Up-regulation of long non-coding RNA H19 ameliorates renal tubulointerstitial fibrosis by reducing lipid deposition and inflammatory response through regulation of the microRNA-130a-3p/long-chain acyl-CoA synthetase 1 axis.

Long non-coding RNA (lncRNA) H19 is an extensively studied lncRNA that is related to numerous pathological changes. Our previous findings have documented that serum lncRNA H19 levels are decreased in patients with chronic kidney disorder and lncRNA H19 reduction is closely correlated with renal tubulointerstitial fibrosis, an essential step in developing end-stage kidney disease. Nonetheless, the precise function and mechanism of lncRNA H19 in renal tubulointerstitial fibrosis are not fully comprehended. The present work utilized a mouse model of unilateral ureteral obstruction (UUO) and transforming growth factor-ß1 (TGF-ß1)-stimulated HK-2 cells to investigate the possible role and mechanism of lncRNA H19 in renal tubulointerstitial fibrosis were investigated. Levels of lncRNA H19 decreased in kidneys of mice with UUO and HK-2 cells stimulated with TGF-ß1. Up-regulation of lncRNA H19 in mouse kidneys remarkably relieved kidney injury, fibrosis and inflammation triggered by UUO. Moreover, the increase of lncRNA H19 in HK-2 cells reduced epithelial-to-mesenchymal transition (EMT) induced by TGF-ß1. Notably, up-regulation of lncRNA H19 reduced lipid accumulation and triacylglycerol content in kidneys of mice with UUO and TGF-ß1-stimulated HK-2 cells, accompanied by the up-regulation of long-chain acyl-CoA synthetase 1 (ACSL1). lncRNA H19 was identified as a sponge of microRNA-130a-3p, through which lncRNA H19 modulates the expression of ACSL1. The overexpression of microRNA-130a-3p reversed the lncRNA H19-induced increases in the expression of ACSL1. The suppressive effects of lncRNA H19 overexpression on the EMT, inflammation and lipid accumulation in HK-2 cells were diminished by ACSL1 silencing or microRNA-130a-3p overexpression. Overall, the findings showed that lncRNA H19 ameliorated renal tubulointerstitial fibrosis by reducing lipid deposition via modulation of the microRNA-130a-3p/ACSL1 axis.

Si-Wu-Tang alleviates metabolic dysfunction-associated fatty liver disease by inhibiting ACSL4-mediated arachidonic acid metabolism and ferroptosis in MCD diet-fed mice.

BACKGROUND: Metabolic dysfunction-associated fatty liver disease (MAFLD) is a prevalent chronic liver disease worldwide. Si-Wu-Tang (SWT), a traditional Chinese medicine decoction has shown therapeutic effects on various liver diseases. However, the hepatoprotective effects and underlying mechanism of SWT on MAFLD remain unclear. METHODS: First, a methionine-choline-deficient (MCD) diet-fed mice model was used and lipidomic analysis and transcriptomic analysis were performed. The contents of total iron ions, ferrous ions, and lipid peroxidation were detected and Prussian blue staining was performed to confirm the protective effects of SWT against ferroptosis. Finally, chemical characterization and network pharmacological analysis were employed to identify the potential active ingredients. RESULTS: Serological and hepatic histopathological findings indicated SWT's discernible therapeutic impact on MCD diet-induced MAFLD. Lipidomic analysis revealed that SWT improved intrahepatic lipid accumulation by inhibiting TG synthesis and promoting TG transport. Transcriptomic analysis suggested that SWT ameliorated abnormal FA metabolism by inhibiting FA synthesis and promoting FA ß-oxidation. Then, ferroptosis phenotype experiments revealed that SWT could effectively impede hepatocyte ferroptosis, which was induced by long-chain acyl-CoA synthetase 4 (ACSL4)-mediated esterification of arachidonic acid (AA). Finally, chemical characterization and network pharmacological analysis identified that paeoniflorin and other active ingredients might be responsible for the regulative effects against ferroptosis and MAFLD. CONCLUSION: In conclusion, our study revealed the intricate mechanism through which SWT improved MCD diet-induced MAFLD by targeting FA metabolism and ferroptosis in hepatocytes, thus offering a novel therapeutic approach for the treatment of MAFLD and its complications.

Deficiency of long-chain acyl-CoA synthetase 4 leads to lipopolysaccharide-induced mortality in a mouse model of septic shock.

Long-chain acyl-CoA synthetase 4 (ACSL4) converts free highly unsaturated fatty acids (HUFAs) into their acyl-CoA esters and is important for HUFA utilization. HUFA-containing phospholipids produced via ACSL4-dependent reactions are involved in pathophysiological events such as inflammatory responses and ferroptosis as a source for lipid mediators and/or a target of oxidative stress, respectively. However, the in vivo role of ACSL4 in inflammatory responses is not fully understood. This study sought to define the effects of ACSL4 deficiency on lipopolysaccharide (LPS)-induced systemic inflammatory responses using global Acsl4 knockout (Acsl4 KO) mice. Intraperitoneal injection of LPS-induced more severe symptoms, including diarrhea, hypothermia, and higher mortality, in Acsl4 KO mice within 24 h compared with symptoms in wild-type (WT) mice. Intestinal permeability induced 3 h after LPS challenge was also enhanced in Acsl4 KO mice compared with that in WT mice. In addition, plasma levels of some eicosanoids in Acsl4 KO mice 6 h post-LPS injection were 2- to 9-fold higher than those in WT mice. The increased mortality observed in LPS-treated Acsl4 KO mice was significantly improved by treatment with the general cyclooxygenase inhibitor indomethacin with a partial reduction in the severity of illness index for hypothermia, diarrhea score, and intestinal permeability. These results suggest that ACSL4 deficiency enhances susceptibility to endotoxin at least partly through the overproduction of cyclooxygenase-derived eicosanoids.

Gene deletion of long-chain acyl-CoA synthetase 4 attenuates xenobiotic chemical-induced lung injury via the suppression of lipid peroxidation.

Long-chain acyl-CoA synthetase (ACSL) 4 converts polyunsaturated fatty acids (PUFAs) into their acyl-CoAs and plays an important role in maintaining PUFA-containing membrane phospholipids. Here we demonstrated decreases in various kinds of PUFA-containing phospholipid species in ACSL4-deficient murine lung. We then examined the effects of ACSL4 gene deletion on lung injury by treating mice with two pulmonary toxic chemicals: paraquat (PQ) and methotrexate (MTX). The results showed that ACSL4 deficiency attenuated PQ-induced acute lung lesion and decreased mortality. PQ-induced lung inflammation and neutrophil migration were also suppressed in ACSL4-deficient mice. PQ administration increased the levels of phospholipid hydroperoxides in the lung, but ACSL4 gene deletion suppressed their increment. We further found that ACSL4 deficiency attenuated MTX-induced pulmonary fibrosis. These results suggested that ACSL4 gene deletion might confer protection against pulmonary toxic chemical-induced lung injury by reducing PUFA-containing membrane phospholipids, leading to the suppression of lipid peroxidation. Inhibition of ACSL4 may be promising for the prevention and treatment of chemical-induced lung injury.

Genome-Wide Identification and Expression Profiling Analysis of the Long-Chain Acyl-CoA Synthetases Reveal Their Potential Roles in Wheat Male Fertility.

Long-chain acyl-CoA synthetase (LACS), responsible for the conversion of free FAs into acyl-CoAs, is involved in multiple pathways of lipid metabolism. Although LACS genes in Arabidopsis have been well characterized, no detailed information concerning this family is available for wheat. In the present study, a systematic analysis was carried out for the wheat LACS family. As a result, 30 putative TaLACSs were identified. Expression analysis revealed that 22 Takacs were expressed in wheat anthers. Two orthologs of AtLACS1, TaLACS2 and TaLACS3, were repressed at the vacuolated stage in the cold-treated BS366 (a temperature-sensitive genic male-sterile line). Thus, TaLACS2 and TaLACS3 may function like AtLACS1 in wax biosynthesis in anthers, and the repression of both genes may be correlated with the male sterility of BS366. TaLACS5 is an ortholog of AtLACS5, which was expressed exclusively in anthers. TaLACS5 was repressed in the cold-treated BS366 at the tetrad, uninucleate, and vacuolated stages. The negative correlation between TaLACS5 and TaGAMYB-B, and the MYB domain found in the promoter sequence suggested that TaLACS5 may be negatively regulated by TaGAMYB-B to participate in wheat fertility. These findings will provide a valuable foundation for the understanding of the wheat LACS gene family in male fertility.

Increased Lipids in Chlamydomonas reinhardtii by Multiple Regulations of DOF, LACS2, and CIS1.

Microalgal lipids are essential for biofuel and dietary supplement production. Lipid engineering for higher production has been studied for years. However, due to the complexity of lipid metabolism, single-gene engineering gradually encounters bottlenecks. Multiple gene regulation is more beneficial to boosting lipid accumulation and further clarifying the complex regulatory mechanism of lipid biosynthesis in the homeostasis of lipids, carbohydrates, and protein metabolism. Here, three lipid-related genes, DOF, LACS2, and CIS, were co-regulated in Chlamydomonas reinhartii by two circles of transformation to overexpress DOF and knock down LACS2 and CIS simultaneously. With the multiple regulations of these genes, the intracellular lipids and FA content increased by 142% and 52%, respectively, compared with CC849, whereas the starch and protein contents decreased by 45% and 24%. Transcriptomic analysis showed that genes in TAG and FA biosynthesis were up-regulated, and genes in starch and protein metabolism were down-regulated. This revealed that more carbon precursor fluxes from starch and protein metabolism were redirected towards lipid synthesis pathways. These results showed that regulating genes in various metabolisms contributed to carbon flux redirection and significantly improved intracellular lipids, demonstrating the potential of multiple gene regulation strategies and providing possible candidates for lipid overproduction in microalgae.

Multiplexed CRISPR/Cas9 editing of the long-chain acyl-CoA synthetase family in the diatom Phaeodactylum tricornutum reveals that mitochondrial ptACSL3 is involved in the synthesis of storage lipids.

Long-chain acyl-CoA synthetases (LACS) play diverse and fundamentally important roles in lipid metabolism. While their functions have been well established in bacteria, yeast and plants, the mechanisms by which LACS isozymes regulate lipid metabolism in unicellular oil-producing microalgae, including the diatom Phaeodactylum tricornutum, remain largely unknown. In P. tricornutum, a family of five genes (ptACSL1-ptACSL5) encodes LACS activities. We generated single lacs knockout/knockdown mutants using multiplexed CRISPR/Cas9 method, and determined their substrate specificities towards different fatty acids (FAs) and subcellular localisations. ptACSL3 is localised in the mitochondria and its disruption led to compromised growth and reduced triacylglycerol (TAG) content when cells were bubbled with air. The ptACSL3 mutants showed altered FA profiles in two galactoglycerolipids and phosphatidylcholine (PC) with significantly reduced distribution of 16:0 and 16:1. ptACSL5 is localised in the peroxisome and its knockdown resulted in reduced growth rate and altered molecular species of PC and TAG, indicating a role in controlling the composition of acyl-CoAs for lipid synthesis. Our work demonstrates the potential of generating gene knockout mutants with the mutation of large fragment deletion using multiplexed CRISPR/Cas9 and provides insight into the functions of LACS isozymes in lipid metabolism in the oleaginous microalgae.

Substrate Specificity of Human Long-Chain Acyl-CoA Synthetase ACSL6 Variants.

Long-chain acyl-CoA synthetases (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their active form, acyl-CoAs. Recent knock-out mouse studies revealed that among ACSL isoenzymes, ACSL6 plays an important role in the maintenance of docosahexaenoic acid (DHA)-containing glycerophospholipids. Several transcript variants of the human ACSL6 gene have been found; the two major ACSL6 variants, ACSL6V1 and V2, encode slightly different short motifs that both contain a conserved structural domain, the fatty acid Gate domain. In the present study, we expressed recombinant human ACSL6V1 and V2 in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus expression system, and then, using our novel ACSL assay system with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we examined the substrate specificities of the recombinant human ACSL6V1 and V2 proteins. The results showed that both ACSL6V1 and V2 could convert various kinds of long-chain fatty acids into their acyl-CoAs. Oleic acid was a good common substrate and eicosapolyenoic acids were poor common substrates for both variants. However, ACSL6V1 and V2 differed considerably in their preferences for octadecapolyenoic acids, such as linoleic acid, and docosapolyenoic acids, such as DHA and docosapentaenoic acid (DPA): ACSL6V1 preferred octadecapolyenoic acids, whereas V2 strongly preferred docosapolyenoic acids. Moreover, our kinetic studies revealed that ACSL6V2 had a much higher affinity for DHA than ACSL6V1. Our results suggested that ACSL6V1 and V2 might exert different physiological functions and indicated that ACSL6V2 might be critical for the maintenance of membrane phospholipids bearing docosapolyenoic acids such as DHA.

Very long-chain acyl-CoA synthetase 3 mediates onco-sphingolipid metabolism in malignant glioma.

Gliomas are the largest category of primary malignant brain tumors in adults, and glioblastomas account for nearly half of malignant gliomas. Glioblastomas are notoriously aggressive and drug-resistant, with a very poor 5 year survival rate of about 5%. New approaches to treatment are thus urgently needed. We previously identified an enzyme of fatty acid metabolism, very long-chain acyl-CoA synthetase 3 (ACSVL3), as a potential therapeutic target in glioblastoma. Using the glioblastoma cell line U87MG, we created a cell line with genomic deletion of ACSVL3 (U87-KO) and investigated potential mechanisms to explain how this enzyme supports the malignant properties of glioblastoma cells. Compared to U87MG cells, U87-KO cells grew slower and assumed a more normal morphology. They produced fewer, and far smaller, subcutaneous xenografts in nude mice. Acyl-CoA synthetases, including ACSVL3, convert fatty acids to their acyl-CoA derivatives, allowing participation in diverse downstream lipid pathways. We examined the effect of ACSVL3 depletion on several such pathways. Fatty acid degradation for energy production was not affected in U87-KO cells. Fatty acid synthesis, and incorporation of de novo synthesized fatty acids into membrane phospholipids needed for rapid tumor cell growth, was not significantly affected by lack of ACSVL3. In contrast, U87-KO cells exhibited evidence of altered sphingolipid metabolism. Levels of ceramides containing 18-22 carbon fatty acids were significantly lower in U87-KO cells. This paralleled the fatty acid substrate specificity profile of ACSVL3. The rate of incorporation of stearate, an 18-carbon saturated fatty acid, into ceramides was reduced in U87-KO cells, and proteomics revealed lower abundance of ceramide synthesis pathway enzymes. Sphingolipids, including gangliosides, are functional constituents of lipid rafts, membrane microdomains thought to be organizing centers for receptor-mediated signaling. Both raft morphology and ganglioside composition were altered by deficiency of ACSVL3. Finally, levels of sphingosine-1-phosphate, a sphingolipid signaling molecule, were reduced in U87-KO cells. We conclude that ACSVL3 supports the malignant behavior of U87MG cells, at least in part, by altering cellular sphingolipid metabolism.

High-fat diet impairs ferroptosis and promotes cancer invasiveness via downregulating tumor suppressor ACSL4 in lung adenocarcinoma.

BACKGROUND: Long-chain acyl-CoA synthetase-4 (ACSL4) is involved in fatty acid metabolism, and aberrant ACSL4 expression could be either tumorigenic or tumor-suppressive in different tumor types. However, the function and clinical significance of ACSL4 in lung adenocarcinoma remain elusive. RESULTS: ACSL4 was frequently downregulated in lung adenocarcinoma when analyzing both the TCGA database and the validation samples, and the lower ACSL4 expression was correlated with a worse prognosis. Using gene set enrichment analysis, we found that high ACSL4 expression was frequently associated with the oxidative stress pathway, especially ferroptosis-related proteins. In vitro functional studies showed that knockdown of ACSL4 increased tumor survival/invasiveness and inhibited ferroptosis, while ACSL4 overexpression exhibited the opposite effects. Moreover, high-fat treatment could also inhibit erastin-induced ferroptosis by affecting ACSL4 expression. The anti-tumor effects of ferroptosis inducers and the anti-ferroptosis effects of the high-fat diet were further validated using the mouse xenograft model. CONCLUSIONS: ACSL4 plays a tumor-suppressive role in lung adenocarcinoma by suppressing tumor survival/invasiveness and promoting ferroptosis. Our study provided a theoretical reference for the application of ferroptotic inducers and dietary guidance for lung adenocarcinoma patients.

Role of Sinorhizobium meliloti and Escherichia coli Long-Chain Acyl-CoA Synthetase FadD in Long-Term Survival.

FadD is an acyl-coenzyme A (CoA) synthetase specific for long-chain fatty acids (LCFA). Strains mutated in fadD cannot produce acyl-CoA and thus cannot grow on exogenous LCFA as the sole carbon source. Mutants in the fadD (smc02162) of Sinorhizobium meliloti are unable to grow on oleate as the sole carbon source and present an increased surface motility and accumulation of free fatty acids at the entry of the stationary phase of growth. In this study, we found that constitutive expression of the closest FadD homologues of S. meliloti, encoded by sma0150 and smb20650, could not revert any of the mutant phenotypes. In contrast, the expression of Escherichia coli fadD could restore the same functions as S. meliloti fadD. Previously, we demonstrated that FadD is required for the degradation of endogenous fatty acids released from membrane lipids. Here, we show that absence of a functional fadD provokes a significant loss of viability in cultures of E. coli and of S. meliloti in the stationary phase, demonstrating a crucial role of fatty acid degradation in survival capacity.

Functional Characterization of Long-Chain Acyl-CoA Synthetase Gene Family from the Oleaginous Alga Chromochloris zofingiensis.

Long-chain acyl-coenzyme A (CoA) synthetase (LACS) catalyzes the formation of acyl-CoAs from free fatty acids, which is pivotal for lipid metabolism. Here, we confirmed the presence of six CzLACS genes in Chromochloris zofingiensis. Functional complementation and in vitro enzymatic assay indicated that CzLACS2 through CzLACS5 rather than CzLACS1 or CzLACS6 are bona fide LACS enzymes and they have overlapping yet distinct substrate preference. The results of the subcellular colocalization experiment and different expression patterns under three triacylglycerol (TAG)-inducing conditions showed that CzLACS2 through CzLACS4 reside at endoplasmic reticulum (ER) and are involved in TAG biosynthesis, while CzLACS5 resides in peroxisome and participates in fatty acid ß-oxidation. The yeast one-hybrid assay using a library of 50 transcription factors (TFs) constructed in our study identified 12 TFs potentially involved in regulating the expression of CzLACSs. Moreover, heterologous expression of CzLACSs demonstrated their engineering potential for modulating TAG synthesis in yeast and algal cells.

Long-Chain acyl-CoA Synthetase LACS2 Contributes to Submergence Tolerance by Modulating Cuticle Permeability in Arabidopsis.

In Arabidopsis thaliana, LONG-CHAIN ACYL-COA SYNTHETASEs (LACSs) catalyze the synthesis of long-chain acyl-CoAs and function in diverse biological processes. We have recently revealed that LACS2 is primarily involved in the production of polyunsaturated linolenoyl-CoA, essential for the activation of ethylene response transcription factors-mediated hypoxia signaling. Here, we further reported the dual role of LACS2 in the regulation of submergence tolerance by modulating cuticle permeability in Arabidopsis cells. LACS2-overexpressors (LACS2-OEs) showed improved tolerance to submergence, with higher accumulation of cuticular wax and cutin in their rosettes. In contrast, knockout of LACS2 in the lacs2-3 mutant resulted in hypersensitivity to submergence with reduced wax crystals and thinner cutin layer. By analyses of plant surface permeability, we observed that the hypoxic sensitivities in the LACS2-OEs and lacs2-3 mutant were physiologically correlated with chlorophyll leaching, water loss rates, ionic leakage, and gas exchange. Thus, our findings suggest the role of LACS2 in plant response to submergence by modulating cuticle permeability in plant cells.

Long-Chain Acyl-CoA Synthetase 1 Role in Sepsis and Immunity: Perspectives From a Parallel Review of Public Transcriptome Datasets and of the Literature.

A potential role for the long-chain acyl-CoA synthetase family member 1 (ACSL1) in the immunobiology of sepsis was explored during a hands-on training workshop. Participants first assessed the robustness of the potential gap in biomedical knowledge identified via an initial screen of public transcriptome data and of the literature associated with ACSL1. Increase in ACSL1 transcript abundance during sepsis was confirmed in several independent datasets. Querying the ACSL1 literature also confirmed the absence of reports associating ACSL1 with sepsis. Inferences drawn from both the literature (via indirect associations) and public transcriptome data (via correlation) point to the likely participation of ACSL1 and ACSL4, another family member, in inflammasome activation in neutrophils during sepsis. Furthermore, available clinical data indicate that levels of ACSL1 and ACSL4 induction was significantly higher in fatal cases of sepsis. This denotes potential translational relevance and is consistent with involvement in pathways driving potentially deleterious systemic inflammation. Finally, while ACSL1 expression was induced in blood in vitro by a wide range of pathogen-derived factors as well as TNF, induction of ACSL4 appeared restricted to flagellated bacteria and pathogen-derived TLR5 agonists and IFNG. Taken together, this joint review of public literature and omics data records points to two members of the acyl-CoA synthetase family potentially playing a role in inflammasome activation in neutrophils. Translational relevance of these observations in the context of sepsis and other inflammatory conditions remain to be investigated.

Long-chain acyl-CoA synthetase 4 participates in the formation of highly unsaturated fatty acid-containing phospholipids in murine macrophages.

Long-chain acyl-coenzyme A synthetases (ACSLs) are a family of enzymes that convert free long-chain fatty acids into their acyl-coenzyme A (CoA) forms. ACSL4, belonging to the ACSL family, shows a preferential use of arachidonic acid (AA) as its substrate and plays a role in the remodeling of AA-containing phospholipids by incorporating free AA. However, little is known about the roles of ACSL4 in inflammatory responses. Here, we assessed the roles of ACSL4 on the effector functions of bone marrow-derived macrophages (BMDMs) obtained from mice lacking ACSL4. Liquid chromatography-tandem mass spectrometry analysis revealed that various highly unsaturated fatty acid (HUFA)-derived fatty acyl-CoA species were markedly decreased in the BMDMs obtained from ACSL4-deficient mice compared with those in the BMDMs obtained from wild-type mice. BMDMs from ACSL4-deficient mice also showed a reduced incorporation of HUFA into phosphatidylcholines. The stimulation of BMDMs with lipopolysaccharide (LPS) elicited the release of prostaglandins (PGs), such as PGE2, PGD2 and PGF2α, and the production of these mediators was significantly enhanced by ACSL4 deficiency. In contrast, neither the LPS-induced release of cytokines, such as IL-6 and IL-10, nor the endocytosis of zymosan or dextran was affected by ACSL4 deficiency. These results suggest that ACSL4 has a crucial role in the maintenance of HUFA composition of certain phospholipid species and in the incorporation of free AA into the phospholipids in LPS-stimulated macrophages. ACSL4 dysfunction may facilitate inflammatory responses by an enhanced eicosanoid storm.

Functional Overlap of Long-Chain Acyl-CoA Synthetases in Arabidopsis.

Long-chain acyl-CoA synthetases (LACSs) play diverse and essential roles in lipid metabolism. The genomes of model eukaryotic organisms encode multiple LACS genes, and the substrate specificities of LACS homologs often overlap substantially. Homologous LACSs tend to differ in their expression patterns, localizations, and, by extension, the metabolic pathways to which they contribute. The Arabidopsis genome encodes a family of nine LACS genes, which have been characterized largely by reverse genetic analysis of mutant phenotypes. Because of redundancy, distinguishing the contributions of some Arabidopsis LACS genes has been challenging. Here, we have attempted to clarify the functions of LACSs that functionally overlap by synopsizing the results of previous work, isolating a suite of higher-order mutants that were previously lacking, and analyzing oil, wax, cutin, cuticle permeability, fertility and growth phenotypes. LACS1, LACS2, LACS4, LACS8 and LACS9 all affect cuticular lipid metabolism, but have different precise roles. Seed set, seed weight and storage oil amounts of higher-order lacs1, lacs2, lacs4, lacs8 and lacs9 mutants vary greatly, with these traits subject to different effects of fertility and oil synthesis defects. LACS4, LACS8 and LACS9 have partially redundant roles in development, as lacs4 lacs8 and lacs4 lacs9 double mutants are dwarf. lacs4 lacs8 lacs9 triple mutants were not recovered, and are assumed to be non-viable. Together, these results sketch a complex network of functions and functional interactions within the Arabidopsis LACS gene family.

Molecular cloning and nutrient regulation analysis of long chain acyl-CoA synthetase 1 gene in grass carp, Ctenopharyngodon idella L.

Long chain acyl-CoA synthetase 1 (ACSL1), a key regulatory enzyme of fatty acid metabolism, catalyzes the conversion of long-chain fatty acids to acyl-coenzyme A. The full-length cDNAs of ACSL1a and ACSL1b were cloned from the liver of a grass carp. Both cDNAs contained a 2094bp open reading frame encoding 697 amino acids. Amino acid sequence alignment showed that ACSL1a shared 73.5% sequence identity with ACSL1b. Each of the two ACSL1s proteins had a transmembrane domain, a P-loop domain, and L-, A-, and G-motifs, which were relatively conserved in comparison to other vertebrates. Relative expression profile of ACSL1 mRNAs in different tissues indicated that ACSL1a is highly expressed in heart, mesenteric adipose, and brain tissues, whereas ACSL1b is highly expressed in heart, white muscle, foregut, and liver tissues. Nutrient regulation research showed that the expression levels of ACSL1a and ACSL1b were significantly down-regulated when 3, 6, and 9% fish oil were added in diet of grass carp as compared to the control group. However, no significant difference in the levels of ACSL1 mRNA was observed between the experimental groups. This study demonstrated the relationship between ACSL1a and ACSL1b genes in grass carp and laid a foundation for further research on ACSL family members in other species.

Hindbrain A2 noradrenergic neuron adenosine 5'-monophosphate-activated protein kinase activation, upstream kinase/phosphorylase protein expression, and receptivity to hormone and fuel reporters of short-term food deprivation are regulated by estradiol.

Estradiol (E) mitigates acute and postacute adverse effects of 12 hr-food deprivation (FD) on energy balance. Hindbrain 5'-monophosphate-activated protein kinase (AMPK) regulates hyperphagic and hypothalamic metabolic neuropeptide and norepinephrine responses to FD in an E-dependent manner. Energy-state information from AMPK-expressing hindbrain A2 noradrenergic neurons shapes neural responses to metabolic imbalance. Here we investigate the hypothesis that FD causes divergent changes in A2 AMPK activity in E- vs. oil (O)-implanted ovariectomized female rats, alongside dissimilar adjustments in circulating metabolic fuel (glucose, free fatty acids [FFA]) and energy deficit-sensitive hormone (corticosterone, glucagon, leptin) levels. FD decreased blood glucose in oil (O)- but not E-implanted ovariectomized female rats and elevated and reduced glucagon levels in O and E, respectively. FD decreased circulating leptin in O and E, but increased corticosterone and FFA concentrations in E only. Western blot analysis of laser-microdissected A2 neurons showed that glucocorticoid receptor type II and very-long-chain acyl-CoA synthetase 3 protein profiles were amplified in FD/E vs. FD/O. A2 total AMPK protein was elevated without change in activity in FD/O, whereas FD/E exhibited increased AMPK activation along with decreased upstream phosphatase expression. The catecholamine biosynthetic enzyme dopamine-ß-hydroxylase (DßH) was increased in FD/O but not FD/E A2 cells. The data show discordance between A2 AMPK activation and glycemic responses to FD; sensor activity was refractory to glucose decrements in FD/O but augmented in FD/E despite stabilized glucose and elevated FFA levels. E-dependent amplification of AMPK activity may reflect adaptive conversion to fatty acid oxidation and/or glucocorticoid stimulation. FD augmentation of A2 DßH protein profiles in FD/O but not FD/E animals suggests that FD may correspondingly regulate NE synthesis vs. metabolism/release in the absence vs. presence of E. Mechanisms underlying translation of E-contingent A2 neuron responses to FD into regulatory signaling remain to be determined. © 2016 Wiley Periodicals, Inc.

Long-chain acyl-CoA synthetase 6 regulates lipid synthesis and mitochondrial oxidative capacity in human and rat skeletal muscle.

KEY POINTS: Long-chain acyl-CoA synthetase 6 (ACSL6) mRNA is present in human and rat skeletal muscle, and is modulated by nutritional status: exercise and fasting decrease ACSL6 mRNA, whereas acute lipid ingestion increase its expression. ACSL6 genic inhibition in rat primary myotubes decreased lipid accumulation, as well as activated the higher mitochondrial oxidative capacity programme and fatty acid oxidation through the AMPK/PGC1-α pathway. ACSL6 overexpression in human primary myotubes increased phospholipid species and decreased oxidative metabolism. ABSTRACT: Long-chain acyl-CoA synthetases (ACSL 1 to 6) are key enzymes regulating the partitioning of acyl-CoA species toward different metabolic fates such as lipid synthesis or ß-oxidation. Despite our understanding of ecotopic lipid accumulation in skeletal muscle being associated with metabolic diseases such as obesity and type II diabetes, the role of specific ACSL isoforms in lipid synthesis remains unclear. In the present study, we describe for the first time the presence of ACSL6 mRNA in human skeletal muscle and the role that ACSL6 plays in lipid synthesis in both rodent and human skeletal muscle. ACSL6 mRNA was observed to be up-regulated by acute high-fat meal ingestion in both rodents and humans. In rats, we also demonstrated that fasting and chronic aerobic training negatively modulated the ACSL6 mRNA and other genes of lipid synthesis. Similar results were obtained following ACSL6 knockdown in rat myotubes, which was associated with a decreased accumulation of TAGs and lipid droplets. Under the same knockdown condition, we further demonstrate an increase in fatty acid content, p-AMPK, mitochondrial content, mitochondrial respiratory rates and palmitate oxidation. These results were associated with increased PGC-1α, UCP2 and UCP3 mRNA and decreased reactive oxygen species production. In human myotubes, ACSL6 overexpression reduced palmitate oxidation and PGC-1α mRNA. In conclusion, ACSL6 drives acyl-CoA toward lipid synthesis and its downregulation improves mitochondrial biogenesis, respiratory capacity and lipid oxidation. These outcomes are associated with the activation of the AMPK/PGC1-α pathway.

Acsl, the Drosophila ortholog of intellectual-disability-related ACSL4, inhibits synaptic growth by altered lipids.

Nervous system development and function are tightly regulated by metabolic processes, including the metabolism of lipids such as fatty acids. Mutations in long-chain acyl-CoA synthetase 4 (ACSL4) are associated with non-syndromic intellectual disabilities. We previously reported that Acsl, the Drosophila ortholog of mammalian ACSL3 and ACSL4, inhibits neuromuscular synapse growth by suppressing bone morphogenetic protein (BMP) signaling. Here, we report that Acsl regulates the composition of fatty acids and membrane lipids, which in turn affects neuromuscular junction (NMJ) synapse development. Acsl mutant brains had a decreased abundance of C16:1 fatty acyls; restoration of Acsl expression abrogated NMJ overgrowth and the increase in BMP signaling. A lipidomic analysis revealed that Acsl suppressed the levels of three lipid raft components in the brain, including mannosyl glucosylceramide (MacCer), phosphoethanolamine ceramide and ergosterol. The MacCer level was elevated in Acsl mutant NMJs and, along with sterol, promoted NMJ overgrowth, but was not associated with the increase in BMP signaling in the mutants. These findings suggest that Acsl inhibits NMJ growth by stimulating C16:1 fatty acyl production and concomitantly suppressing raft-associated lipid levels.