Phosphatidylethanolamine N-methyltransferase Pathway Research Articles

Sex-Specific Perturbation of Systemic Lipidomic Profile in Newborn Lambs Impacted by Prenatal Testosterone Excess.

Gestational hyperandrogenism adversely impacts offspring health. Using an ovine model, we found that prenatal testosterone (T) excess adversely affects growth and cardiometabolic outcomes in female offspring and produces sex-specific effects on fetal myocardium. Since lipids are essential to cardiometabolic function, we hypothesized that prenatal T excess leads to sex-specific disruptions in lipid metabolism at birth. Shotgun lipidomics was performed on the plasma samples collected 48 hours after birth from female (F) and male (M) lambs of control (C) and (T) sheep (CF = 4, TF = 7, CM = 5, TM = 10) and data were analyzed by univariate analysis, multivariate dimensionality reduction modeling followed by functional enrichment, and pathway analyses. Biosynthesis of phosphatidylserine was the major pathway responsible for sex differences in controls. Unsupervised and supervised models showed separation between C and T in both sexes with glycerophospholipids and glycerolipids classes being responsible for the sex differences between C and T. T excess increased cholesterol in females while decreasing phosphatidylcholine levels in male lambs. Specifically, T excess: 1) suppressed the phosphatidylethanolamine N-methyltransferase (PEMT) phosphatidylcholine synthesis pathway overall and in TM lambs as opposed to suppression of carnitine levels overall and TF lambs; and 2) activated biosynthesis of ether-linked (O-)phosphatidylethanolamine and O-phosphatidylcholine from O-diacylglycerol overall and in TF lambs. Higher cholesterol levels could underlie adverse cardiometabolic outcomes in TF lambs, whereas suppressed PEMT pathway in TM lambs could lead to endoplasmic reticulum stress and defective lipid transport. These novel findings point to sex-specific effects of prenatal T excess on lipid metabolism in newborn lambs, a precocial ovine model of translational relevance.

FRI103 Developmental Programming: Sex-specific Differences In Lipid Metabolism Of Newborn Lambs Exposed To Prenatal Testosterone Excess

Abstract Disclosure: N. Saadat: None. J.N. Ciarelli: None. B. Pallas: None. V. Padmanabhan: None. A.K. Vyas: None. Gestational hyperandrogenism such as that seen in Polycystic Ovary Syndrome (PCOS), leads to adverse offspring health outcomes in both humans and animals. Prenatal T excess (T) in a sheep model of PCOS phenotype induces peripheral insulin resistance, dyslipidemia, adipocyte defects, liver steatosis, pathological cardiac remodeling and hypertension in the female offspring. Whether these adverse cardiometabolic outcomes evidenced in the female offspring also extends to male offspring needs to be addressed. We hypothesized that prenatal T excess will induce sex-specific disruptions in lipid metabolism of newborn lambs thereby contributing to the aberrant cardiometabolic phenotype. Shotgun Lipidomics was performed on the plasma samples collected at 48 hours after birth from prenatal T treated (100mg T-propionate in corn oil from days 30 to 90 of gestation intramuscularly; Term: 147days) and control (C, vehicle only) lambs (N=26, Female C=4, T=7 and Male C=5, T=10). The data was analyzed by multivariate dimensionality reduction modeling followed by functional enrichment and pathway analyses. Unsupervised 3D principal component analysis and supervised models showed separation between C and T groupings in female and male lambs. Most lipids responsible for this separation were from glycerophospholipids and glycerolipids classes in both sexes. Prenatal T excess led to an increase in cholesterol levels only in female lambs as opposed to a decrease in levels of carnitine species (CAR) CAR 16:1 and CAR 14:1 and phosphatidylcholine species (PC) PC 39:4, PC 39:5 and PC 38:5 in male lambs. To investigate the impact of prenatal T excess on lipid metabolism pathways and active reactions, 738 lipid species were mapped, of which 147 were found to be active in reactions. The active reactions involving conversion of ether linked diglycerides (DG) to ether linked phosphatidylethanolamines (PE, O-DG→O-PE, z-score 3.367, p <0.05) and ether linked PC (O-DG →O-PC, z-score 2.299, p < 0.05) were increased in female lambs. In contrast, the conversion of PE to PC to phosphatidic acids (PA) was suppressed (PE→PC→PA, z-score 2.133, p <0.05) in prenatal T-treated male lambs pointing towards the suppression of Phosphatidylethanolamine N-methyltransferase (PEMT) phosphatidylcholine synthesis pathway. Higher cholesterol levels in T female lambs could contribute toward the adverse cardiometabolic phenotype evidenced earlier in this model, whereas suppressed PEMT pathway could lead to endoplasmic reticulum stress, defects in membrane permeability and lipid transport that may lead to liver steatosis in T male lambs. These novel findings of sex-specific effects of gestational exposure to T excess on lipid metabolism of newborn lamb, a precocial model of translational relevance, may have sex-specific functional outcomes in cardiometabolic systems. Presentation: Friday, June 16, 2023

Hepatic Choline Transport Is Inhibited During Fatty Acid-Induced Lipotoxicity and Obesity.

Choline is an essential nutrient and a critical component of the membrane phospholipid phosphatidylcholine (PC), the neurotransmitter acetylcholine, while also contributing to the methylation pathway. In the liver specifically, PC is the major membrane constituent and can be synthesized by the cytidine diphosphate–choline or the phosphatidylethanolamine N‐methyltransferase pathway. With the continuing global rise in the rates of obesity and nonalcoholic fatty liver disease, we sought to explore how excess fatty acids on primary hepatocytes and diet‐induced obesity affect choline uptake and metabolism. Our results demonstrate that hepatocytes chronically treated with palmitate, but not oleate or a mixture, had decreased choline uptake, which was associated with lower choline incorporation into PC and lower expression of choline transport proteins. Interestingly, a reduction in the rate of degradation spared PC levels in response to palmitate when compared with control. The effects of palmitate treatment were independent of endoplasmic reticulum stress, which counterintuitively augmented choline transport and transporter expression. In a model of obesity‐induced hepatic steatosis, male mice fed a 60% high‐fat diet for 10 weeks had significantly diminished hepatic choline uptake compared with lean mice fed a control diet. Although the transcript and protein expression of various choline metabolic enzymes fluctuated slightly, we observed reduced protein expression of choline transporter‐like 1 (CTL1) in the liver of mice fed a high‐fat diet. Polysome profile analyses revealed that in livers of obese mice, the CTL1 transcript, despite being more abundant, was translated to a lesser extent compared with lean controls. Finally, human liver cells demonstrated a similar response to palmitate treatment. Conclusion: Our results suggest that the altered fatty acid milieu seen in obesity‐induced fatty liver disease progression may adversely affect choline metabolism, potentially through CTL1, but that compensatory mechanisms work to maintain phospholipid homeostasis.

Methyl-Sensing Nuclear Receptor Liver Receptor Homolog-1 Regulates Mitochondrial Function in Mouse Hepatocytes.

Liver receptor homolog-1 (LRH-1; NR5A2) is a nuclear receptor that regulates metabolic homeostasis in the liver. Previous studies identified phosphatidylcholines as potential endogenous agonist ligands for LRH-1. In the liver, distinct subsets of phosphatidylcholine species are generated by two different pathways: choline addition to phosphatidic acid through the Kennedy pathway and trimethylation of phosphatidylethanolamine through phosphatidylethanolamine N-methyl transferase (PEMT). Here, we report that a PEMT-LRH-1 pathway specifically couples methyl metabolism and mitochondrial activities in hepatocytes. We show that the loss of Lrh-1 reduces mitochondrial number, basal respiration, beta-oxidation, and adenosine triphosphate production in hepatocytes and decreases expression of mitochondrial biogenesis and beta-oxidation genes. In contrast, activation of LRH-1 by its phosphatidylcholine agonists exerts opposite effects. While disruption of the Kennedy pathway does not affect the LRH-1-mediated regulation of mitochondrial activities, genetic or pharmaceutical inhibition of the PEMT pathway recapitulates the effects of Lrh-1 knockdown on mitochondria. Furthermore, we show that S-adenosyl methionine, a cofactor required for PEMT, is sufficient to induce Lrh-1 transactivation and consequently mitochondrial biogenesis. A PEMT-LRH-1 axis regulates mitochondrial biogenesis and beta-oxidation in hepatocytes.

Reproductive state and choline intake influence enrichment of plasma lysophosphatidylcholine-DHA: a post hoc analysis of a controlled feeding trial.

The major facilitator superfamily domain 2a protein was identified recently as a lysophosphatidylcholine (LPC) symporter with high affinity for LPC species enriched with DHA (LPC-DHA). To test the hypothesis that reproductive state and choline intake influence plasma LPC-DHA, we performed a post hoc analysis of samples available through 10weeks of a previously conducted feeding study, which provided two doses of choline (480 and 930mg/d) to non-pregnant (n 21), third-trimester pregnant (n 26), and lactating (n 24) women; all participants consumed 200mg of supplemental DHA and 22% of their daily choline intake as 2H-labelled choline. The effects of reproductive state and choline intake on total LPC-DHA (expressed as a percentage of LPC) and plasma enrichments of labelled LPC and LPC-DHA were assessed using mixed and generalised linear models. Reproductive state interacted with time (P=0·001) to influence total LPC-DHA, which significantly increased by week 10 in non-pregnant women, but not in pregnant or lactating women. Contrary to total LPC-DHA, patterns of labelled LPC-DHA enrichments were discordant between pregnant and lactating women (P<0·05), suggestive of unique, reproductive state-specific mechanisms that result in reduced production and/or enhanced clearance of LPC-DHA during pregnancy and lactation. Regardless of the reproductive state, women consuming 930 v. 480mg choline per d exhibited no change in total LPC-DHA but higher d3-LPC-DHA (P=0·02), indicating that higher choline intakes favour the production of LPC-DHA from the phosphatidylethanolamine N-methyltransferase pathway of phosphatidylcholine biosynthesis. Our results warrant further investigation into the effect of reproductive state and dietary choline on LPC-DHA dynamics and its contribution to DHA status.

Long-term effects of maternal choline supplementation on CA1 pyramidal neuron gene expression in the Ts65Dn mouse model of Down syndrome and Alzheimer's disease.

Choline is critical for normative function of 3 major pathways in the brain, including acetylcholine biosynthesis, being a key mediator of epigenetic regulation, and serving as the primary substrate for the phosphatidylethanolamine N-methyltransferase pathway. Sufficient intake of dietary choline is critical for proper brain function and neurodevelopment. This is especially important for brain development during the perinatal period. Current dietary recommendations for choline intake were undertaken without critical evaluation of maternal choline levels. As such, recommended levels may be insufficient for both mother and fetus. Herein, we examined the impact of perinatal maternal choline supplementation (MCS) in a mouse model of Down syndrome and Alzheimer's disease, the Ts65Dn mouse relative to normal disomic littermates, to examine the effects on gene expression within adult offspring at ∼6 and 11 mo of age. We found MCS produces significant changes in offspring gene expression levels that supersede age-related and genotypic gene expression changes. Alterations due to MCS impact every gene ontology category queried, including GABAergic neurotransmission, the endosomal-lysosomal pathway and autophagy, and neurotrophins, highlighting the importance of proper choline intake during the perinatal period, especially when the fetus is known to have a neurodevelopmental disorder such as trisomy.-Alldred, M. J., Chao, H. M., Lee, S. H., Beilin, J., Powers, B. E., Petkova, E., Strupp, B. J., Ginsberg, S. D. Long-term effects of maternal choline supplementation on CA1 pyramidal neuron gene expression in the Ts65Dn mouse model of Down syndrome and Alzheimer's disease.

Choline: The Underconsumed and Underappreciated Essential Nutrient.

Choline has been recognized as an essential nutrient by the Food and Nutrition Board of the National Academies of Medicine since 1998. Its metabolites have structural, metabolic, and regulatory roles within the body. Humans can endogenously produce small amounts of choline via the hepatic phosphatidylethanolamine N-methyltransferase pathway. However, the nutrient must be consumed exogenously to prevent signs of deficiency. The Adequate Intake (AI) for choline was calculated at a time when dietary intakes across the population were unknown for the nutrient. Unlike the traditional National Academy of Medicine approach of calculating an AI based on observed or experimentally determined approximations or estimates of intake by a group (or groups) of healthy individuals, calculation of the AI for choline was informed in part by a depletion-repletion study in adult men who, upon becoming deficient, developed signs of liver damage. The AI for other gender and life-stage groups was calculated based on standard reference weights, except for infants 0 to 6 months, whose AI reflects the observed mean intake from consuming human breast milk. Recent analyses indicate that large portions of the population (ie, approximately 90% of Americans), including most pregnant and lactating women, are well below the AI for choline. Moreover, the food patterns recommended by the 2015–2020 Dietary Guidelines for Americans are currently insufficient to meet the AI for choline in most age-sex groups. An individual’s requirement for choline is dependent on common genetic variants in genes required for choline, folate, and 1-carbon metabolism, potentially increasing more than one-third of the population’s susceptibly to organ dysfunction. The American Medical Association and American Academy of Pediatrics have both recently reaffirmed the importance of choline during pregnancy and lactation. New and emerging evidence suggests that maternal choline intake during pregnancy, and possibly lactation, has lasting beneficial neurocognitive effects on the offspring. Because choline is found predominantly in animal-derived foods, vegetarians and vegans may have a greater risk for inadequacy. With the 2020–2025 Dietary Guidelines for Americans recommending expansion of dietary information for pregnant women, and the inclusion of recommendations for infants and toddlers 0 to 2 years, better communication of the role that choline plays, particularly in the area of neurocognitive development, is critical. This narrative review summarizes the peer-reviewed literature and discussions from the 2018 Choline Science Summit, held in Washington, DC, in February 2018.

Abnormal liver phosphatidylcholine synthesis revealed in patients with acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is associated with a severe pro-inflammatory response; although decreased plasma cholesterol concentration has been linked to systemic inflammation, any association of phospholipid metabolic pathways with ARDS has not been characterized. Plasma phosphatidylcholine (PC), the major phospholipid of circulating lipoproteins, is synthesized in human liver by two biologically diverse pathways: the cytidine diphosphocholine (CDP):choline and phosphatidylethanolamine N-methyltransferase (PEMT) pathways. Here, we used ESI-MS/MS both to characterize plasma PC compositions and to quantify metabolic fluxes of both pathways using stable isotopes in patients with severe ARDS and in healthy controls. Direct incorporation of methyl-D9-choline estimated CDP:choline pathway flux, while PEMT flux was determined from incorporations of one and two methyl-D3 groups derived from methyl-D9-choline. The results of MS/MS analysis showed significant alterations in plasma PC composition in patients with ARDS versus healthy controls. In particular, the increased overall methyl-D9-PC enrichment and, most importantly, the much lower methyl-D3-PC and methyl-D6-PC enrichments suggest increased flux through the CDP:choline pathway and reduced flux through the PEMT pathway in ARDS. To our knowledge, this study is the first to demonstrate significant plasma PC molecular compositional changes combined with associated alterations in the dynamics of PC synthetic pathways in patients with ARDS.

Genetic Variation in Choline‐Metabolizing Enzymes Alters Choline Dynamics in Women Meeting Current Dietary Recommendations

Choline Kinase-α (CHKA) rs10791957, choline dehydrogenase (CHDH) rs9001, and CHDH rs12676 are genetic risk factors for disease and choline deficiency. While these single nucleotide polymorphisms are known to alter disease risk and the minimum choline intake needed for prevention of acute muscle and liver pathologies, their underlying metabolic signatures are not well characterized and it is unknown whether metabolic differences persist under conditions of nutrient adequacy. We sought to determine if these genetic risk factors alter choline dynamics in pregnant, lactating, and non-pregnant women consuming choline intakes meeting current recommendations. Women consumed 480 or 930 mg choline/d (22% as choline-d9, a metabolic tracer) for 10–12 weeks in a controlled feeding study and stable isotope methodology was used to investigate the effect of genetic differences on choline dynamics. CHKA rs10791957 (Figure 1), CHDH rs9001 (Figure 2), and CHDH rs12676 (Figure 3) all influenced choline dynamics under the study conditions of nutrient adequacy. Variants associated with decreased risk for choline deficiency (CHKA rs10791957, CHDH rs9001) appeared to favor cytidine diphosphate (CDP)-phosphatidylcholine (PC) synthesis relative to phosphatidylethanolamine N-methyltransferase (PEMT)-PC synthesis as compared to non-variants, while the CHDH rs12676 genotype, which is associated with increased risk, favored PEMT-PC synthesis. These results are consistent with the effect of these variants on choline requirements when one considers efficiency of PC production. While each unit of choline directed to the CDP-choline pathway yields one unit of PC, choline converted to betaine (and eventually S-adenosylmethionine) has many metabolic fates besides PEMT-PC production, and three choline-derived methyl groups are needed to yield just one additional unit of PC via the PEMT pathway. Therefore, greater PEMT-PC production may constitute a drain on choline status and exacerbate disease risk during choline deficiency. Our results indicate that these SNPs have metabolic effects at choline intakes relevant to the general population and such metabolic differences may contribute to disease pathogenesis and prognosis over the long-term. In fact, decreased PEMT activity observed among CHKA rs10791957 variant women (decreased turnover of choline-derived methionine → PEMT-PC; p=0.0005) may provide a metabolic basis for the decreased risk of type 2 diabetes among CHKA rs10791957 variant individuals given that PEMT knockout mice are protected from high-fat diet induced obesity and insulin resistance. Overall, these findings highlight the need for further study to examine the effects of genetic differences in choline metabolism on chronic disease outcomes. Support or Funding InformationThis work was supported by the National Institutes of Health under award T32-DK007158 and by the USDA National Institute of Food and Agriculture, Hatch project 231646. The content is the responsibility of the authors and does not necessarily represent the views of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the National Institutes of Health or the National Institute of Food and Agriculture (NIFA) or the United States Department of Agriculture(USDA). Figure 1Open in figure viewerPowerPoint Effect of the CHKA rs10791957 variant on the metabolite flux and partitioning of dietary choline Figure 2Open in figure viewerPowerPoint Effect of the CHDH rs9001 variant on the metabolite flux and partitioning of dietary choline. Figure 3Open in figure viewerPowerPoint Effect of the CHDH rs12676 variant on the metabolite flux and partitioning of dietary choline.

No up-regulation of the phosphatidylethanolamine N-methyltransferase pathway and choline production by sex hormones in cats.

BackgroundFeline hepatic lipidosis (FHL) is a common cholestatic disease affecting cats of any breed, age and sex. Both choline deficiency and low hepatic phosphatidylethanolamine N-methyltransferase (PEMT) activity are associated with hepatic lipidosis (HL) in humans, mice and rats. The PEMT expression is known to be upregulated by oestrogens, protecting the females in these species from the development of HL when exposed to choline deficient diets. The aim of the present study was to evaluate the influence of sex hormones on choline synthesis via the PEMT pathway in healthy male and female cats before and after spaying/neutering, when fed a diet with recommended dietary choline content.ResultsFrom six female and six male cats PEMT activity was assayed directly in liver biopsies taken before and after spaying/neutering, and assessed indirectly by analyses of PEMT–specific hepatic phosphatidylcholine (PC) species and plasma choline levels. Hepatic PEMT activity did not differ between intact female and male cats and no changes upon spaying/neutering were observed. Likewise, no significant differences in liver PC content and PEMT-specific polyunsaturated PC species were found between the sexes and before or after spaying/neutering.ConclusionThese results suggest that choline synthesis in cats differs from what is observed in humans, mice and rats. The lack of evident influence of sex hormones on the PEMT pathway makes it unlikely that spaying/neutering predisposes cats for HL by causing PC deficiency as suggested in other species.

Choline intakes exceeding recommendations during human lactation improve breast milk choline content by increasing PEMT pathway metabolites

Demand for the vital nutrient choline is high during lactation; however, few studies have examined choline metabolism and requirements in this reproductive state. The present study sought to discern the effects of lactation and varied choline intake on maternal biomarkers of choline metabolism and breast milk choline content. Lactating (n=28) and control (n=21) women were randomized to 480 or 930 mg choline/day for 10–12 weeks as part of a controlled feeding study. During the last 4–6 weeks, 20% of the total choline intake was provided as an isotopically labeled choline tracer (methyl-d9-choline). Blood, urine and breast milk samples were collected for choline metabolite quantification, enrichment measurements, and gene expression analysis of choline metabolic genes. Lactating (vs. control) women exhibited higher (P<.001) plasma choline concentrations but lower (P≤.002) urinary excretion of choline metabolites, decreased use of choline as a methyl donor (e.g., lower enrichment of d6-dimethylglycine, P≤.08) and lower (P≤.02) leukocyte expression of most choline-metabolizing genes. A higher choline intake during lactation differentially influenced breast milk d9- vs. d3-choline metabolite enrichment. Increases (P≤.03) were detected among the d3-metabolites, which are generated endogenously via the hepatic phosphatidylethanolamine N-methyltransferase (PEMT), but not among the d9-metabolites generated from intact exogenous choline. These data suggest that lactation induces metabolic adaptations that increase the supply of intact choline to the mammary epithelium, and that extra maternal choline enhances breast milk choline content by increasing supply of PEMT-derived choline metabolites.This trial was registered at clinicaltrials.gov as NCT01127022.

Maternal choline supplementation programs greater activity of the phosphatidylethanolamine N-methyltransferase (PEMT) pathway in adult Ts65Dn trisomic mice.

Maternal choline supplementation (MCS) induces lifelong cognitive benefits in the Ts65Dn mouse, a trisomic mouse model of Down syndrome and Alzheimer's disease. To gain insight into the mechanisms underlying these beneficial effects, we conducted a study to test the hypothesis that MCS alters choline metabolism in adult Ts65Dn offspring. Deuterium-labeled methyl-d9-choline was administered to adult Ts65Dn and disomic (2N) female littermates born to choline-unsupplemented or choline-supplemented Ts65Dn dams. Enrichment of d9-choline metabolites (derived from intact choline) and d3 + d6-choline metabolites [produced when choline-derived methyl groups are used by phosphatidylethanolamine N-methyltransferase (PEMT)] was measured in harvested tissues. Adult offspring (both Ts65Dn and 2N) of choline-supplemented (vs. choline-unsupplemented) dams exhibited 60% greater (P≤0.007) activity of hepatic PEMT, which functions in de novo choline synthesis and produces phosphatidylcholine (PC) enriched in docosahexaenoic acid. Higher (P<0.001) enrichment of PEMT-derived d3 and d6 metabolites was detected in liver, plasma, and brain in both genotypes but to a greater extent in the Ts65Dn adult offspring. MCS also yielded higher (P<0.05) d9 metabolite enrichments in liver, plasma, and brain. These data demonstrate that MCS exerts lasting effects on offspring choline metabolism, including up-regulation of the hepatic PEMT pathway and enhanced provision of choline and PEMT-PC to the brain.

Choline Supplementation Promotes Hepatic Insulin Resistance in Phosphatidylethanolamine N-Methyltransferase-deficient Mice via Increased Glucagon Action

Biosynthesis of hepatic choline via phosphatidylethanolamine N-methyltransferase (PEMT) plays an important role in the development of type 2 diabetes and obesity. We investigated the mechanism(s) by which choline modulates insulin sensitivity. PEMT wild-type (Pemt(+/+)) and knock-out (Pemt(-/-)) mice received either a high fat diet (HF; 60% kcal of fat) or a high fat, high choline diet (HFHC; 4 g of choline/kg of HF diet) for 1 week. Hepatic insulin signaling and glucose and lipid homeostasis were investigated. Glucose and insulin intolerance occurred in Pemt(-/-) mice fed the HFHC diet, but not in their Pemt(-/-) littermates fed the HF diet. Plasma glucagon was elevated in Pemt(-/-) mice fed the HFHC diet compared with Pemt(-/-) mice fed the HF diet, concomitant with increased hepatic expression of glucagon receptor, phosphorylated AMP-activated protein kinase (AMPK), and phosphorylated insulin receptor substrate 1 at serine 307 (IRS1-s307). Gluconeogenesis and mitochondrial oxidative stress were markedly enhanced, whereas glucose oxidation and triacylglycerol biosynthesis were diminished in Pemt(-/-) mice fed the HFHC diet. A glucagon receptor antagonist (2-aminobenzimidazole) attenuated choline-induced hyperglycemia and insulin intolerance and blunted up-regulation of phosphorylated AMPK and IRS1-s307. Choline induces glucose and insulin intolerance in Pemt(-/-) mice through modulating plasma glucagon and its action in liver.

P2‐436: Dose‐dependent effects of dietary folate, vitamin B‐12 and vitamin B‐6 on plasma lipid profile in rats

Folate, vitamins B-12 and B-6 are essential nutritional components in one-carbon metabolism in different ways and typically increase methylation capacity. This could support methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) by PE- N -methyltransferase (PEMT) in the liver. It has been suggested that PC synthesis by PEMT influence circulating levels of polyunsaturated fatty acids, like docosahexaenoic acid (DHA) (Watkins et al., J Nutr 2003; Pynn et al., J Lipid Res 2011). The aim of the present experiment was to investigate whether dietary enrichment with the three B-vitamins could affect plasma lipid composition and particularly plasma DHA levels in rats. After feeding rats one of four diets with increasing levels of folate, vitamins B-12 and B-6 (0%, 100%, 400%, or 1600% of the recommended daily intake) for 4 weeks, plasma fatty acid composition was analyzed using GC and the polar lipid profile was analyzed at the Kansas Lipidomics Research Center using triple quadrupole MS. In addition, plasma homocysteine concentration was measured as an indicator of methylation capacity. The results of the GC analysis indicate that dietary folate, vitamins B-12 and B-6 dose-dependently increased plasma DHA concentration. Plasma DHA and homocysteine showed a significant negative correlation. Data analysis of the polar lipid profile is ongoing. The first results demonstrate the interdependence between folate, vitamins B-12 and B-6 intake and plasma DHA. In this way, intake of supplemental B-vitamins may increase the availability of DHA for processes like neuronal membrane phospholipid synthesis. The increase in plasma DHA may be explained by the effects of B-vitamins on methylation capacity and subsequent enhancement of PC synthesis by the PEMT pathway. Details on this mechanism, implications for Alzheimer's disease, and pending additional results of the MS analysis will be presented.

Characterization of circulating phosphatidylcholine fatty acids in non‐pregnant women with supplemental DHA and variable choline intake

Phosphatidylcholine (PtdCho) synthesized via the phosphatidylethanolamine N‐methyltransferase (PEMT) pathway is enriched with polyunsaturated fatty acids (FAs) including docosahexaenoic acid (DHA). Methyl groups from the choline derivative betaine may be used by PEMT thus choline intake may influence the FA makeup of circulating PtdCho by modulating activity of biosynthetic pathways. DHA intake is associated with concentrations in blood, but it is unknown how varied choline intake may interact with supplemental DHA to impact PtdCho FAs. This study characterized plasma and RBC PtdCho FA species in non‐pregnant women consuming a DHA supplement and two levels of choline. The proportion of plasma and RBC PtdCho DHA increased from 3.4% and 2.6% to 4.9% and 3.4%, respectively. The ratio of other PtdCho FAs decreased concurrently and will be discussed. By study end, alterations of PtdCho FAs were independent of choline level; however, from baseline to week 6 women consuming more choline experienced a spike of plasma PtdCho DHA. Preliminary results indicate a higher choline intake combined with supplementary DHA may increase PEMT activity in the short term. Future studies elucidating the impact of choline intake and supplemental DHA on PtdCho synthesis via the PEMT pathway will include deuterium labeled PtdCho data. Funded in part by USDA, ENC and the Beef Checkoff; AAW is supported by an ENC Dissertation Fellowship.

Hyperhomocysteinemia from trimethylation of hepatic phosphatidylethanolamine during cholesterol cholelithogenesis in inbred mice

Because hyperhomocysteinemia can occur in cholesterol gallstone disease, we hypothesized that this may result from trimethylation of phosphatidylethanolamine (PE), which partakes in biliary phosphatidylcholine (PC) hypersecretion during cholesterol cholelithogenesis. We fed murine strains C57L/J, C57BL/6J, SWR/J, AKR/J, PE N-methyltransferase (PEMT) knockout (KO), PEMT heterozygous (HET), and wildtype (WT) mice a cholesterol/cholic acid lithogenic diet (LD) for up to 56 days and documented biliary lipid phase transitions and secretion rates. We quantified plasma total homocysteine (tHcy), folate, and vitamin B12 in plasma and liver, as well as biliary tHcy and cysteine secretion rates. Rate-limiting enzyme activities of PC synthesis, PEMT and cytidine triphosphate: phosphocholine cytidylyltransferase (PCT), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) were measured in liver homogenates. Other potential sources of plasma tHcy, glycine N-methyltransferase (GNMT) and guanidinoacetate N-methyltransferase (GAMT), were assayed by gene expression. Plasma tHcy and PEMT activities became elevated during cholelithogenesis in gallstone-susceptible C57L, C57BL/6, and SWR mice but not in the gallstone-resistant AKR mice. Persisting in C57L mice, which exhibit the greatest Lith gene burden, these increases were accompanied by elevated hepatic SAM/SAH ratios and augmented biliary tHcy secretion rates. Counter-regulation included remethylation of Hcy to methionine concurrent with decreased folate and vitamin B12 levels and Hcy transsulfuration to cysteine. Concomitantly, methylenetetrahydrofolate reductase (Mthfr), betaine-homocysteine methyltransferase (Bhmt), and cystathionine-β-synthase (Cbs) were up-regulated, but Gnmt and Gamt genes were down-regulated. PEMT KO and HET mice displayed biliary lipid secretion rates and high gallstone prevalence rates similar to WT mice without any elevation in plasma tHcy levels. This work implicates up-regulation of PC synthesis by the PEMT pathway as a source of elevated plasma and bile tHcy during cholesterol cholelithogenesis.

Salinity regulates N-methylation of phosphatidylethanolamine in euryhaline crustaceans hepatopancreas and exchange of newly-formed phosphatidylcholine with hemolymph

Phosphatidylcholine (PC), the main phospholipid in eukaryotes, is synthesized via two different routes, the phosphatidylethanolamine N-methyl transferase (PEMT) and the CDP-choline pathways. We previously showed in euryhaline fish that salinity impacts the relative contribution of the two pathways for PC biosynthesis, with PEMT pathway being activated in the liver of sea water (SW)-adapted animals. To address the occurrence of such phenomenon in other animals we performed in vivo metabolic studies in two crustacean species: the Chinese crab (Eriocheir sinensis) and the green crab (Carcinus maenas). In both species, the levels of PC and phosphatidylethanolamine in hepatopancreas and hemolymph were not modified by SW-adaptation. In E. sinensis, SW-adaptation activated PC labeling from L-(U-(14)C)-serine in the hepatopancreas and resulted in an increased ratio of PC specific activities between hemolymph and hepatopancreas. In C. maenas, incorporation of L-(3-(3)H)-serine and L-(2-(14)C)-ethanolamine into PC of hepatopancreas was strongly inhibited after acclimation to fresh water (FW). The results show that PC synthesis via the PEMT pathway and its subsequent release into hemolymph are both activated in SW- compared to FW-adapted animals. SW-adaptation also resulted in increased tissue concentrations of betaine and labeling from L-(U-(14)C)-serine, suggesting that the PEMT-derived PC is used for the synthesis of organic osmolytes. The physiological relevance of these observations is discussed.

Specificity and rate of human and mouse liver and plasma phosphatidylcholine synthesis analyzed in vivo

Phosphatidylcholine (PC) synthesis by the direct cytidine diphosphate choline (CDP-choline) pathway in rat liver generates predominantly mono- and di-unsaturated molecular species, while polyunsaturated PC species are synthesized largely by the phosphatidylethanolamine-N-methyltransferase (PEMT) pathway. Although altered PC synthesis has been suggested to contribute to development of hepatocarcinoma and nonalcoholic steatohepatitis, analysis of the specificity of hepatic PC metabolism in human patients has been limited by the lack of sensitive and safe methodologies. Here we incorporated a deuterated methyl-D(9)-labled choline chloride, to quantify biosynthesis fluxes through both of the PC synthetic pathways in vivo in human volunteers and compared these fluxes with those in mice. Rates and molecular specificities of label incorporated into mouse liver and plasma PC were very similar and strongly suggest that label incorporation into human plasma PC can provide a direct measure of hepatic PC synthesis in human subjects. Importantly, we demonstrate for the first time that the PEMT pathway in human liver is selective for polyunsaturated PC species, especially those containing docosahexaenoic acid. Finally, we present a multiple isotopomer distribution analysis approach, based on transfer of deuterated methyl groups to S-adenosylmethionine and subsequent sequential methylations of PE, to quantify absolute flux rates through the PEMT pathway that are applicable to studies of liver dysfunction in clinical studies.

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

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 ( approximately 145 and approximately 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.

Physiological Regulation of Phospholipid Methylation Alters Plasma Homocysteine in Mice

Biological methylation reactions and homocysteine (Hcy) metabolism are intimately linked. In previous work, we have shown that phosphatidylethanolamine N-methyltransferase, an enzyme that methylates phosphatidylethanolamine to form phosphatidylcholine, plays a significant role in the regulation of plasma Hcy levels through an effect on methylation demand (Noga, A. A., Stead, L. M., Zhao, Y., Brosnan, M. E., Brosnan, J. T., and Vance, D. E. (2003) J. Biol. Chem. 278, 5952-5955). We have further investigated methylation demand and Hcy metabolism in liver-specific CTP:phosphocholine cytidylyltransferase-alpha (CTalpha) knockout mice, since flux through the phosphatidylethanolamine N-methyltransferase pathway is increased 2-fold to meet hepatic demand for phosphatidylcholine. Our data show that plasma Hcy is elevated by 20-40% in mice lacking hepatic CTalpha. CTalpha-deficient hepatocytes secrete 40% more Hcy into the medium than do control hepatocytes. Liver activity of betaine:homocysteine methyltransferase and methionine adenosyltransferase are elevated in the knockout mice as a mechanism for maintaining normal hepatic S-adenosylmethionine and S-adenosylhomocysteine levels. These data suggest that phospholipid methylation in the liver is a major consumer of AdoMet and a significant source of plasma Hcy.