Choline Biosynthesis Research Articles

Visualizing the Spatial Distribution of Metabolites in Angelica sinensis Roots by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Angelica sinensis is one of the most popular traditional Chinese medicines (TCM) and has been extensively used to treat various diseases. Hundreds of endogenous ingredients have been isolated and identified from this herb, but their spatial distribution within the plant root is largely unknown. In this study, we tried to investigate and map within-tissue spatial distribution of metabolites in Angelica sinensis roots. After optimization of experiment conditions, the 1,5-diaminonaphthalene (1,5-DAN) was chosen as the matrix and was sprayed on the surface of root sections. Then matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was employed to perform insitu detection and obtain detail spatial distribution information of metabolites in Angelica sinensis roots. The spatial distributions of a wide range of metabolites including organic acids, amino acids, oligosaccharides, and phospholipids were characterized and visualized in Angelica sinensis roots. Majority of these metabolites were located in the phloem and xylem, while ferulic acid was mainly present in the cork layer. The results revealed a dramatic metabolic heterogeneity among different regions of the roots and distinct spatial distribution patterns of different metabolites. Additionally, the metabolic pathways involved in the biosynthesis of choline were also successfully localized and visualized. This study comprehensively characterized the spatial distribution of metabolites in Angelica sinensis roots, which would prompt the understanding of its chemical separation, biosynthesis, and pharmacological activities.

Light effects on Lasiodiplodia theobromae metabolome cultured in vitro.

The present work identified and compared intracellular metabolites and metabolic networks in mycelial cultures of Lasiodiplodia theobromae grown under 12 natural light and 24 hours' dark using a 1H NMR-based metabolomics approach. Fungal cultures were grown in potato dextrose media, and metabolites were extracted by sonication with sodium phosphate-buffered saline (pH = 6.0, 10% D2O, 0.1 mM TSP) from mycelium samples collected every week over four weeks. Multivariate analyses revealed that the light exposure group showed a positive correlation within beta-hydroxybutyrate, acetoacetate, acetone, betaine, choline, glycerol, and phosphocholine. On the other hand, phenyl acetate, leucine, isoleucine, valine, and tyrosine were positively correlated with dark conditions. Light favored the oxidative degradation of valine, leucine, and isoleucine, leading to the accumulation of choline, phosphocholine, betaine, and ketone bodies (ketogenesis). Ketogenesis, gluconeogenesis, and the biosynthesis of choline, phosphocholine, and betaine, were considered discriminatory routes for light conditions. The light-sensing pathways were interlinked with fungal development, as verified by the increased production of mycelia biomass without fruiting bodies and stress signaling, as demonstrated by the increased production of pigments.

RF22 | PMON10 Exposure to a Real Life Environmental Chemical Mixture Alters the Metabolite Profile in the Plasma of Sheep: An Untargeted Metabolomics Study.

Abstract Humans are exposed to a mixture of endocrine disrupting chemicals (EDCs) in everyday life, believed to contribute to a range of non-communicable diseases. While the adverse health impacts of exposure to individual EDCs have been extensively investigated, studies on effects of real-world exposures characterized by chronic low doses of a mixture of chemicals, are only recently emerging. Biosolids derived from human wastes, exemplify both the array and dosage of chemicals humans are exposed to in real life and represents a novel model to investigate the risk posed by the EDCs. Previous studies in sheep have demonstrated aberrant reproductive and metabolic phenotypic effects in offsprings, as a result of maternal biosolid exposure. We hypothesize that EDC-mediated disruption in the maternal metabolic milieu is a potential mediator of the aberrant phenotypic outcomes in offsprings in the biosolid-exposure model. Recent untargeted metabolomic studies provide evidence of the technique's ability to unravel biomarkers of both exposure and effect by identifying the disrupted endogenous metabolite profile and xenobiotic mixture, respectively. In this study, we sought to identify biosolid exposure-induced maternal metabolic fingerprints that could influence fetal programming. Ewes were grazed on either organic fertilizer or biosolid-treated pastures from 1 month before mating until close to parturition. Plasma from blood samples collected on day 90 of the 147-day gestation period, from ewes carrying singleton pregnancies (Control n=15, Treatment n=15) were subjected to untargeted metabolomics using liquid chromatography-quadrupole time-of-flight mass spectrometry. Metabolites were identified by matching the retention time, mass and isotope profile to authentic standards or metabolome databases using Agilent's MassHunter Find by Molecular Feature workflow (v7.0). Untargeted metabolomics identified 1006 and 1295 annotated features from the negative and positive mode respectively. Principal component analysis and orthogonal signal correction partial least-squares discriminant analysis discriminated the metabolomes of control sheep and sheep grazed in biosolid-treated pasture. The key differential metabolites identified by variable importance in projection score primarily belonged to the super-class organic acids, fatty acids, benzenoids, glycerophospholipids and organoheterocyclic compounds. These differentially expressed metabolites are implicated in metabolic pathways including carnitine synthesis, alanine metabolism, glucose alanine cycle, phosphatidyl choline biosynthesis and glutathione metabolism. Some of the top differential metabolites- oleamide, pyroglutamic acid and 15-HETrE have been implicated in fetal development. Oleamide, an endocannabinoid, has growth inhibitory properties. 15-HETrE modulates arachidonic acid metabolism and upregulate PPARγ expression, which plays a pivotal role in fetal development and metabolic outcomes. These findings indicate that biosolids exposure results in alterations in the maternal metabolome which likely contributes to the aberrant phenotypic outcomes reported in offspring of biosolid-exposed mothers. This is the first study to apply metabolomics to validate the effect of real-life EDC exposure in sheep, a translationally relevant precocial model with a developmental trajectory similar to humans. Presentation: Sunday, June 12, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

ON THE BIOLOGICAL ROLE OF CHOLINE IN HUMAN AND HIGHER ANIMAL ORGANISMS (A LITERATURE REVIEW)

Purpose. To provide the reader a detailed literature review regarding the history of the discovery of choline, then the history of further research on its biological role in human and higher animal organisms. Describe in detail the mechanisms of choline biosynthesis, absorption, transport and metabolism in human and higher animals, the variety of physiological functions performed by the choline itself and by its derivatives. Describe the various symptoms of dietary choline deficiency. Provide to the reader the data regarding the US and EU minimum recommended dietary intake of choline.
 Methodology. We began our work by studying the literature on the history of medicine, as well as encyclopedic literature on vitamins and vitamin-like compounds, and books on biochemistry. Then we used the search engines PubMed and ScienceDirect to find relevant articles discussing the biological role of choline, its biosynthesis, absorption, transport, and symptoms of its deficiency. We then used materials from the US National Academy of Medicine and the European Food Safety Agency’s RDA for choline from food.
 Results. The data we found and presented in this literature review on the biological role of choline in humans and higher animals convincingly indicate the important role of sufficient consumption of choline in the daily diet for maintaining overall health. Sufficient dietary choline is needed to maintain the normal state of the skin and its appendages, articular cartilage, skeletal muscles, to support the normal functioning of the liver and the endocrine system (especially the cortex and the medulla of the adrenal glands, and the gonads), to prevent stone formation in bile, to improve memory and cognitive functioning, to reduce blood plasma atherogenicity and improve its lipid profile, to delay the development of atherosclerosis.
 Practical implications. The results we presented there deserve the widest application in various fields of both human medicine and veterinary medicine.

An Epigenome Wide Association Study Reveals Timing of Physical Activity During Pregnancy Is Associated With Placental DNA Methylation

ObjectivesPhysical activity (PA) prior to and during pregnancy may influence offspring health through epigenetic modifications in the placenta. Prior studies had a single PA assessment in pregnancy limiting the ability to account for PA changes during pregnancy. We hypothesized that timing of PA may be associated with differential methylation and evaluated associations between multiple assessments of PA and genome-wide methylation changes in the placenta. MethodsPlacental tissues were obtained at delivery and DNA methylation was measured using the Illumina Human Methylation450 Beadchip for 301 mothers in the NICHD Fetal Growth Studies–Singleton cohort. Total PA (metabolic equivalent minutes/week) was assessed using the Pregnancy PA Questionnaire targeted for 8–13 (visit 0), 16–22 (visit 1), 24–29 (visit 2), 30–33 (visit 3), 34–37 (visit 4), 38–41 (visit 5) weeks’ gestation. For associations of PA at each visit with methylation, we conducted linear regression adjusting for potential confounders such as maternal age, race/ethnicity, pre-pregnancy body mass index. Genes annotating significant CpG sites (false discovery rate adjusted P < 0.05) were queried for enrichment of functional pathways using Ingenuity Pathway Analysis. ResultsPA in the 12 months prior to visit 0 was not significantly associated with methylation, whereas PA since last visit for visits 1–5 were associated with methylation of 1, 0, 2, 29, 30 CpG sites, respectively (P values ranging from 3.07 × 10–9 to 3.35 × 10–6). Thirteen CpG sites significantly related to PA overlapped at visits 4 & 5, with the most significant associations at cg21385047 located in sphingosine-1-phosphate receptor 1 (S1PR1, P = 3.07 × 10–9, P = 7.45 × 10–9, respectively). Five enriched pathways overlapped at visits 4 & 5 (P < 0.05): inositol pyrophosphates biosynthesis, gustation pathway, choline biosynthesis III, cAMP-mediated signaling, G-protein coupled receptor signaling. ConclusionsFindings suggest that PA during pregnancy is associated with placental DNA methylation changes at loci potentially related to cardiovascular and neurological system development/function. If replicated, our findings could shed light onto the mechanisms underlying changes in offspring epigenetic profile associated with maternal PA. Funding SourcesEunice Kennedy Shriver National Institute of Child Health and Human Development.

Quantitative Metabonomic Analysis Reveals the Germination-Associated Dynamic and Systemic Biochemical Changes for Mung-Bean (Vigna radiata) Seeds.

Seed germination is essential for plant survival, germplasm resource preservation, and worldwide food supplies, although the germination-associated seed biochemical variations are not fully understood. With the NMR-based metabonomics, we quantitatively analyzed the comprehensive metabolite composition (metabonome) of mung-bean (Vigna radiata) seeds at eight time points of germination covering all three phases. We found that mung-bean seed metabonomes were dominated by 63 metabolites including lipids, amino acids, oligo-/monosaccharides, cyclitols, cholines, organic acids, nucleotides/-sides, nicotinates, and the shikimate pathway-mediated secondary metabolites. During germination, metabolic changes included mainly the degradation of proteins and raffinose family oligosaccharides, glycolysis, tricarboxylic acid (TCA) cycle, anaerobic respiration, biosynthesis of osmolytes and antioxidants together with the metabolisms of nucleotides/-sides, nicotinates, and amino acids. Oligosaccharide degradation was the primary energy source for germination, which coupled with the mobilization of starch and protein storages to produce sugars and amino acids for biomaterial and energy generations. Osmotic and redox regulations were prerequisites for seed germination together with mitochondrial reparations and generations to enable TCA cycle. During the postgermination growth stage (phase-3), the use of small molecules including amino acids and saccharides was switched to meet the growth demands of radicle cells. Small metabolites passed freely through seed testa leaking into the culture media during early germination but were reabsorbed by seed cells around the postgermination growth stage. Extra after-ripening accelerated these metabolic processes of seeds in phase-1, especially the biosynthesis of cyclitols, choline, and nicotinates, increasing the germination uniformity in terms of speed and percentage. Germination-resistant seeds were incapable of activating the germination-associated metabolic processes.

Digestible choline requirement of juvenile yellowtail kingfish (Seriola lalandi)

The present study was conducted to determine the digestible choline requirement of juvenile yellowtail kingfish (Seriola lalandi; YTK) in the presence of 2-amino-2-methyl-1-propanol (AMP), an inhibitor of choline biosynthesis. The second aim of this study was to determine if choline supplementation of a practical diet made from common raw materials can improve the growth performance of YTK and if choline supplementation dosages are affected by water temperature. Two eight-week experiments were conducted. The first was a dose-response experiment in which juvenile YTK (156.3 ± 15.3 g) were reared at 16 °C and fed five isonitrogenous and isoenergetic semi-purified diets containing 0.42 (Diet 1), 1.10 (Diet 2), 1.37 (Diet 3), 2.96 (Diet 4) or 6.05 g digestible choline kg−1 diet (Diet 5) by adding graded concentrations of choline chloride (CC). A sixth diet (Diet 6), comparable to Diet 4, was made without AMP to estimate the de novo synthesis of choline by YTK. The second was a factorial experiment in which juvenile YTK (157.3 ± 11.9 g) were reared at 16 °C and 24 °C and fed a practical diet supplemented with 0.0, 3.0 or 6.0 g of CC kg−1 which equates to a digestible choline concentration of 1.77, 3.54 and 4.66 g kg−1 diet, respectively. Results from experiment 1 indicated the breakpoint (broken-line regression model) in specific growth rate (SGR) and choline deposition rate occurred when digestible choline intake reached 26.1 mg kgBW−1 d−1 and 27.3 mg kgBW−1 d−1, respectively. On a dietary basis, the breakpoint in SGR and choline deposition rate occurred when diets provided 1.93 and 1.94 g digestible choline kg−1 diet, respectively. Experiment 2 results indicated there were no significant interactions between dietary choline and water temperature with respect to production indices such as SGR, food conversion ratio (FCR), whole body composition and nutrient retention. Choline deposition rate, SGR and FCR tended to be better in fish fed the practical diet supplemented with 3.0 g CC kg−1, but there was no additional advantage of raising the content to 6.0 g CC kg−1 diet. The present study confirms the necessity of adding CC to practical diets for YTK given that practical diets with no CC supplementation contain lower digestible choline than the estimated requirement established in experiment 1.

Structure-guided function discovery of an NRPS-like glycine betaine reductase for choline biosynthesis in fungi

Nonribosomal peptide synthetases (NRPSs) and NRPS-like enzymes have diverse functions in primary and secondary metabolisms. By using a structure-guided approach, we uncovered the function of a NRPS-like enzyme with unusual domain architecture, catalyzing two sequential two-electron reductions of glycine betaine to choline. Structural analysis based on the homology model suggests cation-π interactions as the major substrate specificity determinant, which was verified using substrate analogs and inhibitors. Bioinformatic analysis indicates this NRPS-like glycine betaine reductase is highly conserved and widespread in kingdom fungi. Genetic knockout experiments confirmed its role in choline biosynthesis and maintaining glycine betaine homeostasis in fungi. Our findings demonstrate that the oxidative choline-glycine betaine degradation pathway can operate in a fully reversible fashion and provide insight in understanding fungal choline metabolism. The use of an NRPS-like enzyme for reductive choline formation is energetically efficient compared with known pathways. Our discovery also underscores the capabilities of the structure-guided approach in assigning functions of uncharacterized multidomain proteins, which can potentially aid functional discovery of new enzymes by genome mining.

A preclinical evaluation of thiostrepton, a natural antibiotic, in nasopharyngeal carcinoma.

Background Thiostrepton, a natural antibiotic, has recently been shown to be a potential anticancer drug for certain cancers, but its study in nasopharyngeal carcinoma (NPC) is still limited. The aims of this study were to investigate the anticancer effect of thiostrepton on NPC cells and to explore its underlying mechanism. Methods The effects of thiostrepton on the proliferation, migration, and invasion of NPC cells were investigated by a WST-1 assay, wound healing assay, and cell invasion assay, respectively. Microarrays were conducted and further analyzed by Ingenuity Pathways Analysis (IPA) to determine the molecular mechanism by which thiostrepton affects NPC cells. Results Our results showed that thiostrepton reduced NPC cell viability in a dose-dependent manner. Thiostrepton inhibited the migration and invasion of NPC cells in wound healing and cell invasion assays. The microarray data analyzed by IPA indicated the top 5 ingenuity canonical pathways, which were unfolded protein response, NRF2-mediated oxidative stress response, retinoate biosynthesis I, choline biosynthesis III, and pancreatic adenocarcinoma signaling. Conclusion Thiostrepton effectively suppressed NPC cell proliferation, migration, and invasion, likely by several mechanisms. Thiostrepton may be a potential therapeutic agent for treating NPC in the future.

A Putative Zn2Cys6 Transcription Factor Is Associated With Isoprothiolane Resistance in Magnaporthe oryzae.

Isoprothiolane (IPT), a systemic fungicide, has been applied to control rice blast since the 1970s. Although resistance to IPT has been observed, the mechanism of resistance still has not been fully elucidated. In this study, nucleotide polymorphisms were detected between two IPT-resistant mutants generated in the lab, and their parental wild type isolates using a whole-genome sequencing approach. In the genomes of the two resistant mutants, single point mutations were identified in a gene encoding a Zn2Cys6 transcription factor-like protein. Notably, either knocking out the gene or replacing the wild type allele with the mutant allele (R343W) in a wild type isolate resulted in resistance to IPT, indicating that the gene is associated with IPT resistance, and thus was designated as MoIRR (Magnaporthe oryzae isoprothiolane resistance related). Along with point mutations R343W in mutant 1a_mut, and R345C in 1c_mut, a 16 bp insertion in 6c_mut was also located in the Fungal_TF_MHR domain of MoIRR, revealing that this domain may be the core element for IPT resistance. In addition, IPT-resistant mutants and transformants showed cross-resistance with iprobenfos (IBP), which was consistent with previous observations. These results indicated that MoIRR is strongly connected to resistance to choline biosynthesis inhibitor (CBI), and further work should focus on investigating downstream effects of MoIRR.

Transcriptomic profiling of maize (Zea mays L.) seedlings in response to Pseudomonas putida stain FBKV2 inoculation under drought stress

Several mechanisms have been proposed for plant growth-promoting rhizobacteria (PGPR)-mediated drought stress tolerance in plants, but little is known about the molecular pathways involved in the drought tolerance promoted by PGPR. We, therefore, aim to study the differential gene response between Pseudomonas putida strain FBKV2 and maize interaction under drought stress using Illumina sequencing. RNA Seq libraries were generated from leaf tissue of maize seedlings with and without strain FBKV2 subjected to drought stress. The libraries were mapped with maize genome database for the identification of differentially expressed genes (DEGs). The expression studies confirmed the downregulation of ethylene biosynthesis (ET), abscisic acid (ABA) and auxin signaling, superoxide dismutase, catalase, and peroxidase in FBKV2-inoculated seedlings. On the other hand, genes involved in β-alanine and choline biosynthesis, heat shock proteins, and late embryogenesis abundant (LEA) proteins were upregulated, which could act as key elements in the drought tolerance conferred by P. putida strain FBKV2. Another remarkable expression was observed in genes encoding benzoxazinoid (BX) biosynthesis which act as the chemoattractant, which was further confirmed by gfp-labeled P. putida strain FBKV2 root colonization studies. Overall, these results indicate that secretion of BXs attracted P. putida strain FBKV2 resulted in root colonization and mediated drought tolerance by modulating metabolic, signaling, and stress-responsive genes.

Choline Essentiality and Its Requirement in Diets for Juvenile Parrot Fish (Oplegnathus fasciatus).

A 12-wk feeding trial was conducted to evaluate the essentiality of choline supplementation in diets for parrot fish. Five isonitrogenous and isocaloric diets were supplemented with 0 (as control), 500, 1,000, and 2,000 mg choline per kg diet, and a positive control diet without choline contained 0.3% of 2-amino-2-methyl-1-propanol as choline biosynthesis inhibitor (designated as Con, C500, C1000, C2000 and Con+, respectively). Triplicate groups of fish (body weight, 8.8±0.01 g) were fed one of the experimental diets at a rate of 4% body weight twice daily. The fish fed Con+ diet revealed significantly lower growth performance and feed utilization efficiency than other fish groups. Supplementation of choline to the basal diet did not significantly influence fish growth. The highest liver lipid content was observed in fish fed the Con+ diet and inversely correlated with liver choline concentration although the differences were not significant. Also, significantly higher liver linoleic, eicosapentaenoic and docosahexaenoic acid contents were found in fish fed the Con+ diet. Innate immune parameters including respiratory burst and myeloperoxidase activities were not significantly affected by dietary choline levels. The findings in this study conclude that choline concentration of approximately 230 mg kg−1 diet meets the requirement of parrot fish.

Integration of a constraint-based metabolic model of Brassica napus developing seeds with (13)C-metabolic flux analysis.

The use of large-scale or genome-scale metabolic reconstructions for modeling and simulation of plant metabolism and integration of those models with large-scale omics and experimental flux data is becoming increasingly important in plant metabolic research. Here we report an updated version of bna572, a bottom-up reconstruction of oilseed rape (Brassica napus L.; Brassicaceae) developing seeds with emphasis on representation of biomass-component biosynthesis. New features include additional seed-relevant pathways for isoprenoid, sterol, phenylpropanoid, flavonoid, and choline biosynthesis. Being now based on standardized data formats and procedures for model reconstruction, bna572+ is available as a COBRA-compliant Systems Biology Markup Language (SBML) model and conforms to the Minimum Information Requested in the Annotation of Biochemical Models (MIRIAM) standards for annotation of external data resources. Bna572+ contains 966 genes, 671 reactions, and 666 metabolites distributed among 11 subcellular compartments. It is referenced to the Arabidopsis thaliana genome, with gene-protein-reaction (GPR) associations resolving subcellular localization. Detailed mass and charge balancing and confidence scoring were applied to all reactions. Using B. napus seed specific transcriptome data, expression was verified for 78% of bna572+ genes and 97% of reactions. Alongside bna572+ we also present a revised carbon centric model for 13C-Metabolic Flux Analysis (13C-MFA) with all its reactions being referenced to bna572+ based on linear projections. By integration of flux ratio constraints obtained from 13C-MFA and by elimination of infinite flux bounds around thermodynamically infeasible loops based on COBRA loopless methods, we demonstrate improvements in predictive power of Flux Variability Analysis (FVA). Using this combined approach we characterize the difference in metabolic flux of developing seeds of two B. napus genotypes contrasting in starch and oil content.

Biochemical and metabolomic effects of perinatal choline deficiency in the piglet

Choline plays an essential role in tissue development during gestation and early postnatal life, and the piglet (Sus scrofa) may serve as a translational animal model to study perinatal effects of choline deficiency. Our objective was to determine developmental changes in piglets exposed to differing levels of choline both prenatally and postnatally. A factorial arrangement of choline sufficient (CS; 50% above requirement) and choline deficient (CD; 50% below requirement) diets were provided during the prenatal (last 65 d of gestation) and postnatal (48 h after birth through 28 d of age) periods. An interactive effect of choline level and perinatal period was observed for body weight gain (P &lt; 0.001), and prenatal CD piglets had smaller (P = 0.014) brains than prenatal CS piglets. Blood chemistry panels revealed generalized impairments in liver function in postnatal CD piglets. Overall, metabolomic analysis identified 276 biochemicals and revealed that choline deficiency in the pre‐ and postnatal periods altered plasma levels of metabolites related to choline biosynthesis, glycolysis, and lipid metabolism. Additionally, interactive effects were noted for glycolytic intermediates, carnitine‐related metabolites, and many choline‐containing lysolipids. We conclude that the piglet is a sensitive animal model for studying developmental aspects of perinatal choline deficiency.U.S. Department of Agriculture Hatch Project ILLU‐538–319

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.

Aberrant Estrogen Regulation of PEMT Results in Choline Deficiency-associated Liver Dysfunction

When dietary choline is restricted, most men and postmenopausal women develop multiorgan dysfunction marked by hepatic steatosis (choline deficiency syndrome (CDS)). However, a significant subset of premenopausal women is protected from CDS. Because hepatic PEMT (phosphatidylethanolamine N-methyltransferase) catalyzes de novo biosynthesis of choline and this gene is under estrogenic control, we hypothesized that there are SNPs in PEMT that disrupt the hormonal regulation of PEMT and thereby put women at risk for CDS. In this study, we performed transcript-specific gene expression analysis, which revealed that estrogen regulates PEMT in an isoform-specific fashion. Locus-wide SNP analysis identified a risk-associated haplotype that was selectively associated with loss of hormonal activation. Chromatin immunoprecipitation, analyzed by locus-wide microarray studies, comprehensively identified regions of estrogen receptor binding in PEMT. The polymorphism (rs12325817) most highly linked with the development of CDS (p < 0.00006) was located within 1 kb of the critical estrogen response element. The risk allele failed to bind either the estrogen receptor or the pioneer factor FOXA1. These data demonstrate that allele-specific ablation of estrogen receptor-DNA interaction in the PEMT locus prevents hormone-inducible PEMT expression, conferring risk of CDS in women.

THE ROLE OF GENDER AND GENETIC POLYMORPHISMS ON CHOLINE BIOSYNTHESIS

The requirement for choline, an essential nutrient for humans, is met in part by endogenous synthesis catalyzed by phosphatidylethanolamine N‐methyltransferase (PEMT). In contrast to post‐menopausal women and men, a subset of pre‐menopausal women exhibits resistance to hepatic steatosis after being placed on an experimental choline‐deficient diet. We identified a common (78% of the study population has at least 1 variant allele) single‐nucleotide polymorphism (SNP) in the PEMT gene (rs12325817) that is significantly associated with increased susceptibility to choline deficiency (odds ratio 25, p= 0.002) in this group. Since estrogen production is unique to premenopausal women, we investigated whether PEMT gene expression is regulated by estrogen and whether the presence of the rs12325817 SNP affects estrogen regulation of the PEMT gene. RT‐PCR revealed a dose‐dependent increase in PEMT expression and activity in human hepatocytes treated with 17b‐estradiol. Using chromatin immunoprecipitation, we found that the estrogen receptor complex binds the human PEMT locus at sites in close proximity to those SNPs in linkage disequilibrium with rs12325817. This study is the first to explore the mechanism underlying the susceptibility of women to organ dysfunction under conditions of low dietary choline.

Cloning, characterization, and transformation of the phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) in maize (Zea mays L.)

N-methylation of phosphoethanolamine, the committing step in choline (Cho) biosynthesis in plants, is catalyzed by S-adenosyl-L-methionine: phosphoethanolamine N-methyltransferase (PEAMT, EC 2.1.1.103). Herein we report the cloning and characterization of the novel maize phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) using a combination of bioinformatics and a PCR-based allele mining strategy. The cDNA sequence of ZmPEAMT1 gene is 1,806 bp in length and translates a 495 amino acids peptide. The upstream promoter sequence of ZmPEAMT1 were obtained by TAIL-PCR, and contained four kinds of putative cis-acting regulatory elements, including stress-responsive elements, phytohormone-responsive elements, pollen developmental special activation elements, and light-induced signal transduction elements, as well as several other structural features in common with the promoter of rice and Arabidopsis homologies. RT-PCR analysis showed that expression of ZmPEAMT1 was induced by salt stress and suppressed by high temperature. Over-expression of ZmPEAMT1 enhanced the salt tolerance, root length, and silique number in transgenic Arabidopsis. These data indicated that ZmPEAMT1 maybe involved in maize root development and stress resistance, and maybe having a potential application in maize genetic engineering.

Posttranscriptional Regulation by the Upstream Open Reading Frame of the PhosphoethanolamineN-Methyltransferase Gene

Phosphoethanolamine N-methyltransferase (PEAMT) is involved in choline biosynthesis in plants. The 5' untranslated region (UTR) of several PEAMT genes was found to contain an upstream open reading frame (uORF). We generated transgenic Arabidopsis calli that expressed a chimeric gene constructed by fusing the 5' UTR of the Arabidopsis PEAMT gene (AtNMT1) upstream of the beta-glucuronidase gene. The AtNMT1 uORF was found to be involved in declining levels of the chimeric gene mRNA and repression of downstream beta-glucuronidase gene translation in the calli when the cells were treated with choline. Further, we discuss the role of the uORF.

Phosphatidylethanolamine N‐methyltransferase (PEMT) gene polymorphisms and risk of spina bifida

Recently, our group demonstrated a reduction in risk of neural tube defects (NTDs) in infants of mothers who had elevated intakes of dietary choline [Shaw et al., 2004]. We hypothesized that disturbance in choline metabolism that are secondary to either a choline deficiency, or choline pathway genes mutations, may represent risk factors for NTDs. Evidence to further support this hypothesis is derived from in vitro studies. These studies showed that inhibition of choline uptake and metabolism during murine neurulation result in growth retardation and developmental defects, including an increase in the prevalence of NTDs in mouse embryos [Fisher et al., 2001, 2002]. Choline or its derivatives, such as acetylcholine, betaine, phosphocholine, phosphatidylcholine (PtdCho), and sphingomyelin, are nutrients critical to many cellular processes. These include the maintenance of the structural integrity of cell membranes, phospholipid biosynthesis, cholinergic neurotransmission, transmembrane signaling, and lipid-cholesterol transport and metabolism. Choline is also one of the major sources of methyl groups in the diet and has essential roles in methyl-metabolism [Zeisel and Blusztajn, 1994]. The phosphatidylethanolamine N-methyltransferase (PEMT, EC 2.1.1.17) pathway contributes approximately one-third of the synthesis of PtdCho, which is the most abundant mammalian phospholipids [Walkey et al., 1999; Reo et al., 2002]. In the absence of dietary supplementation, the PEMT pathway provides the only de novo biosynthesis of choline. This reaction requires S-adenosylmethionine (SAM) as the methyl donor [Bremer and Greenberg, 1961], generating S-adenosylhomocysteine (SAH), which is subsequently hydrolyzed yielding adenosine and homocysteine (Hcy) [Finkelstein, 1998]. In mice, PEMT expression enhances not only plasma Hcy levels, but also Hcy secretion from hepatocytes [Schneider and Vance, 1978]. Hyperhomocysteinemia has been identified as a risk factor for NTDs [Rosenquist and Finnell, 2001]. Knowing the regulatory role of PEMT gene in choline metabolism and ultimately in determining Hcy levels, we examined two non-synonymous SNPs, rs7946 (Met212Val) and rs897453 (Val95Ile). We investigated whether PEMT gene variants would disturb PEMT function, rendering affected individuals more susceptible to spina bifida due to their increased dietary requirements for choline and the dysregulation of Hcy homeostasis. Our analytic strategy investigated potential spina bifida risks associated with PEMT gene polymorphisms in a population-based case-control study.