Incorporation Of Fatty Acids Research Articles

Electrophysiological Characterization of Monoolein-Fatty Acid Bilayers.

Understanding the evolution of protocells, primitive compartments that distinguish self from nonself, is crucial for exploring the origin of life. Fatty acids and monoglycerides have been proposed as key components of protocell membranes due to their ability to self-assemble into bilayers and vesicles capable of nutrient exchange. In this study, we investigate the electrophysiological properties of planar bilayers composed of monoglyceride and fatty acid mixtures, using a droplet interface bilayer system. Three fatty acids with varying hydrocarbon chain lengths─oleic acid (C18), palmitoleic acid (C16), and myristoleic acid (C14)─in combination with monoolein (C18) are examined to evaluate the influence of chain length and composition on bilayer stability, thickness, and ion permeability. The results show that pure monoolein bilayers exhibit enhanced ion permeability compared to phospholipid bilayers, which are characteristic of modern cellular membranes. Furthermore, the incorporation of fatty acids into monoolein bilayers destabilizes the membrane structure and further increases ion permeability. We consider that this increased permeability is likely driven by three molecular characteristics. First, the wedge-like shape of monoolein may disrupt bilayer packing and induce transient pore formation. Second, the rapid flip-flop of fatty acids between bilayer leaflets likely facilitates ion transport. Third, the chain-length mismatch between monoolein and myristoleic acid further destabilizes the bilayer, promoting the formation of structural defects. These findings suggest that compositional motifs in monoglyceride-fatty acid bilayers may provide an alternative ion transport mechanism, such as the flip-flop of amphiphilic molecules, in early protocell membranes before the evolution of protein-based transporters.

Synthetic-bioinformatic natural product-inspired peptides.

Covering: 2016 to 2024Natural products, particularly cyclic peptides, are a promising source of bioactive compounds. Nonribosomal peptide synthetases (NRPSs) play a key role in biosynthesizing these compounds, which include antibiotic and anticancer agents, immunosuppressants, and others. Traditional methods of discovering natural products have limitations including cryptic biosynthetic gene clusters (BGCs), low titers, and currently unculturable organisms. This has prompted the exploration of alternative approaches. Synthetic-bioinformatic natural products (syn-BNPs) are one such alternative that utilizes bioinformatics techniques to predict nonribosomal peptides (NRPs) followed by chemical synthesis of the predicted peptides. This approach has shown promise, resulting in the discovery of a variety of bioactive compounds including peptides with antibacterial, antifungal, anticancer, and proteasome-stimulating activities. Despite the success of this approach, challenges remain especially in the accurate prediction of fatty acid incorporation, tailoring enzyme modifications, and peptide release mechanisms. Further work in these areas will enable the discovery of many bioactive peptides that are currently inaccessible.

De novo lipogenesis protects dormant breast cancer cells from ferroptosis and promotes metastasis.

Dormant disseminated tumor cells (DTCs) remain viable for years to decades before establishing a clinically overt metastatic lesion. DTCs are known to be highly resilient and able to overcome the multiple biological hurdles imposed along the metastatic cascade. However, the specific metabolic adaptations of dormant DTCs remain to be elucidated. Here, we reveal that dormant DTCs upregulate de novo lipogenesis and favor the activation and incorporation of monounsaturated fatty acids (MUFAs) to their cellular membranes through the activation of acyl-coenzyme A synthetase long-chain family member 3 (ACSL3). Pharmacologic inhibition of de novo lipogenesis or genetic knockdown of ACSL3 results in lipid peroxidation and non-apoptotic cell death through ferroptosis. Clinically, ACSL3 was found to be overexpressed in quiescent DTCs in the lymph nodes of breast cancer patients and to significantly correlate with shorter disease-free and overall survival. Our work provides new insights into the molecular mechanisms enabling the survival of dormant DTCs and supports the use of de novo lipogenesis inhibitors to prevent breast cancer metastasis.

Characterization and functional properties of walnut protein fibrils for enhanced bioaccessibility of CoQ10 and ALA

Emulsion-based delivery systems have garnered significant attention in terms of encapsulation and delivery of the hydrophobic bioactive compounds in recent years. This study investigated the formation of walnut protein fibrils (WPF) through acid-heat treatment for varying durations. Emulsions stabilized by WPF were prepared for the codelivery of CoQ10 and ALA. The emulsifying properties and interfacial distribution characteristics of WPF were compared and the differences in the stability of the emulsions were studied. Prolonged treatment duration resulted in secondary structural alterations, including an increase in β-sheet content (from 39.42 % to 45.87 %). Fibrillation increased protein interfacial adsorption, leading to WPF stabilized emulsions with better storage stability, resilience to environmental stress fluctuations and oxidative stability. In summary, compared to unmodified walnut protein, WPF10 stabilized emulsion already significantly enhanced the bioaccessibility of CoQ10 and ALA. The potential delivery system may facilitate the incorporation of hydrophobic active substances and functional fatty acids into beverage products.

CEACAM6 facilitates gastric cancer progression through upregulating SLC27A2.

Gastric cancer (GC) is one of the most lethal cancers. However, the underlying mechanisms are not yet fully understood. Here, we investigated the role of carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) in tumor initiation and progression in GC and proposed therapeutic strategies for CEACAM6-positive patients. In this article, we found that CEACAM6 overexpression promoted GC initiation and progression by overactivating FAO. CEACAM6 promotes SLC27A2 expression, contributing to enhanced fatty acid incorporation. CEACAM6 interacts with both SLC27A2 and USP29, facilitating the deubiquitination of USP29 on SLC27A2. Pharmacological inhibition of SLC27A2 attenuates the tumor-initiating ability of GC. Taken together, CEACAM6 overexpression facilitates GC progression by upregulating fatty acid uptake through SLC27A2, thereby contributing to FAO. Genetic ablation of SLC27A2 is a promising therapeutic strategy for patients with CEACAM6-positive GC.

Lipid droplet-associated proteins in alcohol-associated fatty liver disease: A proteomic approach.

The earliest manifestation of alcohol-associated liver disease (ALD) is steatosis characterized by deposition of fat in specialized organelles called lipid droplets (LDs). While alcohol administration causes a rise in LD numbers in the hepatocytes, little is known regarding their characteristics that allow their accumulation and size to increase. The aim of the present study is to gain insights into underlying pathophysiological mechanisms by investigating the ethanol-induced changes in hepatic LD proteome as a function of LD size. Adult male Wistar rats (180-200 g BW) were fed with ethanol liquid diet for 6 weeks. At sacrifice, large-, medium-, and small-sized hepatic LD subpopulations (LD1, LD2, and LD3, respectively) were isolated and subjected to morphological and proteomic analyses. Morphological analysis of LD1-LD3 fractions of ethanol-fed rats clearly demonstrated that LD1 contained larger LDs compared with LD2 and LD3 fractions. Our preliminary results from principal component analysis showed that the proteome of different-sized hepatic LD fractions was distinctly different. Proteomic data analysis identified over 2000 proteins in each LD fraction with significant alterations in protein abundance among the three LD fractions. Among the altered proteins, several were related to fat metabolism, including synthesis, incorporation of fatty acid, and lipolysis. Ingenuity pathway analysis revealed increased fatty acid synthesis, fatty acid incorporation, LD fusion, and reduced lipolysis in LD1 compared to LD3. Overall, the proteomic findings indicate that the increased level of protein that facilitates fusion of LDs combined with an increased association of negative regulators of lipolysis dictates the generation of large-sized LDs during the development of alcohol-associated hepatic steatosis. Several significantly altered proteins were identified in different-sized LDs isolated from livers of ethanol-fed rats. Ethanol-induced increases in specific proteins that hinder LD lipid metabolism led to the accumulation and persistence of large-sized LDs in the liver.

Integrated cellular response of the zebrafish (Danio rerio) heart to temperature change.

A decrease in environmental temperature represents a challenge to the cardiovascular system of ectotherms. To gain insight into the cellular changes that occur during cold exposure and cold acclimation we characterized the cardiac phosphoproteome and proteome of zebrafish following 24 h or 1 week exposure to 20°C from 27°C; or at multiple points during 6 weeks of acclimation to 20°C from 27°C. Our results indicate that cold exposure causes an increase in mitogen-activated protein kinase signalling, the activation of stretch-sensitive pathways, cellular remodelling via ubiquitin-dependent pathways and changes to the phosphorylation state of proteins that regulate myofilament structure and function including desmin and troponin T. Cold acclimation (2-6 weeks) led to a decrease in multiple components of the electron transport chain through time, but an increase in proteins for lipid transport, lipid metabolism, the incorporation of polyunsaturated fatty acids into membranes and protein turnover. For example, there was an increase in the levels of apolipoprotein C, prostaglandin reductase-3 and surfeit locus protein 4, involved in lipid transport, lipid metabolism and lipid membrane remodelling. Gill opercular movements suggest that oxygen utilization during cold acclimation is reduced. Neither the amount of food consumed relative to body mass nor body condition was affected by acclimation. These results suggest that while oxygen uptake was reduced, energy homeostasis was maintained. This study highlights that the response of zebrafish to a decrease in temperature is dynamic through time and that investment in the proteomic response increases with the duration of exposure.

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.

Phospholipase A2 group IVD mediates the transacylation of glycerophospholipids and acylglycerols

In mammalian cells, glycerolipids are mainly synthesized using acyl-CoA–dependent mechanisms. The acyl-CoA–independent transfer of fatty acids between lipids, designated as transacylation reaction, represents an additional mechanism for lipid remodeling and synthesis pathways. Here, we demonstrate that human and mouse phospholipase A2 group IVD (PLA2G4D) catalyzes transacylase reactions using both phospholipids and acylglycerols as substrates. In the presence of monoglycerol and diacylglycerol (MAG and DAG), purified PLA2G4D generates DAG and triacylglycerol, respectively. The enzyme also transfers fatty acids between phospholipids and from phospholipids to acylglycerols. Overexpression of PLA2G4D in COS7 cells enhances the incorporation of polyunsaturated fatty acids into triacylglycerol stores and induces the accumulation of lysophospholipids. In the presence of exogenously added MAG, the enzyme strongly increases cellular DAG formation, while MAG levels are decreased. PLA2G4D is not or poorly detectable in commonly used cell lines. It is expressed in keratinocytes, where it is strongly upregulated by proinflammatory cytokines. Pla2g4d-deficient mouse keratinocytes exhibit complex lipidomic changes in response to cytokine treatment, indicating that PLA2G4D is involved in the remodeling of the lipidome under inflammatory conditions. Transcriptomic analysis revealed that PLA2G4D modulates fundamental biological processes including cell proliferation, differentiation, and signaling. Together, our observations demonstrate that PLA2G4D has broad substrate specificity for fatty acid donor and acceptor lipids, allowing the acyl-CoA-independent synthesis of both phospholipids and acylglycerols. Loss-of-function studies indicate that PLA2G4D affects metabolic and signaling pathways in keratinocytes, which is associated with complex lipidomic and transcriptomic alterations.

Examining the impact from incorporation of fatty acid in nitrile-based synthetic rubber: film properties and biodegradability

Examining the impact from incorporation of fatty acid in nitrile-based synthetic rubber: film properties and biodegradability

Toxoplasma acyl-CoA synthetase TgACS3 is crucial to channel host fatty acids in lipid droplets and for parasite propagation

Apicomplexa comprise important pathogenic parasitic protists that heavily depend on lipid acquisition to survive within their human host cells. Lipid synthesis relies on the incorporation of an essential combination of fatty acids (FAs) either generated by a metabolically adaptable de novo synthesis in the parasite or by scavenging from the host cell. The incorporation of FAs into membrane lipids depends on their obligate metabolic activation by specific enzyme groups, acyl-CoA synthetases (ACSs). Each ACS has its own specificity, so it can fulfill specific metabolic functions. Whilst such functionalities have been well studied in other eukaryotic models, their roles and importance in Apicomplexa are currently very limited, especially for Toxoplasma gondii. Here, we report the identification of seven putative ACSs encoded by the genome of T. gondii (TgACS), which localize to different sub-cellular compartments of the parasite, suggesting exclusive functions. We show that the perinuclear/cytoplasmic TgACS3 regulates the replication and growth of Toxoplasma tachyzoites. Conditional disruption of TgACS3 shows that the enzyme is required for parasite propagation and survival, especially under high host nutrient content. Lipidomic analysis of parasites lacking TgACS3 reveals its role in the activation of host-derived FAs that are used for i) parasite membrane phospholipid and ii) storage triacylglycerol (TAG) syntheses, allowing proper membrane biogenesis of parasite progenies. Altogether, our results reveal the role of TgACS3 as the bulk FA activator for membrane biogenesis allowing intracellular division and survival in T. gondii tachyzoites, further pointing to the importance of ACS and FA metabolism for the parasite.

501 The effect of dietary camelina, flaxseed, and canola oil supplementation on skin fatty acid profile, inflammation and immune responses in healthy adult horses

Abstract As n-6 and n-3 fatty acids (FA) have pro- and anti-inflammatory effects, respectively, supplemental oils have the potential to impact inflammation, the immune response, and skin and coat health. This study aimed to evaluate the effects of camelina (CAM), in comparison to canola (OLA) and flaxseed (FLX) oil on the skin FA profile, inflammation, and the immune response in horses. Horses [n = 24; 14.2 ± 4.8 yr; 518 ± 63 kg body weight (BW)] were acclimated to sunflower oil over 4 wk. Groups were balanced by location, age, weight, sex, and breed and randomly assigned one of the three treatments (CAM, OLA, FLX). Horses were fed their assigned oil at a dose of 0.37 g/kg BW for 16 wk. Skin samples were collected on wk 16, and the skin FA composition was analyzed by gas chromatography. On wk 10 and 12, keyhole limpet hemocyanin (KLH) was administered intramuscularly to each horse to stimulate a systemic immune response. Blood was collected on wk 14 and 16 for the evaluation of plasma KLH immunoglobulin G (IgG) concentrations using a modified ELISA protocol and results were presented as optical density units. On wk 12, after the first KLH injection, a delayed-type hypersensitivity (DTH) test was performed by intradermally injecting KLH and the area of swelling was measured using the software ImageJ at 0.5-, 1-, 8-, 24-, 48-, 72-, and 96-h post-injection. Data were analyzed using the GLIMMIX procedure of SAS considering the effects of treatment, time, and their interaction as fixed, and the effects of horse and location as random. Repeated measures on horses were modelled using a spatial power covariance structure. Significance was declared at P ≤ 0.05 and when fixed effects were significant, means were separated using Tukey-Kramer adjustments. Immune and inflammatory responses were produced by the KLH injections; however, there were no differences among treatment groups regarding KLH antibody and DTH response (P > 0.44). The percentages of palmitic (P = 0.02) and stearic acid (P = 0.03) in the skin at wk 16 were greater in FLX as compared with CAM but neither differed from the OLA. The percentage of linoleic acid (P = 0.01) was greater in CAM than OLA but neither differed from FLX at wk 16. The percentage of all other FA was similar among treatment groups (P > 0.06). These results suggest that CAM is comparable to FLX and OLA regarding support of an immune and inflammatory response, and the oil FA composition was not reflected in the skin FA profile after feeding for 16 wk as hypothesized. In conclusion, at the dose provided, CAM supports a similar immune and inflammatory response to KLH as FLX and OLA; however, the lack of incorporation of FA from the diet into the skin should be further explored.

The role of lysophosphatidylcholine acyltransferase 2 in osteoblastic differentiation of C2C12 cells.

Glycerophospholipids, a primary component of cellular membranes, play important structural and functional roles in cells. In the remodelling pathway (Lands' cycle), the concerted actions of phospholipase As and lysophospholipid acyltransferases (LPLATs) contribute to the incorporation of diverse fatty acids in glycerophospholipids in an asymmetric manner, which differ between cell types. In this study, the role of LPLATs in osteoblastic differentiation of C2C12 cells was investigated. Gene and protein expression levels of lysophosphatidylcholine acyltransferase 2 (LPCAT2), one of the LPLATs, increased during osteoblastic differentiation in C2C12 cells. LPCAT2 knockdown in C2C12 cells downregulated the expression of osteoblastic differentiation markers and the number and size of lipid droplets (LDs) and suppressed the phosphorylation of Smad1/5/9. In addition, LPCAT2 knockdown inhibited Snail1 and the downstream target of Runx2 and vitamin D receptor (VDR). These results suggest that LPCAT2 modulates osteoblastic differentiation in C2C12 cells through the bone morphogenetic protein (BMP)/Smad signalling pathway.

The brominated flame retardant hexabromocyclododecane causes systemic changes in polyunsaturated fatty acid incorporation in mouse lipids.

Brominated flame retardants are used in many household products to reduce flammability, but often leach into the surrounding environment over time. Hexabromocyclododecane (HBCD) is a brominated flame retardant detected in human blood across the world. HBCD exposure can result in neurological problems and altered lipid metabolism, but to date, the two remain unlinked. As lipids constitute ∼50% of brain dry weight, lipid metabolism plays a critical role in neuronal function and homeostasis. To determine the effect of HBCD exposure on brain lipid metabolism, young adult male C57BL/6 mice were exposed to 1 mg/kg HBCD every 3 d for 28 d. Major lipid classes were found to change across brain regions, including membrane glycerolipids such as phosphatidylcholine and phosphatidylethanolamine, and sphingolipids such as hexosylceramide. In addition, saturated, monounsaturated, and polyunsaturated fatty acids were enriched within brain lipid species. To understand the source of the brain lipidomic alterations, the blood and liver lipidomes and the cecal microbiome were evaluated. The liver and blood demonstrated changes amongst multiple lipid classes, including triacylglycerol suppression, as well as altered esterified fatty acid content. Significant alterations were also detected in the cecal microbiome, with decreases in the Firmicutes to Bacteriodetes ratio, changes in beta diversity, and pathway alterations associated with metabolic pathways and amino acid biosynthesis. These data demonstrate that HBCD can induce lipidomic alterations across brain regions and organs and support a potential role of the microbiome in these alterations.

Phospholipid Acyltransferases: Characterization and Involvement of the Enzymes in Metabolic and Cancer Diseases.

This review delves into the enzymatic processes governing the initial stages of glycerophospholipid (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) and triacylglycerol synthesis. The key enzymes under scrutiny include GPAT and AGPAT. Additionally, as most AGPATs exhibit LPLAT activity, enzymes participating in the Lands cycle with similar functions are also covered. The review begins by discussing the properties of these enzymes, emphasizing their specificity in enzymatic reactions, notably the incorporation of polyunsaturated fatty acids (PUFAs) such as arachidonic acid and docosahexaenoic acid (DHA) into phospholipids. The paper sheds light on the intricate involvement of these enzymes in various diseases, including obesity, insulin resistance, and cancer. To underscore the relevance of these enzymes in cancer processes, a bioinformatics analysis was conducted. The expression levels of the described enzymes were correlated with the overall survival of patients across 33 different types of cancer using the GEPIA portal. This review further explores the potential therapeutic implications of inhibiting these enzymes in the treatment of metabolic diseases and cancer. By elucidating the intricate enzymatic pathways involved in lipid synthesis and their impact on various pathological conditions, this paper contributes to a comprehensive understanding of these processes and their potential as therapeutic targets.

Dynamics of acyl carrier protein in de novo fatty acid synthesis by Enterococcus faecalis based on NMR spectroscopy and molecular dynamics simulation

Fatty acid synthesis (FAS) is essential for the production of biological components such as cell membrane building blocks and metabolism-related compounds. There are two types of bacterial FAS: de novo FAS and FAS through the incorporation of external fatty acids. Enterococcus faecalis possesses two distinct acyl carrier proteins (ACPs), AcpA (EfAcpA) and AcpB (EfAcpB), which serve as cofactors in the two types of FAS. We previously showed through NMR spectroscopy that EfAcpA comprises only three long helices, while EfAcpB consists of four helices, including a short α3 helix, similar to other bacterial ACPs. An increase in melting temperature (Tm) from 64.0 to 76.1 °C confirmed that protein structural stability increased in the presence of calcium ions. Using NMR spectroscopy, two metal binding sites were identified in EfAcpA: site A was located at the start of the α2 helix while site B was situated near the α2 helix and α2α3 loop. To understand the importance of structural flexibility of EfAcpA in de novo FAS, we investigated its motional properties using backbone spin relaxation and molecular dynamics simulations. The α2α3 loop in EfAcpA displayed high flexibility, as indicated by low heteronuclear NOE values. The residues Val51, Glu54, and Gly58 exhibited significant R2 values, likely due to the movement of this loop. EfAcpA created a novel cavity towards the α1α2 loop, in contrast to conventional cavity formation in most bacterial ACPs. This unique behavior was attributed to the flexibility exhibited by the α2α3 loop. The structural and motional characteristics of EfAcpA confirmed that its conformational plasticity is a crucial factor influencing acyl chain transfers in de novo FAS. Given the increasing antibiotic resistance observed for E. faecalis in clinical settings, the findings of this study may contribute to the development of more effective pathogen management strategies targeting FAS.

#1955 Ferroptotic proteins ACSL4 and ALOX15 as therapeutic targets in AKI

Abstract Background and Aims Ferroptosis is a regulated form of necrosis which is dependent on cellular iron and is characterized by the accumulation of lipid peroxides and failure of cellular antioxidant defences. We have previously described in vivo that ferroptosis is the primary cause of folic acid-induced acute kidney injury (FA-AKI) and that necroinflammation secondary to ferroptosis may further worsen kidney injury, since ferroptosis inhibition improved kidney function and decreased tubular cell death and oxidative stress. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is involved in the incorporation of polyunsaturated fatty acid (PUFA) into membranes, while 15-Lipoxygenase (Alox15) catalyzes the regio- and enantioselective peroxidation of membrane-esterified PUFAs, forming the ultimate peroxidized species that induce ferroptosis. In the present work, we aim to explore the potential of both proteins as therapeutic targets in AKI, as well as the molecular characterization of kidney ferroptosis in cultured tubular cells. Method Animal model: Female 12- to 14-week-old C57BL/6J wild type mice received a single intraperitoneal (i.p) injection of folic acid or vehicle and were sacrificed 48 hours later. Troglitazone was intravenously administered for in vivo ACSL4 inhibition, and Compound 1 was i.p administered for in vivo ALOX15 inhibition. In vitro characterization of ferroptosis: HK2 human tubular kidney cells were incubated with arachidonic acid (AA) prior to sublethal RSL3 administration for sensitization to ferroptosis. We used Ferrostatin-1 (Fer-1) and Liproxstatin-1 (Lpx-1) as inhibitors of ferroptosis. The thiazolidinedione family members Rosiglitazone, Pioglitazone and Troglitazone were used as pharmacological inhibitors of ACSL4. We also performed transcriptional silencing of ACSL4 using a specific siRNA. Compound 1 and PD1646 were administered for in vitro Alox15 inhibition. Cellular lipid peroxidation was analyzed by flow cytometry with Bodipy 581/591 staining. To characterize the lipid signature of ferroptotic cells, supernatants were collected and 15-HETEs levels were measured by ELISA. Oxido-lipidomic analysis of ferroptotic cells was performed by LC-MS. Real Time PCR and Western Blot of whole kidneys and cultured cells were performed for the detection and quantification of ACSL4 and ALOX15. Cell death was assessed by measurement of cell viability (MTT assay) and cytotoxicity (LDH assay). Tissue cell death was assessed by TUNEL. Results Kidney transcriptomics identified Acsl4 as the most upregulated member of Acsl family during FA-AKI. This was validated at mRNA and protein levels. Supplementation with AA, the preferred ACSL4 substrate, sensitized HK2 tubular cells to ferroptosis under sublethal RSL3 conditions, and this was prevented with specific ferroptosis inhibitors Fer-1 and Lpx-1. Pharmacological inhibition of ACSL4 with Troglitazone and a specific siRNA protected from cell death and lipid peroxidation induced by co-stimulation of AA and RSL3 in HK2 cells. Likewise, ALOX15 proteins levels were also increased in FA-AKI at 48 hours. PD1646 and Compound 1, both used to inhibit ALOX15, protected from ferroptotic cell death and lipid peroxidation in HK2 cells. Lipidomic analysis on HK2 tubular cells stimulated with AA+RSL3 uncovered an increased content of key peroxidized lipid species involved in ferroptosis execution, which was alleviated by targeting ACSL4 or ALOX15. Additionally, renal function of FA-AKI in mice was improved by ACSL4 pharmacological inhibition with Troglitazone and with ALOX15 inhibition with Compound 1. Conclusion Our preliminary results suggest therapeutic potential of ACSL4 and ALOX15 as ferroptotic targes during AKI. High PUFA content sensitizes human tubular HK2 cells to cell death and lipid peroxidation in the presence of sublethal ferroptotic triggers, and this was prevented by targeting ACSL4 or ALOX15 both in vitro and in vivo in FA-AKI. Closely related lipid peroxide species were decreased by ACSL4 or ALOX15 inhibitors.

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.

Overexpression of the Phosphatidylcholine:DiacylglycerolCholinephosphotransferase (PDCT) gene increases carbon flux toward triacylglycerol (TAG) synthesis in Camelinasativa seeds

Camelinasativa has considerable promise as a dedicated industrial oilseed crop. Its oil-based blends have been tested and approved as liquid transportation fuels. Previously, we utilized metabolomic and transcriptomic profiling approaches and identified metabolic bottlenecks that control oil production and accumulation in seeds. Accordingly, we selected candidate genes for the metabolic engineering of Camelina. Here we targeted the overexpression of Camelina PDCT gene, which encodes the phosphatidylcholine: diacylglycerol cholinephosphotransferase enzyme. PDCT is proposed as a gatekeeper responsible for the interconversions of diacylglycerol (DAG) and phosphatidylcholine (PC) pools and has the potential to increase the levels of TAG in seeds. To confirm whether increased CsPDCT activity in developing Camelina seeds would enhance carbon flux toward increased levels of TAG and alter oil composition, we overexpressed the CsPDCT gene under the control of the seed-specific phaseolin promoter. Camelina transgenics exhibited significant increases in seed yield (19–56%), seed oil content (9–13%), oil yields per plant (32–76%), and altered polyunsaturated fatty acid (PUFA) content compared to their parental wild-type (WT) plants. Results from [14C] acetate labeling of Camelina developing embryos expressing CsPDCT in culture indicated increased rates of radiolabeled fatty acid incorporation into glycerolipids (up to 64%, 59%, and 43% higher in TAG, DAG, and PC, respectively), relative to WT embryos. We conclude that overexpression of PDCT appears to be a positive strategy to achieve a synergistic effect on the flux through the TAG synthesis pathway, thereby further increasing oil yields in Camelina.

Moringa oleifera Oil Nutritional and Safety Impact on Deep-Fried Potatoes.

Aiming to understand the nutritional impact of Moringa oleifera oil (MOO) on the quality of fried potatoes as consumed, a frying study using intermittent frying at 180 °C was conducted over 5 days, with a total heating time of 15 h, against olive (OO) and sunflower (SFO) oils. Additionally, due to MOO's higher costs, a SFO/MOO blend (80/20 w/w) was tested. With similar fat incorporation and moisture contents, potato lipid composition revealed the impact of oil oxidation over the frying time, gradually decreasing the content of unsaturated fatty acids and antioxidants, including vitamin E, carotenoids and ascorbic acid, and increasing the incorporation of trans fatty acids (TFAs) and volatile aldehydes. When the potatoes fried at the ninth hour of heating are compared, MOO and OO were still able to protect potato ascorbic acid better than SFO, due to the low oxidative stress imposed by their fatty-acid composition. SFO, on the contrary, with linoleic acid as the main fatty acid, and despite its higher content of vitamin E, demonstrated higher oxidative stress and increased incorporation of alkenals and alkadienals. Acrylamide content was generally low, as were the trans fatty acids formed and incorporated with frying time, with MOO fried potatoes having lower amounts of all these process contaminants. Interestingly, the blend SFO/MOO (80/20 w/w) doubled the amount of vitamin E in fried potatoes when compared with SFO alone, increased the ascorbic acid protection and reduced by half the amounts of volatile aldehydes, indicative of an efficient reduction of the oxidative status of the SFO-fried potatoes, with benefits to the consumer from a health point of view.