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Metabolomics

Phosphatidylethanolamine-N-methyltransferase Activity and Dietary Choline Regulate Liver-Plasma Lipid Flux and Essential Fatty Acid Metabolism in Mice¹

Lipomics Technologies, Incorporated, West Sacramento, CA 95691 and ^* Department of Nutrition, School of Public Health, School of Medicine, University of North Carolina, Chapel Hill, NC 27599-7400

²To whom correspondence should be addressed. E-mail: steve.watkins{at}lipomics.com.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION DISCUSSION LITERATURE CITED

 
Phosphatidylethanolamine-N-methyltransferase (PEMT) catalyzes the methylation of phosphatidylethanolamine to form phosphatidylcholine (PC) and represents one of the two major pathways for PC biosynthesis. Mice with a homozygous disruption of the PEMT gene are dependent on the 1,2-diacylglycerol cholinephosphotransferase (CDP-choline) pathway for the synthesis of PC and develop severe liver steatosis when fed a diet deficient in choline. The present study used quantitative lipid metabolite profiling to characterize lipid metabolism in PEMT-deficient mice fed diets containing varying concentrations of choline. Choline supplementation restored liver, but not plasma PC concentrations of PEMT-deficient mice to levels commensurate with control mice. Choline supplementation also restored plasma triglyceride concentrations to normal levels, but did not restore plasma cholesterol ester concentrations in the PEMT-deficient mice to those equal to control mice. PEMT-deficient mice also had substantially diminished concentrations of docosahexaenoic acid [22:6(n-3)] and arachidonic acid [20:4(n-6)] in plasma, independent of choline status. Thus, choline supplementation rescued some but not all of the phenotypes induced by the knockout. These findings indicate that PEMT activity functions beyond its recognized role as a compensatory pathway for PC biosynthesis and that, in contrast, PEMT activity is involved in many physiologic processes including the flux of lipid between liver and plasma and the delivery of essential fatty acids to blood and peripheral tissues via the liver-derived lipoproteins.

KEY WORDS: • essential fatty acids • lipid metabolism • metabolomics • phosphatidylcholine

Phosphatidylcholine (PC)² is the most abundant phospholipid in mammalian cells; it is synthesized by the transfer of phosphocholine to the sn-3 position of 1,2-diacylglycerol (DAG) via cytidine diphosphate-choline (CDP-choline): 1,2-diacylglycerol cholinephosphotransferase activity (1) or by the methylation of phosphatidylethanolamine (PE) via phosphatidylethanolamine-N-methyltransferase (PEMT) activity (2). Although the production of phosphatidylcholine (PC) via the CDP-choline pathway is dependent on dietary choline, the production of PC by PEMT activity is not, and requires only PE and S-adenosylmethionine (2). The CDP-choline and PEMT pathways operate in concert to maintain normal liver PC concentrations in response to variations in choline and methyl status (2–6). Many agents modulate the rate of liver PC biosynthesis including ethanol (7), estrogen (8), norepinephrine and propranolol (9) and fibrates (10–12), but the effects of these modulations on global lipid metabolism are largely unknown.

Although liver PC concentrations are maintained by both the PEMT and the CDP-choline pathways, recent work demonstrated that PEMT is essential for the incorporation of neutral lipid into VLDL particles in liver cells (13) and for normal concentrations and compositions of VLDL and VLDL components in plasma in mice (14,15). Moreover, PEMT-deficient mice had gender-specific differences in the concentrations of PC, triglyceride (TG) and cholesterol esters (CE) in plasma (15). Thus, it appears that PC synthesized by the two pathways does not fully mix within the cell, and that the two pathways have distinct physiologic roles not confined to compensating for decreased PC concentrations. Several studies evaluated the relative contribution of the PEMT and CDP-choline pathways to liver PC concentration and composition. NMR analysis of liver extracts from rats treated with ¹³C-labeled choline and ethanolamine demonstrated that 30% of liver PC was synthesized by PEMT (16). The composition of liver PC derived from PEMT activity was also shown to differ from that produced by the CDP-choline pathway. PC molecules produced from the CDP-choline pathway were comprised mainly of medium-chain, saturated fatty acids, whereas PC produced via PEMT activity were comprised of longer-chain PUFA (17). These data suggest that changes in PEMT activity can cause changes in lipid metabolism and physiology that are not linked solely to liver PC concentrations. PEMT activity is thus critical to many physiologic processes, but roles for PEMT pathway activity in disease processes have not been extensively studied.

The PEMT-deficient mice used in this study have two selectively disrupted alleles of the pemt2 gene at exon 2 (18), which encodes PEMT; these mice do not express any PEMT activity in liver. Therefore, these mice depend completely on dietary choline intake to meet daily choline requirements. When fed a diet deficient in choline and methionine, PEMT knockout mice developed decreased PC concentrations in hepatic membranes, severe liver damage and die; a choline-supplemented diet prevented this and can reverse hepatic damage if begun early enough (6).

The present study used comprehensive and quantitative lipid metabolome profiling to determine the effects of a disruption of PEMT activity and of varied dietary choline concentrations on structural and energetic lipid metabolism. The quantitative data were evaluated to characterize the effect of PEMT deletion and of choline status on the concentration and composition of individual lipids from liver and plasma, and to determine the role of the PEMT and CDP-choline pathways in determining PC concentration and fatty acid composition.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION DISCUSSION LITERATURE CITED

 
Mice and diets.

PEMT knockout mice were a generous gift from Dr. Dennis Vance, University of Alberta, Edmonton, Canada. All mice were housed in cages in a climate-controlled room (24°C), exposed to light from 0600 to 1800 h daily. Six PEMT -/- and 6 wild-type adult male mice (mixed genetic background of 129/J and C57BL/6), weighing 20–25 g at the start of study, were a fed choline-deficient diet (AIN76A diet without choline: CD diet; Dyets, Bethlehem, PA) plus choline in their drinking water [choline deficient (CD), 0 mmol/L choline chloride; control (CT), 6.4 mmol/L choline chloride; choline supplemented (CS), 28.9 mmol/L choline chloride; n = 6 per group] for 12 d. Mice ingested a mean of 3 mg choline/d in the control group, and 12 mg/d in the supplemented group. Livers and plasma were collected after mice were anesthetized intraperitoneally with 200 mg/kg ketamine (Fort Dodge Animal Health, Fort Dodge, IA) and 16 mg/kg xylazine (Ben Venue Laboratories, Bedford, OH).

Lipid metabolome data.

The lipids from plasma and liver were extracted in the presence of authentic internal standards by the method of Folch et al. (19) using chloroform/methanol (2:1, v/v). Either 25 mg liver tissue or 200 µL plasma was used for each analysis. Individual lipid classes within the extract were separated by preparative TLC as described by Watkins et al. (20). Isolated lipid classes were transesterified in 3 mol/L methanolic-HCl in a sealed vial under a nitrogen atmosphere at 100°C for 45 min. The resulting FAME were extracted with hexane containing 0.05% BHT and prepared for GC by sealing the hexane extracts under nitrogen.

FAME were separated and quantified by capillary GC using a gas chromatograph (Hewlett-Packard model 6890, Wilmington, DE) equipped with a 30 m DB-225MS capillary column (J&W Scientific, Folsom, CA) and a flame-ionization detector as described previously (20).

Data processing and statistics.

Lipid metabolome data were calculated as quantitative data and as mol% data. Quantitative data, expressed as nmol or µmol/g, were assembled in tables as means and SD for each group. Mol% data were calculated as the percentage that each individual fatty acid represented relative to the sum of the concentrations of all fatty acids within that lipid class. The concentration of lipid metabolites in each of the groups was evaluated to determine the effect of choline status and PEMT activity on the lipid metabolome. Additionally, the data were evaluated to determine whether there was an interaction between choline status and PEMT activity. Significant differences in the concentration of lipid metabolites were determined using a two-way ANOVA (NCSS, Kaysville, UT) at an < 0.05.

Compositional data from the study were visualized using the Lipomics Surveyor software system, which creates a "heat-map" graph of the difference between the treated and control data. The Surveyor software system was described in a previous report (21). The Surveyor data can be read as follows: the column headers display the fatty acid and the family of fatty acids present in each lipid class, which are in turn described in the row headers. The lipid classes are grouped by tissue. The heat map displays an increase in each metabolite in a selected treatment group relative to a selected control group as a green square and a decrease in a metabolite as a red square. The brightness of the square indicates the magnitude of the difference, as described in the figure legend. Metabolites not meeting P < 0.05 (assessed by a Student’s t test) are displayed in black.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION DISCUSSION LITERATURE CITED

 
The concentrations of individual lipid classes in liver and plasma are shown in Table 1and Table 2, respectively.³

PEMT-deficient mice fed either CD or CT diets had significantly lower concentrations of PC in liver than control mice fed CD or CT diets, respectively (Table 1). However, PEMT-deficient mice fed the CS diet had PC concentrations that did not differ from those of control mice fed a CS diet (Table 1). Hence, the concentration of PC in PEMT-deficient mice was responsive to choline and PEMT status, and under conditions of choline supplementation, the CDP-choline pathway produced normal liver concentrations of PC. The choline content of the diet did not affect the PC concentration in liver in control mice (Table 1), indicating that the normal hepatic PC concentrations were not increased by supplementary choline, and that PEMT activity compensated for diminished CDP-choline synthesized PC in the choline-deficient state.

Plasma PC concentrations were lower in PEMT-deficient mice relative to control mice fed each of the three diets (Table 2). The concentration of PC in plasma from PEMT-deficient mice fed the CS diet was significantly greater than that in PEMT-deficient mice fed the CD or CT diets, demonstrating that plasma PC concentrations were responsive to choline status (Table 2). The choline-responsive nature of PC in plasma reflected that of PC in the liver; however, in contrast to liver, choline supplementation did not restore plasma PC concentrations to the level of their corresponding wild-type controls. These data demonstrate that although the CDP-choline pathway can compensate for liver PC concentrations, it cannot fully compensate for PEMT deficiency in restoring the concentrations of PC in plasma.

TG and CE metabolism.

PEMT-deficient mice fed all three diets had significantly greater concentrations of TG in liver relative to control mice (Table 1). Feeding the CS diet relative to the CD or CT diets significantly diminished the magnitude of the increased TG. Thus, choline supplementation partially relieved the TG accumulation induced by the PEMT deficiency. These data demonstrate that the accumulation of TG in liver is responsive to both choline and PEMT status. The concentration of TG in plasma was inversely related to the concentration of TG in liver because PEMT-deficient mice fed CD or CT diets had significantly lower plasma TG concentrations (Table 2), relative to their control groups fed the same diet. Additionally, PEMT-deficient mice fed CS diets had plasma TG concentrations that did not differ from those of their wild-type controls (Table 2), indicating that choline supplementation was sufficient to fundamentally restore the normal flux of TG between the liver and plasma.

Livers from PEMT-deficient mice fed all three diets had greater concentrations of CE relative to control mice (Table 1). Similar to TG, the magnitude of the increased CE was lower when the CS diet was fed relative to mice fed CD or CT diets. Thus, the hepatic CE concentrations were responsive to both PEMT and choline status. In plasma, CE concentrations were responsive to choline status in PEMT-deficient mice; increasing dietary choline concentrations increased plasma CE concentrations (Table 2). As with TG concentrations, plasma CE concentrations were inversely related to hepatic CE concentrations, indicating that choline status affected the flux of CE between liver and plasma in PEMT-deficient mice. In contrast to plasma TG concentrations, none of the three diets restored plasma CE concentrations to the levels of control mice.

The dysregulation of PEMT and the depletion of choline from the diet each decreased plasma CE concentrations and concomitantly increased hepatic CE concentrations. Thus, although CE concentrations demonstrated a reciprocal relationship between plasma and liver, CE concentrations in plasma were correlated directly with the concentration of plasma PC (r = 0.86, P < 0.0001). These data demonstrate a relationship between PC metabolism and CE metabolism that is different from that between PC and TG metabolism.

Other lipid classes.

Hepatic PE concentrations in PEMT-deficient mice were altered by the three diets, with CD-fed mice having significantly lower concentrations of PE, and CS-fed mice significantly higher concentrations of PE relative to mice fed CT-diets (Table 1). Thus, there was an accumulation of PE in liver in response to PEMT deficiency, but only under conditions of choline supplementation. Hepatic PE concentrations were not affected by choline status in control mice (Table 1). The concentration of other lipid classes in liver including cardiolipin, free fatty acids and phosphatidylserine + inositol were not altered by either PEMT or choline status (Table 1).

Compositional analysis.

Because the PEMT and CDP-choline pathways use distinct substrates for the synthesis of PC, the compositions of hepatic and plasma PC were analyzed to determine whether liver and plasma lipid compositions were affected by PEMT deficiency. Each of the fatty acids comprising hepatic PC was quantified independently and reported in Figure 1as a mol% composition of total PC fatty acids. PEMT deficiency caused a significant depletion of docosahexaenoic acid [22:6(n-3)] from both hepatic and plasma PC (Fig. 1). Additionally, plasma PC from PEMT-deficient mice was comprised of significantly less stearic acid (18:0) and arachidonic acid [20:4(n-6)] and significantly more linoleic acid [18:2(n-6)] than in control mice, independent of choline status (Fig. 1). The fatty acids 22:6(n-3) and 20:4(n-6) are essential for many biological processes including neural function and eicosanoid metabolism, respectively, and the substantial depletion of these fatty acids from plasma in the PEMT-deficient mice indicates a key role for PEMT in essential fatty acid metabolism.

View larger version (83K): [in this window] [in a new window]   FIGURE 1 The effect of phosphatidylethanolamine-N-methyltransferase (PEMT) and choline status on liver and plasma lipid fatty acid compositions of PEMT-deficient and wild-type mice fed choline-deficient (CD), control (CT) or choline-supplemented (CS) diets for 12 d. The fatty acid composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), cholesterol ester (CE) and triglycerides (TG) are shown. The mean mol% concentration of each fatty acid in each lipid class was compared between the PEMT-deficient mice and wild-type controls. Data are presented in a heat map format (color range bar shown) as described in Materials and Methods. The heat map displays the percentage difference between the concentration of each metabolite in the PEMT-deficient mice and control mice fed the same diet (as shown in the row headers on the left of the graph). Fatty acids that were diminished in the PEMT-deficient mice relative to control mice are displayed in red, whereas fatty acids that were greater in the PEMT-deficient mice relative to control mice are shown in green. Fatty acids that did not differ between the groups (Student’s t test > 0.05) are shown in black.

The composition of plasma CE from PEMT-deficient mice was significantly depleted of 22:6(n-3) and 20:4(n-6) relative to that from control mice (Fig. 1). Hepatic CE 22:6(n-3) and 20:4(n-6) concentrations did not differ.

Composition of hepatic and plasma TG.

Hepatic TG from PEMT-deficient mice was comprised of significantly more 18:0, 22:6(n-3) and long-chain (n-6) PUFA than that from control mice when the mice were fed the CD diets. TG from PEMT-deficient mice fed CD diets contained significantly less 16:0, 16:1(n-7) and 18:1(n-9) than TG from their control mice. Except for a lower 18:1(n-9) concentration, hepatic TG from PEMT-deficient mice fed the CS diet was indistinguishable from the hepatic TG produced by the control mice (Fig. 1).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION DISCUSSION LITERATURE CITED

 
Two pathways, the CDP-choline pathway and the PEMT pathway, synthesize hepatic PC. These pathways use two previously acylated glycerolipids, 1,2-DAG in the CDP-choline pathway and PE in the PEMT pathway, as substrate for the biosynthesis of PC. Because 1,2-DAG and PE have widely dissimilar fatty acid compositions, the PC produced by the CDP-choline and PEMT pathways may also have distinct compositions. Delong et al. (17) presented evidence that hepatic PC synthesized by the PEMT pathway was comprised of more PUFA than PC produced by the CDP-choline pathway, whereas the molecular species of PC synthesized via the CDP-choline pathway were comprised mainly of saturated (e.g., 16:0/18:0) species. Other reports describe no acyl-chain–specific differences between bile PC synthesized exclusively by the CDP-choline pathway relative to PC synthesized by both the CDP-choline pathway and the PEMT pathway (22). This report presents quantitative evidence from PEMT-deficient mice that PEMT activity does in fact produce a PC molecular species that is distinct from the species produced by the CDP-choline pathway, and that PEMT activity is essential to maintain normal concentrations of 18:0 and 22:6(n-3) in hepatic PC and 18:0, 22:6(n-3) and 20:4(n-6) in plasma PC. This indicates that PEMT activity is critical for mobilizing the essential fatty acids 20:4(n-6) and 22:6(n-3) from liver into plasma.

Because PC is by far the most prevalent phospholipid in plasma, factors that influence hepatic PEMT activity influence the concentration of fatty acid components of plasma PC and their distribution to peripheral tissues. The results presented here show that the disruption of PEMT activity significantly diminishes the concentration of the essential fatty acid 22:6(n-3) in both hepatic and plasma PC. This depletion of 22:6(n-3) occurred independently of choline status, demonstrating the key role for PEMT in mobilizing essential fatty acids from the liver and plasma. The disruption of the PEMT pathway also caused an accumulation of 22:6(n-3) in hepatic PE, indicating that the conversion of 22:6(n-3)–containing PE to PC via PEMT activity is the normal pathway for incorporating 22:6(n-3) into PC. These data strongly suggest that PEMT activity is almost solely responsible for the mobilization of 22:6(n-3) into plasma, and factors that modulate PEMT activity may also alter the essential fatty acid status. This may have important implications for neurodevelopment, cardiovascular disease, immune dysfunction and other diseases related to tissue and plasma essential fatty acid concentrations. Furthermore, although the disruption of the PEMT pathway had little effect on the concentration of 20:4(n-6) in hepatic PC, 20:4(n-6) was significantly depleted from plasma PC in the PEMT-disrupted mice independent of choline status. These data indicated a critical role for PEMT in the mobilization of 20:4(n-6) into plasma and suggested that PEMT status may be important in phenotypes influenced by 20:4(n-6)–derived regulatory lipids, including eicosanoids, epoxides and diols.

The depletion of 22:6(n-3) from plasma PC also has secondary effects on essential fatty acid metabolism in plasma. The major carrier of 22:6(n-3) in plasma other than PC, CE, was also significantly depleted of 22:6(n-3). The basis for this depletion was likely a drastic decrease in the concentration of 22:6(n-3) in plasma PC because the enzyme responsible for esterifying cholesterol in plasma, lecithin:cholesterol acyltransferase, requires fatty acids from the sn-2 position of plasma PC as substrate. Thus, PEMT appears to exert substantial control over the concentration of 22:6(n-3) in plasma.

Another effect of the knockout was a large accumulation of PUFA in hepatic TG, a condition that is quite unusual. The deletion of PEMT caused a substantial and specific accumulation of 18:0, 22:6(n-3) and long-chain (n-6) PUFA in TG that was exacerbated by choline deficiency (Fig. 1). Interestingly, these fatty acids are major components of PE, but not PC, indicating a conversion of PE into (ultimately) TG via a phospholipase C or D pathway. This conversion of PE to neutral lipid may implicate phospholipases as a strategy for maintaining normal PE concentrations in liver. In the absence of the conversion of PE to PC and its subsequent export into plasma, several compensatory mechanisms, including decreased synthesis of PE via the CDP-ethanolamine and phosphatidylserine decarboxylase pathways and increased clearance of PE from membranes by phospholipase C or D reactions may be induced.

The present data indicate that choline supplementation is sufficient to restore normal hepatic PC concentrations, but not sufficient to restore plasma PC, CE or TG concentrations (Table 2). This finding is in partial disagreement with a recent report by Noga and Vance (15), who found no effect of the PEMT deficiency on plasma PC and CE concentrations in male mice fed a standard diet. It is not entirely clear why the present data differ from the previous report. However, it should be noted that decreases in PC and CE similar to those in the present study occurred in mice fed a high fat diet (15), indicating a relationship between PEMT activity and CE metabolism that is similar to that reported here. In the present study, the pattern of CE accumulation in the liver suggests that both the PEMT pathway and the CDP-choline pathway have to be active for proper flux of CE between liver and plasma. This stands in contrast with TG metabolism in which the disruption of PEMT activity or inhibition of the CDP-choline pathway alone was not sufficient to significantly diminish plasma TG concentrations. Thus, although choline supplementation may be sufficient to ameliorate TG accumulation in the liver, it may not be sufficient to ameliorate CE accumulation in the liver.

The quantitative profiling of lipid metabolites described in this study indicated that the PEMT pathway has several functions critical to normal physiologic function beyond its recognized role as a compensatory pathway for PC biosynthesis. These functions include a key role for PEMT in the flux of CE and TG between liver and plasma and the regulation of the distribution of 22:6(n-3) and 20:4(n-6) to tissues other than liver. The present data also suggest that diseases related to essential fatty acid metabolism could be caused by dysregulation of PEMT activity; under such conditions, dietary (n-3) fatty acids could fail to rescue this deficiency because PEMT activity is still required for the inclusion of 22:6(n-3) in lipoproteins. It is clear that the PEMT pathway plays a role in lipid metabolism beyond its recognized role as a compensatory pathway for PC biosynthesis under conditions of choline deficiency and that the pathway may prove central to many aspects of physiology.

	ACKNOWLEDGMENTS

 
The authors gratefully acknowledge the writing support of C. J. Dillard. We thank Dennis E. Vance for kindly providing us with PEMT-/- mice.

	FOOTNOTES

 
¹ Supported in part by the National Institutes of Health (DK55865, AG09525). Support for this work was also provided by grants from the NIH to the UNC Clinical Nutrition Research Unit (DK56350) and the Center for Environmental Health (ES10126).

³ Abbreviations used: CD, choline-deficient; CDP-choline, cytidine diphosphate-choline; CE, cholesterol ester; CS, choline-supplemented; CT, control diet; DAG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PEMT, phosphatidylethanolamine-N-methyltransferase; TG, triglyceride.

⁴ The quantitative fatty acid compositions of each lipid class in liver and plasma are in supplementary Tables 3 and 4, respectively, in the online posting of this article at www.nutrition.org.

Manuscript received 4 May 2003. Initial review completed 28 May 2003. Revision accepted 11 August 2003.

	LITERATURE CITED

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