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.

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.

Hepatic levels of S-adenosylmethionine regulate the adaptive response to fasting.

There has been an intense focus to uncover the molecular mechanisms by which fasting triggers the adaptive cellular responses in the major organs of the body. Here, we show that in mice, hepatic S-adenosylmethionine (SAMe)-the principal methyl donor-acts as a metabolic sensor of nutrition to fine-tune the catabolic-fasting response by modulating phosphatidylethanolamine N-methyltransferase (PEMT) activity, endoplasmic reticulum-mitochondria contacts, ß-oxidation, and ATP production in the liver, together with FGF21-mediated lipolysis and thermogenesis in adipose tissues. Notably, we show that glucagon induces the expression of the hepatic SAMe-synthesizing enzyme methionine adenosyltransferase α1 (MAT1A), which translocates to mitochondria-associated membranes. This leads to the production of this metabolite at these sites, which acts as a brake to prevent excessive ß-oxidation and mitochondrial ATP synthesis and thereby endoplasmic reticulum stress and liver injury. This work provides important insights into the previously undescribed function of SAMe as a new arm of the metabolic adaptation to fasting.

PEMT rs7946 Polymorphism and Sex Modify the Effect of Adequate Dietary Choline Intake on the Risk of Hepatic Steatosis in Older Patients with Metabolic Disorders.

In humans, PEMT rs7946 polymorphism exerts sex-specific effects on choline requirement and hepatic steatosis (HS) risk. Few studies have explored the interaction effect of the PEMT rs7946 polymorphism and sex on the effect of adequate choline intake on HS risk. In this cross-sectional study, we investigated the association between PEMT polymorphism and adequate choline intake on HS risk. We enrolled 250 older patients with metabolic disorders with (n = 152) or without (n = 98; control) ultrasonically diagnosed HS. An elevated PEMT rs7946 A allele level was associated with a lower HS risk and body mass index in both men and women. Dietary choline intake-assessed using a semiquantitative food frequency questionnaire-was associated with reduced obesity in men only (p for trend < 0.05). ROC curve analysis revealed that the cutoff value of energy-adjusted choline intake for HS diagnosis was 448 mg/day in women (AUC: 0.62; 95% CI: 0.57-0.77) and 424 mg/day in men (AUC: 0.63, 95% CI: 0.57-0.76). In women, GG genotype and high choline intake (>448 mg/day) were associated with a 79% reduction in HS risk (adjusted OR: 0.21; 95% CI: 0.05-0.82); notably, GA or AA genotype was associated with a reduced HS risk regardless of choline intake (p < 0.05). In men, GG genotype and high choline intake (>424 mg/day) were associated with a 3.7-fold increase in HS risk (OR: 3.7; 95% CI: 1.19-11.9). Further adjustments for a high-density lipoprotein level and body mass index mitigated the effect of choline intake on HS risk. Current dietary choline intake may be inadequate for minimizing HS risk in postmenopausal Taiwanese women carrying the PEMT rs7946 GG genotype. Older men consuming more than the recommended amount of choline may have an increased risk of nonalcoholic fatty liver disease; this risk is mediated by a high-density lipoprotein level and obesity.

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.

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.

Construction of a novel choline metabolism-related signature to predict prognosis, immune landscape, and chemotherapy response in colon adenocarcinoma.

Background: Colon adenocarcinoma (COAD) is a common digestive system malignancy with high mortality and poor prognosis. Accumulating evidence indicates that choline metabolism is closely related to tumorigenesis and development. However, the efficacy of choline metabolism-related signature in predicting patient prognosis, immune microenvironment and chemotherapy response has not been fully clarified. Methods: Choline metabolism-related differentially expressed genes (DEGs) between normal and COAD tissues were screened using datasets from The Cancer Genome Atlas (TCGA), Kyoto Encyclopedia of Genes and Genomes (KEGG), AmiGO2 and Reactome Pathway databases. Two choline metabolism-related genes (CHKB and PEMT) were identified by univariate and multivariate Cox regression analyses. TCGA-COAD was the training cohort, and GSE17536 was the validation cohort. Patients in the high- and low-risk groups were distinguished according to the optimal cutoff value of the risk score. A nomogram was used to assess the prognostic accuracy of the choline metabolism-related signature. Calibration curves, decision curve analysis (DCA), and clinical impact curve (CIC) were used to improve the clinical applicability of the prognostic signature. Gene Ontology (GO) and KEGG pathway enrichment analyses of DEGs in the high- and low-risk groups were performed. KEGG cluster analysis was conducted by the KOBAS-i database. The distribution and expression of CHKB and PEMT in various types of immune cells were analyzed based on single-cell RNA sequencing (scRNA-seq). The CIBERSORT and ESTIMATE algorithms evaluated tumor immune cell infiltration in the high- and low-risk groups. Evaluation of the half maximal inhibitory concentration (IC50) of common chemotherapeutic drugs based on the choline metabolism-related signature was performed. Small molecule compounds were predicted using the Connectivity Map (CMap) database. Molecular docking is used to simulate the binding conformation of small molecule compounds and key targets. By immunohistochemistry (IHC), Western blot, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) experiments, the expression levels of CHKB and PEMT in human, mouse, and cell lines were detected. Results: We constructed and validated a choline metabolism-related signature containing two genes (CHKB and PEMT). The overall survival (OS) of patients in the high-risk group was significantly worse than that of patients in the low-risk group. The nomogram could effectively and accurately predict the OS of COAD patients at 1, 3, and 5 years. The DCA curve and CIC demonstrate the clinical utility of the nomogram. scRNA-seq showed that CHKB was mainly distributed in endothelial cells, while PEMT was mainly distributed in CD4+ T cells and CD8+ T cells. In addition, multiple types of immune cells expressing CHKB and PEMT differed significantly. There were significant differences in the immune microenvironment, immune checkpoint expression and chemotherapy response between the two risk groups. In addition, we screened five potential small molecule drugs that targeted treatment for COAD. Finally, the results of IHC, Western blot, and qRT-PCR consistently showed that the expression of CHKB in human, mouse, and cell lines was elevated in normal samples, while PMET showed the opposite trend. Conclusion: In conclusion, we constructed a choline metabolism-related signature in COAD and revealed its potential application value in predicting the prognosis, immune microenvironment, and chemotherapy response of patients, which may lay an important theoretical basis for future personalized precision therapy.

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.

PEMT variants are associated with nonsyndromic cleft lip with or without cleft palate in Chile.

Aim: To assess the association between PEMT variants and nonsyndromic cleft lip with or without cleft palate in Chile and the effects of these variants on global DNA methylation. Subjects & methods: The authors obtained genotypes for nine variants from 247 cases and 453 controls for genotype-phenotype associations. The effect of significant polymorphisms on global DNA methylation (percentage of long interspersed element-1 methylation) was evaluated in a subsample of 95 controls. Results: After multiple comparison corrections, variants rs7649 and rs4646409 were associated with nonsyndromic cleft lip with or without cleft palate. Carriers of risk alleles presented lower DNA methylation levels than noncarriers. Conclusion: According to functional analysis for risk variants from previous reports, the authors infer that a decrease of methyl group availability is occurring in affected subjects.

This study evaluated if variants in the gene named PEMT confers an increased risk for nonsyndromic cleft lip with or without cleft palate in Chile and its possible effects on methylation of DNA, a variable linked to gene expression modulation. The study found that the variants recognized as rs7649 and rs4646409 increase the risk of nonsyndromic cleft lip with or without cleft palate in the Chilean population and decrease DNA methylation. The authors conclude that this gene may be involved in this birth defect. New studies are needed to confirm the relation between this condition and DNA methylation mediated by these genetic variants.

Phosphatidylethanolamine N-Methyltransferase Knockout Modulates Metabolic Changes in Aging Mice.

Phospholipid metabolism, including phosphatidylcholine (PC) biosynthesis, is crucial for various biological functions and is associated with longevity. Phosphatidylethanolamine N-methyltransferase (PEMT) is a protein that catalyzes the biosynthesis of PC, the levels of which change in various organs such as the brain and kidneys during aging. However, the role of PEMT for systemic PC supply is not fully understood. To address how PEMT affects aging-associated energy metabolism in tissues responsible for nutrient absorption, lipid storage, and energy consumption, we employed NMR-based metabolomics to study the liver, plasma, intestine (duodenum, jejunum, and ileum), brown/white adipose tissues (BAT and WAT), and skeletal muscle of young (9-10 weeks) and old (91-132 weeks) wild-type (WT) and PEMT knockout (KO) mice. We found that the effect of PEMT-knockout was tissue-specific and age-dependent. A deficiency of PEMT affected the metabolome of all tissues examined, among which the metabolome of BAT from both young and aged KO mice was dramatically changed in comparison to the WT mice, whereas the metabolome of the jejunum was only slightly affected. As for aging, the absence of PEMT increased the divergence of the metabolome during the aging of the liver, WAT, duodenum, and ileum and decreased the impact on skeletal muscle. Overall, our results suggest that PEMT plays a previously underexplored, critical role in both aging and energy metabolism.

Genetic Variants in One-Carbon Metabolism and Their Effects on DHA Biomarkers in Pregnant Women: A Post-Hoc Analysis.

The delivery of docosahexanoic acid (DHA) to the fetus is dependent on maternal one-carbon metabolism, as the latter supports the hepatic synthesis and export of a DHA-enriched phosphatidylcholine molecule via the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. The following is a post-hoc analysis of a choline intervention study that sought to investigate whether common variants in one-carbon metabolizing genes associate with maternal and/or fetal blood biomarkers of DHA status. Pregnant women entering their second trimester were randomized to consume, until delivery, either 25 (n = 15) or 550 (n = 15) mg choline/d, and the effects of genetic variants in the PEMT, BHMT, MTHFD1, and MTHFR genes on DHA status were examined. Variant (vs. non-variant) maternal PEMT rs4646343 genotypes tended to have lower maternal RBC DHA (% total fatty acids) throughout gestation (6.9% vs. 7.4%; main effect, p = 0.08) and lower cord RBC DHA at delivery (7.6% vs. 8.4%; main effect, p = 0.09). Conversely, variant (vs. non-variant) maternal MTHFD1 rs2235226 genotypes exhibited higher cord RBC DHA (8.3% vs. 7.3%; main effect, p = 0.0003) and higher cord plasma DHA (55 vs. 41 µg/mL; main effect, p = 0.05). Genotype tended to interact with maternal choline intake (p < 0.1) to influence newborn DHA status for PEMT rs4646343 and PEMT rs7946. These data support the need to consider variants in one-carbon metabolic genes in studies assessing DHA status and requirements during pregnancy.

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.

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.

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.

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.

Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.

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.

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.

Duodenal-jejunal bypass maintains hepatic S-adenosylmethionine/S-homocysteine ratio in diet-induced obese rats.

We previously reported that the duodenal-jejunal bypass (DJB) surgery altered transsulfuration and purine metabolism via flux changes in 1-carbon metabolism in the liver. In this study, we extended our study to gain further insight into mechanistic details of how the DJB-induced flux changes in 1-carbon metabolism contributes to the improvement of diet-induced nonalcoholic fatty liver disease. Rodents were subjected to surgical (sham operation and DJB) or dietary (reduced food supply to follow the weight changes in the DJB group) interventions. The microscopic features of the liver were examined by immunohistochemistry. The expressions of genes in lipid synthesis and in 1-carbon cycle in the liver were analyzed by real-time polymerase chain reaction and western blotting. Metabolic changes in the liver were determined. We observed that DJB reduces hepatic steatosis and improves insulin sensitivity in both high-fat diet-fed rats and mice. Metabolic analyses revealed that the possible underlying mechanism may involve decreased S-adenosylmethionine (SAM)-to-S-adenosylhomocysteine ratio via downregulation of SAM synthesizing enzyme and upregulation of SAM catabolizing enzyme. We also found in mice that DJB-mediated attenuation of hepatic steatosis is independent of weight loss. DJB also increased hepatic expression levels of GNMT while decreasing those of PEMT and BHMT, a change in 1-carbon metabolism that may decrease the ratio of SAM to S-adenosylhomocysteine, thereby resulting in the prevention of fat accumulation in the liver. Thus, we suggest that the change in 1-carbon metabolism, especially the SAM metabolism, may contribute to the improvement of diet-induced fatty liver disease after DJB surgery.

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.