Fatty Acid Elongase Research Articles

Abstract 4121104: Fish Oil-Derived N-3 Very-Long-Chain Polyunsaturated Fatty Acids Improve Retinal Function in Aged Mice

Introduction: Epidemiological studies and clinical trials have revealed cardiometabolic benefits associated with dietary fish oil-derived omega-3 polyunsaturated fatty acids, particularly EPA and DHA. Omega-3 very-long-chain polyunsaturated fatty acids (VLCPUFA), synthesized in vivo by very-long-chain fatty acid elongase (ELOVL), e.g. ELOVL2 and 4, have recently garnered interest for their role in eye and cardiometabolic diseases. In our previous study, aging mice fed a VLCPUFA-rich diet for 8 weeks exhibited lowered plasma lipid levels, and repeated gavage of VLCPUFA in young mice resulted in improved retinal function. However, it is largely not known if VLCPUFA intake for a short term in aged mice would also improve retinal function and have beneficial effects in cardiometabolic risk factors. Hypothesis: We hypothesized that VLCPUFA may delay or rescue age-related vision loss by supplementing the products of ELOVL2, which decrease in quantity with age. We also hypothesize that short-term VLCPUFA intake may also confer favorable cardiometabolic benefits. Aims: We aimed to evaluate the effects of exogenous VLCPUFA on visual function and lipoprotein profiles in aged mice. Methods: Sixteen-month-old mice received a daily oral gavage of C24-28-rich VLCPUFA-oil (80 mg/kg BW) or an Intralipid vehicle for 15 consecutive days (n=8). The electroretinogram (ERG) instrument used was a UTAS E-3000 with gold corneal and stainless-steel scalp electrodes. Fast Protein Liquid Chromatography was performed using pooled plasma. Results: Consumption of VLCPUFA for 15 days increased scotopic a- (p < 0.05 at −5, 0, 5, 10, and 15 dB) and b-wave (p < 0.05 at 0 and 5 dB, p = 0.08 at 10 dB) amplitudes on ERG in both male and female mice compared with control. Although photopic ERG results did not exhibit statistically significant differences, likely due to low sample size, the overall results were consistent with those in our previous studies in young mice. We report no statistically significant difference in the composition of pro-atherogenic VLDL and LDL fractions due to VLCPUFA gavage. Increasing the duration and samples size in future gavage studies may be critical to determine the effects of VLCPUFA on eye and cardiovascular risk factors. Conclusions: This study revealed that a short-term oral administration of VLCPUFA was effective in improving ERG response overall, but not lipoprotein profiles.

Abstract 4128491: Dietary N-3 Very-Long-Chain Polyunsaturated Fatty Acids Improve Retinal Function And Reduce Aortic Atherosclerosis In ApoE-Deficient Mice

Introduction: N-3 very-long-chain polyunsaturated fatty acids (VLCPUFA; C≥24), which are found primarily in retina and a few other select tissues, are known to play critical roles in specific biological systems. Although n-3 PUFA, such as eicosapentanoic acid (EPA, C20:5 n-3) and docosahexaenoic acid (DHA, C22:6 n-3), may confer cardiovascular benefits, they did not improve age-related macular degeneration (AMD), a leading cause of blindness worldwide, in clinical trials. The activity of ELOVL fatty acid elongase 2 (ELOVL2), an enzyme that converts EPA into tetracosapentaenoic acid (TPA, C24:5 n-3), is known to decrease in the retina with age due to promoter methylation. Hypothesis: We hypothesized that dietary VLCPUFA may delay or prevent AMD, by bypassing the ELOVL2-mediated lipid elongation step. We also hypothesize that VLCPUFA may benefit cardiometabolic health like shorter-chain n-3 PUFA through similar mechanisms. Aims: We aimed to investigate the effect of dietary VLCPUFA on retinal function and cardiometabolic risk factors in mice. Methods: We have produced a new fish oil that contains ~40% (w/w) of C24-C28-rich VLCPUFA. We fed 9-month-old ApoE -/- mice with normal or VLCPUFA fortified diet (1% or 3% (w/w)) for 8 weeks, and age-matched C57BL/6J mice were used as control. We conducted electroretinography (ERG) and cognitive ability tests at the end of feeding period. In in vitro studies, we performed PPAR reporter assay and investigated the anti-inflammatory effects of TPA in lipopolysaccharide-stimulated RAW264.7 cells. Results: Supplementation of VLCPUFA showed a significant and dose-dependent improvement in ERG response. Like EPA and DHA, we also observed favorable cardiometabolic changes and decreased atherosclerotic plaque area due to dietary VLCPUFA. Intriguingly, VLCPUFA supplemented aging mice exhibit better cognitive performance compared with control. Transcriptome analysis revealed that VLCPUFA-enriched fish oil favorably regulated genes involved in nuclear receptor signaling pathways, lipid metabolism and inflammation. Furthermore, purified TPA potently activates PPARs, and suppressed inflammation in macrophage cells. Conclusions: Overall, our studies revealed for the first time several potential health benefits for our new VLCPUFA-enriched fish oil in several age-related diseases and support its future development as a new dietary supplement.

Targeted engineering of camelina and pennycress seeds for ultrahigh accumulation of acetyl-TAG

Acetyl-TAG (3-acetyl-1,2-diacylglycerol), unique triacylglycerols (TAG) possessing an acetate group at the sn-3 position, exhibit valuable properties, such as reduced viscosity and freezing points. Previous attempts to engineer acetyl-TAG production in oilseed crops did not achieve the high levels found in naturally producing Euonymus seeds. Here, we demonstrate the successful generation of camelina and pennycress transgenic lines accumulating nearly pure acetyl-TAG at 93 mol% and 98 mol%, respectively. These ultrahigh acetyl-TAG synthesizing lines were created using gene-edited FATTY ACID ELONGASE1 (FAE1) mutant lines as an improved genetic background to increase levels of acetyl-CoA available for acetyl-TAG synthesis mediated by the expression of EfDAcT, a high-activity diacylglycerol acetyltransferase isolated from Euonymus fortunei. Combining EfDAcT expression with suppression of the competing TAG-synthesizing enzyme DGAT1 further enhanced acetyl-TAG accumulation. These ultrahigh levels of acetyl-TAG exceed those in earlier engineered oilseeds and are equivalent or greater than those in Euonymus seeds. Imaging of lipid localization in transgenic seeds revealed that the low amounts of residual TAG were mostly confined to the embryonic axis. Similar spatial distributions of specific TAG and acetyl-TAG molecular species, as well as their probable diacylglycerol (DAG) precursors, provide additional evidence that acetyl-TAG and TAG are both synthesized from the same tissue-specific DAG pools. Remarkably, this ultrahigh production of acetyl-TAG in transgenic seeds exhibited minimal negative effects on seed properties, highlighting the potential for production of designer oils required for economical biofuel industries.

ELO-6 expression predicts longevity in isogenic populations of Caenorhabditis elegans

Variations of individual lifespans within genetically identical populations in homogenous environments are remarkable, with the cause largely unknown. Here, we show the expression dynamic of the Caenorhabditis elegans fatty acid elongase ELO-6 during aging predicts individual longevity in isogenic populations. elo-6 expression is reduced with age. ELO-6 expression level exhibits obvious variation between individuals in mid-aged worms and is positively correlated with lifespan and health span. Interventions that prolong longevity enhance ELO-6 expression stability during aging, indicating ELO-6 is also a populational lifespan predictor. Differentially expressed genes between short-lived and long-lived isogenic worms regulate lifespan and are enriched for PQM-1 binding sites. pqm-1 in young to mid-aged adults causes individual ELO-6 expression heterogeneity and restricts health span and life span. Thus, our study identifies ELO-6 as a predictor of individual and populational lifespan and reveals the role of pqm-1 in causing individual health span variation in the mid-aged C. elegans.

Identification and validation of core genes associated with polycystic ovary syndrome and metabolic syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder affecting women of reproductive age, affecting reproductive health, and increasing the incidence of diabetes mellitus and hypertension. Metabolic syndrome (MetS) is the most common metabolic disorder. Although clinical studies have shown a close association between PCOS and MetS, the molecular mechanisms are unknown. In this study, datasets of PCOS and MetS were obtained from the Gene Expression Omnibus database; differential expression analysis and weighted gene coexpression network analysis (WGCNA) were performed; and gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses also performed of differentially expressed genes (DEGs). The PCOS- and MetS-coexpressed DEGs were subsequently intersected with the coexpressed genes in the WGCNA module to obtain the core genes. By constructing receiver operating characteristic curves, we verified the predictive effects of the core genes. We also validated the expression of the core genes in the datasets. Finally, we verified the expression of the core genes by quantitative polymerase chain reaction in human follicular fluid granulosa cells. In addition, we used Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts to analyze the immune infiltration of immune cells in PCOS and MetS. Finally, we obtained 52 coexpressed DEGs of PCOS and MetS and 3 coexpressed genes in the WGCNA module. By taking the intersection of coexpressed DEGs and coexpressed genes of the WGCNA module, we get ELOVL fatty acid elongase 7 (ELOVL7) as the core gene. Receiver operating characteristic curve analysis showed that ELOVL7 is a reliable biological marker for PCOS and MetS. The expression level of ELOVL7 in human follicular fluid granulosa cells from PCOS patients was significantly higher than that of controls, as verified by quantitative polymerase chain reaction. This study provides the first evidence of the role of ELOVL7 in developing PCOS and MetS. This gene may serve as a potential diagnostic marker and therapeutic target for both conditions.

Regulatory Changes in the Fatty Acid Elongase eloF Underlie the Evolution of Sex-specific Pheromone Profiles in Drosophila prolongata.

Pheromones play a key role in regulating sexual behavior throughout the animal kingdom. In Drosophila and other insects, many cuticular hydrocarbons (CHCs) are sexually dimorphic, and some are known to perform pheromonal functions. However, the genetic control of sex-specific CHC production is not understood outside of the model species D. melanogaster . A recent evolutionary change is found in D. prolongata , which, compared to its closest relatives, shows greatly increased sexual dimorphism in both CHCs and the chemosensory system responsible for their perception. A key transition involves a male-specific increase in the proportion of long-chain CHCs. Perfuming D. prolongata females with the male-biased CHCs reduces copulation success, suggesting that these compounds function as sex pheromones. The evolutionary change in CHC profiles correlates with a male-specific increase in the expression of multiple genes involved in CHC biosynthesis, including fatty acid elongases and reductases and other key enzymes. In particular, elongase F , which is responsible for producing female-specific pheromones in D. melanogaster , is strongly upregulated in D. prolongata males compared both to females and to males of the sibling species. Induced mutations in eloF reduce the amount of long-chain CHCs, resulting in a partial feminization of pheromone profiles in D. prolongata males while having minimal effect in females. Transgenic experiments show that sex-biased expression of eloF is caused in part by a putative transposable element insertion in its regulatory region. These results reveal one of the genetic mechanisms responsible for a recent evolutionary change in sexual communication.

The Spinocerebellar Ataxia 34-Causing W246G ELOVL4 Mutation Does Not Alter Cerebellar Neuron Populations in a Rat Model.

Spinocerebellar ataxia 34 (SCA34) is an autosomal dominant disease that arises from point mutations in the fatty acid elongase, Elongation of Very Long Chain Fatty Acids 4 (ELOVL4), which is essential for the synthesis of Very Long Chain-Saturated Fatty Acids (VLC-SFA) and Very Long Chain-Polyunsaturated Fatty Acids (VLC-PUFA) (28-34 carbons long). SCA34 is considered a neurodegenerative disease. However, a novel rat model of SCA34 (SCA34-KI rat) with knock-in of the W246G ELOVL4 mutation that causes human SCA34 shows early motor impairment and aberrant synaptic transmission and plasticity without overt neurodegeneration. ELOVL4 is expressed in neurogenic regions of the developing brain, is implicated in cell cycle regulation, and ELOVL4 mutations that cause neuroichthyosis lead to developmental brain malformation, suggesting that aberrant neuron generation due to ELOVL4 mutations might contribute to SCA34. To test whether W246G ELOVL4 altered neuronal generation or survival in the cerebellum, we compared the numbers of Purkinje cells, unipolar brush cells, molecular layer interneurons, granule and displaced granule cells in the cerebellum of wildtype, heterozygous, and homozygous SCA34-KI rats at four months of age, when motor impairment is already present. An unbiased, semi-automated method based on Cellpose 2.0 and ImageJ was used to quantify neuronal populations in cerebellar sections immunolabeled for known neuron-specific markers. Neuronal populations and cortical structure were unaffected by the W246G ELOVL4 mutation by four months of age, a time when synaptic and motor dysfunction are already present, suggesting that SCA34 pathology originates from synaptic dysfunction due to VLC-SFA deficiency, rather than aberrant neuronal production or neurodegeneration.

The cytotoxic activities of the major diterpene extracted from Salvia multicaulis (Bardakosh) are mediated by the regulation of heat-shock response and fatty acid metabolism pathways in human leukemia cells

BackgroundLeukemia is one of the most lethal cancers worldwide and represents the sixth-leading cause of cancer deaths. The results of leukemia treatment have not been as positive as desired, and recurrence is common. PurposeThus, there is an urgent requirement for the development of new therapeutic drugs. Salvia multicaulis (Bardakosh) is a widespread species that contains multiple phytochemical components with anti-cancer activities. MethodsWe isolated and characterized the major diterpene candesalvone B methyl ester from S. multicaulis and investigated its action as a cytotoxic agent towards sensitive and drug-resistant leukemia cells by the resazurin reduction assay. Additionally, the targeted genes and the affected molecular mechanisms attributed to the potent cytotoxic activities were discovered by transcriptome-wide mRNA expression profiling. The targets predicted to be regulated by candesalvone B methyl ester in each cell line were confirmed by qRT-PCR, molecular docking, microscale thermophoresis, and western blotting. Moreover, cell cycle distribution and apoptosis were analyzed by flow cytometry. ResultsCandesalvone B methyl ester was cytotoxic with IC50 values of 20.95 ± 0.15 µM against CCRF-CEM cells and 4.13 ± 0.10 µM against multidrug-resistant CEM/ADR5000 leukemia cells. The pathway enrichment analysis disclosed that candesalvone B methyl ester could regulate the heat-shock response signaling pathway via targeting heat shock factor 1 (HSF1) in CCRF-CEM cells and ELOVL fatty acid elongase 5 (ELOVL5) controls the fatty acid metabolism pathway in CEM/ADR5000 cells. Microscale thermophoresis showed the binding of candesalvone B methyl ester with HSF1 and ELOVL5, confirming the results of molecular docking analysis. Down-regulation of both HSF1 and ELOVL5 by candesalvone B methyl ester as detected by both western blotting and RT-qPCR was related to the reversal of drug resistance in the leukemia cells. Furthermore, candesalvone B methyl ester increased the arrest in the sub-G1 phase of the cell cycle in a dose-dependent manner from 1.3 % to 32.3 % with concomitant induction of apoptosis up to 29.0 % in CCRF-CEM leukemic cells upon inhibition of HSF1. ConclusionCandesalvone B methyl ester isolated from S. multicaulis exerted cytotoxicity by affecting apoptosis, cell division, and modulation of expression levels of genes contributing to the heat stress signaling and fatty acid metabolism pathways that could relieve drug resistance of leukemia cells.

Recent advances on the physiological and pathophysiological roles of polyunsaturated fatty acids and their biosynthetic pathway

Polyunsaturated fatty acids (PUFAs)—fatty acids containing multiple double bonds within their carbon chain—are an indispensable component of the cell membrane. PUFAs, including the omega-6 PUFA arachidonic acid (ARA; C20:4n-6) and the omega-3 PUFAs eicosapentaenoic acid (EPA; C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3), have been implicated in various (patho)physiological events. These PUFAs are either obtained from the diet or biosynthesized from the essential fatty acids linoleic acid (LA; C18:2n-6) and α-linolenic acid (ALA; C18:3n-3) via enzymatic reactions that are catalyzed by fatty acid elongases (ELOVL2 and ELOVL5) and fatty acid desaturases (FADS1 and FADS2). In this review, we summarize the recent literature studying the role of PUFAs, placing a special emphasis on the newly discovered functions of PUFAs and their biosynthetic pathway as revealed by studies using animal models targeting the PUFA biosynthetic pathway and genetic approaches including genome-wide association studies.

Evolutionary dynamics in gut-colonizing Candida glabrata during caspofungin therapy: Emergence of clinically important mutations in sphingolipid biosynthesis.

Invasive fungal infections are associated with high mortality, which is exacerbated by the limited antifungal drug armamentarium and increasing antifungal drug resistance. Echinocandins are a frontline antifungal drug class targeting β-glucan synthase (GS), a fungal cell wall biosynthetic enzyme. Echinocandin resistance is generally low but increasing in species like Candida glabrata, an opportunistic yeast pathogen colonizing human mucosal surfaces. Mutations in GS-encoding genes (FKS1 and FKS2 in C. glabrata) are strongly associated with clinical echinocandin failure, but epidemiological studies show that other, as yet unidentified factors also influence echinocandin susceptibility. Furthermore, although the gut is known to be an important reservoir for emergence of drug-resistant strains, the evolution of resistance is not well understood. Here, we studied the evolutionary dynamics of C. glabrata colonizing the gut of immunocompetent mice during treatment with caspofungin, a widely-used echinocandin. Whole genome and amplicon sequencing revealed rapid genetic diversification of this C. glabrata population during treatment and the emergence of both drug target (FKS2) and non-drug target mutations, the latter predominantly in the FEN1 gene encoding a fatty acid elongase functioning in sphingolipid biosynthesis. The fen1 mutants displayed high fitness in the gut specifically during caspofungin treatment and contained high levels of phytosphingosine, whereas genetic depletion of phytosphingosine by deletion of YPC1 gene hypersensitized the wild type strain to caspofungin and was epistatic to fen1Δ. Furthermore, high resolution imaging and mass spectrometry showed that reduced caspofungin susceptibility in fen1Δ cells was associated with reduced caspofungin binding to the plasma membrane. Finally, we identified several different fen1 mutations in clinical C. glabrata isolates, which phenocopied the fen1Δ mutant, causing reduced caspofungin susceptibility. These studies reveal new genetic and molecular determinants of clinical caspofungin susceptibility and illuminate the dynamic evolution of drug target and non-drug target mutations reducing echinocandin efficacy in patients colonized with C. glabrata.

MiR-206 Suppresses Triacylglycerol Accumulation via Fatty Acid Elongase 6 in Dairy Cow Mammary Epithelial Cells.

Cow milk possesses high nutritional value due to its rich array of beneficial fatty acids. It is important to understand the mechanisms involved in lipid metabolism in dairy cows. These mechanisms are driven by a complex molecular regulatory network. In addition, there are many regulatory factors involved in the process of fatty acid metabolism, including transcription factors and non-coding RNAs, amongst others. MicroRNAs (miRNAs) can regulate the expression of target genes and modulate various biological processes, including lipid metabolism. Specifically, miR-206 has been reported to impair lipid accumulation in nonruminant hepatocytes. However, the effects and regulatory mechanisms of miR-206 on lipid metabolism in bovine mammary cells remain unclear. In the present study, we investigated the effects of miR-206 on lipid-related genes and TAG accumulation. The direct downstream gene of miR-206 was subsequently determined via a dual-luciferase assay. Finally, the fatty acid content of bovine mammary epithelial cells (BMECs) upon ELOVL6 inhibition was examined. The results revealed that miR-206 overexpression significantly decreased triacylglycerol (TAG) concentration and abundances of the following: acetyl-coenzyme A carboxylase alpha (ACACA); fatty acid synthase (FASN); sterol regulatory element binding transcription factor 1 (SREBF1); diacylglycerol acyltransferase 1 (DGAT1); 1-acylglycerol-3-phosphate O-acyltransferase 6 (AGPAT6); lipin 1 (LPIN1); and fatty acid elongase 6 (ELOVL6). Overexpression of miR-206 was also associated with an increase in patatin-like phospholipase domain-containing 2 (PNPLA2), while inhibition of miR-206 promoted milk fat metabolism in vitro. In addition, we found that ELOVL6 is a direct target gene of miR-206 through mutation of the binding site. Furthermore, ELOVL6 intervention significantly decreased the TAG levels and elongation indexes of C16:0 and C16:1n-7 in BMECs. Finally, ELOVL6 siRNA partially alleviated the increased TAG accumulation caused by miR-206 inhibition. In summary, we found that miR-206 inhibits milk fatty acid synthesis and lipid accumulation by targeting ELOVL6 in BMECs. The results presented in this paper may contribute to the development of strategies for enhancing the quality of cow milk and its beneficial fatty acids, from the perspective of miRNA-mRNA networks.

Potential and Metabolic Impacts of Double Enrichments of Docosahexaenoic Acid and 25-Hydroxy Vitamin D3 in Tissues of Broiler Chickens

BackgroundChicken may be enriched with 25-hydroxy D3 [25(OH)D3] and docosahexaenoic acid (DHA) to enhance the dietary intake of the public. ObjectivesTwo experiments (Expt.) were conducted to determine the potential and metabolic impacts of enriching both DHA and 25(OH)D3 in the tissues of broiler chickens. MethodsIn Expt. 1, 144 chicks (6 cages/treatment and 6 birds/cage) were fed a corn–soybean meal basal diet (BD), BD + 10,000 IU 25(OH)D3/kg [BD + 25(OH)D3], BD + 1% DHA-rich Aurantiochytrium (1.2 g DHA/kg; BD + DHA), or BD + 25(OH)D3+DHA for 6 wk. In Expt. 2, 180 chicks were fed the BD, BD + DHA-rich microalgal oil (1.5–3.0 g DHA/kg, BD + DHA), BD + DHA + eicosapentaenoic acid (EPA)-rich microalgae (0.3–0.6 g EPA/kg, BD + DHA + EPA), BD + DHA + 25(OH)D3 [6000 to 12,000 IU/kg diet; BD + DHA + 25(OH)D3], and BD + DHA + EPA + 25(OH)D3 for 6 wk. ResultsSupranutrition of these 2 nutrients resulted in 57–62 mg DHA and 1.9–3.3 μg of 25(OH)D3/100 g of breast or thigh muscles. The DHA enrichment was independent of dietary EPA or 25(OH)D3, but that of 25(OH)D3 in the liver was decreased (68%, P < 0.05) by dietary DHA in Expt. 1. Compared with BD, BD + 25(OH)D3 enhanced (P < 0.05) gene expression related to D3 absorption (scavenger receptor class B type 1 and Niemann-pick c1 like 1) in the liver and D3 degradation (cytochrome P450 24A1) in the breast, and decreased mRNA or protein concentrations of vitamin D binding protein in the adipose tissue or thigh muscle. Supranutrition of DHA decreased mRNA concentrations of lipid metabolism-related genes (fatty acid desaturase 1,2, ELOVL fatty acid elongase 5, fatty acid desaturase 2, fatty acid synthase, and sterol regulatory element-binding protein 1). ConclusionsBoth DHA and 25(OH)D3 were enriched in the muscles up to meeting 50%–100% of the suggested intakes of these nutrients by consuming 2 servings of 100 g of fortified chicken. The enrichments altered gene expression related to lipid biosynthesis and vitamin D transport or storage.

Characterization of Entamoeba fatty acid elongases; validation as targets and provision of promising leads for new drugs against amebiasis.

Entamoeba histolytica is a protozoan parasite belonging to the phylum Amoebozoa that causes amebiasis, a global public health problem. E. histolytica alternates its form between a proliferative trophozoite and a dormant cyst. Trophozoite proliferation is closely associated with amebiasis symptoms and pathogenesis whereas cysts transmit the disease. Drugs are available for clinical use; however, they have issues of adverse effects and dual targeting of disease symptoms and transmission remains to be improved. Development of new drugs is therefore urgently needed. An untargeted lipidomics analysis recently revealed structural uniqueness of the Entamoeba lipidome at different stages of the parasite's life cycle involving very long (26-30 carbons) and/or medium (8-12 carbons) acyl chains linked to glycerophospholipids and sphingolipids. Here, we investigated the physiology of this unique acyl chain diversity in Entamoeba, a non-photosynthetic protist. We characterized E. histolytica fatty acid elongases (EhFAEs), which are typically components of the fatty acid elongation cycle of photosynthetic protists and plants. An approach combining genetics and lipidomics revealed that EhFAEs are involved in the production of medium and very long acyl chains in E. histolytica. This approach also showed that the K3 group herbicides, flufenacet, cafenstrole, and fenoxasulfone, inhibited the production of very long acyl chains, thereby impairing Entamoeba trophozoite proliferation and cyst formation. Importantly, none of these three compounds showed toxicity to a human cell line; therefore, EhFAEs are reasonable targets for developing new anti-amebiasis drugs and these compounds are promising leads for such drugs. Interestingly, in the Amoebazoan lineage, gain and loss of the genes encoding two different types of fatty acid elongase have occurred during evolution, which may be relevant to parasite adaptation. Acyl chain diversity in lipids is therefore a unique and indispensable feature for parasitic adaptation of Entamoeba.

Engineering erucic acid biosynthesis in camelina (Camelina sativa) via FAE1 gene cloning and antisense technology.

Oil seeds now make up the world's second-largest food source after cereals. In recent years, the medicinal- oil plant Camelina sativa has attracted much attention for its high levels of unsaturated fatty acids and low levels of saturated fatty acids as well as its resistanceto abiotic stresses. Improvement of oil quality is considered an important trait in this plant. Erucic acid is one of the fatty acids affecting the quality of camelina oil. Altering the fatty acid composition in camelina oil through genetic manipulation requires the identification, isolation, and cloning of genes involved in fatty acid biosynthesis. The Fatty Acid Elongase 1 (FAE1) gene encodes the enzyme β-ketoacyl CoA synthase (KCS), a crucial enzyme in the biosynthesis of erucic acid. In this study, the isolation and cloning of the FAE1 gene from Camelina sativa were conducted to construct an antisense structure. The molecular homology modeling of DFAE1 proteins using the SWISS-MODEL server on ExPASy led to the generation of the 3D structures of FAE1 and DFAE1 proteins. The GMQE values of 0.44 for FAE1 and 0.08 for DFAE1 suggest high accuracy in the structural estimation of these genes. The fragments were isolated from the DNA source of the genomic Soheil cultivar with an erucic acid content of about 3% (in matured seeds) using PCR. After cloning the FAE1 gene into the Bluescript II SK+ vector and sequencing, the resulting fragments were utilized to construct the antisense structure in the pBI121 plant expression vector. The approved antisense structure was introduced into the Camelina plant using the Agrobacterium-mediated method, with optimization of tissue culture and gene transfer conditions. This approach holds potential to advance our knowledge of fat biosynthesis, leading to potential improvements in oil quality in Camelina sativa.

3-ketoacyl-CoA synthase 19 contributes to the biosynthesis of seed lipids and cuticular wax in Arabidopsis and abiotic stress tolerance.

Very-long-chain fatty acids (VLCFAs) are essential precursors for plant membrane lipids, cuticular waxes, suberin, and storage oils. Integral to the fatty acid elongase (FAE) complex, 3-ketoacyl-CoA synthases (KCSs) function as crucial enzymes in the VLCFA pathway, determining the chain length of VLCFA. This study explores the in-planta role of the KCS19 gene. KCS19 is predominantly expressed in leaves and stem epidermis, sepals, styles, early silique walls, beaks, pedicels, and mature embryos. Localized in the endoplasmic reticulum, KCS19 interacts with other FAE proteins. kcs19 knockout mutants displayed reduced total wax and wax crystals, particularly alkanes, while KCS19 overexpression increased these components and wax crystals. Moreover, the cuticle permeability was higher for the kcs19 mutants compared to the wild type, rendering them more susceptible to drought and salt stress, whereas KCS19 overexpression enhanced drought and salt tolerance. Disrupting KCS19 increased C18 species and decreased C20 and longer species in seed fatty acids, indicating its role in elongating C18 to C20 VLCFAs, potentially up to C24 for seed storage lipids. Collectively, KCS19-mediated VLCFA synthesis is required for cuticular wax biosynthesis and seed storage lipids, impacting plant responses to abiotic stress.

Testosterone Inhibits Lipid Accumulation in Porcine Preadipocytes by Regulating ELOVL3.

Castration is commonly used to reduce stink during boar production. In porcine adipose tissue, castration reduces androgen levels resulting in metabolic disorders and excessive fat deposition. However, the underlying detailed mechanism remains unclear. In this study, we constructed porcine preadipocyte models with and without androgen by adding testosterone exogenously. The fluorescence intensity of lipid droplet (LD) staining and the fatty acid synthetase (FASN) mRNA levels were lower in the testosterone-treated cells than in the untreated control cells. In contrast, the mRNA levels of adipose triglycerides lipase (ATGL) and androgen receptor (AR) were higher than in the testosterone-treated cells than in the control cells. Subsequently, transcriptomic sequencing of porcine preadipocytes incubated with and without testosterone showed that the mRNA expression levels of very long-chain fatty acid elongase 3 (ELOVL3), a key enzyme involved in fatty acids synthesis and metabolism, were high in control cells. The siRNA-mediated knockdown of ELOVL3 reduced LD accumulation and the mRNA levels of FASN and increased the mRNA levels of ATGL. Next, we conducted dual-luciferase reporter assays using wild-type and mutant ELOVL3 promoter reporters, which showed that the ELOVL3 promoter contained an androgen response element (ARE); furthermore, its transcription was negatively regulated by AR overexpression. In conclusion, our study reveals that testosterone inhibits fat deposition in porcine preadipocytes by suppressing ELOVL3 expression. Moreover, our study provides a theoretical basis for further studies on the mechanisms of fat deposition caused by castration.

A fatty acid elongase complex regulates cell membrane integrity and septin-dependent host infection by the rice blast fungus.

Very-long-chain fatty acids (VLCFAs) regulate biophysical properties of cell membranes to determine growth and development of eukaryotes, such as the pathogenesis of the rice blast fungus Magnaporthe oryzae. The fatty acid elongase Elo1 regulates pathogenesis of M. oryzae by modulating VLCFA biosynthesis. However, it remains unknown whether and how Elo1 associates with other factors to regulate VLCFA biosynthesis in fungal pathogens. Here, we identified Ifa38, Phs1 and Tsc13 as interacting proteins of Elo1 by proximity labelling in M. oryzae. Elo1 associated with Ifa38, Phs1 and Tsc13 on the endoplasmic reticulum (ER) membrane to control VLCFA biosynthesis. Targeted gene deletion mutants Δifa38, Δphs1 and Δtsc13 were all similarly impaired as Δelo1 in vegetative growth, conidial morphology, stress responses in ER, cell wall and membrane. These deletion mutants also displayed severe damage in cell membrane integrity and failed to organize the septin ring that is essential for penetration peg formation and pathogenicity. Our study demonstrates that M. oryzae employs a fatty acid elongase complex to regulate VLCFAs for maintaining or remodelling cell membrane structure, which is important for septin-mediated host penetration.

ALKBH5 regulates chicken adipogenesis by mediating LCAT mRNA stability depending on m6A modification

BackgroundPrevious studies have demonstrated the role of N6-methyladenosine (m6A) RNA methylation in various biological processes, our research is the first to elucidate its specific impact on LCAT mRNA stability and adipogenesis in poultry.ResultsThe 6 100-day-old female chickens were categorized into high (n = 3) and low-fat chickens (n = 3) based on their abdominal fat ratios, and their abdominal fat tissues were processed for MeRIP-seq and RNA-seq. An integrated analysis of MeRIP-seq and RNA-seq omics data revealed 16 differentially expressed genes associated with to differential m6A modifications. Among them, ELOVL fatty acid elongase 2 (ELOVL2), pyruvate dehydrogenase kinase 4 (PDK4), fatty acid binding protein 9 (PMP2), fatty acid binding protein 1 (FABP1), lysosomal associated membrane protein 3 (LAMP3), lecithin-cholesterol acyltransferase (LCAT) and solute carrier family 2 member 1 (SLC2A1) have ever been reported to be associated with adipogenesis. Interestingly, LCAT was down-regulated and expressed along with decreased levels of mRNA methylation methylation in the low-fat group. Mechanistically, the highly expressed ALKBH5 gene regulates LCAT RNA demethylation and affects LCAT mRNA stability. In addition, LCAT inhibits preadipocyte proliferation and promotes preadipocyte differentiation, and plays a key role in adipogenesis.ConclusionsIn conclusion, ALKBH5 mediates RNA stability of LCAT through demethylation and affects chicken adipogenesis. This study provides a theoretical basis for further understanding of RNA methylation regulation in chicken adipogenesis.

SPDYC serves as a prognostic biomarker related to lipid metabolism and the immune microenvironment in breast cancer.

Breast cancer remains the most common malignant carcinoma among women globally and is resistant to several therapeutic agents. There is a need for novel targets to improve the prognosis of patients with breast cancer. Bioinformatics analyses were conducted to explore potentially relevant prognostic genes in breast cancer using The Cancer Genome Atlas (TCGA) and The Gene Expression Omnibus (GEO) databases. Gene subtypes were categorized by machine learning algorithms. The machine learning-related breast cancer (MLBC) score was evaluated through principal component analysis (PCA) of clinical patients' pathological statuses and subtypes. Immune cell infiltration was analyzed using the xCell and CIBERSORT algorithms. Kyoto Encyclopedia of Genes and Genomes enrichment analysis elucidated regulatory pathways related to speedy/RINGO cell cycle regulator family member C (SPDYC) in breast cancer. The biological functions and lipid metabolic status of breast cancer cell lines were validated via quantitative real-time polymerase chain reaction (RT‒qPCR) assays, western blotting, CCK-8 assays, PI‒Annexin V fluorescence staining, transwell assays, wound healing assays, and Oil Red O staining. Key differentially expressed genes (DEGs) in breast cancer from the TCGA and GEO databases were screened and utilized to establishthe MLBC score. Moreover, the MLBC score we established was negatively correlated with poor prognosis in breast cancer patients. Furthermore, the impacts of SPDYC on the tumor immune microenvironment and lipid metabolism in breast cancer were revealed and validated. SPDYC is closely related to activated dendritic cells and macrophages and is simultaneously correlated with the immune checkpoints CD47, cytotoxic T lymphocyte antigen-4 (CTLA-4), and poliovirus receptor (PVR). SPDYC strongly correlated with C-C motif chemokine ligand 7 (CCL7), a chemokine that influences breast cancer patient prognosis. A significant relationship was discovered between key genes involved in lipid metabolism and SPDYC, such as ELOVL fatty acid elongase 2 (ELOVL2), malic enzyme 1 (ME1), and squalene epoxidase (SQLE). Potent inhibitors targeting SPDYC in breast cancer were also discovered, including JNK inhibitor VIII, AICAR, and JW-7-52-1. Downregulation of SPDYC expression in vitro decreased proliferation, increased the apoptotic rate, decreased migration, and reduced lipid droplets. SPDYC possibly influences the tumor immune microenvironment and regulates lipid metabolism in breast cancer. Hence, this study identified SPDYC as a pivotal biomarker for developing therapeutic strategies for breast cancer.

The effects of diet, salinity and temperature on HUFA accumulation in Artemia

Artemia nauplii is an important live food item for marine fish and crustaceans. However, the HUFA content of Artemia from different origins varies greatly and is generally low. Compared with other species or strains, Artemia cysts in Tibet salt lakes have very high HUFA content, which may be related to their unique living conditions. This study determined the effects of dietary HUFA (P. tricornutum and C. zofingiensis), salinity (4%, 8%, and 12%), and temperature (14 °C, 19 °C, and 24 °C) on the HUFA accumulation of Lagkor Co Artemia tibetiana after 14-day culture, in comparison with Great Salt Lake Artemia franciscana. The results showed that the HUFA profile of Artemia was closely related to the dietary HUFA level, and the low temperature and low salinity could significantly increase the ARA, EPA, and DHA content. PCA results showed that the fatty acid distribution patterns of the two Artemia species were mostly affected by dietary fatty acids, which were greater than those of temperature, salinity, and Artemia species. RNA-seq analysis and qRT-PCR verification revealed that low temperature and low salinity could promote the gene expression of fatty acid binding protein gene, long chain fatty acid elongase, and desaturase, suggesting these genes may be involved in the process of HUFA accumulation in A. franciscana. In conclusion, diet is a key factor affecting HUFA accumulation in Artemia, while low temperature and low salinity led to higher HUFA retention. The findings of this study provide a reference for revealing the mechanism of HUFA synthesis and metabolism of Artemia, and to develop the Artemia products with high HUFA content through manipulation of culture conditions.