Exogenous Fatty Acids Research Articles

The intracellular growth of the vacuolar pathogen Legionella pneumophila is dependent on the acyl chain composition of host membranes

Legionella pneumophila is an accidental human bacterial pathogen that infects and replicates within alveolar macrophages causing a severe atypical pneumonia known as Legionnaires’ disease. As a prototypical vacuolar pathogen L. pneumophila establishes a unique endoplasmic reticulum (ER)-derived organelle within which bacterial replication takes place. Bacteria-derived proteins are deposited in the host cytosol and in the lumen of the pathogen-occupied vacuole via a type IVb (T4bSS) and a type II (T2SS) secretion system respectively. These secretion system effector proteins manipulate multiple host functions to facilitate intracellular survival of the bacteria. Subversion of host membrane glycerophospholipids (GPLs) by the internalized bacteria via distinct mechanisms feature prominently in trafficking and biogenesis of the Legionella-containing vacuole (LCV). Conventional GPLs composed of a glycerol backbone linked to a polar headgroup and esterified with two fatty acids constitute the bulk of membrane lipids in eukaryotic cells. The acyl chain composition of GPLs dictates phase separation of the lipid bilayer and therefore determines the physiochemical properties of biological membranes - such as membrane disorder, fluidity and permeability. In mammalian cells, fatty acids esterified in membrane GPLs are sourced endogenously from de novo synthesis or via internalization from the exogenous pool of lipids present in serum and other interstitial fluids. Here, we exploited the preferential utilization of exogenous fatty acids for GPL synthesis by macrophages to reprogram the acyl chain composition of host membranes and investigated its impact on LCV homeostasis and L. pneumophila intracellular replication. Using saturated fatty acids as well as cis- and trans- isomers of monounsaturated fatty acids we discovered that under conditions promoting lipid packing and membrane rigidification L. pneumophila intracellular replication was significantly reduced. Palmitoleic acid – a C16:1 monounsaturated fatty acid – that promotes membrane disorder when enriched in GPLs significantly increased bacterial replication within human and murine macrophages but not in axenic growth assays. Lipidome analysis of infected macrophages showed that treatment with exogenous palmitoleic acid resulted in membrane acyl chain reprogramming in a manner that promotes membrane disorder and live-cell imaging revealed that the consequences of increasing membrane disorder impinge on several LCV homeostasis parameters. Collectively, we provide experimental evidence that L. pneumophila replication within its intracellular niche is a function of the lipid bilayer disorder and hydrophobic thickness.

Elucidating the impact of bacterial lipases, human serum albumin, and FASII inhibition on the utilization of exogenous fatty acids by Staphylococcus aureus.

Incorporation of host-derived exogenous fatty acids (eFAs), particularly unsaturated fatty acids (UFAs), by Staphylococcus aureus could affect the bacterial membrane fluidity and susceptibility to antimicrobials. In this work, we found that glycerol ester hydrolase (Geh) is the primary lipase hydrolyzing cholesteryl esters and, to a lesser extent, triglycerides and that human serum albumin (HSA) could serve as a buffer of eFAs, where low levels of HSA facilitate the utilization of eFAs but high levels of HSA inhibit it. The fact that the type II fatty acid synthesis (FASII) inhibitor, AFN-1252, leads to an increase in UFA content even in the absence of eFA suggests that membrane property modulation is part of its mechanism of action. Thus, Geh and/or the FASII system look to be promising targets to enhance S. aureus killing in a host environment by restricting eFA utilization or modulating membrane properties, respectively.

TMET-15. THE COMBINATIONAL EFFECT OF INHIBITOR FOR A FATTY ACID DESATURASE AND A FATTY ACID TRANSPORTER ON GLIOMA GROWTH

Abstract BACKGROUND It is not well understood how much exogenous fatty acids uptake and de novo fatty acid synthesis affect the cell proliferation in glioma. In this study, we examined the combinational effect of inhibitor for a fatty acid desaturase (SCD) and a fatty acid transporter (CD36) on glioma growth. METHODS Normal human astrocytes (NHA) were used as glial cells, and U251 transfected with IDH wild-type or IDH mutant gene and patient-derived glioma cells (BT142, TS603, GSC923S) were used as glioma cells. The function of SCD was suppressed by SCD inhibitor and SCD siRNA, and Sulfo-N-succinimidyl oleate (SSO) was used to inhibit the CD36 function. RESULTS We treated U251 cells with palmitic acid (C16:0), stearic acid (C18:0), palmitoleic acid (C16:1) or oleic acid (C18:1) after SCD inhibitors treatment or SCD knockdown, and cell proliferation was rescued only after the treatment with C16:1 and C18:1. NHA was not affected by SCD inhibitor, although CD36 inhibitor suppressed cell proliferation of both U251 and NHA. The combination of SCD inhibitor and CD36 inhibitor under the addition of exogenous fatty acid, oleic acid, suppressed the proliferation of U251 cells more effectively than SCD inhibitor or CD36 inhibitor alone. Discussion: The SCD inhibitory effect on gliomas was attenuated by the presence of exogenous monounsaturated fatty acids, whereas the CD36 inhibitor enhanced the cell-killing effect. On the other hand, it should be noted that CD36 inhibitors affect the proliferation of glial cells.

ACC1 Inhibition Enhances Treg Gvhd Treatment Efficacy through Regulation of Mitochondrial Fusion and Elongation

Regulatory T-cells (Treg) play a critical role in preventing autoimmune and alloimmune reactions. Clinical trials demonstrated that Treg adoptive transfer significantly reduced but did not uniformly eliminate grades II-IV graft-vs-host disease (GVHD). Treg more dominantly rely on fatty acid oxidation (FAO) as a fuel source, in contrast to glycolytic conventional T-cells. We hypothesized that increasing FAO would augment Treg efficacy by increasing potency and persistence, thereby ultimately decreasing the Treg cell number required to enhance Treg-mediated GVHD reduction. The FAO and fatty acid synthesis (FAS) pathways compete for acetyl-CoA use. Acetyl-CoA carboxylase-1 (ACC1) skews acetyl-CoA toward FAS and results in downstream inhibition of CPT1a, the rate-limiting enzyme in FAO. ACC1 deletion or inhibition skews naive CD4 T-cell toward FAO, favoring Treg over Th17 differentiation. To precisely determine the role of ACC1 on in vivo Treg-mediated suppression of GVHD, we tested the effects of a 2-hour treatment of murineTreg with ND630, a pharmacological inhibitor of ACC1 currently in clinical trials for hepatic steatosis, or Treg-specific ACC1 knockout (KO). ACC1 inhibition or KO increased in vitro Treg suppressive function, and when infused into mice with established bronchiolitis obliterans (BOS) and chronic GVHD (cGVHD), ACC1KO Treg were more potent than wild-type Treg in reversing BOS pulmonary resistance (FigA), a finding consistent in each of 3 replicate transplants. Mechanistically, both ND630 treated Treg and ACC1KO Treg demonstrated an increased oxygen consumption rate (OCR), a surrogate for mitochondrial oxidative phosphorylation (OXPHOS), a critical source of ATP for Treg. This was associated with an increase in uptake of BoDipyC16, a synthetic fatty acid, suggesting exogenous fatty acids may drive the increase in OXPHOS through FAO. IACS, an OXPHOS inhibitor, abrogated OCR augmentation and precluded increased suppressive function after ND630 treatment, indicating a critical role for OXPHOS in driving Treg suppressor function. To directly evaluate effects on Treg mitochondria, where OXPHOS occurs, Treg were stained with MitoTracker dyes that measure mitochondrial mass and membrane potential, two key surrogate measurements for mitochondrial function. Compared to controls, ND630 treated and ACC1KO Treg each showed increased mitochondrial mass and membrane potential, as well as enhanced mitochondrial polarization (membrane potential to mass ratio), which is required for full Treg functionality. Expansion and scanning electron microscopic analysis of Treg mitochondria revealed enhanced mitochondrial fusion and elongation after ND630 treatment ( FigB). This mitochondrial structural change boosts OXPHOS, consistent with a direct effect of ACC1 inhibition/KO on mitochondrial fitness. In light of these mitochondrial morphology changes, we hypothesized that directly modifying the mitochondria itself, without inhibiting ACC1, might result in augmented Treg function. Consistent with this hypothesis, a 2-hour treatment with the combination of M1 (mitochondrial fusion promoter) and mDIVI1 (mitochondrial fission inhibitor), significantly enhanced Treg function in vitro, and enhanced Treg OCR to a similar degree as ND630. Electron microscopy confirmed that M1/mDIVI1 treatment resulted in a similar degree of mitochondrial fusion as ND630 treatment. Similar to ACC1KO Treg, M1/mDIVI1 treated Treg were more potent than control Treg in reversing established cGVHD. To further explore mitochondrial fusion effects on Treg function, we used siRNA to knockdown the critical mitochondrial fusion protein mitofusin 1 (MFN1). A 50% decrease in MFN1 protein significantly decreased Treg in vitro functionality and OCR, and treatment of MFN1 knockdown Treg with ND630 had no functional or metabolic effect (FigC), pointing to mitochondrial fusion as a critical component of the mechanism for ACC1 inhibition/KO. In summary, ACC1 inhibition or deletion amplifies murine Treg potency resulting in superior metabolic fitness, and greater efficacy in treating cGVHD mice with established BOS, in part through enhancing mitochondrial fusion. Since ND630 is already under investigation in patients, ACC1 inhibition with ND630 could be readily translatable for clinical trials with a goal of utilizing Tregs with inhibited or deleted ACC1 to improve GVHD therapy.

Acyl-AcpB, a FabT corepressor in Streptococcus pyogenes.

Membranes are a universal barrier to all cells. Phospholipids, essential bacterial membrane components, are composed of a polar head and apolar fatty acid (FA) chains. Most bacterial FAs are synthesized by the Type II FA synthesis pathway (FASII). In Streptococcaceae, Enterococci, and Lactococcus lactis, a unique feedback mechanism controls the FASII gene expression. FabT, encoded in the FASII main locus, is the repressor, and it is activated by long-chain acyl-acyl carrier protein (acyl-ACP). Many Streptococci, Enterococcus faecalis, but not L. lactis, possess two ACPs. The AcpA-encoding gene is within the FASII locus and is coregulated with the FASII genes. Acyl-AcpA is the end product of FASII. The AcpB-encoding gene is in operon with plsX encoding an acyl-ACP:phosphate acyltransferase. The role of acyl-AcpB as FabT corepressor is controversial. Streptococcus pyogenes, which causes a wide variety of diseases ranging from mild non-invasive to severe invasive infections, possesses AcpB. In this study, by comparing the expression of FabT-controlled genes in an acpB-deleted mutant with those in a wild-type and in a fabT mutant strain, grown in the presence or absence of exogenous FAs, we show that AcpB is the S. pyogenes FabT main corepressor. Its deletion impacts membrane FA composition and bacterial adhesion to eucaryotic cells, highlighting the importance of FASII control. Importance Membrane composition is crucial for bacterial growth or interaction with the environment. Bacteria synthesize fatty acids (FAs), membrane major constituents, via the Type II FAS (FASII) pathway. Streptococci control the expression of the FASII genes via a transcriptional repressor, FabT, with acyl-acyl carrier proteins (ACPs) as corepressor. Streptococcus pyogenes that causes a wide variety of diseases ranging from mild non-invasive to severe invasive infections possesses two ACPs. acpA, but not acpB, is a FASII gene. In this study, we show that acyl-AcpBs are FabT main corepressors. Also, AcpB deletion has consequences on the membrane FA composition and bacterial adhesion to host cells. In addition to highlighting the importance of FASII control in the presence of exogeneous FAs for the adaptation of bacteria to their environment, our data indicate that FASII gene repression is mediated by a corepressor whose gene expression is not repressed in the presence of exogenous FAs.

Important Features for Protein Foldings in Two Acyl Carrier Proteins from Enterococcus faecalis.

The emergence of multi-drug resistant Enterococcus faecalis raises a serious threat to global public health. E. faecalis is a gram-positive intestinal commensal bacterium found in humans. E. faecalis can endure extreme environments such as high temperature, pressure, and high salt, which facilitates them to cause infection in hospitals. E. faecalis has two acyl carrier proteins, AcpA (EfAcpA) in de novo fatty acid synthesis (FAS) and AcpB (EfAcpB) which utilizes exogenous fatty acids. Previously, we determined the tertiary structures of these two ACPs and investigated their structure-function relationships. Solution structures revealed that overall folding of these two ACPs is similar to those of other bacterial ACPs. However, circular dichroism (CD) experiments showed that the melting temperature of EfAcpA is 76.3°C and that of EfAcpB is 79.2°C, which are much higher than those of other bacterial ACPs. In this study, to understand the origin of their structural stabilities, we verified the important residues for stable folding of these two ACPs by monitoring thermal and chemical denaturation. Hydrogen/deuterium exchange and chemical denaturation experiments on wild-type and mutant proteins revealed that Ile10 of EfAcpA and Ile14 of EfAcpB mediate compact intramolecular packing and promote high thermostability and stable folding. E. faecalis may maximize efficiency of FAS and increase adaptability to the environmental stress by having two thermostable ACPs. This study may provide insight into bacterial adaptability and development of antibiotics against multi-drug-resistant E. faecalis.

Oral Intake of Inosine 5'-Monophosphate in Mice Promotes the Absorption of Exogenous Fatty Acids and Their Conversion into Triglycerides though Enhancing the Phosphorylation of Adenosine 5'-Monophosphate-Activated Protein Kinase in the Liver, Leading to Lipohyperplasia.

Inosine 5'-monophoaphate (IMP) is a food additive that promotes serious lipohyperplasia in the liver of C57/KsJ-db/db (db/db) mice. Thus, IMP taken orally by healthy mice might also damage their health. To date, how IMP affects health after being taken by healthy animals is still unclear. Therefore, we investigated the health of C57BL/6J mice affected by IMP intake. Our data revealed that C57BL/6J mice administered 255 μM IMP daily via oral gavage for 4 months caused hyperlipidemia and an increase in body fat rate. The expressions of acetyl-CoA carboxylase 1 (ACC1) and phosphorylated acetyl-CoA carboxylase 2 (ACC2) in hepatocytes increased though the administration of IMP, promoting the phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). The conversion of acetyl-CoA into triglycerides (TGs) was promoted by ACC1. These TGs were transported from the hepatocytes to avoid the development of non-alcoholic fatty liver disease (NAFLD), causing a deficiency of acetyl-CoA in the liver, and then, the increased phosphorylated ACC2 promoted the cytoplasm fatty acids entering the mitochondria and conversion into acetyl-CoA through the fatty acid β-oxidation pathway, causing a deficiency in fatty acids. Therefore, the liver showed enhanced absorption of exogenous fatty acids, which were converted into TGs, causing lipohyperplasia. In conclusion, an excessive IMP intake promotes metabolic dysfunction in adipose tissue.

Growth of Enterococcus faecalis ∆plsX strains is restored by increased saturated fatty acid synthesis.

The Enterococcus faecalis acyl-acyl carrier protein (ACP) phosphate acyltransferase PlsX plays an important role in phospholipid synthesis and exogenous fatty acid incorporation. Loss of plsX almost completely blocks growth by decreasing de novo phospholipid synthesis, which leads to abnormally long-chain acyl chains in the cell membrane phospholipids. The ∆plsX strain failed to grow without supplementation with an appropriate exogenous fatty acid. Introduction of a ∆fabT mutation into the ∆plsX strain to increase fatty acid synthesis allowed very weak growth. The ∆plsX strain accumulated suppressor mutants. One of these encoded a truncated β-ketoacyl-ACP synthase II (FabO) which restored normal growth and restored de novo phospholipid acyl chain synthesis by increasing saturated acyl-ACP synthesis. Saturated acyl-ACPs are cleaved by a thioesterase to provide free fatty acids for conversion to acyl-phosphates by the FakAB system. The acyl-phosphates are incorporated into position sn1 of the phospholipids by PlsY. We report the tesE gene encodes a thioesterase that can provide free fatty acids. However, we were unable to delete the chromosomal tesE gene to confirm that it is the responsible enzyme. TesE readily cleaves unsaturated acyl-ACPs, whereas saturated acyl-ACPs are cleaved much more slowly. Overexpression of an E. faecalis enoyl-ACP reductase either FabK or FabI which results in high levels of saturated fatty acid synthesis also restored the growth of the ∆plsX strain. The ∆plsX strain grew faster in the presence of palmitic acid than in the presence of oleic acid with improvement in phospholipid acyl chain synthesis. Positional analysis of the acyl chain distribution in the phospholipids showed that saturated acyl chains dominate the sn1-position indicating a preference for saturated fatty acids at this position. High-level production of saturated acyl-ACPs is required to offset the marked preference of the TesE thioesterase for unsaturated acyl-ACPs and allow the initiation of phospholipid synthesis.

Free Fatty Acids Induce Apoptosis of Mammary Epithelial Cells of Ketotic Dairy Cows via the Mito-ROS/NLRP3 Signaling Pathway.

Early lactation increases metabolic stress in ketotic dairy cows, leading to mitochondrial damage, apoptosis, and inflammatory response in mammary epithelial cells. The pyrin domain 3 (NLRP3) pathway involving the mitochondrial reactive oxygen species (Mito-ROS)-induced nucleotide-binding oligomerization domain-like receptor has been recognized as a key mechanism in this inflammatory response and cell apoptosis. This study aimed to elucidate the underlying regulatory mechanism of Mito-ROS-NLRP3 pathway-mediated mammary epithelial cell apoptosis in dairy cows with ketosis. Mitochondrial damage and cellular apoptotic program and NLRP3 inflammasome activation were observed in the mammary gland of ketotic cows. Similar damage was detected in MAC-T cells treated with exogenous fatty acids (FFAs). However, NLRP3 inhibitor MCC950 pretreatment or Mito-ROS scavenging by MitoTEMPO attenuated apoptosis in FFA-induced MAC-T cells by inhibiting the NLRP3 inflammasome pathway. These findings reveal that the Mito-ROS-NLRP3 pathway activation is a potent mechanism underlying mammary epithelial cell apoptosis in response to metabolic stress in ketotic dairy cows, which further contributes to reduced milk yield.

Tracking the Metabolic Fate of Exogenous Arachidonic Acid in Ferroptosis Using Dual-Isotope Labeling Lipidomics.

Lipid metabolism is implicated in a variety of diseases, including cancer, cell death, and inflammation, but lipidomics has proven to be challenging due to the vast structural diversity over a narrow range of mass and polarity of lipids. Isotope labeling is often used in metabolomics studies to follow the metabolism of exogenously added labeled compounds because they can be differentiated from endogenous compounds by the mass shift associated with the label. The application of isotope labeling to lipidomics has also been explored as a method to track the metabolism of lipids in various disease states. However, it can be difficult to differentiate a single isotopically labeled lipid from the rest of the lipidome due to the variety of endogenous lipids present over the same mass range. Here we report the development of a dual-isotope deuterium labeling method to track the metabolic fate of exogenous polyunsaturated fatty acids, e.g., arachidonic acid, in the context of ferroptosis using hydrophilic interaction-ion mobility-mass spectrometry (HILIC-IM-MS). Ferroptosis is a type of cell death that is dependent on lipid peroxidation. The use of two isotope labels rather than one enables the identification of labeled species by a signature doublet peak in the resulting mass spectra. A Python-based software, D-Tracer, was developed to efficiently extract metabolites with dual-isotope labels. The labeled species were then identified with LiPydomics based on their retention times, collision cross section, and m/z values. Changes in exogenous AA incorporation in the absence and presence of a ferroptosis inducer were elucidated.

The fadXDEBA locus of Staphylococcus aureus is required for metabolism of exogenous palmitic acid and in vivo growth.

In Staphylococcus aureus, genes that should confer the capacity to metabolize fatty acids by β-oxidation occur in the fadXDEBA locus, but their function has not been elucidated. Previously, incorporation into phospholipid through the fatty acid kinase FakA pathway was thought to be the only option available for S. aureus to metabolize exogenous saturated fatty acids. We now find that in S. aureus USA300, a fadX::lux reporter was repressed by glucose and induced by palmitic acid but not stearic acid, while in USA300ΔfakA basal expression was significantly elevated, and enhanced in response to both fatty acids. When cultures were supplemented with palmitic acid, palmitoyl-CoA representing the first metabolite in the β-oxidation pathway was detected in USA300, but not in a fadXDEBA deletion mutant USA300Δfad, which relative to USA300 exhibited increased incorporation of palmitic acid into phospholipid accompanied by a rapid loss of viability. USA300Δfad also exhibited significantly reduced viability in a murine tissue abscess infection model. Our data are consistent with FakA-mediated incorporation of fatty acids into phospholipid as a preferred pathway for metabolism of exogenous fatty acids, while the fad locus is critical for metabolism of palmitic acid, which is the most abundant free fatty acid in human plasma.

Efficient megakaryopoiesis and platelet production require phospholipid remodeling and PUFA uptake through CD36.

Lipids contribute to hematopoiesis and membrane properties and dynamics; however, little is known about the role of lipids in megakaryopoiesis. Here we show that megakaryocyte progenitors, megakaryocytes and platelets present a unique lipidome progressively enriched in polyunsaturated fatty acid (PUFA)-containing phospholipids. In vitro, inhibition of both exogenous fatty acid functionalization and uptake as well as de novo lipogenesis impaired megakaryocyte differentiation and proplatelet production. In vivo, mice on a high saturated fatty acid diet had significantly lower platelet counts, which was prevented by eating a PUFA-enriched diet. Fatty acid uptake was largely dependent on CD36, and its deletion in mice resulted in low platelets. Moreover, patients with a CD36 loss-of-function mutation exhibited thrombocytopenia and increased bleeding. Our results suggest that fatty acid uptake and regulation is essential for megakaryocyte maturation and platelet production and that changes in dietary fatty acids may be a viable target to modulate platelet counts.

Multi-Omics Reveals the Impact of Exogenous Short-Chain Fatty Acid Infusion on Rumen Homeostasis: Insights into Crosstalk between the Microbiome and the Epithelium in a Goat Model.

Emerging data have underscored the significance of exogenous supplementation of butyrate in the regulation of rumen development and homeostasis. However, the effects of other short-chain fatty acids (SCFAs), such as acetate or propionate, has received comparatively less attention, and the consequences of extensive exogenous SCFA infusion remain largely unknown. In our study, we conducted a comprehensive investigation by infusion of three SCFAs to examine their respective roles in regulating the rumen microbiome, metabolism, and epithelium homeostasis. Data demonstrated that the infusion of sodium acetate (SA) increased rumen index while also promoting SCFA production and absorption through the upregulation of SCFA synthetic enzymes and the mRNA expression of SLC9A1 gene. Moreover, both SA and sodium propionate infusion resulted in an enhanced total antioxidant capacity, an increased concentration of occludin, and higher abundances of specific rumen bacteria, such as "Candidatus Saccharimonas," Christensenellaceae R-7, Butyrivibrio, Rikenellaceae RC9 gut, and Alloprevotella. In addition, sodium butyrate (SB) infusion exhibited positive effects by increasing the width of rumen papilla and the thickness of the stratum basale. SB infusion further enhanced antioxidant capacity and barrier function facilitated by cross talk with Monoglobus and Incertae Sedis. Furthermore, metabolome and transcriptome data revealed distinct metabolic patterns in rumen contents and epithelium, with a particular impact on amino acid and fatty acid metabolism processes. In conclusion, our data provided novel insights into the regulator effects of extensive infusion of the three major SCFAs on rumen fermentation patterns, antioxidant capacity, rumen barrier function, and rumen papilla development, all achieved without inducing rumen epithelial inflammation. IMPORTANCE The consequences of massive exogenous supplementation of SCFAs on rumen microbial fermentation and rumen epithelium health remain an area that requires further exploration. In our study, we sought to investigate the specific impact of administering high doses of exogenous acetate, propionate, and butyrate on rumen homeostasis, with a particular focus on understanding the interaction between the rumen microbiome and epithelium. Importantly, our findings indicated that the massive infusion of these SCFAs did not induce rumen inflammation. Instead, we observed enhancements in antioxidant capacity, strengthening of rumen barrier function, and promotion of rumen papilla development, which were facilitated through interactions with specific rumen bacteria. By addressing existing knowledge gaps and offering critical insights into the regulation of rumen health through SCFA supplementation, our study holds significant implications for enhancing the well-being and productivity of ruminant animals.

Phylogenetic investigation of Gammaproteobacteria proteins involved in exogenous long-chain fatty acid acquisition and assimilation

BackgroundThe incorporation of exogenous fatty acids into the cell membrane yields structural modifications that directly influence membrane phospholipid composition and indirectly contribute to virulence. FadL and FadD are responsible for importing and activating exogenous fatty acids, while acyltransferases (PlsB, PlsC, PlsX, PlsY) incorporate fatty acids into the cell membrane. Many Gammaproteobacteria species possess multiple homologs of these proteins involved in exogenous fatty acid metabolism, suggesting the evolutionary acquisition and maintenance of this transport pathway. MethodsThis study developed phylogenetic trees based on amino acid and nucleotide sequences of homologs of FadL, FadD, PlsB, PlsC, PlsX, and PlsY via Mr. Bayes and RAxML algorithms. We also explored the operon arrangement of genes encoding for FadL. Additionally, FadL homologs were modeled via SWISS-MODEL, validated and refined by SAVES, Galaxy Refine, and GROMACS, and docked with fatty acids via AutoDock Vina. Resulting affinities were analyzed by 2-way ANOVA test and Tukey's post-hoc test. ResultsOur phylogenetic trees revealed grouping based on operon structure, original homolog blasted from, and order of the homolog, suggesting a more ancestral origin of the multiple homolog phenomena. Our molecular docking simulations indicated a similar binding pattern for the fatty acids between the different FadL homologs. General significanceOur study is the first to illustrate the phylogeny of these proteins and to investigate the binding of various FadL homologs across orders with fatty acids. This study helps unravel the mystery surrounding these proteins and presents topics for future research.

Effects of myonectin on porcine intramuscular adipocyte differentiation and exogenous free fatty acid utilization

As an important factor secreted by skeletal muscle, myonectin can regulate lipid metabolism and energy metabolism, but its role in the utilization of peripheral free fatty acids (FFAs) by porcine intramuscular fat cells remains to be further investigated. In this study, porcine intramuscular adipocytes were treated with recombinant myonectin and palmitic acid (PA), either alone or in combination, and then were examined for their uptake of exogenous FFAs, intracellular lipid synthesis and catabolism, and mitochondrial oxidation of fatty acids. The results showed that myonectin decreased the area of lipid droplets in intramuscular adipocytes (p < 0.05) and significantly increased (p < 0.05) the expression levels of hormone-sensitive lipase (HSL) and lipoprotein lipase (LPL). Moreover, myonectin can up-regulate the expression of p38 mitogen-activated protein kinase (p38 MAPK). Myonectin significantly promoted the uptake of peripheral FFAs (p < 0.01), improved (p < 0.05) the expression of fatty transport protein 1 (FATP1) and fatty acid binding protein 4 (FABP4) in intramuscular adipocytes. Myonectin also significantly increased (p < 0.05) the expression levels of fatty acid oxidation markers: transcription factor (TFAM), uncoupling protein-2 (UCP2) and oxidative respiratory chain marker protein complex I (NADH-CoQ) in mitochondria of intramuscular adipocytes. In summary, myonectin promoted the absorption, transport, and oxidative metabolism of exogenous FFAs in mitochondria, thereby inhibiting lipid deposition in porcine intramuscular adipocytes.

Optimization of a hybrid bacterial/Arabidopsis thaliana fatty acid synthase system II in Saccharomyces cerevisiae

Fatty acids are produced by eukaryotes like baker's yeast Saccharomyces cerevisiae mainly using a large multifunctional type I fatty acid synthase (FASI) where seven catalytic steps and a carrier domain are shared between one or two protein subunits. While this system may offer efficiency in catalysis, only a narrow range of fatty acids are produced. Prokaryotes, chloroplasts and mitochondria rely instead on a FAS type II (FASII) where each catalytic step is carried out by a monofunctional enzyme encoded by a separate gene. FASII is more flexible and capable of producing a wider range of fatty acid structures, such as the direct production of unsaturated fatty acids. An efficient FASII in the preferred industrial organism S. cerevisiae could provide a platform for developing sustainable production of specialized fatty acids. We functionally replaced either yeast FASI genes (FAS1 or FAS2) with a FASII consisting of nine genes from Escherichia coli (acpP, acpS and fab -A, -B, -D, -F, -G, -H, -Z) as well as three from Arabidopsis thaliana (MOD1, FATA1 and FATB). The genes were expressed from an autonomously replicating multicopy vector assembled using the Yeast Pathway Kit for in-vivo assembly in yeast. Two rounds of adaptation led to a strain with a maximum growth rate (μmax) of 0.19 h−1 without exogenous fatty acids, twice the growth rate previously reported for a comparable strain. Additional copies of the MOD1 or fabH genes resulted in cultures with higher final cell densities and three times higher lipid content compared to the control.

Oxylipin secretion by human CD3+ T lymphocytes in vitro is modified by the exogenous essential fatty acid ratio and life stage.

Immune function changes across the life stages; for example, senior adults exhibit a tendency towards a weaker cell-mediated immune response and a stronger inflammatory response than younger adults. This might be partly mediated by changes in oxylipin synthesis across the life course. Oxylipins are oxidation products of polyunsaturated fatty acids (PUFAs) that modulate immune function and inflammation. A number of PUFAs are precursors to oxylipins, including the essential fatty acids (EFAs) linoleic acid (LA) and α-linolenic acid (ALA). LA and ALA are also substrates for synthesis of longer chain PUFAs. Studies with stable isotopes have shown that the relative amounts of LA and ALA can influence their partitioning by T lymphocytes between conversion to longer chain PUFAs and to oxylipins. It is not known whether the relative availability of EFA substrates influences the overall pattern of oxylipin secretion by human T cells or if this changes across the life stages. To address this, the oxylipin profile was determined in supernatants from resting and mitogen activated human CD3+ T cell cultures incubated in medium containing an EFA ratio of either 5:1 or 8:1 (LA : ALA). Furthermore, oxylipin profiles in supernatants of T cells from three life stages, namely fetal (derived from umbilical cord blood), adults and seniors, treated with the 5:1 EFA ratio were determined. The extracellular oxylipin profiles were affected more by the EFA ratio than mitogen stimulation such that n-3 PUFA-derived oxylipin concentrations were higher with the 5:1 EFA ratio than the 8:1 ratio, possibly due to PUFA precursor competition for lipoxygenases. 47 oxylipin species were measured in all cell culture supernatants. Extracellular oxylipin concentrations were generally higher for fetal T cells than for T cells from adult and senior donors, although the composition of oxylipins was similar across the life stages. The contribution of oxylipins towards an immunological phenotype might be due to the capacity of T cells to synthesize oxylipins rather than the nature of the oxylipins produced.

Ceramide analog C2-cer induces a loss in insulin sensitivity in muscle cells through the salvage/recycling pathway

Ceramides have been shown to play a major role in the onset of skeletal muscle insulin resistance and therefore in the prevalence of type 2 diabetes. However, many of the studies involved in the discovery of deleterious ceramide actions used a nonphysiological, cell-permeable, short-chain ceramide analog, the C2-ceramide (C2-cer). In the present study, we determined how C2-cer promotes insulin resistance in muscle cells. We demonstrate that C2-cer enters the salvage/recycling pathway and becomes deacylated, yielding sphingosine, re-acylation of which depends on the availability of long chain fatty acids provided by the lipogenesis pathway in muscle cells. Importantly, we show these salvaged ceramides are actually responsible for the inhibition of insulin signaling induced by C2-cer. Interestingly, we also show that the exogenous and endogenous monounsaturated fatty acid oleate prevents C2-cer to be recycled into endogenous ceramide species in a diacylglycerol O-acyltransferase 1-dependent mechanism, which forces free fatty acid metabolism towards triacylglyceride production. Altogether, the study highlights for the first time that C2-cer induces a loss in insulin sensitivity through the salvage/recycling pathway in muscle cells. This study also validates C2-cer as a convenient tool to decipher mechanisms by which long-chain ceramides mediate insulin resistance in muscle cells and suggests that in addition to the de novo ceramide synthesis, recycling of ceramide could contribute to muscle insulin resistance observed in obesity and type 2 diabetes.

Interactions between exogenous free fatty acids and maize starches varying in amylose content at high heating temperatures

This study investigated the effects of adding 8.0% (w/w, dsb) free fatty acids (FFAs), including myristic acid (MA), palmitic acid (PA), stearic acid (SA), oleic acid (OA), and linoleic acid (LA), on the physicochemical properties of waxy (WMS), normal (NMS), and high-amylose maize (HMS) starches over 95–140 °C heating. After cooking the starch + FFA blends in Rapid Visco Analyser (RVA), the fully gelatinized NMS and HMS formed amylose-lipid complexes (ALC) with all the added FFAs. The developed ALC displayed melting temperatures of MA, PA, SA > OA > LA but discrepant trends in the melting enthalpy changes (ΔH) between the two starches. The FFAs noticeably elevated the final viscosity of NMS at 95 and 120 °C heating and that of HMS at 120 °C heating, but the increasing effect was not found at 140 °C heating. Most of the added FFAs significantly diminished the gel strength of NMS and HMS after 95–140 °C cooking. Among all the FFAs, SA – with the highest melting temperature (70.6 °C) – exhibited the poorest complexing ability with NMS and HMS and the least impact on their pasting and gelling properties; however, this FFA significantly improved the gelling ability of HMS after 120 and 140 °C cooking. The FFA addition generally exhibited negligible effect on the pasting and gelling properties of WMS. Our research revealed that, besides ALC formation, physical properties of the exogenous FFAs (e.g., melting temperature), the presence of endogenous ALC in starch, and the gelatinization temperature of starch significantly influenced the functional attributes of the different starch-FFA blends during 95–140 °C heating.

A Streptococcus pyogenes DegV protein regulates the membrane lipid content and limits the formation of extracellular vesicles.

Membranes contain lipids that are composed of fatty acids (FA) and a polar head. Membrane homeostasis is crucial for optimal bacterial growth and interaction with the environment. Bacteria synthesize their FAs via the FASII pathway. Gram-positive bacteria can incorporate exogenous FAs which need to be phosphorylated to become substrate of the lipid biosynthetic pathway. In many species including staphylococci, streptococci and enterococci, this phosphorylation is carried out by the Fak complex, which is composed of two subunits, FakA and FakB. FakA is the kinase. FakB proteins are members of the DegV family, proteins known to bind FAs. Two or three FakB types have been identified depending on the bacterial species and characterized by their affinity for saturated and/or unsaturated FAs. Some species such as Streptococcus pyogenes, which causes a wide variety of diseases ranging from mild non-invasive to severe invasive infections, possess an uncharacterized additional DegV protein. We identify here this DegV member as a fourth FakB protein, named FakB4. The fakB4 gene is co-regulated with FASII genes suggesting an interaction with endogenous fatty acids. fakB4 deletion has no impact on membrane phospholipid composition nor on the percentage of other major lipids. However, the fakB4 mutant strain produced more lipids and more extracellular membrane vesicles than the wild-type strain. This suggests that FakB4 is involved in endogenous FA binding and controls FA storage or catabolism resulting in a limitation of extracellular FA release via membrane vesicles.