Phosphatidylcholine Synthesis Research Articles

EFFICIENCY OF APPLICATION OF THE DRUG "HEPTRAL-VET" IN THE COMPLEX TREATMENT OF DOGS WITH ACUTE COURSE OF PYROPLASMOSIS

The article presents the results of clinical trials of the drug, the active substance of which is ademethionine 1,4-butanedisulfonate (ademethionine salt), on dogs with an acute course of piroplasmosis. The drug is one of hepatoprotectors designed to normalize the morpho-functional state of the liver and metabolic processes in the body. It should be noted that hepatoprotectors don’t affect the cause of the disease, but the pathogenesis. Ademethionine is a natural amino acid, which is present in all body tissues and fluids. It acts as a coenzyme and methyl group donor in transmethylation reactions, is a precursor in the formation of physiological sulfurated (thiol) compounds (cysteine, taurine, glutathione, coenzyme A, etc.) in transsulfurization reactions.
 Due to the restoration of the synthesis of glutathione, phosphatidylcholine and taurine, ademethionine provides antioxidant and detoxification effects, restores the structure of hepatocyte membranes and promotes the removal of toxic bile acids.
 According to the system of preclinical and clinical testing of veterinary medicinal products, which meets international requirements and is the main guarantee for the development of effective, safe and competitive drugs, we conducted clinical studies of the hepatoprotective drug "HEPTRAL-VET", manufactured by "BIOTESTLAB" LLC, the active ingredient of which is ademethionine 1,4-butanedisulfonate, on dogs with an acute piroplasmosis, in order to study its hepatoprotective and detoxifying action.
 According to the results of clinical examinations of dogs by doctors of veterinary clinics in Lviv and blood laboratory tests 8 animals were selected with signs of acute pyroplasmosis. After specific therapy, the animals were administered intravenously the drug "Heptral-vet" at a dose of 20 mg/kg body weight once a day for 3-6 days, depending on the clinical state of animals.
 Before the treatment of animals, a decrease in the concentration of hemoglobin, the number of erythrocytes and the value of hematocrit, an increase of ESR, in leucoformula – an increase in the number of rod-nuclear neutrophils and a decrease in the number of lymphocytes were revealed in their blood. From the biochemical indicators – a decrease in the concentration of total protein, an increase in the content of total bilirubin, and a significant increase in the activity of liver transaminases were established.
 On the 7th day after the treatment, hematological studies have established an increase in the concentration of hemoglobin, the number of erythrocytes, value of hematocrit, a decrease in ESR and the number of rod-nuclear neutrophils, compared to the initial values. At the same time, biochemical studies of blood serum established a tendency to increase the content of total protein and albumin, decrease the content of bilirubin against the background of normalization of enzyme activity. The increase in the content of urea and creatinine in the dog’s blood serum occurred within the physiological norm. Thus, during this period, we observed the recovery of the dogs' bodies after disease. According to the observations of treating doctors, the drug "HEPTRAL-VET" contributed to the faster recovery of dogs, the normalization of their physiological indicators, and was well tolerated by animals.

Decreased Hyocholic Acid and Lysophosphatidylcholine Induce Elevated Blood Glucose in a Transgenic Porcine Model of Metabolic Disease.

(1) Background: This work aims to investigate the metabolomic changes in PIGinH11 pigs and investigate differential compounds as potential therapeutic targets for metabolic diseases. (2) Methods: PIGinH11 pigs were established with a CRISPR/Cas9 system. PNPLA3I148M, hIAPP, and GIPRdn were knocked in the H11 locus of the pig genome. The differential metabolites between and within groups were compared at baseline and two months after high-fat-high-sucrose diet induction. (3) Results: 72.02% of the 815 detected metabolites were affected by the transgenic effect. Significantly increased metabolites included isoleucine, tyrosine, methionine, oxoglutaric acid, acylcarnitine, glucose, sphinganines, ceramides, and phosphatidylserines, while fatty acids and conjugates, phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins were decreased. Lower expression of GPAT3 and higher expression of PNPLA3I148M decreased the synthesis of diacylglycerol and phosphatidylcholines. Accumulated ceramides that block Akt signaling and decrease hyocholic acid and lysophosphatidylcholines might be the main reason for increased blood glucose in PIGinH11 pigs, which was consistent with metabolomic changes in patients. (4) Conclusions: Through serum metabolomics and lipidomics studies, significant changes in obesity and diabetes-related biomarkers were detected in PIGinH11 pigs. Excessive fatty acids β-oxidation interfered with glucose and amino acids catabolism and reduced phosphatidylcholines. Decreased hyocholic acid, lysophosphatidylcholine, and increased ceramides exacerbated insulin resistance and elevated blood glucose. Phosphatidylserines were also increased, which might promote chronic inflammation by activating macrophages.

Flow Synthesis of L-α-Glycerylphosphorylcholine: Studies on Synthetic Routes Applicable to a Flow Reactor and Optimization of Reaction Conditions.

L-α-Glycerylphosphorylcholine (L-α-GPC) has mainly been produced by two methods: extraction from plants rich in phosphatidylcholine and chemical synthesis. However, production through extraction involves difficult processes, such as fermentation, extractions and ripening, and conventional chemical synthesis methods with high-cost reactants and a batch reactor. These methods are not ideal for large-quantity production. Thus, it is important to develop a simple production method of L-α-GPC, which is suitable for mass production without the need for expensive reactants. Here, we studied synthetic L-α-GPC methods that are applicable to a flow synthesis system, which can provide selectivity, reproducibility, scalability, and a high yield in short reaction time using inexpensive starting materials. We developed a two-step synthetic route to produce L-α-GPC, including the synthesis of phosphoryl choline using choline chloride and phosphoryl oxychloride (POCl3) as a first step and synthesis of L-α-GPC by reacting phosphoryl choline with (R)-(-)-3-chloro-1,2-propanediol (CPD) as a second step under basic conditions. Both steps were separately performed in a customized flow reactor, and reaction conditions were optimized. Finally, phosphoryl choline and L-α-GPC, the products first and second reactions, were successfully synthesized with high conversion yields of 97% and 79%, respectively.

Human placental lipid content and lipid metabolic enzyme abundance in obesity and across gestation.

Changes in placental lipid metabolism influence the delivery of lipids critical for fetal development and fetal requirements for lipids change across gestation. We hypothesized that placental lipid content and metabolic enzyme protein levels increase across gestation and are elevated in obesity. Placentas (4-40 weeks' gestation) were collected from control (body mass index, BMI = 18.5-24.9, n=37) and obese (BMI > 30, n=19) pregnant women. Trophoblast villous tissue was homogenized and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) for phospholipid and triacylglycerol (TAG) analysis and western blot for protein quantification. The placental content of TAG species and nine of 35 identified phosphatidylcholines (PC) were significantly higher (P<0.05) in first trimester (28-79%, 10-47%, respectively). Furthermore, two TAG and three PC differed by maternal BMI and were significantly increased (P<0.05) in the obese group in first trimester (72-87%, 88-119%, respectively). Placental protein abundance of glycerol-2-phosphate (GPAT3) and 1-acyl-sn-glycerol-3-phosphate acyltransferase 2 (AGPAT2), involved in de novo synthesis of PC and TAG, were higher (P<0.05) in the first trimester (66 and 74%, respectively). The protein abundance of the PC-remodeling enzyme PLA2G4c was also higher (63%) in first trimester (P<0.05). In conclusion, the placental content of many phospholipid and TAG species and the protein level of associated synthesis enzymes are higher in first-trimester human placenta. The high PC content may be related to the rapid membrane expansion in early pregnancy and the low placental oxygen tension may promote the accumulation of tissue TAGs in first trimester. Maternal obesity had only limited impact on placental lipid content and metabolic enzyme protein abundance.

Less is more: seipin, phospholipids, and embryogenesis

Abstract In a recent paper published in Life Metabolism, Zhu et al. revealed that mutations that reduce phosphatidylcholine (PC) synthesis rescue embryonic lethality but exacerbate lipid droplet (LD) abnormalities in C. elegans seipin mutants, demonstrating distinct roles for Seipin in embryogenesis and LD formation.

Dietary choline prevents high fat-induced disorder of hepatic cholesterol metabolism through SREBP-2/HNF-4α/CYP7A1 pathway in a freshwater teleost yellow catfish Pelteobagrus fulvidraco

Lipid overload-induced hepatic cholesterol accumulation is a major public health problem worldwide, and choline has been reported to ameliorate cholesterol accumulation, but its mechanism remains unclear. Our study found that choline prevented high-fat diet (HFD)-induced cholesterol metabolism disorder and enhanced choline uptake and phosphatidylcholine synthesis in the liver tissues; choline incubation prevented fatty acid (FA)-induced cholesterol accumulation and FA-induced inhibition of bile acid synthesis. Moreover, compared to single FA incubation, choline incubation or FA + choline co-incubation increased the mRNA abundances and protein levels of HNF4α and up-regulated the degradation of cholesterol into bile acids. Mechanistically, choline prevented the FA-induced accumulation of SREBP2 protein and the interaction between SREBP2 and HNF4α, thereby enhancing the DNA binding capacity of the HNF4α to the CYP7A1 promoter, and promoting the degradation of cholesterol into bile acids. Our study elucidated the novel regulatory mechanisms of choline preventing HFD-induced cholesterol accumulation and increasing bile acid synthesis by SREBP-2/HNF-4α/CYP7A1 pathway.

Reduced phosphatidylcholine synthesis suppresses the embryonic lethality of seipin deficiency

AbstractSeipin plays a vital role in lipid droplet homeostasis, and its deficiency causes congenital generalized lipodystrophy type II in humans. It is not known whether the physiological defects are all caused by cellular lipid droplet defects. Loss-of-function mutation of seip-1, the Caenorhabditis elegans seipin ortholog, causes embryonic lethality and lipid droplet abnormality. We uncover nhr-114 and spin-4 as two suppressors of seip-1 embryonic lethality. Mechanistically, nhr-114 and spin-4 act in the “B12-one-carbon cycle-phosphatidylcholine (PC)” axis, and reducing PC synthesis suppresses the embryonic lethality of seip-1 mutants. Conversely, PC deficiency enhances the lipid droplet abnormality of seip-1 mutants. The suppression of seip-1 embryonic lethality by PC reduction requires polyunsaturated fatty acid. In addition, the suppression is enhanced by the knockdown of phospholipid scramblase epg-3. Therefore, seipin and PC exhibit opposite actions in embryogenesis, while they function similarly in lipid droplet homeostasis. Our results demonstrate that seipin-mediated embryogenesis is independent of lipid droplet homeostasis.

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 (β = −1.497; p = 0.005). PEMT levels also declined with increasing severity of fibrosis with cirrhosis < incomplete cirrhosis < bridging fibrosis (β = −1.185; p = 0.011). Hepatic PEMT expression was reduced in postmenopausal women with NASH compared to those with normal liver histology (β = −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.

P73α1, an Isoform of the p73 Tumor Suppressor, Modulates Lipid Metabolism and Cancer Cell Growth via Stearoyl-CoA Desaturase-1.

Altered lipid metabolism is a hallmark of cancer. p73, a p53 family member, regulates cellular processes and is expressed as multiple isoforms. However, the role of p73 in regulating lipid metabolism is not well-characterized. Previously, we found that loss of p73 exon 12 (E12) leads to an isoform switch from p73α to p73α1, the latter of which has strong tumor suppressive activity. In this study, comprehensive untargeted metabolomics was performed to determine whether p73α1 alters lipid metabolism in non-small cell lung carcinoma cells. RNA-seq and molecular biology approaches were combined to identify lipid metabolism genes altered upon loss of E12 and identify a direct target of p73α1. We found that loss of E12 leads to decreased levels of phosphatidylcholines, and this was due to decreased expression of genes involved in phosphatidylcholine synthesis. Additionally, we found that E12-knockout cells had increased levels of phosphatidylcholines containing saturated fatty acids (FAs) and decreased levels of phosphatidylcholines containing monounsaturated fatty acids (MUFAs). We then found that p73α1 inhibits cancer cell viability through direct transcriptional suppression of Stearoyl-CoA Desaturase-1 (SCD1), which converts saturated FAs to MUFAs. Finally, we showed that p73α1-mediated suppression of SCD1 leads to increased ratios of saturated FAs to MUFAs.

The Amount of Dietary Methionine Required to Maximize Synthesis of Transmethylated Products Is Higher Than That Needed for Protein Synthesis in Neonatal Piglets

ObjectivesMethionine is an indispensable sulfur-containing amino acid required for synthesizing protein as well as other critical nutrients. Methionine is the primary methyl donor for >50 transmethylation reactions to produce metabolites including creatine, phosphatidylcholine (PC), and methylated DNA. In piglets, because only one-third of methionine flux is incorporated into protein compared to transmethylation reactions (∼2/3), non-protein demands need to be considered when determining methionine requirement. Our objective was to quantify how much dietary methionine is required to maximize synthesis of protein as well as key transmethylated products. We hypothesized that more dietary methionine will be needed to maximize creatine synthesis, with lower levels required to maximize PC and protein synthesis. MethodsTwenty surgically altered Yucatan miniature piglets (∼8 d old; ∼1.6 kg) were fed complete diets for 5 d and then randomized to 20 test diets with methionine intakes ranging from 20%-220% of requirement (0.05–0.55 g/kg/d). After 24 h of test diet, [3H-methyl]-methionine was infused for 6 h to measure methyl incorporation into creatine, PC, and methylated DNA, and then a3H-phenylalanine flooding dose was given to measure protein synthesis. Break-point analysis was used to identify the methionine required to maximize product synthesis. ResultsPlasma methionine increased above 0.3 g methionine/kg/d, reflecting the whole body requirement. Creatine synthesis maximized at ∼0.3 g methionine/kg/d, while muscle protein synthesis was maximized at 0.2 g methionine/kg/d, suggesting growth is prioritized when methionine is limiting. PC synthesis was low up to 0.35 g methionine/kg/d and increased linearly with higher intakes, suggesting PC synthesis was not maximized at 200% of requirement.3H-DNA did not change with dietary methionine, and plasma homocysteine was positively correlated with dietary methionine. ConclusionsThese data suggest that more methionine is required to accommodate demands for creatine and PC synthesis than that for protein synthesis. Therefore, methionine requirement estimates for infants, which rely on protein synthesis outcomes, are likely insufficient to achieve maximal synthesis of key methylated metabolites creatine and PC. Funding SourcesNSERC.

Synthesis of Lysophosphatidylcholine and Mixed Phosphatidylcholine.

Lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) are important membrane constituents implicated in signaling and immune regulation. Synthesis of LPCs is challenging due to rapid acyl migration, e.g., induced by chromatography. We here report a highly regioselective synthesis of LPC and mixed PC via an intermediate allowing specific terminal acyl introduction, yielding the pure LPC without chromatography by an exceedingly mild TBS deprotection, using 1 equiv of TFA in aqueous solution. The method enabled the synthesis of glycerol-, acyl-, and choline-labeled LPC.

Cover Picture: Controlled Tetradeuteration of Straight‐Chain Fatty Acids: Synthesis, Application, and Insight into the Metabolism of Oxidized Linoleic Acid (Angew. Chem. Int. Ed. 25/2022)

The light of deuterium shines in the darkness of the molecular mechanism of the oxidized phosphatidylcholine synthesis. A concise protocol for the controlled tetradeuteration of straight-chain fatty acids is reported by Kotaro Hama, Ryo Takita, and co-workers (e202202779). The use of four deuterium atoms in fatty acids as well as a diagnostic peak of phosphatidylcholines thus obtained enabled the identification of acyltransferases that transfer the acyl moiety derived from an oxidized linoleic acid.

Choline deficiency in the body, clinical manifestations and long-term consequences

Choline, a substance essential for the existence of any organism, is the basis for the synthesis of phosphatidylcholine and sphingomyelin, the two main phospholipids of cell membranes. Acetylcholine is the main neurotransmitter of the parasympathetic nervous system, i.e. part of the autonomic nervous system. It affects smooth muscles, vascular wall tone, heart rate and regulates metabolism as a source of methyl groups. Choline enters the body through food and is partially synthesized endogenously. Choline plays an important role in gene expression, cell membrane signalling, lipid transport and metabolism, and early infant brain development. Choline deficiency increases the risk of cardiovascular and metabolic disorders. Current scientific evidence suggests a negative effect of choline deficiency on the development of non-alcoholic fatty liver disease. Choline deficiency is associated with impaired memory, concentration, and cognitive functions. This article deals with the mechanisms of choline influence on the organism and possibility of choline deficiency correction in the organism.

Unusual Lipid Components of Legionella gormanii Membranes.

Legionella spp. cause Legionnaires’ disease with pneumonia as the predominant clinical symptom. L. gormanii is the second most prevalent causative agent of community-acquired pneumonia after L. pneumophila. The study aimed to characterize the lipidome of L. gormanii membranes and the importance of these analyses in bacterial chemotaxonomy. Lipidomic analyses based on ultra-high performance liquid chromatography-mass spectrometry allowed the detection of individual molecular species of a wide range of L. gormanii membrane lipids contained in the outer (OM) and inner membranes (IM). The lipid profile comprised glycerolipids (triglycerides, diglycerides), phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin), and sphingolipids (ceramides, hexosylceramides). The most abundant lipid fraction in the IM and OM were phospholipids. The lipidomic analysis showed that two independent phosphatidylcholine (PC) synthesis pathways operating in L. gormanii: the PE-methylation (PmtA) pathway and the PC synthase (Pcs) pathway. Comparison of the molecular profile of PC species contained in the lipids of L. gormanii membranes cultured on the medium, with and without exogenous choline, showed quantitative differences in the PC pool. An unusual feature of the L. gormanii lipids was the presence of ceramides and hexosylceramides, which are typical components of eukaryotic cells and a very small group of bacteria. To the best of our knowledge, this is the first report of the occurrence of ceramides in Legionella bacteria.

Integrated lipidomics and RNA sequencing analysis reveal novel changes during 3T3-L1 cell adipogenesis.

After adipogenic differentiation, key regulators of adipogenesis are stimulated and cells begin to accumulate lipids. To identify specific changes in lipid composition and gene expression patterns during 3T3-L1 cell adipogenesis, we carried out lipidomics and RNA sequencing analysis of undifferentiated and differentiated 3T3-L1 cells. The analysis revealed significant changes in lipid content and gene expression patterns during adipogenesis. Slc2a4 was up-regulated, which may enhance glucose transport; Gpat3, Agpat2, Lipin1 and Dgat were also up-regulated, potentially to enrich intracellular triacylglycerol (TG). Increased expression levels of Pnpla2, Lipe, Acsl1 and Lpl likely increase intracellular free fatty acids, which can then be used for subsequent synthesis of other lipids, such as sphingomyelin (SM) and ceramide (Cer). Enriched intracellular diacylglycerol (DG) can also provide more raw materials for the synthesis of phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE), ether-PE, and ether-PC, whereas high expression of Pla3 may enhance the formation of lysophophatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Therefore, in the process of adipogenesis of 3T3-L1 cells, a series of genes are activated, resulting in large changes in the contents of various lipid metabolites in the cells, especially TG, DG, SM, Cer, PI, PC, PE, etherPE, etherPC, LPC and LPE. These findings provide a theoretical basis for our understanding the pathophysiology of obesity.

The endoplasmic reticulum membrane protein complex localizes to the mitochondrial - endoplasmic reticulum interface and its subunits modulate phospholipid biosynthesis in Trypanosoma brucei.

The endoplasmic reticulum membrane complex (EMC) is a versatile complex that plays a key role in membrane protein biogenesis in the ER. Deletion of the complex has wide-ranging consequences including ER stress, disturbance in lipid transport and organelle tethering, among others. Here we report the function and organization of the evolutionarily conserved EMC (TbEMC) in the highly diverged eukaryote, Trypanosoma brucei. Using (co-) immunoprecipitation experiments in combination with mass spectrometry and whole cell proteomic analyses of parasites after depletion of select TbEMC subunits, we demonstrate that the TbEMC is composed of 9 subunits that are present in a high molecular mass complex localizing to the mitochondrial-endoplasmic reticulum interface. Knocking out or knocking down of single TbEMC subunits led to growth defects of T. brucei procyclic forms in culture. Interestingly, we found that depletion of individual TbEMC subunits lead to disruption of de novo synthesis of phosphatidylcholine (PC) or phosphatidylethanolamine (PE), the two most abundant phospholipid classes in T. brucei. Downregulation of TbEMC1 or TbEMC3 inhibited formation of PC while depletion of TbEMC8 inhibited PE synthesis, pointing to a role of the TbEMC in phospholipid synthesis. In addition, we found that in TbEMC7 knock-out parasites, TbEMC3 is released from the complex, implying that TbEMC7 is essential for the formation or the maintenance of the TbEMC.

The rate‐limiting enzyme in the CDP‐choline pathway is regulated by phosphorylation‐domain charge density

Eukaryotic cell membranes are primarily composed of phospholipids, the most abundant of which is phosphatidylcholine (PC). De novo synthesis of PC by the CDP‐choline pathway is under the control of the rate‐limiting enzyme CTP:phosphocholine cytidylyltransferase (CCT). The nuclear CCTa isoform is regulated by translocation to the inner nuclear membrane (INM) and nuclear lipid droplets (nLD) in response to lipid activators, such as oleate, or deficiency in PC content. CCTa association with the INM is mediated by a positively‐charged membrane‐binding M‐domain and a phosphorylated P‐domain containing 16 serine phospho‐sites. Mutation of all P‐domain serine residues to alanine (dephosphorylated mimic) enhanced association with nLDs in oleate‐treated U2OS cells, while mutation to glutamate (phosphorylated mimic) or mutation of M‐domain lysine residues ablated nLD association. However, it is unknown if phosphorylation regulation of CCTa association with the INM and nLDs involves specific serine residues or the net charge of the P‐domain. To address this question, we mutated 4 sets of 2‐3 serine residues in the P‐domain to alanine or aspartate. CCTa cDNAs expressing different combinations of these mutations were expressed in oleate‐treated U2OS cells and the frequency of nLD association was measured. The mutants were also expressed in CHO cells with a temperature‐sensitive CCTα isoform and INM translocation in response to oleate was determined. The greatest inhibitory effect on INM and nLD association was observed when all 4 sets of serine residues were mutated to aspartate and not by any one site, suggesting that net charge density of the P‐domain regulates affinity for the NE and nLD. Additionally, CCTa serine‐to‐alanine mutants expressed in U2OS cells were unstable compared to the corresponding aspartate mutants. We conclude that CCTa association with the INM and nLD is inhibited as overall P‐domain phosphorylation increases. In its active membrane‐associated, dephosphorylated state CCTa protein has decreased stability, a potently negative feedback mechanism to control PC synthesis. These results provide novel insight into how PC synthesis is regulated that will be essential for identifying kinases and phosphatases that act on CCTα.

Choline metabolism underpins macrophage IL-4 polarization in vitro and in vivo

Abstract Choline homeostasis in macrophage biology is important for LPS-polarized inflammation. In macrophages, choline mainly supports phosphatidylcholine (PC) synthesis, which is crucial for membrane production and cytokine secretion. Here, we examined choline metabolism in IL-4 polarized macrophages in vitro and in vivo. Like LPS, IL-4 increased choline transporter-like protein 1 (CTL1) expression, choline uptake and the incorporation of choline into PC. Targeted lipidomics analysis revealed increased PC content in IL-4-polarized macrophages, with an enrichment in low-saturated species. Pharmacological inhibition of choline uptake/choline kinase with hemicholinium-3 or RSM-932a showed no effect on certain hallmark IL-4-induced macrophage genes (Chil3, Mrc1, Arg1) but significantly reduced the transcript and protein expression of RELMα. Consistent with the function of RELMα in wound healing, 3T3-L1 cells healed more slowly in a scratch wound assay with conditioned media from IL-4 polarized macrophages in which choline metabolism was inhibited compared to vehicle-treated conditioned media. In addition, inhibiting choline metabolism completely prevented PD-L2 upregulation and increased PD-L1 expression on IL-4-polarized macrophages, together with suppressed cellular respiration and increased glycolysis. Furthermore, in vivo administration of RSM-932a (3 mg/kg i.p.) in C57BL/6J mice lowered RELMα in macrophages, decreased PD-L2 and increased PD-L1, and resulted in a loss of resident peritoneal F4/80hi macrophages. Choline represents an underappreciated regulator of macrophage immunometabolism and strategies to target macrophage choline uptake or choline metabolism may be therapeutically valuable.

CTP:phosphocholine cytidylyltransferase alpha regulates nLD biogenesis in Caco2 cells

Recent investigations reveal that nuclear lipid droplets (nLDs) stimulate lipid metabolic enzymes in response to excess fatty acid loading of cells. For example, in oleate‐treated hepatocytes the rate‐limiting enzyme in the CDP‐choline pathway, CTP:phosphocholine cytidylyltransferase alpha (CCT alpha), is recruited to the surface of nLDs and stimulates the synthesis of phosphatidylcholine (PC). Hepatocyte nLDs derive from ApoB‐free LDs in the lumen of the endoplasmic reticulum that migrate into the nucleoplasm through type I nucleoplasmic reticulum invaginations of the inner nuclear membrane (INM). Once formed inside the nucleus, nLDs recruit proteins CCT alpha, Lipin1 and promyelocytic leukemia (PML) to their surfaces. Endothelial cells of the colon also express ApoB lipoproteins for chylomicron assembly and secretion, so we hypothesized that these cells might also form nLDs in response to excess lipid loading. Indeed, using immunofluorescence confocal microscopy we found that oleate‐treated Caco2 cells formed between 5 to 10 nLDs per cell that recruited both CCT alpha and PML to their surface. CRISPR/Cas9‐mediated CCT alpha knockout Caco2 (CCT alphaKO) cells had significantly fewer but larger cLDs and nLDs compared to wild‐type cells. The percentage of PML‐positive nLDs increased in CCT alphaKO cells, suggesting that CCT alpha and PML compete for access to the nLD surface following oleate treatment. To determine if phosphorylation of CCT alpha regulates translocation to nLDs, phospho‐mimetic and phospho‐null S315/S319 and Y359/S362 mutants were expressed in CCT alphaKO cells and localization to nLDs was measured. In vitrophosphorylation assays of recombinant, dephosphorylated CCT alpha indicated that AMP‐dependent kinase phosphorylated CCT alpha S319, which could be involved in regulating CCT alpha activity on nLDs. In summary, we have demonstrated that oleate treatment induces nLD biogenesis in Caco2 cells and that CCT alpha activity regulates the biogenesis of both nLDs and cLDs.

Dietary lipids as modulators of fatty acid profile and gene expression patterns on body compartments of Octopus vulgaris paralarvae

The common octopus, Octopus vulgaris, is a promising mollusc species for marine aquaculture diversification due to its high growth rates and commercial value. Yet, the elevated mortalities mainly related to lipid-linked nutritional deficiencies during the planktonic stage (paralarvae), hinder the development of efficient protocols for a complete rearing cycle. Although the effect of dietary lipids, especially fatty acids (FA), has been the subject of intense research, the available information is essentially restricted to their impact on the composition of the whole parlarval organism. In contrast, little is known about the effects of dietary signature and the specific requirements of each anatomical structure through the paralarvae development. In addition, knowledge about the endogenous capacity in each paralarvae body structure for adaptation to different dietary scenarios is necessary. In the present work, a series of experiments were carried out based on newly hatched paralarvae (PH) and paralarvae fed (30 days post-hatch (DPH)) either with marine crustacean zoeae (PZ) or with Artemia metanauplii (PA). At the end of the trials, the paralarvae were dissected into functional (mantle, head, and arms) and digestive (digestive gland (DG)) body compartment and the FA profile, as well as the expression patterns of genes involved in the long chain polyunsaturated FA (LC-PUFA) (stearoyl-CoA desaturase (scd), ωx2 desaturase (ωx2), ωx1 desaturase (ωx1), fatty acyl desaturase (fad), elovl2/5, elovl4), and glycerophospholipid biosynthetic pathways (agpta, lpin, chpt, and dgat) were analysed. Results showed a positive effect of the PZ diet on growth and development of paralarvae as compared to PA, and distinct FA composition for the 3 experimental groups. The digestive gland was associated to 18C FA (18:3n-3, 18:4n3, 18:1n9, and 18:2n6), while n-6 and n-3 LC-PUFA (20:4n6, 20:5n3, 22:5n3, and 22:6n3) were found in a higher proportion in functional body compartments. The expression of LC-PUFA biosynthesis-linked genes increased significantly during development, with the functional body compartments of the PA treatment being up-regulated as compared to PZ, pointing at a putative compensatory mechanism. In addition, a higher amount of transcripts linked to the triggering of the phosphatidylcholine synthesis (chpt) was found in the digestive gland of PA and PZ and arms of PZ; whereas genes related with triacylglycerol (TAG) synthesis (lpin and dgat) were enhanced in the digestive gland of PA and PH. Dietary treatments affected the FA profile and the gene expression patterns in both digestive (more similar FA profile and glycerophospholipid biosynthesis) and functional (different FA profile and LC-PUFA biosynthesis) compartments of the paralarvae. Furthermore, LC-PUFA biosynthesis-related genes in the head and glycerophospholipid target genes in DG could be used as biomarkers of nutritional deficiencies in paralarvae.