A Metabolic Function for Phospholipid and Histone Methylation.

S-adenosylmethionine (SAM) is the methyl donor for biological methylation modifications that regulate protein and nucleic acid functions. Here, we show that methylation of a phospholipid, phosphatidylethanolamine (PE), is a major consumer of SAM. The induction of phospholipid biosynthetic genes is accompanied by induction of the enzyme that hydrolyzes S-adenosylhomocysteine (SAH), a product and inhibitor of methyltransferases. Beyond its function for the synthesis of phosphatidylcholine (PC), the methylation of PE facilitates the turnover of SAM for the synthesis of cysteine and glutathione through transsulfuration. Strikingly, cells that lack PE methylation accumulate SAM, which leads to hypermethylation of histones and the major phosphatase PP2A, dependency on cysteine, and sensitivity to oxidative stress. Without PE methylation, particular sites on histones then become methyl sinks to enable the conversion of SAM to SAH. These findings reveal an unforeseen metabolic function for phospholipid and histone methylation intrinsic to the life of a cell.

Role of circadian clock system in the mitochondrial trans-sulfuration pathway and tissue remodeling.

Previous studies from our laboratory revealed that the gaseous molecule hydrogen sulfide (H2S), a metabolic product of epigenetics, involves trans-sulfuration pathway for ensuring metabolism and clearance of homocysteine (Hcy) from body, thereby mitigating the skeletal muscle's pathological remodeling. Although the master circadian clock regulator that is known as brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1 (i.e., BMAL 1) is associated with S-adenosylhomocysteine hydrolase (SAHH) and Hcy metabolism but how trans-sulfuration pathway is influenced by the circadian clock remains unexplored. We hypothesize that alterations in the functioning of circadian clock during sleep and wake cycle affect skeletal muscle's biology. To test this hypothesis, we measured serum matrix metalloproteinase (MMP) activities using gelatin gels for analyzing the MMP-2 and MMP-9. Further, employing casein gels, we also studied MMP-13 that is known to be influenced by the growth arrest and DNA damage-45 (GADD45) protein during sleep and wake cycle. The wild type and cystathionine ß synthase-deficient (CBS-/+) mice strains were treated with H2S and subjected to measurement of trans-sulfuration factors from skeletal muscle tissues. The results suggested highly robust activation of MMPs in the wake mice versus sleep mice, which appears somewhat akin to the "1-carbon metabolic dysregulation", which takes place during remodeling of extracellular matrix during muscular dystrophy. Interestingly, the levels of trans-sulfuration factors such as CBS, cystathionine γ lyase (CSE), methyl tetrahydrofolate reductase (MTHFR), phosphatidylethanolamine N-methyltransferase (PEMT), and Hcy-protein bound paraoxonase 1 (PON1) were attenuated in CBS-/+ mice. However, treatment with H2S mitigated the attenuation of the trans-sulfuration pathway. In addition, levels of mitochondrial peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC 1-α) and mitofusin-2 (MFN-2) were significantly improved by H2S intervention. Our findings suggest participation of the circadian clock in trans-sulfuration pathway that affects skeletal muscle remodeling and mitochondrial regeneration.

Hepatic oleate regulates one-carbon metabolism during high carbohydrate feeding.

Obesity is a major risk factor for type 2 diabetes, coronary heart disease, and strok. These diseases are associated with profound alterations in gene expression in metabolic tissues. Epigenetic-mediated regulation of gene expression is one mechanism through which environmental factors, such as diet, modify gene expression and disease predisposition. However, epigenetic control of gene expression in obesity and insulin resistance is not fully characterized. We discovered that liver-specific stearoyl-CoA desaturase-1 (Scd1) knockout mice (LKO) fed a high-carbohydrate low-fat diet exhibit dramatic changes in hepatic gene expression and metabolites of the folate cycle and one-carbon metabolism respectively for the synthesis of S-adenosylmethionine (SAM). LKO mice show an increased ratio of S-adenosylmethionine to S-adenosylhomocysteine, a marker for increased cellular methylation capacity. Furthermore, expression of DNA and histone methyltransferase genes is up-regulated while the mRNA and protein levels of the non-DNA methyltransferases including phosphatidylethanolamine methyltransferase (PEMT), Betaine homocysteine methyltransferase (Bhmt), and the SAM-utilizing enzymes such as glycine-N-methyltransferase (Gnmt) and guanidinoacetate methyltransferase (Gamt) are generally down-regulated. Feeding LKO mice a high carbohydrate diet supplemented with triolein, but not tristearin, and increased endogenous hepatic synthesis of oleate but not palmitoleate in Scd1 global knockout mice normalized one carbon gene expression and metabolite levels. Additionally, changes in one carbon gene expression are independent of the PGC-1α-mediated ER stress response previously reported in the LKO mice. Together, these results highlight the important role of oleate in maintaining one-carbon cycle homeostasis and point to observed changes in one-carbon metabolism as a novel mediator of the Scd1 deficiency-induced liver phenotype.

Ferrostatin-1 modulates dysregulated kidney lipids in acute kidney injury.

Ferroptosis, a form of regulated necrosis characterized by peroxidation of lipids such as arachidonic acid-containing phosphatidylethanolamine (PE), contributes to the pathogenesis of acute kidney injury (AKI). We have characterized the kidney lipidome in an experimental nephrotoxic AKI induced in mice using folic acid and assessed the impact of the ferroptosis inhibitor Ferrostatin-1. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was used to assess kidney lipidomics and it discriminated between glomeruli, medulla, and cortex in control kidneys, AKI kidneys, and AKI + Ferrostatin-1 kidneys. Out of 139 lipid species from 16 classes identified, 29 (20.5%) showed significant differences between control and AKI at 48 h. Total PE and lyso-sulfatide species decreased, while phosphatidylinositol (PI) species increased in AKI. Dysregulated mRNA levels for Pemt, Pgs1, Cdipt, and Tamm41, relevant to lipid metabolism, were in line with the lipid changes observed. Ferrostatin-1 prevented AKI and some AKI-associated changes in lipid levels, such as the decrease in PE and lyso-sulfatide species, without changing the gene expression of lipid metabolism enzymes. In conclusion, changes in the kidney lipid composition during nephrotoxic AKI are associated with differential gene expression of lipid metabolism enzymes and are partially prevented by Ferrostatin-1. © 2022 The Pathological Society of Great Britain and Ireland.

The Role of Phosphatidylethanolamine N-Methyltransferase (PEMT) and Its Waist-Hip-Ratio-Associated Locus rs4646404 in Obesity-Related Metabolic Traits and Liver Disease.

In previous genome-wide association studies (GWAS), genetic loci associated with obesity and impaired fat distribution (FD) have been identified. In the present study, we elucidated the role of the PEMT gene, including the waist-hip-ratio-associated single nucleotide polymorphism rs4646404, and its influence on obesity-related metabolic traits. DNA from 2926 metabolically well-characterized subjects was used for genotyping. PEMT expression was analyzed in paired visceral (vis) and subcutaneous (sc) adipose tissue (AT) from a subset of 574 individuals. Additionally, PEMT expression was examined in vis, sc AT and liver tissue in a separate cohort of 64 patients with morbid obesity and liver disease. An in vitro Pemt knockdown was conducted in murine epididymal and inguinal adipocytes. Our findings highlight tissue-specific variations in PEMT mRNA expression across the three studied tissues. Specifically, vis PEMT mRNA levels correlated significantly with T2D and were implicated in the progression of non-alcoholic steatohepatitis (NASH), in contrast to liver tissue, where no significant associations were found. Moreover, sc PEMT expression showed significant correlations with several anthropometric- and metabolic-related parameters. The rs4646404 was associated with vis AT PEMT expression and also with diabetes-related traits. Our in vitro experiments supported the influence of PEMT on adipogenesis, emphasizing its role in AT biology. In summary, our data suggest that PEMT plays a role in regulating FD and has implications in metabolic diseases.

PEMT Mediates Hepatitis C Virus-Induced Steatosis, Explains Genotype-Specific Phenotypes and Supports Virus Replication.

The hepatitis C virus (HCV) relies on cellular lipid pathways for virus replication and also induces liver steatosis, but the mechanisms involved are not clear. We performed a quantitative lipidomics analysis of virus-infected cells by combining high-performance thin-layer chromatography (HPTLC) and mass spectrometry, using an established HCV cell culture model and subcellular fractionation. Neutral lipid and phospholipids were increased in the HCV-infected cells; in the endoplasmic reticulum there was an ~four-fold increase in free cholesterol and an ~three-fold increase in phosphatidyl choline (p < 0.05). The increase in phosphatidyl choline was due to the induction of a non-canonical synthesis pathway involving phosphatidyl ethanolamine transferase (PEMT). An HCV infection induced expression of PEMT while knocking down PEMT with siRNA inhibited virus replication. As well as supporting virus replication, PEMT mediates steatosis. Consistently, HCV induced the expression of the pro-lipogenic genes SREBP 1c and DGAT1 while inhibiting the expression of MTP, promoting lipid accumulation. Knocking down PEMT reversed these changes and reduced the lipid content in virus-infected cells. Interestingly, PEMT expression was over 50% higher in liver biopsies from people infected with the HCV genotype 3 than 1, and three times higher than in people with chronic hepatitis B, suggesting that this may account for genotype-dependent differences in the prevalence of hepatic steatosis. PEMT is a key enzyme for promoting the accumulation of lipids in HCV-infected cells and supports virus replication. The induction of PEMT may account for virus genotype specific differences in hepatic steatosis.

Impairment of transcription factor Gcr1p binding motif perturbs OPI3 transcription in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the transcription factor GCR1 plays a vital role in carbohydrate metabolism and in the current study we tried to elucidate its role in lipid metabolism. In silico analysis revealed the upstream activation sequence (UAS) in the promoter region of OPI3 possessed six conserved recognition sequences for Gcr1p and the ChIP assay confirmed the binding of Gcr1p on the OPI3 promoter region. The real-time quantitative polymerase chain reaction and promoter-reporter activity revealed a substantial reduction in OPI3 expression and was supported with decreased phosphatidylcholine (PC) level that is rescued with exogenous choline supplementation in gcr1∆ cells. Simultaneously, there was an increase in triacylglycerol level, accompanied with increased number and size of lipid droplets in gcr1∆ cells. The expression of pT1, pT2 truncations in opi3∆ cells revealed the -1 to -500 bp in the promoter region is essential for the activation of OPI3 transcription. The mutation specifically at UASCT box (-265) in the OPI3 promoter region displayed a reduction in the PC level and the additional mutation at UASINO (-165) further reduced the PC level. Collectively, our data suggest that the GCR1 transcription factor also regulates the OPI3 expression and has an impact on lipid homeostasis.

Effects of acute intravenous lipopolysaccharide administration on the plasma lipidome and metabolome in lactating Holstein cows experiencing hyperlipidemia.

INTRODUCTION: The effects of lipopolysaccharides (i.e., endotoxin; LPS) on metabolism are poorly defined in lactating dairy cattle experiencing hyperlipidemia. OBJECTIVES: Our objective was to explore the effects of acute intravenous LPS administration on metabolism in late-lactation Holstein cows experiencing hyperlipidemia induced by intravenous triglyceride infusion and feed restriction. METHODS: Ten non-pregnant lactating Holstein cows (273 ± 35 d in milk) were administered a single bolus of saline (3 mL of saline; n [Formula: see text] 5) or LPS (0.375 [Formula: see text]g of LPS/kg of body weight; n [Formula: see text] 5). Simultaneously, cows were intravenously infused a triglyceride emulsion and feed restricted for 16 h to induce hyperlipidemia in an attempt to model the periparturient period. Blood was sampled at routine intervals. Changes in circulating total fatty acid concentrations and inflammatory parameters were measured. Plasma samples were analyzed using untargeted lipidomics and metabolomics. RESULTS: Endotoxin increased circulating serum amyloid A, LPS-binding protein, and cortisol concentrations. Endotoxin administration decreased plasma lysophosphatidylcholine (LPC) concentrations and increased select plasma ceramide concentrations. These outcomes suggest modulation of the immune response and insulin action. Lipopolysaccharide decreased the ratio of phosphatidylcholine to phosphatidylethanomanine, which potentially indicate a decrease in the hepatic activation of phosphatidylethanolamine N-methyltransferase and triglyceride export. Endotoxin administration also increased plasma concentrations of pyruvic and lactic acids, and decreased plasma citric acid concentrations, which implicate the upregulation of glycolysis and downregulation of the citric acid cycle (i.e., the Warburg effect), potentially in leukocytes. CONCLUSION: Acute intravenous LPS administration decreased circulating LPC concentrations, modified ceramide and glycerophospholipid concentrations, and influenced intermediary metabolism in dairy cows experiencing hyperlipidemia.

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 2 weeks. 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.

Hepatic PEMT Expression Decreases with Increasing NAFLD Severity.

Choline deficiency causes hepatic fat accumulation, and is associated with a higher risk of nonalcoholic fatty liver disease (NAFLD) and more advanced NAFLD-related hepatic fibrosis. Reduced expression of hepatic phosphatidylethanolamine N-methyltransferase (PEMT), which catalyzes the production of phosphatidylcholine, causes steatosis, inflammation, and fibrosis in mice. In humans, common PEMT variants impair phosphatidylcholine synthesis, and are associated with NAFLD risk. We investigated hepatic PEMT expression in a large cohort of patients representing the spectrum of NAFLD, and examined the relationship between PEMT genetic variants and gene expression. Hepatic PEMT expression was reduced in NAFLD patients with inflammation and fibrosis (i.e., nonalcoholic steatohepatitis or NASH) compared to participants with normal liver histology (ß = −1.497; p = 0.005). PEMT levels also declined with increasing severity of fibrosis with cirrhosis < incomplete cirrhosis < bridging fibrosis (ß = −1.185; p = 0.011). Hepatic PEMT expression was reduced in postmenopausal women with NASH compared to those with normal liver histology (ß = −3.698; p = 0.030). We detected a suggestive association between rs7946 and hepatic fibrosis (p = 0.083). Although none of the tested variants were associated with hepatic PEMT expression, computational fine mapping analysis indicated that rs4646385 may impact PEMT levels in the liver. Hepatic PEMT expression decreases with increasing severity of NAFLD in obese individuals and postmenopausal women, and may contribute to disease pathogenesis in a subset of NASH patients.

ATP8B1 Deficiency Results in Elevated Mitochondrial Phosphatidylethanolamine Levels and Increased Mitochondrial Oxidative Phosphorylation in Human Hepatoma Cells.

ATP8B1 is a phospholipid flippase that is deficient in patients with progressive familial intrahepatic cholestasis type 1 (PFIC1). PFIC1 patients suffer from severe liver disease but also present with dyslipidemia, including low plasma cholesterol, of yet unknown etiology. Here we show that ATP8B1 knockdown in HepG2 cells leads to a strong increase in the mitochondrial oxidative phosphorylation (OXPHOS) without a change in glycolysis. The enhanced OXPHOS coincides with elevated low-density lipoprotein receptor protein and increased mitochondrial fragmentation and phosphatidylethanolamine levels. Furthermore, expression of phosphatidylethanolamine N-methyltransferase, an enzyme that catalyzes the conversion of mitochondrial-derived phosphatidylethanolamine to phosphatidylcholine, was reduced in ATP8B1 knockdown cells. We conclude that ATP8B1 deficiency results in elevated mitochondrial PE levels that stimulate mitochondrial OXPHOS. The increased OXPHOS leads to elevated LDLR levels, which provides a possible explanation for the reduced plasma cholesterol levels in PFIC1 disease.

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.

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

BACKGROUND AND AIMS: 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). APPROACH AND RESULTS: 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. CONCLUSIONS: A PEMT-LRH-1 axis regulates mitochondrial biogenesis and beta-oxidation in hepatocytes.

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

BACKGROUND: 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. CLINICAL IMPACT: CF patients with exocrine pancreas insufficiency frequently develop choline deficiency, due to loss of bile Ptd'Cho via feces. ~ 50% (11-12 g) 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.

Comparison of Associations between One-Carbon Metabolism, Lipid Metabolism, and Fatty Liver Markers in Normal-Weight and Overweight People Aged 20-40 Years.

The aim of the present study was to compare biomarkers of one-carbon metabolism (OCM), lipid metabolism, and fatty liver in people with normal and increased body weight. The study was performed on 421 participants, aged 20-40 years, enrolled in Poznan, Poland, in 2016-2018. Choline and betaine intakes were assessed. DNA samples were genotyped for polymorphisms of phosphatidylethanolamine N-methyltransferase (PEMT; rs7946 and rs12325817), methylene tetrahydrofolate reductase (MTHFR; rs180113), methylenetetrahydrofolate dehydrogenase (MTHFD1; rs2236225), and dihydrofolate reductase (DHFR; rs70991108). To assess the associations between blood metabolites (choline, betaine, folate, L-carnitine, o-acetyl-L-carnitine, and trimethylamine N-oxide]), circulating lipids, and fatty liver indices, multiple logistic regression analyses were performed. Overweight/obese participants had 5.8% higher choline (p < 0.05) and 10% higher L-carnitine (p < 0.001) levels than normal-weight subjects. Serum folate and betaine levels were associated with lower total cholesterol (p < 0.001 and p < 0.05), low-density lipoprotein (LDL) cholesterol (p < 0.001 and p < 0.05, respectively), triacylglycerols (p < 0.01 and p < 0.001), and triglyceride glucose index (p < 0.001 and p < 0.01, respectively), though only in overweight/obese people. The PEMT rs12325817 CC genotype was associated with higher levels of high-density lipoprotein (HDL) cholesterol (p < 0.01) in overweight/obese people. The associations between OCM markers, fatty liver indices, and blood lipids differ in subjects with normal and excessive body weight.

An engineered mutant of a host phospholipid synthesis gene inhibits viral replication without compromising host fitness.

Viral infections universally rely on numerous hijacked host factors to be successful. It is therefore possible to control viral infections by manipulating host factors that are critical for viral replication. Given that host genes may play essential roles in certain cellular processes, any successful manipulations for virus control should cause no or mild effects on host fitness. We previously showed that a group of positive-strand RNA viruses enrich phosphatidylcholine (PC) at the sites of viral replication. Specifically, brome mosaic virus (BMV) replication protein 1a interacts with and recruits a PC synthesis enzyme, phosphatidylethanolamine methyltransferase, Cho2p, to the viral replication sites that are assembled on the perinuclear endoplasmic reticulum (ER) membrane. Deletion of the CHO2 gene inhibited BMV replication by 5-fold; however, it slowed down host cell growth as well. Here, we show that an engineered Cho2p mutant supports general PC synthesis and normal cell growth but blocks BMV replication. This mutant interacts and colocalizes with BMV 1a but prevents BMV 1a from localizing to the perinuclear ER membrane. The mislocalized BMV 1a fails to induce the formation of viral replication complexes. Our study demonstrates an effective antiviral strategy in which a host lipid synthesis gene is engineered to control viral replication without comprising host growth.

Hepatic PEMT activity mediates liver health, weight gain, and insulin resistance.

Phosphatidylethanolamine (PE) N-methyltransferase (PEMT) accounts for ∼30% of hepatic phosphatidylcholine (PC) biosynthesis. Pemt-/- mice fed a high-fat diet are protected against diet-induced obesity (DIO) and insulin resistance (IR) but develop nonalcoholic fatty liver disease (NAFLD) associated with a decreased PC:PE ratio. We investigated whether the lack of hepatic PEMT or the lack of PEMT in other tissues (where it is expressed at low levels) is responsible for or contributes to the protection against DIO and IR in Pemt-/- mice. Furthermore, we investigated whether decreasing PEMT expression with antisense oligonucleotides (ASOs) would result in metabolic benefits in both lean and obese mice without negatively impacting liver health. We both restored hepatic PEMT in Pemt-/- mice via adeno-associated virus delivery and decreased hepatic PEMT with ASOs in wild-type and ob/ob mice. Weight gain, insulin sensitivity, and indices of liver function were determined. We report that the protection against DIO and IR and the development of NAFLD is dependent on hepatic PEMT activity. NAFLD, associated with a significant decrease in the hepatic PC:PE ratio, was exacerbated by PEMT deficiency in obese mice, suggesting that phospholipid insufficiency promotes NAFLD progression during obesity or overnutrition. Hepatic PEMT is critical for maintaining phospholipid balance, which is crucial for a healthy liver.-Wan, S., van der Veen, J. N., Bakala N'Goma, J.-C., Nelson, R. C., Vance, D. E., Jacobs, R. L. Hepatic PEMT activity mediates liver health, weight gain, and insulin resistance.

A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt-/- mice fed a high-fat diet are protected from obesity and whole-body insulin resistance. However, Pemt-/- mice develop severe nonalcoholic steatohepatitis (NASH). Because NASH is often associated with hepatic insulin resistance, we investigated whether the increased insulin sensitivity in Pemt-/- mice was restricted to nonhepatic tissues or whether the liver was also insulin sensitive. Strikingly, the livers of Pemt-/- mice compared with those of Pemt+/+ mice were not insulin resistant, despite elevated levels of hepatic triacylglycerols and diacylglycerols, as well as increased hepatic inflammation and fibrosis. Endogenous glucose production was lower in Pemt-/- mice under both basal and hyperinsulinemic conditions. Experiments in primary hepatocytes and hepatoma cells revealed improved insulin signaling in the absence of PEMT, which was not due to changes in diacylglycerols, ceramides, or gangliosides. On the other hand, the phospholipid composition in hepatocytes seems critically important for insulin signaling such that lowering the PC:phosphatidylethanolamine (PE) ratio improves insulin signaling. Thus, treatments to reduce the PC:PE ratio in liver may protect against the development of hepatic insulin resistance.-Van der Veen, J. N., Lingrell, S., McCloskey, N., LeBlond, N. D., Galleguillos, D., Zhao, Y. Y., Curtis, J. M., Sipione, S., Fullerton, M. D., Vance, D. E., Jacobs, R. L. A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.

The membrane stress response buffers lethal effects of lipid disequilibrium by reprogramming the protein homeostasis network.

Lipid composition can differ widely among organelles and even between leaflets of a membrane. Lipid homeostasis is critical because disequilibrium can have disease outcomes. Despite their importance, mechanisms maintaining lipid homeostasis remain poorly understood. Here, we establish a model system to study the global effects of lipid imbalance. Quantitative lipid profiling was integral to monitor changes to lipid composition and for system validation. Applying global transcriptional and proteomic analyses, a dramatically altered biochemical landscape was revealed from adaptive cells. The resulting composite regulation we term the "membrane stress response" (MSR) confers compensation, not through restoration of lipid composition, but by remodeling the protein homeostasis network. To validate its physiological significance, we analyzed the unfolded protein response (UPR), one facet of the MSR and a key regulator of protein homeostasis. We demonstrate that the UPR maintains protein biogenesis, quality control, and membrane integrity-functions otherwise lethally compromised in lipid dysregulated cells.

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.