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Department of Biochemistry and CIHR Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, AB, T6G 2S2, Canada
4To whom correspondence should be addressed. E-mail: dennis.vance{at}ualberta.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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KEY WORDS: • phosphatidylcholine • choline deficiency • phosphatidylethanolamine-N-methyltransferase knockout mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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) is the most abundant phospholipid in mammalian cellular membranes. In all nucleated cells, PC is made primarily through the cytidinediphosphocholine (CDP-choline) pathway (1
). In addition to its important role in signal transduction as a source of lipid second messengers (2
), PC has an important structural role in membranes and lipoproteins. In hepatic tissue, an alternative pathway exists to synthesize PC; this pathway utilizes phosphatidylethanolamine N-methyltransferase (PEMT), which converts phosphatidylethanolamine to PC via three sequential methylation events (3
).
There are several possible reasons why hepatic cells would maintain two pathways for PC synthesis. One possibility is that PEMT activity is necessary to maintain levels of endogenous choline, which may have several metabolic fates, including the biosynthesis of acetylcholine and as a source for betaine (4
), an important source of methyl groups for other metabolic pathways. Other studies have suggested that PEMT-derived PC may be preferentially secreted with lipoproteins (5
). A recent study, utilizing the Pemt-/-mouse (6
) has suggested that the PEMT gene may have been conserved during evolution as a protective mechanism to provide PC when dietary choline is insufficient during such times as starvation, pregnancy or lactation (7
).
Pemt-/-mice fed a choline-deficient (CD) diet developed severe lipid pathology after 3–4 d of consuming the CD diet (7
), had a significant decrease in hepatic and plasma PC levels and had an increase in hepatic triacylglycerol (TG) levels. Pemt-/-mice fed a choline-supplemented (CS) diet did not display liver damage and had normal hepatic and plasma PC levels as well as normal hepatic TG levels (7
). Hence, choline and/or PC is necessary for survival of mice. In this study we investigated whether the liver damage induced in Pemt-/- mice by the CD diet could be reversed by the addition of dietary choline.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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PC and TG standards were purchased from Avanti Polar Lipids (Alabaster, AL). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) detection kits were obtained from Sigma Chemical (St. Louis, MO). The CD semipurified diet6 was obtained from ICN (Cosa Mesa, CA). All other chemicals and reagents were obtained from standard commercial sources.
Care and feeding of mice.
The Pemt-/-mouse (6
) colony was maintained by homozygous breeding; the mice had a mixed genetic background of 129/J and C57bl/6 (6
). At 8 wk of age, the mice were placed in wire-bottomed cages with no bedding and consumed, ad libitum, the CD purified diet for 4 d, a CS diet (CD diet + 0.4% choline chloride) for 4 d, or the CD diet for 3 d and the CS diet for 1 d (CD/CS). The only difference in the diets was the addition of choline. Normal mouse nonpurified diet contains 0.4% choline. On d 4 of the study, plasma was obtained after collection of blood from a leg vein. The mice were deprived of food the night of d 4 and killed on d 5; blood and tissues were collected and processed as described below. Mice were anesthetized by inhalation of Metavane. All procedures were preformed with the approval of the University of Alberta Animal Welfare Committee.
Determination of PC, TG and protein.
Hepatic tissue was homogenized in 5 mL of homogenization buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol and 0.1 mmol/L phenylmethylsulfonyl fluoride). Lipids from liver tissue (1 mg of protein) or plasma (100 µL), were extracted (8
) and separated by TLC on Silica Gel G60 plates. For analysis of PC and TG, phospholipids were first separated with chloroform/methanol/acetic acid/formic acid/water (70:30:12:4:1) as a developing solvent. The solvent was allowed to migrate halfway up the TLC plate, the plate was dried and the neutral lipids were separated by developing in heptane/diisopropyl ether/acetic acid (60:40:4) to the top of the plate. After visualization with iodine vapor, the bands of interest were scraped and analyzed. PC mass was determined by measuring the phosphorous content (9
), whereas TG mass was determined using the hydroxylamine method (10
). Protein concentrations were determined using the Coomasie Plus protein protocol from Pierce (Rockford, IL), which is based on the Bradford assay (11
). Bovine serum albumin was used as a standard.
Measurement of plasma aminotransferase activity.
Blood was collected in the presence of EDTA, from a leg vein on d 4 of the study or from the inferior vena cava on d 5 of the study and separated by centrifugation (1000 x g for 5 min). AST and ALT activities were measured as previously described (12
,13
). The data are presented as Sigma Frankel units/mL, with one Sigma Frankel unit of AST and ALT equal to the formation of 4.82 µmol/L glutamate/min at pH 7.5 and 25°C.
Statistical analysis.
Student’s t test was performed between the two groups analyzed, as indicated. Differences were considered significant at P < 0.05.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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We investigated whether the addition of dietary choline could rescue the liver damage in Pemt-/-mice after consumption of the CD diet. Pemt-/-mice fed the CD diet for 4 d appeared indolent and uncoordinated; they were terminated at 5 d to avoid distress. Livers from these mice were enlarged and distinctly paler in color compared with livers from CS-fed Pemt-/-mice. Pemt-/-mice fed the CS diet for 4 d appeared healthy and had normal livers. Pemt-/-mice fed the CD diet for 3 d and then the CS diet for 1 d (CD/CS) showed signs of indolence and lack of coordination at the start of d 4, but had recovered on d 5 after consuming the CS diet for 1 d. The livers from the CD/CS-fed Pemt-/-mice appeared normal and were similar to the livers from Pemt-/-mice fed the CS diet for 4 d.
Elevated plasma AST and ALT activities are hallmarks of liver damage. Mice fed the CS diet had the anticipated low AST and ALT activities. Mice fed the CD diet for 4 d had elevated AST and ALT activities compared with CS-fed Pemt-/-mice (Fig. 1
). However, Pemt-/-mice fed the CD/CS diet had AST and ALT levels, on the day of termination, that were little different from those of CS-fed Pemt-/-mice. We expected the AST and ALT activities from CD/CS fed mice to be lower than the activities from CD fed Pemt-/-mice based on the appearance of their livers.
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, inset). AST and ALT activities were higher on d 4 in the Pemt-/-mice fed the CD diet compared with the day of killing. This was most likely due to the initial release of AST and ALT into the plasma due to liver damage. However, AST and ALT were still elevated significantly in the CD-fed Pemt-/-mice compared with the CS- and CD/CS-fed Pemt-/-mice on d 5 (P < 0.005). Taken together, these data indicate that liver damage had occurred or was starting to occur in Pemt-/-mice fed the CD diet for 3 d. This liver damage would be fatal if the CD diet were maintained but can be reversed by dietary choline. Dietary choline restores hepatic PC and TG to normal levels in Pemt-/-mice fed the CD diet.
Because the liver damage induced by the CD diet in Pemt-/-mice could be reversed by the addition of dietary choline for 1 d, we analyzed the levels of hepatic PC and TG. Hepatic PC decreased in Pemt-/-mice fed the CD diet compared with Pemt-/-mice fed the CS diet as previously reported (Fig. 2
) (7
). In contrast, hepatic PC concentrations in CD/CS-fed Pemt-/-mice were not lower than PC concentrations in Pemt-/-mice fed the CS diet. These data indicate that the addition of choline in the diet for only 1 d can increase hepatic PC concentrations to above normal conditions.
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) showed previously that hepatic TG levels increase in Pemt-/-mice fed a CD diet. In this study, hepatic TG levels in CD fed mice increased 
eightfold compared with mice fed the CS diet (Fig. 2)
. This is a greater increase than previously reported (7
) but was most likely due to a difference in the composition of the CD diets. The diet used in this study contained safflower oil. The diet used in Walkey’s work was a nonpurified diet that contained soybean oil. TG levels decreased 66% in Pemt-/-mice fed the CD/CS diet compared with mice fed the CD diet (P < 0.005), although levels were still slightly higher than in mice fed the CS diet. This indicates that the addition of dietary choline to the diet begins to restore TG levels to normal in mice that were previously fed a choline-free diet. These results were expected because we and others (14
,15
) have shown that TG levels increase in cells in culture when de novo phospholipid synthesis is inhibited. The increase in TG may be due to the diversion of newly synthesized diacylglycerol into the TG pool. When the CD diet is consumed, de novo synthesis of PC is inhibited in Pemt-/-mice and the increase in TG levels is observed (Fig. 2)
. When choline is introduced into the diet, as in the CD/CS-fed Pemt-/-mice, the de novo pathway for PC biosynthesis is stimulated (Fig. 2)
and TG levels decrease. An additional explanation for an increase in hepatic TG is that VLDL secretion may be impaired, which might be indicated by a decrease in serum PC levels. However, because most of the PC in plasma is associated with HDL, and not VLDL or LDL, we think decreased secretion of VLDL may be quantitatively less important than the inhibition of PC synthesis in the liver. Plasma PC concentrations are restored by dietary choline in Pemt-/-mice.
Pemt-/-mice fed the CD diet had lower concentrations of PC in plasma than CS-fed mice (Fig. 3
). This was probably due to the decrease in the hepatic pool of PC found in these mice. Plasma PC levels were restored in the CD/CS fed mice (Fig. 3)
. On the basis of the results in Figure 2
, which showed that hepatic PC levels increased when choline was restored to the diet, normal plasma PC is not a surprising result. Pemt-/-mice apparently are able to produce enough PC to restore the levels in the plasma.
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) demonstrated that the CD diet induced liver damage in Pemt-/-mice. However, the reversibility of the damage was not examined.
We demonstrated that the liver damage induced by the CD diet in Pemt-/-mice can be reversed by the readdition of dietary choline after only 1 d. Pemt-/-mice fed the CD diet for 3 d had high plasma aminotransferase activities on d 4 (Fig. 1)
. The addition of dietary choline, to levels found in a nonpurified diet, dramatically decreased the plasma aminotransferase activity to control (CS-fed Pemt-/-mice) levels. In addition, the mice appeared healthier after the addition of choline and their livers appeared normal. Furthermore, we found that hepatic PC and TG levels (Fig. 2)
as well as plasma PC levels (Fig. 3)
were restored to normal levels.
Inhibition of PC biosynthesis via a temperature-sensitive mutation in CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary cells (16
) or by removal of choline from the medium of SV-40 immortalized CWSV-1 rat hepatocytes (17
) results in cell death via apoptosis. In neither of these studies was there an attempt to reverse the process. Our results are consistent with the initiation of an apoptotic process in the livers of the CD mice that was reversed after 3 d even though there was substantial liver damage as assessed by plasma aminotransferase activities. The rapidity of the reversal of the liver damage induced by the CD diet in Pemt-/-mice may not be surprising based upon the rapid onset of the liver damage in these mice. The results show the importance of PC and choline levels in maintaining hepatic homeostasis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Postdoctoral Fellow of the Alberta Heritage Foundation for Medical Research.
3 Medical Scientist of the Alberta Heritage Foundation for Medical Research.
5 Abbreviations used: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CD, choline deficient; CDP-choline, cytidinediphosphocholine; CS, choline supplemented; PC, phosphatidylcholine; PEMT, phosphatidylethanolamine-N-methyltransferase; TG, triacylglycerol.
6 CD diet contained (g/kg): vitamin-free casein, 100; alpha protein, 100; sucrose, 510; alphacel nonnutritive bulk, 50; safflower oil, 100; cornstarch, 100; Wesson salt mix, 40 (consisting of calcium carbonate, 210; copper sulfate, 0.39; ferric phosphate, 14.7; magnesium sulfate, 90; potassium aluminum sulfate, 0.09; potassium chloride, 120; potassium phosphate monobasic, 310; potassium iodide, 0.05; sodium chloride, 105; sodium fluoride, 0.57; tricalcium phosphate, 149; manganese sulfate·H2O, 0.15); zinc chloride, 0.02; chromium potassium sulfate·12H2O, 0.055; sodium selenite, 0.001; plus ICN vitamin diet fortification mixture (1 kg/100 lbs) containing (g/kg unless stated otherwise): vitamin A acetate, 1.8; vitamin D, 0.125; dl-
-tocopherol acetate, 22; ascorbic acid, 45; inositol, 5 g/mg; menadione, 2.25; p-aminobenzoic acid, 5; niacin, 4.25; riboflavin, 1; pyridoxine hydrochloride, 1; thiamine hydrochloride, 1; calcium panothenate, 3; biotin, 0.02; folic acid, 0.09; and vitamin B-12, 0.00135.
Manuscript received 3 May 2001. Revision accepted 28 September 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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1. Kennedy, E. P. (1986) The biosynthesis of phospholipids. Op-den-Kamp, J.A.F. Roelofsen, B. Wirtz, K.W.A. eds. Lipids and Membranes: Past Present and Future 1986:171-206 Elsevier Sciences Publishers B.V Amsterdam, The Netherlands. .
2. Exton, J. H. (1994) Phosphatidylcholine breakdown and signal transduction. Biochim. Biophys. Acta 1212:26-42.[Medline]
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4. Zeisel, S. H. & Blusztajn, J. K. (1994) Choline and human nutrition. Annu. Rev. Nutr. 14:269-296.[Medline]
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Vance, J. E. & Vance, D. E. (1986) Specific pools of phospholipids are used for lipoprotein secretion by cultured rat hepatocytes. J. Biol. Chem. 261:4486-4491.
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Walkey, C. J., Donohue, L. R., Bronson, R., Agellon, L. B. & Vance, D. E. (1997) Disruption of the murine gene encoding phosphatidylethanolamine-N-methyltransferase. Proc. Natl. Acad. Sci. U.S.A. 94:12880-12885.
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Walkey, C. J., Yu, L., Agellon, L. B. & Vance, D. E. (1998) Biochemical and evolutionary significance of phospholipid methylation. J. Biol. Chem. 273:27043-27046.
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Sundler, R., Akesson, B. & Nilsson, A. (1974) Effect of different fatty acids on glycerolipid synthesis in isolated rat hepatocytes. J. Biol. Chem. 249:5102-5107.
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10. Snyder, F. & Stephens, N. (1959) A simplified spectrophotometric determination of ester groups in lipids. Biochim. Biophys. Acta 34:244-245.[Medline]
11. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[Medline]
12. Tonhanzy, N. E., White, N. G. & Umbreit, W. W. (1950) A rapid method for the estimation of glutamic-aspartic transaminase in tissues and its application to radiation sickness. Arch. Biochem. Biophys. 28:36-40.
13. Amador, E. & Wacker, W.E.C. (1962) Serum glutamic-oxalacetic transaminase activity—a new modification and an analytical assessment of current assay techniques. Clin. Chem. 8:343-359.[Abstract]
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Waite, K. A. & Vance, D. E. (2000) Why expression of phosphatidylethanolamine N-methyltransferase does not rescue Chinese hamster ovary cells that have an impaired CDP-choline pathway. J. Biol. Chem. 275:21197-21202.
15. Jackowski, S., Wang, J. & Baburina, I. (2000) Activity of the phosphatidylcholine biosynthetic pathway modulates the distribution of fatty acids into glycerolipids in proliferating cells. Biochim. Biophys. Acta 1483:310-315.
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Cui, Z., Houweling, M., Chen, M. H., Record, M., Chap, H., Vance, D. E. & Tercé, F. (1996) A genetic defect in phosphatidylcholine biosynthesis triggers apoptosis in Chinese hamster ovary cells. J. Biol. Chem. 271:14668-14671.
17. Albright, C. D., Liu, R., Bethea, T. C., DaCosta, K.-A., Salganik, R. I. & Zeisel, S. H. (1996) Choline deficiency induces apoptosis in SV40-immortalized rat hepatocytes in culture. FASEB J 10:510-516.[Abstract]
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different from d-4 data (inset), P < 0.005.
