Exogenous Fatty Acids Research Articles

Prophages divert Staphylococcus aureus defenses against host lipids

Phages are ubiquitous in bacteria, including clinical Staphylococcus aureus, where Sfi 21/Sa3 phages often integrate into the hlb gene, which encodes Hlb sphingomyelinase. This integration acts as a rapid regulatory switch for Hlb production. Our findings suggest that Sfi 21/Sa3 prophages and Hlb activity influence S. aureus fitness by modulating the incorporation of the toxic linoleic acid (C18:2) from serum into the bacterial membrane. This process relies on C18:2 derived from 1,3-diglyceride, facilitated by the FakB1 kinase subunit. Palmitic acid (C16), primarily released from serum through Hlb activity, competes with C18:2 for FakB1. This mechanism contributes to adaptation to AFN-1252, an antibiotic inhibiting the fatty acid synthesis pathway (anti-FASII). Since S. aureus relies on exogenous fatty acids for growth, AFN-1252 treatment leads to increased proportion of C18:2 in the membrane. Furthermore, Hlb inhibition, whether by prophage insertion, gene inactivation, or enzyme inhibition, delays S. aureus adaptation, resulting in a higher proportion of C18:2 in the membrane. This study sheds light on the role of lipid environments in infections and may contribute to the accurate prediction of infection risks and therapeutic efficacy. Moreover, since both anti-FASII agent and Hlb inhibitor enhance C18:2 incorporation, they represent potential candidates for combined strategies against S. aureus.

Different responses of larval fatty acid profiles to cryopreservation in two commercially important bivalves.

Larval cryopreservation techniques have been established in Pacific oysters and Mediterranean mussels. Although initial cryopreservation protocols for both species differed slightly in Ficoll PM 70 (FIC) concentration, better post-thaw larval survivability was produced in mussels than oysters. Furthermore, the post-thaw survivability in the latter could be improved significantly by the addition of lipids and antioxidants to the cryoprotectant agent. These findings have generated a unique opportunity to investigate the cryo-functions of both endogenous and exogenous fatty acids (FAs) in bivalves as mammalian studies indicated that lipid compositions could contribute the interspecific difference in gamete and embryo cryopreservation. Our study showed that the higher survivability of post-thaw larvae in mussels (65% vs. 34% in oysters) could be attributed to their higher proportion of total polyunsaturated fatty acids (PUFAs), especially C22:6, and higher resistance of FA profiles to cryopreservation. In oysters, on the other hand, their FA profiles were very sensitive to cryopreservation, with saturated FAs and PUFAs being significantly increased (from 37 to 41%) and decreased (from 35% to ~ 32%), respectively. Although exogenous lipids could significantly improve the survivability of post-thaw oyster larvae from 34 to 51%, their supplementation did not alter the response pattern of endogenous FA profiles to cryopreservation.

Metabolomic Profiling Reveals Potential of Fatty Acids as Regulators of Stem-like Exhausted CD8 T Cells During Chronic Viral Infection.

This study examines systemic metabolite changes during acute and chronic viral infections. Notably, we identified an early, transient nutrient shift in chronic infection, marked by an increase in medium- and long-chain fatty acid related species. Concomitantly, a virus-specific stem-like T cell population, essential for maintaining other T cells, displayed high lipid avidity and was capable of metabolizing exogenous fatty acids. Administering fatty acids late in chronic infection, when endogenous lipid levels had normalized, expanded this stem-like T cell population and enhanced their mitochondrial fitness. These findings highlight the potential role of fatty acids in regulating stem-like T cells in chronic settings and offer a valuable resource for studying other metabolic signatures in both acute and persistent infections.

The Enteric Bacterium Enterococcus faecalis Elongates and Incorporates Exogenous Short and Medium Chain Fatty Acids Into Membrane Lipids.

Enterococcus faecalis incorporates and elongates exogeneous short- and medium-chain fatty acids to chains sufficiently long to enter membrane phospholipid synthesis. The acids are activated by the E. faecalis fatty acid kinase (FakAB) system and converted to acyl-ACP species that can enter the fatty acid synthesis cycle to become elongated. Following elongation the acyl chains are incorporated into phospholipid by the PlsY and PlsC acyltranferases. This process has little effect on de novo fatty acid synthesis in the case of short-chain acids, but a greater effect with medium-chain acids. Incorporation of exogenous short-chain fatty acids in E. faecalis was greatly increased by overexpression of either AcpA, the acyl carrier protein of fatty acid synthesis, or the phosphate acyl transferase PlsX. The PlsX of Lactococcus lactis was markedly superior to the E. faecalis PlsX in incorporation of short-chain but not long-chain acids. These manipulations also allowed unsaturated fatty acids of lengths too short for direct transfer to the phospholipid synthesis pathway to be elongated and support growth of E. faecalis unsaturated fatty acid auxotrophic strains. Short- and medium-chain fatty acids can be abundant in the human gastrointestinal tract and their elongation by E. faecalis would conserve energy and carbon by relieving the requirement for total de novo synthesis of phospholipid acyl chains.

Organelle-Targeted Laurdans Measure Heterogeneity in Subcellular Membranes and Their Responses to Saturated Lipid Stress.

Organelles feature characteristic lipid compositions that lead to differences in membrane properties. In cells, membrane ordering and fluidity are commonly measured using the solvatochromic dye Laurdan, whose fluorescence is sensitive to lipid packing. As a general lipophilic dye, Laurdan stains all hydrophobic environments in cells; therefore, it is challenging to characterize membrane properties in specific organelles or assess their responses to pharmacological treatments in intact cells. Here, we describe the synthesis and application of Laurdan-derived probes that read out the membrane packing of individual cellular organelles. The set of organelle-targeted Laurdans (OTL) localizes to the ER, mitochondria, lysosomes, and Golgi compartments with high specificity while retaining the spectral resolution needed to detect biological changes in membrane ordering. We show that ratiometric imaging with OTLs can resolve membrane heterogeneity within organelles as well as changes in lipid packing resulting from inhibition of trafficking or bioenergetic processes. We apply these probes to characterize organelle-specific responses to saturated lipid stress. While the ER and lysosomal membrane fluidity is sensitive to exogenous saturated fatty acids, that of mitochondrial membranes is protected. We then use differences in ER membrane fluidity to sort populations of cells based on their fatty acid diet, highlighting the ability of organelle-localized solvatochromic probes to distinguish between cells based on their metabolic state. These results expand the repertoire of targeted membrane probes and demonstrate their application in interrogating lipid dysregulation.

Cardiolipin remodeling maintains the inner mitochondrial membrane in cells with saturated lipidomes

Cardiolipin (CL) is a unique, four-chain phospholipid synthesized in the inner mitochondrial membrane (IMM). The acyl chain composition of CL is regulated through a remodeling pathway, whose loss causes mitochondrial dysfunction in Barth syndrome (BTHS). Yeast has been used extensively as a model system to characterize CL metabolism, but mutants lacking its two remodeling enzymes, Cld1p and Taz1p, exhibit mild structural and respiratory phenotypes compared to mammalian cells. Here, we show an essential role for CL remodeling in the structure and function of the IMM in yeast grown under reduced oxygenation. Microaerobic fermentation, which mimics natural yeast environments, caused the accumulation of saturated fatty acids and, under these conditions, remodeling mutants showed a loss of IMM ultrastructure. We extended this observation to HEK293 cells, where phospholipase A2 inhibition by Bromoenol lactone resulted in respiratory dysfunction and cristae loss upon mild treatment with exogenous saturated fatty acids. In microaerobic yeast, remodeling mutants accumulated unremodeled, saturated CL, but also displayed reduced total CL levels, highlighting the interplay between saturation and CL biosynthesis and/or breakdown. We identified the mitochondrial phospholipase A1 Ddl1p as a regulator of CL levels, and those of its precursors phosphatidylglycerol and phosphatidic acid, under these conditions. Loss of Ddl1p partially rescued IMM structure in cells unable to initiate CL remodeling and had differing lipidomic effects depending on oxygenation. These results introduce a revised yeast model for investigating CL remodeling and suggest that its structural functions are dependent on the overall lipid environment in the mitochondrion.

An inhibitory mechanism of AasS, an exogenous fatty acid scavenger: Implications for re-sensitization of FAS II antimicrobials.

Antimicrobial resistance is an ongoing "one health" challenge of global concern. The acyl-ACP synthetase (termed AasS) of the zoonotic pathogen Vibrio harveyi recycles exogenous fatty acid (eFA), bypassing the requirement of type II fatty acid synthesis (FAS II), a druggable pathway. A growing body of bacterial AasS-type isoenzymes compromises the clinical efficacy of FAS II-directed antimicrobials, like cerulenin. Very recently, an acyl adenylate mimic, C10-AMS, was proposed as a lead compound against AasS activity. However, the underlying mechanism remains poorly understood. Here we present two high-resolution cryo-EM structures of AasS liganded with C10-AMS inhibitor (2.33 Å) and C10-AMP intermediate (2.19 Å) in addition to its apo form (2.53 Å). Apart from our measurements for C10-AMS' Ki value of around 0.6 μM, structural and functional analyses explained how this inhibitor interacts with AasS enzyme. Unlike an open state of AasS, ready for C10-AMP formation, a closed conformation is trapped by the C10-AMS inhibitor. Tight binding of C10-AMS blocks fatty acyl substrate entry, and therefore inhibits AasS action. Additionally, this intermediate analog C10-AMS appears to be a mixed-type AasS inhibitor. In summary, our results provide the proof of principle that inhibiting salvage of eFA by AasS reverses the FAS II bypass. This facilitates the development of next-generation anti-bacterial therapeutics, esp. the dual therapy consisting of C10-AMS scaffold derivatives combined with certain FAS II inhibitors.

Lipidomics of homeoviscous adaptation to low temperatures in Staphylococcus aureus utilizing exogenous straight-chain unsaturated fatty acids.

We show that Staphylococcus aureus can use its known ability to incorporate exogenous fatty acids to enhance its growth at low temperatures. Individual species of phosphatidylglycerols and diglycosyldiacylglycerols bearing one or two degrees of unsaturation derived from the incorporation of C18:1Δ9 at 12°C are described for the first time. In addition, enhanced production of the carotenoid staphyloxanthin occurs at low temperatures. The studies describe a biochemical reality underlying membrane biophysics. This is an example of homeoviscous adaptation to low temperatures utilizing exogenous fatty acids over the regulation of the biosynthesis of endogenous fatty acids. The studies have likely relevance to food safety in that unsaturated fatty acids may enhance the growth of S. aureus in the food environment.

Arsenic exposure at environmentally relevant levels induced metabolic toxicity in development mice: Mechanistic insights from integrated transcriptome and metabolome

Emerging evidence has linked arsenic exposure and metabolic homeostasis, but the mechanism is incompletely understood, especially at relatively low concentrations. In this study, we used a mouse model to evaluate the health impacts and metabolic toxicity of arsenic exposure in drinking water at environmentally relevant levels (0.25 and 1.0 ppm). Our results indicated that arsenic damaged intestinal barrier and induced arsenic accumulation, oxidative stress, and pathological changes in the liver and illum. Interestingly, arsenic increased the hepatic triglyceride (TG) and total cholesterol (TC), while reduced serum TG and TC levels. The liver transcriptome found that arsenic exposure caused transcriptome perturbation and promoted hepatic lipid accumulation by regulating the exogenous fatty acids degradation and apolipoproteins related genes. The serum metabolomics identified 74 and 88 differential metabolites in 0.25 and 1.0 ppm, respectively. The KEGG disease and subcellular location analysis indicated that arsenic induced liver and intestinal diseases, and the mitochondrion might be the target organelle for arsenic-induced toxicity. Co-enrichment of transcriptome and metabolome identified 24 metabolites and 9 genes as metabolic toxicity biomarkers. Moreover, 40 male (20 nonalcoholic fatty liver disease (NAFLD) cases and 20 healthy controls) was further selected to validate our findings. Importantly, the significantly changed L-palmitoylcarnitine, 3-hydroxybutyric acid, 2-hydroxycaproic acid and 6 genes of Hadha, Acadl, Aldh3a2, Cpt1a, Cpt2, and Acox1 were found in the NAFLD cases. The results from integrated multi-omics and chemical-protein network analysis indicated that L-palmitoylcarnitine played a critical role in metabolic toxicity by regulating mitochondrial fatty acids β-oxidation genes (Cpt1a, Cpt2). In conclusion, these findings provided new clues for the metabolic toxicity of arsenic exposure at environmentally relevant levels, which involved in the late-life NAFLD development. Our results also contribute to understanding the human responses and phenotypic changes to this hazardous material exposure in the environment.

Diacylglycerol O-acyltransferase isoforms play a role in peridroplet mitochondrial fatty acid metabolism in bovine liver

Hepatocellular lipid accumulation characterizes fatty liver in dairy cows. Lipid droplets (LD), specialized organelles that store lipids and maintain cellular lipid homeostasis, are responsible for the ectopic storage of lipids associated with several metabolic disorders. In recent years, nonruminant studies have reported that LD-mitochondria interactions play an important role in lipid metabolism. Due to the role of diacylglycerol acyltransferase isoforms (DGAT1 and DGAT2) in LD synthesis, we explored mechanisms of mitochondrial fatty acid transport in ketotic cows using liver biopsies and isolated primary hepatocytes. Compared with healthy cows, cows with fatty liver had massive accumulation of LD and high protein expression of the triglyceride (TAG) synthesis-related enzymes DGAT1 and DGAT2, LD synthesis-related proteins perilipin 2 (PLIN2) and perilipin 5 (PLIN5), and the mitochondrial fragmentation-related proteins dynamin-related protein 1 (DRP1) and fission 1 (FIS1). In contrast, factors associated with fatty acid oxidation, mitochondrial fusion, and mitochondrial electron transport chain complex were lower compared with those in the healthy cows. In addition, transmission electron microscopy revealed significant contacts between LD-mitochondria in liver tissue from cows with fatty liver. Compared with isolated cytoplasmic mitochondria, expression of carnitine palmitoyl transferase 1A (CPT1A) and DRP1 was lower, but mitofusin 2 (MFN2) and mitochondrial electron transport chain complex was greater in isolated peridroplet mitochondria from hepatic tissue of cows with fatty liver. In vitro data indicated that exogenous free fatty acids (FFA) induced hepatocyte LD synthesis and mitochondrial dynamics consistent with in vivo results. Furthermore, DGAT2 inhibitor treatment attenuated the FFA-induced upregulation of PLIN2 and PLIN5 and rescued the impairment of mitochondrial dynamics. Inhibition of DGAT2 also restored mitochondrial membrane potential and reduced hepatocyte reactive oxygen species production. The present in vivo and in vitro results indicated functional differences are present among different types of mitochondria in the liver tissue of dairy cows with ketosis. Activity of DGAT2 may play a key role in maintaining liver mitochondrial function and lipid homeostasis in dairy cows during the transition period.

Assessment of membrane labelling mechanisms with exogenous fatty acids and detergents in bacteria

Labelling of bacterial membranes using exogenous fatty acids has proven to be a valuable tool to investigate molecular interactions by in-cell solid-state nuclear magnetic resonance (ssNMR) spectroscopy, notably with antimicrobial peptides. However, the mechanism by which this labelling takes place in non-mutated bacteria has not yet been investigated. In this work, we propose a rapid method to assess the fate of the fatty acids during the labelling of bacteria, involving two different methylation schemes and gas chromatography coupled to mass spectrometry. We applied this approach to Gram(+) and Gram(−) bacteria grown with deuterated palmitic acid under different conditions. We assessed the extent of labelling, then the resulting membrane rigidity by 2H ssNMR. Our results reveal that the labelling mechanism depends on the detergent used to micellize the fatty acids. This labelling can be either active or passive, whether the fatty acids are metabolized and used in the phospholipids biosynthesis, or remain unmodified in the membrane. We discuss the best labelling protocol for studying peptide-membrane interactions.

Bile promotes Lactobacillus johnsonii N6.2 extracellular vesicle production with conserved immunomodulatory properties

Recently, Lactobacillus johnsonii N6.2-derived extracellular vesicles (EVs) were shown to reduce apoptosis in human beta cell lines and stimulate insulin secretion in human islets. Our goal was to identify a physiologically relevant environmental condition that induces a hypervesiculation phenotype in L. johnsonii N6.2 and to evaluate if transcriptional changes are involved in this process. Culturing this strain in the presence of 0.2% bovine bile, which mimics a stressor encountered by the bacterium in the small intestine, resulted in approximately a 100-fold increase in EVs relative to cells grown in media without bile. Whole transcriptome analysis of cells grown with bile revealed upregulation of several peptidoglycan hydrolases as well as several genes involved in fatty acid utilization. These results suggest that the hypervesiculation phenotype may be the result of increased cell wall turnover combined with increased accumulation of phospholipids, in agreement with our previous proteomic and lipidomics results. Additionally, EVs isolated from L. johnsonii N6.2 grown in presence of bile maintained their immunomodulatory properties in host-derived βlox5 pancreatic and THP-1 macrophage cell lines. Our findings suggest that in L. johnsonii N6.2 vesiculogenesis is significantly impacted by the expression of cell wall modifying enzymes and proteins utilized for exogenous fatty acid uptake that are regulated at the transcriptional level. Furthermore, this data suggests that vesiculogenesis could be stimulated in vivo using small molecules thereby maximizing the beneficial interactions between bacteria and their hosts.

Biosynthetic deficiency of docosahexaenoic acid causes nonalcoholic fatty liver disease and ferroptosis-mediated hepatocyte injury

Exogenous omega-3 fatty acids, particularly docosahexaenoic acid (DHA), have shown to exert beneficial effects on nonalcoholic fatty liver disease (NAFLD), which is characterized by the excessive accumulation of lipids and chronic injury in the liver. However, the effect of endogenous DHA biosynthesis on the lipid homeostasis of liver is poorly understood. In this study, we used a DHA biosynthesis-deficient zebrafish model, elovl2 mutant, to explore the effect of endogenously biosynthesized DHA on hepatic lipid homeostasis. We found the pathways of lipogenesis and lipid uptake were strongly activated, while the pathways of lipid oxidation and lipid transport were inhibited in the liver of elovl2 mutants, leading to lipid droplet accumulation in the mutant hepatocytes and NAFLD. Furthermore, the elovl2 mutant hepatocytes exhibited disrupted mitochondrial structure and function, activated endoplasmic reticulum (ER) stress, and hepatic injury. We further unveiled that the hepatic cell death and injury was mainly mediated by ferroptosis, rather than apoptosis, in elovl2 mutants. Elevating DHA content in elovl2 mutants, either through introduction of an omega-3 desaturase (fat1) transgene or feeding with a DHA-rich diet, could strongly alleviate NAFLD features and ferroptosis-mediated hepatic injury. Together, our study elucidates the essential role of endogenous DHA biosynthesis in maintaining hepatic lipid homeostasis and liver health, highlighting that DHA deficiency can lead to NAFLD and ferroptosis-mediated hepatic injury.

Probing into the impacts of endogenous and exogenous short chain fatty acids (SCFAs) in fish health and growth

Abstract Short-chain fatty acids (SCFAs) are small organic acids exist in the fish gastrointestinal tract, mainly acetate, propionate, and butyrate. These SCFAs are produced through anaerobic fermentation of dietary non-digestible carbohydrates by gut microbes. Though enough studies have been devoted to showcasing the potential of SCFAs in human nutrition, the research on their production, potential, and existence in fish has been comparatively limited until recently. This review aims to provide a comprehensive overview of the current knowledge regarding SCFAs in aquaculture. It starts with commenting on the different sources, such as substrate and microbial species that are responsible for the production of SCFAs and their transportation and ends up depicting the relationship between biofloc technology and SCFAs production. This review also discusses the factors influencing SCFA production, such as environment, diet, and feeding habits of fish. Furthermore, the biological functions of SCFAs have been highlighted, and critical findings were compiled, including immunomodulatory effects, physiological response, gut health, and immunity.

Recognition and acquisition of FakB2-loaded exogenous fatty acid (eFA) by a streptococcal FakA kinase

Recognition and acquisition of FakB2-loaded exogenous fatty acid (eFA) by a streptococcal FakA kinase

Raman Imaging-A Valuable Tool for Tracking Fatty Acid Metabolism-Normal and Cancer Human Colon Single-Cell Study.

Altered metabolism of lipids is a key factor in many diseases including cancer. Therefore, investigations into the impact of unsaturated and saturated fatty acids (FAs) on human body homeostasis are crucial for understanding the development of lifestyle diseases. In this paper, we focus on the impact of palmitic (PA), linoleic (LA), and eicosapentaenoic (EPA) acids on human colon normal (CCD-18 Co) and cancer (Caco-2) single cells using Raman imaging and spectroscopy. The label-free nature of Raman imaging allowed us to evaluate FAs dynamics without modifying endogenous cellular metabolism. Thanks to the ability of Raman imaging to visualize single-cell substructures, we have analyzed the changes in chemical composition of endoplasmic reticulum (ER), mitochondria, lipid droplets (LDs), and nucleus upon FA supplementation. Analysis of Raman band intensity ratios typical for lipids, proteins, and nucleic acids (I1656/I1444, I1444/I1256, I1444/I750, I1304/I1256) proved that, using Raman mapping, we can observe the metabolic pathways of FAs in ER, which is responsible for the uptake of exogenous FAs, de novo synthesis, elongation, and desaturation of FAs, in mitochondria responsible for energy production via FA oxidation, in LDs specialized in cellular fat storage, and in the nucleus, where FAs are transported via fatty-acid-binding proteins, biomarkers of human colon cancerogenesis. Analysis for membranes showed that the uptake of FAs effectively changed the chemical composition of this organelle, and the strongest effect was noticed for LA. The spectroscopy studies have been completed using XTT tests, which showed that the addition of LA or EPA for Caco-2 cells decreases their viability with a stronger effect observed for LA and the opposite effect observed for PA. For normal cells, CCD-18 Co supplementation using LA or EPA stimulated cells for growing, while PA had the opposite impact.

Evaluation of Fusobacterium nucleatum Enoyl-ACP Reductase (FabK) as a Narrow-Spectrum Drug Target.

Fusobacterium nucleatum, a pathobiont inhabiting the oral cavity, contributes to opportunistic diseases, such as periodontal diseases and gastrointestinal cancers, which involve microbiota imbalance. Broad-spectrum antimicrobial agents, while effective against F. nucleatum infections, can exacerbate dysbiosis. This necessitates the discovery of more targeted narrow-spectrum antimicrobial agents. We therefore investigated the potential for the fusobacterial enoyl-ACP reductase II (ENR II) isoenzyme FnFabK (C4N14_ 04250) as a narrow-spectrum drug target. ENRs catalyze the rate-limiting step in the bacterial fatty acid synthesis pathway. Bioinformatics revealed that of the four distinct bacterial ENR isoforms, F. nucleatum specifically encodes FnFabK. Genetic studies revealed that fabK was indispensable for F. nucleatum growth, as the gene could not be deleted, and silencing of its mRNA inhibited growth under the test conditions. Remarkably, exogenous fatty acids failed to rescue growth inhibition caused by the silencing of fabK. Screening of synthetic phenylimidazole analogues of a known FabK inhibitor identified an inhibitor (i.e., 681) of FnFabK enzymatic activity and F. nucleatum growth, with an IC50 of 2.1 μM (1.0 μg/mL) and a MIC of 0.4 μg/mL, respectively. Exogenous fatty acids did not attenuate the activity of 681 against F. nucleatum. Furthermore, FnFabK was confirmed as the intracellular target of 681 based on the overexpression of FnFabK shifting MICs and 681-resistant mutants having amino acid substitutions in FnFabK or mutations in other genetic loci affecting fatty acid biosynthesis. 681 had minimal activity against a range of commensal flora, and it was less active against streptococci in physiologic fatty acids. Taken together, FnFabK is an essential enzyme that is amenable to drug targeting for the discovery and development of narrow-spectrum antimicrobial agents.

Characterization of GPAT gene family in Euphorbia lathyris L. and elucidating the role of ElGPAT9 in the biosynthesis of oils and pollen viability

Caper spurge (Euphorbia lathyris L.), known for its high content of oleic acid-enriched seed oils, has gained recognition as a valuable non-edible feedstock for biodiesel production. However, there is limited understanding of the biosynthesis and regulation of these desirable seed oils. In this study, an omics-based approach and genetic transformation were employed to investigate the glycerol-3-phosphate acyltransferase (GPAT) enzyme, which catalyzes the initial acylation reaction in triacylglycerol (TAG) biosynthesis, in caper spurge. Ten genes encoding ElGPAT, identified from the E. lathyris genome, were classified into three phylogenetic groups, exhibiting similar protein motifs and gene structures. RNA-seq data revealed distinct expression patterns of these ElGPAT genes in various tissues and different stages of seed development. Notably, ElGPAT9 showed the highest expression in flowers and developing seeds, with its encoded protein localized in the endoplasmic reticulum. Experiments using a yeast (Saccharomyces cerevisiae) expression system demonstrated that ElGPAT9 exhibited strong GPAT enzyme activity, crucial for TAG assembly, and preference for oleic acid (C18:1), as confirmed by feeding tests with exogenous fatty acids (FA). Similarly, stable transgenic lines of Nicotiana tabacum over-expressing ElGPAT9 showed increased levels of total oil and oleic acid, as well as improved pollen viability, without any growth penalty compared to wild-type and empty-vector controls. The transcriptome based WGCNA also identified genes related to lipid synthesis that are co-expressed with ElGPAT9. These findings provide a foundation for further understanding the roles of ElGPAT family members in E. lathyris and suggest the potential of ElGPAT9 as a desirable target for genetic engineering to enhance the production of vegetable oils enriched with C18:1 in caper spurge and other oil crops, contributing to sustainable biodiesel and lipid product production.

Determination of fatty acid uptake and desaturase activity in mammalian cells by NMR-based stable isotope tracing

BackgroundMammalian cells both import exogenous fatty acids and synthesize them de novo. Palmitate, the end product of fatty acid synthase (FASN) is a substrate for stearoyl-CoA desaturases (Δ-9 desaturases) that introduce a single double bond into fatty acyl-CoA substrates such as palmitoyl-CoA and stearoyl-CoA. This process is particularly upregulated in lipogenic tissues and cancer cells. Tracer methodology is needed to determine uptake versus de novo synthesis of lipids and subsequent chain elongation and desaturation. Here we describe an NMR method to determine the uptake of 13C-palmitate from the medium into HCT116 human colorectal cancer cells, and the subsequent desaturation and incorporation into complex lipids. ResultsExogenous 13C16-palmitate was absorbed from the medium by HCT116 cells and incorporated primarily into complex glycerol lipids. Desaturase activity was determined from the quantification of double bonds in acyl chains, which was greatly reduced by ablation of the major desaturase SCD1. SignificanceThe NMR approach requires minimal sample preparation, is non-destructive, and provides direct information about the level of saturation and incorporation of fatty acids into complex lipids.

Fatty Acids Play a Critical Role in Mitochondrial Oxidative Phosphorylation in Effector T Cells in Graft-versus-Host Disease.

Although the role of aerobic glycolysis in activated T cells has been well characterized, whether and how fatty acids (FAs) contribute to donor T cell function in allogeneic hematopoietic stem cell transplantation is unclear. Using xenogeneic graft-versus-host disease (GVHD) models, this study demonstrated that exogenous FAs serve as a crucial source of mitochondrial respiration in donor T cells in humans. By comparing human T cells isolated from wild-type NOD/Shi-scid-IL2rγnull (NOG) mice with those from MHC class I/II-deficient NOG mice, we found that donor T cells increased extracellular FA uptake, the extent of which correlates with their proliferation, and continued to increase FA uptake during effector differentiation. Gene expression analysis showed the upregulation of a wide range of lipid metabolism-related genes, including lipid hydrolysis, mitochondrial FA transport, and FA oxidation. Extracellular flux analysis demonstrated that mitochondrial FA transport was required to fully achieve the mitochondrial maximal respiration rate and spare respiratory capacity, whereas the substantial disruption of glucose supply by either glucose deprivation or mitochondrial pyruvate transport blockade did not impair oxidative phosphorylation. Taken together, FA-driven mitochondrial respiration is a hallmark that differentiates TCR-dependent T cell activation from TCR-independent immune response after hematopoietic stem cell transplant.