Phosphatidylethanolamine N-methyltransferase Research Articles

Magnetosome membrane engineering to improve G protein-coupled receptor activities in the magnetosome display system

Magnetotactic bacterium, Magnetospirillum magneticum, produces biogenic magnetic nanoparticles termed magnetosomes, which are primarily composed of a magnetite core and a surrounding lipid bilayer membrane. We have fabricated human transmembrane protein-magnetosome complexes by genetic engineering with embedding the transmembrane proteins of interest, in particular G protein-coupled receptors (GPCRs), in the magnetosome membrane. The magnetosomes provide a promising platform for high throughput ligand screening towards drug discovery, and this is a critical advantage of the magnetosome display system beyond conventional membrane platforms such as liposomes and lipid nano-discs. However, the human GPCRs expressed on the magnetosomes were not fully functionalized in bacterial membranes the most probably due to the lack of essential phospholipids such as phosphatidylcholine (PC) for GPCR functionalization. To overcome this issue, we expressed two types of PC-producing enzymes, phosphatidylcholine synthase (PCS) and phosphatidylethanolamine N-methyltransferase (PMT) in M. magneticum. As a result, generation and incorporation of PC in cell- and magnetosome-membranes were demonstrated. To the best of our knowledge, M. magneticum is the second bacterial species which had the PC-incorporated lipid membrane by genetic engineering. Subsequently, a GPCR, thyroid-stimulating hormone receptor (TSHR) and PCS were simultaneously expressed. We found that PC in the magnetosome membrane assisted the binding of TSHR and its ligand, indicating that the genetic approach demonstrated in this study is useful to enhance the function of the GPCRs displayed on the magnetosomes.

Relaxin has beneficial effects on liver lipidome and metabolic enzymes.

Relaxin is an insulin-like hormone with pleiotropic protective effects in several organs, including the liver. We aimed to characterize its role in the control of hepatic metabolism in healthy rats. Sprague-Dawley rats were treated with human recombinant relaxin-2 for 2weeks. The hepatic metabolic profile was analyzed using UHPLC-MS platforms. Hepatic gene expression of key enzymes of desaturation (Fads1/Fads2) of n-6 and n-3 polyunsaturated fatty acids (PUFAs), of phosphatidylethanolamine (PE) N-methyltransferase (Pemt), of fatty acid translocase Cd36, and of glucose-6-phosphate isomerase (Gpi) were quantified by Real Time-PCR. Activation of 5'AMP-activated protein kinase (AMPK) was analyzed by Western Blot. Relaxin-2 significantly modified the hepatic levels of 19 glycerophospholipids, 2 saturated (SFA) and 1 monounsaturated (MUFA) fatty acids (FA), 3 diglycerides, 1 sphingomyelin, 2 aminoacids, 5 nucleosides, 2 nucleotides, 1 carboxylic acid, 1 redox electron carrier, and 1 vitamin. The most noteworthy changes corresponded to the substantially decreased lysoglycerophospholipids, and to the clearly increased FA (16:1n-7/16:0) and MUFA + PUFA/SFA ratios, suggesting enhanced desaturase activity. Hepatic gene expression of Fads1, Fads2, and Pemt, which mediates lipid balance and liver health, was increased by relaxin-2, while mRNA levels of the main regulator of hepatic FA uptake Cd36, and of the essential glycolysis enzyme Gpi, were decreased. Relaxin-2 augmented the hepatic activation of the hepatoprotector and master regulator of energy homeostasis AMPK. Relaxin-2 treatment also rised FADS1, FADS2, and PEMT gene expression in cultured Hep G2 cells. Our results bring to light the hepatic metabolic features stimulated by relaxin, a promising hepatoprotective molecule.

Saccharomyces cerevisiae Δ9‐desaturase Ole1 Interacts with Lipid Biosynthetic Enzymes that Produce Storage Lipid, Phospholipid, and Sterol‐esters

Lipids are essential both for integrity of membranes and energy storage in a variety of organisms, from yeast to humans. The chemical properties of membrane phospholipids and triacylglycerol are determined by the composition and organization of their acyl chain components. Acyl-CoA molecules are delivered to the enzymes involved in lipid biosynthesis in a specific order, as is evidenced by the high prevalence of unsaturated acyl chains in the second position of phosphatidic acid and triacylglycerol in Saccharomyces cerevisiae. The mechanism controlling the specific nature of these lipids is not yet known, but we have previously demonstrated that acyltransferases that produce both storage and membrane lipid interact with the Δ9-desaturase, Ole1, in a complex we have called the desaturasome. We propose that these interactions determine the incorporation of unsaturated acyl chains into phospholipid and triacylglycerol and thus control the composition of membrane and storage lipids. Yeast two-hybrid and coimmunoprecipitation studies have revealed novel protein-protein interactions between Ole1 and storage lipid, phospholipid, and sterol-ester biosynthesis enzymes. Notably, Ole1 has been found to interact with the acyl-CoA:sterol acyltransferases Are1 and Are2, phosphatidate cytidylyltransferase Cds1, phosphatidylserine synthase Cho1, phosphatidylinositol synthase Pis1, phosphatidylethanolamine methyltransferase Cho2, and the rate limiting step in sterol synthesis, HMG-CoA reductase Hmg1. This research has determined the composition of an interactome that may exist to regulate the order of acyl chain incorporation into phospholipid, triacylglycerol, and sterol-esters. Future research aims to determine exactly how these interactions control lipid synthesis and composition via metabolic flux analysis and gene deletion studies. Investigating this lipid biosynthesis complex will have implications for a wide variety of applied research, from treating lipid dysregulation in humans to biofuel production by microbes.

Stable Isotopic Tracer Phospholipidomics Reveals Contributions of Key Phospholipid Biosynthetic Pathways to Low Hepatocyte Phosphatidylcholine to Phosphatidylethanolamine Ratio Induced by Free Fatty Acids.

There is a strong association between hepatocyte phospholipid homeostasis and non-alcoholic fatty liver disease (NAFLD). The phosphatidylcholine to phosphatidylethanolamine ratio (PC/PE) often draws special attention as genetic and dietary disruptions to this ratio can provoke steatohepatitis and other signs of NAFLD. Here we demonstrated that excessive free fatty acid (1:2 mixture of palmitic and oleic acid) alone was able to significantly lower the phosphatidylcholine to phosphatidylethanolamine ratio, along with substantial alterations to phospholipid composition in rat hepatocytes. This involved both a decrease in hepatocyte phosphatidylcholine (less prominent) and an increase in phosphatidylethanolamine, with the latter contributing more to the lowered ratio. Stable isotopic tracer phospholipidomic analysis revealed several previously unidentified changes that were triggered by excessive free fatty acid. Importantly, the enhanced cytidine diphosphate (CDP)-ethanolamine pathway activity appeared to be driven by the increased supply of preferred fatty acid substrates. By contrast, the phosphatidylethanolamine N-methyl transferase (PEMT) pathway was restricted by low endogenous methionine and consequently low S-adenosylmethionine, which resulted in a concomitant decrease in phosphatidylcholine and accumulation of phosphatidylethanolamine. Overall, our study identified several previously unreported links in the relationship between hepatocyte free fatty acid overload, phospholipid homeostasis, and the development of NAFLD.

Distribution of perfluorooctane sulfonate in mice and its effect on liver lipidomic

Perfluorooctane sulfonate (PFOS) is an emerging persistent organic pollutant (POP), and the harm caused by the enrichment of PFOS in living organism has attracted more and more attention. In this work, animal exposure model to PFOS was established. Mass spectrometry (MS), mass spectrometry imaging (MSI), hematoxylin and eosin (H&E) staining and lipidomics were combined for the study of the organ targeting of PFOS, the toxicity and possible mechanism caused by PFOS. PFOS most accumulated in the liver, followed by the lungs, kidneys, spleen, heart and brain. Combined with H&E staining and matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) results, it was found that the accumulation of PFOS indeed caused damage in particular areas of specific organ, like in the liver and in the marginal area of the heart. This work found that PFOS could cross the blood-brain barrier, entered the brain and caused the neurotoxicity, which was surprising and might be the reason that high dose of PFOS could cause convulsions. From the liver lipidomic analysis, we found that PFOS exposure mainly affected glycerophospholipid metabolism and sphingolipid metabolism. The up-regulated ceramide and lysophosphatidylcholine (LPC) might lead to liver cell apoptosis, and the decrease in liver triglyceride (TG) content might result in insufficient energy in mice and cause liver morphological damage. Phosphatidylcholine (PC) synthesis via phosphatidylethanolamine N-methyltransferase (PEMT) pathway might be a mechanism of self-protection in animals against PFOS induced inflammation. This study might provide new insight into underlying toxicity mechanism after exposure to PFOS.

Dietary Choline Intake during Pregnancy and PEMT rs7946 Polymorphism on Risk of Preterm Birth: A Case-Control Study

Introduction and Aims: Choline-metabolizing genetic variation may interact with choline intake on fetal programming and pregnancy outcome. This case-control study aims to explore the association of maternal choline consumption and phosphatidylethanolamine N-methyltransferase (PEMT) gene polymorphism rs7946 with preterm birth risk. Methods: 145 Han Chinese women with preterm delivery and 157 Han Chinese women with term delivery were recruited in Shanghai. Dietary choline intake during pregnancy was assessed using a validated food frequency questionnaire. Additionally, DNA samples were genotyped for PEMT rs7946 (G5465A) with plasma homocysteine (Hcy) levels measured. Results: Compared with the lowest quartile of choline intake, women within the highest consumption quartile had adjusted odds ratio (aOR) for preterm birth of 0.48 (95% confidence interval, CI [0.24, 0.95]). There was a significant interaction between maternal choline intake and PEMT rs7946 (p for interaction = 0.04), where the AA genotype carriers who consumed the energy-adjusted choline <255.01 mg/day had aOR for preterm birth of 3.75 (95% CI [1.24, 11.35]), compared to those with GG genotype and choline intake >255.01 mg/day during pregnancy. Additionally, the greatest elevated plasma Hcy was found in the cases with AA genotype and choline consumption <255.01 mg/day (p < 0.001). Conclusion: The AA genotype of PEMT rs7946 may be associated with increased preterm birth in these Han Chinese women with low choline intake during pregnancy.

Postnatal adaptations of phosphatidylcholine metabolism in extremely preterm infants: implications for choline and PUFA metabolism

ABSTRACTBackgroundLipid metabolism in pregnancy delivers PUFAs from maternal liver to the developing fetus. The transition at birth to diets less enriched in PUFA is especially challenging for immature, extremely preterm infants who are typically supported by total parenteral nutrition.ObjectiveThe aim was to characterize phosphatidylcholine (PC) and choline metabolism in preterm infants and demonstrate the molecular specificity of PC synthesis by the immature preterm liver in vivo.MethodsThis MS-based lipidomic study quantified the postnatal adaptations to plasma PC molecular composition in 31 preterm infants <28 weeks’ gestational age. Activities of the cytidine diphosphocholine (CDP-choline) and phosphatidylethanolamine-N-methyltransferase (PEMT) pathways for PC synthesis were assessed from incorporations of deuterated methyl-D9-choline chloride.ResultsThe concentration of plasma PC in these infants increased postnatally from median values of 481 (IQR: 387–798) µM at enrollment to 1046 (IQR: 616–1220) µM 5 d later (P < 0.001). Direct incorporation of methyl-D9-choline demonstrated that this transition was driven by an active CDP-choline pathway that synthesized PC enriched in species containing oleic and linoleic acids. A second infusion of methyl-D9-choline chloride at day 5 clearly indicated continued activity of this pathway. Oxidation of D9-choline through D9-betaine resulted in the transfer of 1 deuterated methyl group to S-adenosylmethionine. A very low subsequent transfer of this labeled methyl group to D3-PC indicated that liver PEMT activity was essentially inactive in these infants.ConclusionsThis study demonstrated that the preterm infant liver soon after birth, and by extension the fetal liver, was metabolically active in lipoprotein metabolism. The low PEMT activity, which is the only pathway for endogenous choline synthesis and is responsible for hormonally regulated export of PUFAs from adult liver, strongly supports increased supplementation of preterm parenteral nutrition with both choline and PUFAs.

Pharmacologic downregulation of protein arginine methyltransferase1 expression by adenosine dialdehyde increases cell senescence in breast cancer

We investigated the role of protein arginine methylation (PAM) in estrogen receptor (ER)-positive breast cancer cells through pharmacological intervention. Tamoxifen (TAM) or adenosine dialdehyde (ADOX), independently, triggered cell cycle arrest and down-regulated PAM, as reduced protein arginine methyltransferase1 (PRMT1) mRNA and asymmetric dimethylarginine (ADMA) levels. Synergistic effect of these compounds elicited potent anti-cancer effect. However, reduction in ADMA was not proportionate with the compound-induced down-regulation of PRMT1 mRNA. We hypothesized that the disproportionate effect is due to the influence of the compounds on other methyltransferases, which catalyze the arginine dimethylation reaction and the diversity in the degree of drug-protein interaction among these methyltransferases. In silico analyses revealed that independently, ADOX or TAM, binds with phosphatidylethanolamine-methyltransferase (PEMT) or betaine homocysteine-methyl transferase (BHMT); and that the binding affinity of ADOX with PEMT or BHMT is prominent than TAM. These observations suggest that in breast cancer, synergistic effect of ADOX + TAM elicits impressive protective function by regulating PAM; and plausibly, restoration of normal enzyme activities of methyltransferases catalyzing arginine dimethylation could have clinical benefits.

Distinct infrastructure of lipid networks in visceral and subcutaneous adipose tissues in overweight humans

ABSTRACTBackgroundAdipose tissue plays important roles in health and disease. Given the unique association of visceral adipose tissue with obesity-related metabolic diseases, the distribution of lipids between the major fat depots located in subcutaneous and visceral regions may shed new light on adipose tissue–specific roles in systemic metabolic perturbations.ObjectiveWe sought to characterize the lipid networks and unveil differences in the metabolic infrastructure of the 2 adipose tissues that may have functional and nutritional implications.MethodsPaired visceral and subcutaneous adipose tissue samples were obtained from 17 overweight patients undergoing elective abdominal surgery. Ultra-performance LC-MS was used to measure 18,640 adipose-derived features; 520 were putatively identified. A stem cell model for adipogenesis was used to study the functional implications of the differences found.ResultsOur analyses resulted in detailed lipid metabolic maps of the 2 major adipose tissues. They point to a higher accumulation of phosphatidylcholines, triacylglycerols, and diacylglycerols, although lower ceramide concentrations, in subcutaneous tissue. The degree of unsaturation was lower in visceral adipose tissue (VAT) phospholipids, indicating lower unsaturated fatty acid incorporation into adipose tissue. The differential abundance of phosphatidylcholines we found can be attributed at least partially to higher expression of phosphatidylethanolamine methyl transferase (PEMT). PEMT-deficient embryonic stem cells showed a dramatic decrease in adipogenesis, and the resulting adipocytes exhibited lower accumulation of lipid droplets, in line with the lower concentrations of glycerolipids in VAT. Ceramides may inhibit the expression of PEMT by increased insulin resistance, thus potentially suggesting a functional pathway that integrates ceramide, PEMT, and glycerolipid biosynthetic pathways.ConclusionsOur work unveils differential infrastructure of the lipid networks in visceral and subcutaneous adipose tissues and suggests an integrative pathway, with a discriminative flux between adipose tissues.

Choline in cystic fibrosis: relations to pancreas insufficiency, enterohepatic cycle, PEMT and intestinal microbiota.

Cystic Fibrosis (CF) is an autosomal recessive disorder with life-threatening organ manifestations. 87% of CF patients develop exocrine pancreas insufficiency, frequently starting in utero and requiring lifelong pancreatic enzyme substitution. 99% develop progressive lung disease, and 20-60% CF-related liver disease, from mild steatosis to cirrhosis. Characteristically, pancreas, liver and lung are linked by choline metabolism, a critical nutrient in CF. Choline is a tightly regulated tissue component in the form of phosphatidylcholine (Ptd'Cho) and sphingomyelin (SPH) in all membranes and many secretions, particularly of liver (bile, lipoproteins) and lung (surfactant, lipoproteins). Via its downstream metabolites, betaine, dimethylglycine and sarcosine, choline is the major one-carbon donor for methionine regeneration from homocysteine. Methionine is primarily used for essential methylation processes via S-adenosyl-methionine. CF patients with exocrine pancreas insufficiency frequently develop choline deficiency, due to loss of bile Ptd'Cho via feces. ~ 50% (11-12g) of hepatic Ptd'Cho is daily secreted into the duodenum. Its re-uptake requires cleavage to lyso-Ptd'Cho by pancreatic and small intestinal phospholipases requiring alkaline environment. Impaired CFTR-dependent bicarbonate secretion, however, results in low duodenal pH, impaired phospholipase activity, fecal Ptd'Cho loss and choline deficiency. Low plasma choline causes decreased availability for parenchymal Ptd'Cho metabolism, impacting on organ functions. Choline deficiency results in hepatic choline/Ptd'Cho accretion from lung tissue via high density lipoproteins, explaining the link between choline deficiency and lung function. Hepatic Ptd'Cho synthesis from phosphatidylethanolamine by phosphatidylethanolamine-N-methyltransferase (PEMT) partly compensates for choline deficiency, but frequent single nucleotide polymorphisms enhance choline requirement. Additionally, small intestinal bacterial overgrowth (SIBO) frequently causes intraluminal choline degradation in CF patients prior to its absorption. As adequate choline supplementation was clinically effective and adult as well as pediatric CF patients suffer from choline deficiency, choline supplementation in CF patients of all ages should be evaluated.

Abstract 3740: Role of phosphatidylcholine in β-catenin-driven hepatocellular carcinoma

Abstract Introduction: This study aimed to clarify the role of phosphatidylcholine (PC) and choline metabolites in β-catenin-driven hepatocellular carcinoma (HCC) using human HCC cell lines and transgenic zebrafish expressing hepatocyte-specific activated β-catenin. Background: β-catenin is widely believed to be a major oncogene in HCC. PC is one of the major constituents of biological membranes and is synthesized via the phosphatidylethanolamine N-methyltransferase (PEMT) and cytidine diphosphate (CDP)-choline pathways in the liver. Several studies have reported that changes in gene expression and enzyme activity lead to altered PC synthesis in cancer, which contributes to malignant growth. However, the role of altered PC synthesis in HCC and the contribution of activated β-catenin to changes in PC metabolism have not been fully explored. Methods and Results: Transgenic zebrafish expressing hepatocyte-specific activated β-catenin develop HCC as adults and show significant liver enlargement during larval development due to increased cell proliferation. We measured PC levels in transgenic zebrafish HCC and non-transgenic control livers using liquid chromatography-mass spectrometry (LC-MS). PC levels in transgenic zebrafish HCC were significantly higher than in controls. Using a colorimetric assay kit, we found that PC levels were significantly higher in human HCC cell lines (SNU398, C3A and PLC/PRF/5) than in immortalized human hepatocytes (THLE2). To clarify the role of PC and choline metabolites in β-catenin-driven liver overgrowth, we treated larval zebrafish with bezafibrate (PEMT inhibitor) or meclofenoxate (CPT/CEPT inhibitor). We found that both of these inhibitors of PC lipid synthesis reduced larval liver size and decreased the number of EdU-labeled proliferative cells in transgenic zebrafish. Histologically, nonalcoholic steatohepatitis (NASH)-like changes, such as steatosis, ballooning, inflammation, and hepatocellular necrosis, were detected in the livers of transgenic larvae treated with bezafibrate or meclofenoxate. To characterize the role of PC in human HCC cell lines, we treated SNU398, C3A and PLC/PRF/5 cells with bezafibrate or meclofenoxate and measured PC levels by colorimetric assay and viability by CellTiter-Glo Assay. We found that both drugs decreased PC levels and viability in these HCC cell lines. Furthermore, SNU398 cells overexpressing activated β-catenin using a Tet-on system demonstrated higher PC levels and greater viability than SNU398 control cells. Conclusion: Our results suggest that increased PC synthesis is associated with cell proliferation, zebrafish larval liver overgrowth, and hepatocarcinogenesis. These results also suggest that activated β-catenin may regulate choline metabolites and contribute to an increase in PC production. Citation Format: Junko Kuramoto, Richard Smith, Sharanya M. Kalasekar, Alexis Fulbright, Gregory S. Ducker, Kimberley J. Evason. Role of phosphatidylcholine in β-catenin-driven hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3740.

Plasma phospholipid dysregulation in patients with cystathionine-β synthase deficiency

Background & aimsPatients with cystathionine β-synthase deficiency (CBSD) exhibit high circulating levels of homocysteine and enhanced lipid peroxidation. We have characterized the plasma lipidome in CBSD patients and related lipid abnormalities with reactions underlying enhanced homocysteine levels. Methods and resultsUsing an ultra-high-performance liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry method, plasma lipids were determined with an untargeted lipidomics approach in 11 CBSD patients and 11 matched healthy subjects (CTRL). Compared to CTRL, CBSD patients had a higher medium and long-chain polyunsaturated fatty acids (PUFA) content in phosphatidylethanolamine (PE) and lysophosphatidylethanolamine (LPE) species (p < 0.02), and depletion of phosphatidylcholine (PC; p = 0.02) and of lysophosphatidylcholine (LPC; p = 0.003) species containing docosahexaenoic acid (DHA), suggesting impaired phosphatidylethanolamine-N-methyltransferase (PEMT) activity. PEMT converts PE into PC using methyl group by S-adenosylmethionine (SAM) thus converted in S-adenosylhomocysteine (SAH). Whole blood SAM and SAH concentrations by liquid chromatography tandem mass spectrometry were 1.4-fold (p = 0.015) and 5.3-fold (p = 0.003) higher in CBSD patients than in CTRL. A positive correlation between SAM/SAH and PC/PE ratios (r = 0.520; p = 0.019) was found. ConclusionsA novel biochemical abnormality in CBSD patients consisting in depletion of PC and LPC species containing DHA and accumulation of PUFA in PE and LPE species is revealed by this lipidomic approach. Changes in plasma SAM and SAH concentrations are associated with such phospholipid dysregulation. Given the key role of DHA in thrombosis prevention, depletion of PC species containing DHA in CBSD patients provides a new direction to understand the poor cardiovascular outcome of patients with homocystinuria.

Dysregulation of Lipid Metabolism in Macrophages Is Responsible for Severe Endotoxin Tolerance in FcgRIIB-Deficient Lupus Mice.

FcgRIIB dysfunction is commonly found in patients with lupus, especially in Asia. LPS-tolerance is prominent in FcgRIIB–/– lupus mice. LPS-tolerant macrophages demonstrate cell energy depletion, which might affect lipid metabolism. Therefore, to explore lipid metabolism, LPS-tolerance was induced twice by LPS administration in macrophages and in mice. LPS-tolerant FcgRIIB–/– macrophages demonstrated lesser mitochondrial DNA (mtDNA), more severe ATP depletion, lower cytokine production, and higher lipid accumulation (oil red O staining) compared to LPS-tolerant WT cells. Mass-spectrometry-based lipidomic analysis demonstrated a higher abundance of phosphatidylethanolamine (PE) phospholipid in LPS-tolerant FcgRIIB–/– macrophages than WT cells. This was at least in part due to the lower expression of phosphatidylethanolamine N-methyltransferase (pemt), an enzyme that converts PE to phosphatidylcholine (PC). Aminoimidazole-4-carboxamide ribonucleotide (AICAR), a pemt inhibitor, worsens LPS-tolerance in WT macrophages and supports the impact of pemt upon LPS-tolerant FcgRIIB–/– macrophages. Additionally, phosphorylated AMP-activated protein kinase (AMPK-p), a molecule for ATP-restoration associated with pemt, and phosphorylated acetyl CoA carboxylase, a downstream signaling of AMPK-p, were higher in LPS-tolerant FcgRIIB–/– macrophages than WT. Furthermore, Compound C, an AMPK inhibitor, attenuated LPS-tolerance in both FcgRIIB–/– macrophages and mice. Taken together, the intense decrease in cytokine production after the second LPS stimulation (LPS-tolerance) in FcgRIIB–/– macrophages was possibly due to the impact of an immense cytokine synthesis after the first dose of LPS. This includes using up PEMT, an enzyme of phospholipid synthesis during cytokine production, and AMPK-p induction in response to profound ATP-depletion. Therefore, the manipulation of the AMPK/PEMT axis provides a novel therapeutic candidate for the treatment of severe LPS-tolerance in lupus.

Implication of phosphatidylethanolamine N-methyltransferase in adipocyte differentiation

Phosphatidylethanolamine N-methyltransferase (PEMT) is a small integral membrane protein that converts phosphatidylethanolamine (PE) into phosphatidylcholine (PC). It has been previously reported that, unexpectedly, PEMT deficiency protected from high-fat diet (HFD)-induced obesity and insulin resistance, pointing to a possible role of this enzyme in the regulation of adipose cell metabolism. Using mouse 3T3-L1 preadipocytes as a biological system, we demonstrate that PEMT expression is strongly increased during the differentiation of preadipocytes into mature adipose cells. Knockdown of PEMT reduced the expression of early and late adipogenic markers, inhibited lipid droplet formation, reduced triacylglycerol content and decreased the levels of leptin release from the adipocytes, suggesting that PEMT is a novel and relevant regulator of adipogenesis. Investigation into the mechanisms whereby PEMT regulates adipocyte differentiation revealed that extracellularly regulated kinases (ERK1/2) and AKT are essential factors in this process. Specifically, the activities of ERK1/2 and AKT, which are decreased during adipocyte differentiation, were elevated upon Pemt knockdown. Moreover, treatment of cells with exogenous ceramide 1-phosphate (C1P), which we reported to be a negative regulator of adipogenesis, decreased PEMT expression, suggesting that PEMT is also a relevant factor in the anti-adipogenic action of C1P. Altogether, the data presented here identify PEMT as a novel regulator of adipogenesis and a mediator of the anti-adipogenic action of C1P.

Dietary Choline Intake During Pregnancy and PEMT rs7946 Polymorphism on Risk of Preterm Birth: A Case-Control Study

Abstract Objectives Choline-metabolizing genetic variation may interact with choline intake on fetal programming and pregnancy outcome. This case-control study aims to explore the association of maternal choline consumption and phosphatidylethanolamine N-methyltransferase (PEMT) gene polymorphism rs7946 with incident preterm birth. Methods 165 Chinese Han women with preterm delivery and 165 Chinese Han women with term delivery were recruited in Shanghai, China. Dietary choline intake during pregnancy was assessed using a validated food frequency questionnaire. Additionally, DNA samples were genotyped for PEMT rs7946 (G5465A) by a real-time polymerase chain reaction method with plasma homocysteine (Hcy) levels measured by an enzymatic cycling method. Odds ratio (OR) and 95% CI were estimated using unconditional logistic regression with adjustment of the covariates which were selected based on a comparison of social-democratic and clinical characteristics between two groups. Results There was a significant interaction between maternal choline intake and PEMT rs7946 (P for interaction = 0.04), where the AA genotype carriers who consumed the energy-adjusted choline &amp;lt;255.01 mg/d had an adjusted OR for preterm birth of 3.75 (95% CI 1.24–11.35), compared to those with GG genotype and choline intake &amp;gt;255.01 mg/d during pregnancy. Additionally, the highest plasma Hcy was found in the cases with AA genotype and choline consumption &amp;lt;255.01 mg/d (P &amp;lt; 0.001). The AA genotype was also associated with a greater risk of preterm birth among the women when their plasma Hcy is at the highest tertile level (AA vs. GA + GG: adjusted OR = 1.56, 95% CI 1.02–2.88). Conclusions The AA genotype of PEMT rs7946 may be associated with increased preterm birth in these Chinese Han women with low choline intake during pregnancy. The observed association may be partially explained by mechanisms related to the increasing Hcy level induced by PEMT rs7946 gene polymorphism. This study was registered at ClinicalTrials.gov (#NCT02841813). Funding Sources The Danone Nutrition Research and Education Grant (DIC2017–09); Research Project of Shanghai University of Traditional Chinese Medicine (2019LK042).

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.

The topology of the ER-resident phospholipid methyltransferase Opi3 of Saccharomyces cerevisiae is consistent with in trans catalysis

Phospholipid N-methyltransferases (PLMTs) synthesize phosphatidylcholine by methylating phosphatidylethanolamine using S-adenosylmethionine as a methyl donor. Eukaryotic PLMTs are integral membrane enzymes located in the endoplasmic reticulum (ER). Recently Opi3, a PLMT of the yeast Saccharomyces cerevisiae was proposed to perform in trans catalysis, i.e. while localized in the ER, Opi3 would methylate lipid substrates located in the plasma membrane at membrane contact sites. Here, we tested whether the Opi3 active site is located at the cytosolic side of the ER membrane, which is a prerequisite for in trans catalysis. The membrane topology of Opi3 (and its human counterpart, phosphatidylethanolamine N-methyltransferase, expressed in yeast) was addressed by topology prediction algorithms and by the substituted cysteine accessibility method. The results of these analyses indicated that Opi3 (as well as phosphatidylethanolamine N-methyltransferase) has an N-out C-in topology and contains four transmembrane domains, with the fourth forming a re-entrant loop. On the basis of the sequence conservation between the C-terminal half of Opi3 and isoprenyl cysteine carboxyl methyltransferases with a solved crystal structure, we identified amino acids critical for Opi3 activity by site-directed mutagenesis. Modeling of the structure of the C-terminal part of Opi3 was consistent with the topology obtained by the substituted cysteine accessibility method and revealed that the active site faces the cytosol. In conclusion, the location of the Opi3 active site identified here is consistent with the proposed mechanism of in trans catalysis, as well as with conventional catalysis in cis.

The associations between phosphatidylethanolamine N-methyltransferase gene, dietary choline intake and anxiety and depression in healthy UK adults

The associations between phosphatidylethanolamine N-methyltransferase gene, dietary choline intake and anxiety and depression in healthy UK adults

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.

The Role of Phosphatidylethanolamine-N-methyltransferase (PEMT) Gene rs12449964 Polymorphism in the Development of Acute Pancreatitis and its Complications

Background. Acute pancreatitis is considered to be an important issue in modern medicine. The phosphatidylethanolamine-N-methyltransferase enzyme plays a significant role in the regulation of lipid metabolism by catalyzing the process of methylation of phosphatidylethanolamine to phosphatidylcholine. These lipids are key components of mitochondrial and cell membranes, providing their fluid and plastic properties and participating in the transport of fats, fatty acids and cholesterol. Along with its function in the synthesis of phosphatidylcholine, the methylation of phosphatidylethanolamine promotes the turnover of S-adenosylmethionine for the synthesis of cysteine and glutathione through transulphurisation. PEMT is a gene encoding the phosphatidylethanolamine-N-methyltransferase enzyme.Aim. To determine the role of PEMT C/T rs12449964 polymorphism in the risk of developing acute pancreatitis and its complications among Russian residents in Central Russia.Materials and methods. Whole blood samples were collected from 502 unrelated patients with acute non-biliary pancreatitis (97 women and 405 men) of Russian nationality who had been admitted to the surgical departments of the city of Kursk from 2015 to 2018, as well as from 513 unrelated individuals of Russian nationality without gastrointestinal diseases (101 women and 412 men). The average age of patients and healthy individuals was 48.9 ± 13.1 and 47.89 ± 12.1 years, respectively. Genomic DNA was isolated by a standard phenol-chloroform extraction method. Genotyping of rs12449964 polymorphism was performed using real-time PCR by allelic discrimination using a CFX96 Bio-Rad Laboratories amplifier (USA) with TaqMan probes and commercial TaqMan SNP Genotyping Assays reagents purchased from Applied Biosystems (USA).Results. The study has shown that the frequency of the C allele and the C/C PEMT C/T rs12449964 genotype was higher in the group of patients with acute pancreatitis, while the C/T genotype was predominant in the control group. C/T — T/T genotypes demonstrated a protective effect on the development of infected pancreatic necrosis, purulent necrotic peripancreatitis and severe acute pancreatitis.Conclusions. The disruption of phosphatidylethanolamine methylation processes increases the sensitivity of cells to oxidative stress, which can lead to the development of acute pancreatitis.