriate forward and reverse primers (Invitrogen) and a template-specific 3'-TAMRA (6-carboxytetramethylrhodamine)/5'-FAM (6-carboxyfluorescein)-labeled double dye oligonucleotide probe (Eurogentec). The primers used for ß-actin and Mdr2 mRNA quantitation were identical to those described previously (15). Calibration curves were run in the same experiments. The data obtained were processed using the ABI Sequence Detector software (version 1.6.3; Applied Biosystems). All quantified expression levels were within the linear part of the calibration curves and were calculated using these curves. PCR results were normalized to ß-actin mRNA levels.

Calculations and statistics
All values are given as means ± SD for the number of animals indicated. Results obtained in Pemt–/– and wild-type mice were analyzed by the Mann-Whitney U-test, using SPSS 10.1 software, with P < 0.05 as the threshold for significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Bile formation in mice under basal physiological conditions
The deficiency of the PEMT pathway did not significantly affect body weight or liver weight (absolute or relative) in mice on the basal diet (Table 1 ). In addition, bile flow and biliary secretion rates of bile salts and phospholipids were similar in wild-type and Pemt–/– mice fed the basal diet during the first 30 min after interruption of the enterohepatic circulation by gallbladder cannulation (Table 1). Accordingly, the PC-to-bile salt molar ratio was similar in bile of wild-type and Pemt–/– mice (0.19 ± 0.11 and 0.22 ± 0.07, respectively). The cholesterol secretion rate was higher in the latter.


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  		TABLE 1. Liver and bile parameters in wild-type and Pemt–/– mice fed the control diet

 
The capacity of biliary PC secretion can be determined by administration of a bile salt in stepwise increasing doses (3). Figure 1 shows the relationship between the biliary secretion rates of bile salts and of phospholipid or cholesterol in wild-type and Pemt–/– mice during the infusion of TUDC in a stepwise increasing dose. Bile salt secretion rates under these conditions were similar between Pemt–/– and wild-type mice. At any bile salt secretion rate, Pemt–/– mice secreted similar amounts of PC into the bile as wild-type mice. Similar to the results on biliary PC secretion, biliary cholesterol secretion rates were similar in Pemt–/– and wild-type mice (Fig. 1).


Figure 1
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  		Fig. 1. Biliary lipid secretion during acute metabolic stress. Relationships between biliary secretion rates of bile salts (BS) and phosphatidylcholine (PC; top panel) or cholesterol (bottom panel) in wild-type mice (open symbols) and phosphatidylethanolamine N-methyltransferase-deficient mice (Pemt–/–) (closed symbols) fed the control diet during intravenous infusion of tauroursodeoxycholate (TUDC) in step-wise increasing dosages. Each dot represents an individual bile sample during the course of the experiment. As detailed in Materials and Methods, a maximum of 20 bile samples were collected per mouse. The relationships can be characterized by the following equations: phospholipids, y = 36.8Ln(x) – 143.2, r2 = 0.82 (wild type; n = 8) and y = 35.2Ln(x) – 119.7, r2 = 0.66 (Pemt–/–; n = 9); cholesterol, y = 5.2Ln(x) – 19.9, r2 = 0.70 (wild type; n = 8) and y = 4.6Ln(x) – 15.7, r2 = 0.61 (Pemt–/–; n = 9).

 
Mdr2 P-glycoprotein is known to function as a PC flippase in the bile canalicular membrane, and its expression can be rate-limiting for biliary PC secretion (16). Mdr2 mRNA abundance and Mdr2 protein content in hepatic plasma membrane fractions were similar in Pemt–/– and wild-type mice (Fig. 2 ). Bile flow and biliary bile salt secretion rate were also virtually identical in Pemt–/– and wild-type mice under basal conditions (Table 1), indicating similar bile salt-independent fractions of the bile flow. CT is the rate-limiting enzyme for the CDP-choline pathway of PC biosynthesis. The CT activity in liver homogenates and in microsomal subfractions was 20% higher in Pemt–/– mice compared with controls (P < 0.05) (Table 2 ).


Figure 2
Figure 2
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  		Fig. 2. Hepatic multi-drug-resistant protein 2 (Mdr2) mRNA abundance and Mdr2 protein content in hepatic plasma membrane fractions. A: mRNA abundance of Mdr2 was determined by real-time PCR in liver tissue from wild-type and Pemt–/– mice fed the basal (control) diet or for 1 week the same diet supplemented with 0.5% taurocholate (TC; w/w). PCR results were normalized to ß-actin mRNA levels. B: Protein immunoblots using an anti-MDR3 antibody (cross-reacting against mouse Mdr2) on plasma membranes from wild-type and Pemt–/– mice fed the basal (control) diet or for 1 week the same diet supplemented with 0.5% taurocholate (w/w). Liver tissue was pooled from three or four mice per group. C: Densitometry of Mdr2 protein immunoblots for the four groups, normalized to wild-type mice fed the basal (control) diet (set at 1.0). The results were similar upon loading equal protein amounts or upon correction for plasma membrane enrichments (Na+K+-ATPase). Further experimental details are provided in Materials and Methods. The differences between the groups did not reach statistical significance for Mdr2 mRNA (n = 5 per group) or for Mdr2 protein (n = 3 per group). Values shown are means ± SD.

 

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  		TABLE 2. Cytidylyltransferase activity in livers from wild-type and Pemt–/– mice fed the control diet

 
Bile formation in mice under chronic metabolic stress
Recently, we found that Abcb4–/–;Pemt–/– mice (lacking both Mdr2 and PEMT) can efficiently adapt to chronic metabolic stress in the form of choline deprivation (9). Enhancing biliary PC secretion is another form of metabolic stress, as indicated by the deleterious effects for the liver of the inhibition of PC biosynthesis via the CDP-choline pathway in Pemt–/– mice but not in Abcb4–/–;Pemt–/– mice (8). We investigated whether bile formation differed between wild-type and Pemt–/– mice exposed to bile salt feeding for 1 week. Interestingly, no significant differences in the secretion rates of biliary bile salt, PC, or cholesterol were found between Pemt–/– and wild-type mice after acute interruption of the enterohepatic circulation (Table 3 ). Both in Pemt–/– and wild-type mice, basal bile flow and biliary secretion rates of bile salts, phospholipids, and cholesterol were at least 2- to 4-fold higher during chronic metabolic stress, compared with the condition in which mice were fed the control diet (Table 1). Similar to the observations after acute metabolic stress, the abundance of Mdr2 protein in isolated liver plasma membrane fractions was similar in Pemt–/– and wild-type mice (Fig. 2), as were steady-state Mdr2 mRNA values (Mdr2/ß-actin ratio, 0.99 ± 0.11 and 1.26 ± 0.39, respectively; NS). Under chronic metabolic stress, CT activity was comparable in Pemt–/– and wild-type mice (Table 4 ).


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  		TABLE 3. Hepatic and bile parameters in wild-type and Pemt–/– mice fed a taurocholate-supplemented (0.5 wt%) diet for 1 week

 

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  		TABLE 4. Cytidylyltransferase activity in livers from wild-type and Pemt–/– mice fed a taurocholate-supplemented (0.5 wt%) diet for 1 week

 
Bile flow and biliary secretion rates of bile salts, phospholipids, and cholesterol were also similar when the mice fed the taurocholate-containing diet were exposed to intravenous TUDC infusion in stepwise increasing dosages (Fig. 3 ). TUDC infusion in mice under chronic metabolic stress approximately doubled the biliary bile salt secretion rates, compared with the rates immediately after interruption of the enterohepatic circulation (during the last hour, the secretion rate of bile salt was 1,050 ± 447 and 905 ± 323 nmol/min/100 g, and that of PC was 146 ± 46 and 135 ± 33 nmol/min/100 g, in Pemt–/– and wild-type mice, respectively; NS). Cholesterol secretion showed some variability and was not profoundly different between Pemt–/– and wild-type mice (Fig. 3).


Figure 3
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  		Fig. 3. Biliary lipid secretion during acute metabolic stress in mice pretreated with a taurocholate-containing diet. Relationships between biliary secretion rates of bile salts (BS) and of phosphatidyl-choline (PC; top panel) or cholesterol (bottom panel) in wild-type mice (open symbols) and Pemt–/– mice (closed symbols) fed the challenge diet (taurocholate 0.5%, w/w) for 1 week during intravenous infusion of tauroursodeoxycholate in step-wise increasing dosages. The relationships can be characterized by the following equations: phospholipids, y = 26.5Ln(x) – 39.7, r2 = 0.13 (wild type; n = 7) and y = 12.9Ln(x) + 39.2, r2 = 0.03 (Pemt–/–; n = 7); cholesterol, y = 2.27Ln(x) – 9.5, r2 = 0.22 (wild type; n = 7) and y = 0.67Ln(x) – 0.09, r2 = 0.03 (Pemt–/–; n = 7).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The availability of the Pemt–/– mouse strain permitted the determination of the quantitative requirement of PEMT for PC secretion into bile. The present data on bile secretion rates, in combination with literature data, allowed calculation of the actual fractional turnover of liver PC toward bile secretion. Under basal (physiological) conditions, wild-type mice secreted 9 µmol of PC into bile per day (this study). The total amount of PC in the liver of a wild-type mouse was ~11 µmol (this study). These estimates imply that the daily biliary PC secretion equals ~82% of the total PC present in the liver. The biosynthesis rate of PC in rat liver via the CDP-choline pathway is ~0.35 µmol/h/g liver and that via the PEMT pathway is ~0.11 µmol/h/g liver, determined by 1H-NMR (17). Assuming similar hepatic PC biosynthetic rates in rats and mice, this would account for 11 µmol of PC synthesized daily (for an adult mouse of 25 g body weight, 1 g liver). Based on these calculations, we anticipated that an important quantitative role of PEMT for the repletion of biliary PC would manifest as a decreased capacity to secrete PC into bile.

Interruption of the enterohepatic circulation of wild-type and Pemt–/– mice on a basal diet showed that the biliary secretion rates of bile salts and PC were very similar. This observation suggests that the CDP-choline pathway of PC biosynthesis (i.e., the only pathway for PC biosynthesis available in Pemt–/– mice) can provide sufficient PC for biliary secretion under basal conditions. As demonstrated by the increased enzyme activity of CT, the rate-limiting enzyme for the CDP-choline pathway, the liver of Pemt–/– mice does undergo metabolic adaptation to maintain PC homeostasis (Table 2).

Immediately after interruption of the enterohepatic circulation, the biliary cholesterol secretion was higher in Pemt–/– mice fed the basal diet compared with controls (Table 1), but not after feeding the bile salt-containing diet (Table 3). The TUDC infusion after either diet did not indicate that the capacity to secrete cholesterol differed between the genotypes (Figs. 1, 3). These observations together suggest, in our opinion, that the observed difference (Table 1) does not reflect a physiologically relevant population difference between the genotypes.

To determine the versatility of the metabolic adaptations, an acute metabolic stress was imposed in the form of the administration of TUDC in stepwise increasing doses. Bile salt secretion rates under these conditions increased similarly in Pemt–/– and wild-type mice fed the basal diet (~15-fold). Even at high bile salt secretion rates, Pemt–/– mice secreted similar amounts of PC in the bile as wild-type mice. Also, after the TUDC infusions, the hepatic PC contents were similar in the two genotypes (data not shown). Apparently, the metabolic adaptations under basal conditions in Pemt–/– mice are sufficiently versatile to provide an adequate amount of PC when the demand acutely exceeds the basal PC supply by a factor of seven. Moreover, the PEMT pathway does not seem to be required for repletion of the pool of PC for bile secretion under chronic stress (i.e., dietary bile salt feeding for 1 week) (18, 19). In the taurocholate experiment, the biliary PC secretion rate obtained immediately after interruption of the enterohepatic circulation (Table 3) was in the same range as the maximal secretion rate upon TUDC infusion in Pemt–/– mice fed the basal diet (Fig. 1). TUDC infusion did not further increase PC secretion in either of the two genotypes of taurocholate-fed mice. Based on these observations, we conclude that the PEMT pathway does not regulate the amount of PC secretion in mice under conditions of acute or chronic bile salt administration. Previously, we demonstrated that chronic metabolic stress in Pemt–/– mice induces a wide range of compensatory, PC-conserving processes both intrahepatically and at the interorgan level (9). Because VLDL secretion is a major pathway for PC secretion from the liver, it is reasonable to assume that a decreased loss of PC to lipoprotein secretion would maintain the versatility to cope with demands to supply PC for biliary secretion.

The amount of PC secreted into bile is influenced by several factors (20): 1) the intracanalicular bile salt concentration (21, 22); 2) the hydrophobicity of the intracanalicular bile salts (23); 3) the magnitude of the bile salt-independent fraction of the bile flow (24); 4) the concentration of hydrophilic organic anions (25); 5) the abundance of Mdr2 at the bile canalicular membrane (3, 16, 26); and 6) the lipid composition of the bile canalicular membrane (27–30). Taurocholate feeding did not induce detectable changes in Mdr2 mRNA or protein levels (Fig. 2). Despite the fact that Mdr2 is a farnesoid X receptor target, our present results are consistent with previous mouse studies (including our own) showing that Mdr2 mRNA levels do not change or are only slightly stimulated by bile salt feeding (31–33). Theoretically, one could have hypothesized that PC biosynthesis via the PEMT pathway is an additional, independent factor regulating the amount of PC secreted into bile. Our present results, however, indicate that the PEMT pathway is quantitatively not essential for biliary PC secretion.


   ACKNOWLEDGMENTS
 
The authors thank Sandy Ungarian, Lena Li, and Le Luong for technical assistance. Grant support for these studies was provided by the Canadian Institutes of Health Research, the Royal Netherlands Society for Arts and Sciences (KNAW), and NATO (Collaborative Grant). D.E.V. holds the Canada Research Chair on Molecular and Cell Biology of Lipids and is a Heritage Scientist of the Alberta Heritage Foundation for Medical Research.

Manuscript received September 29, 2006 and in revised form June 14, 2007.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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