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Alzheimer’s disease (AD) is associated with high incidence of cardiovascular events but the mechanism remains elusive. Our previous study reveals a tight correlation between cardiac dysfunction and low mitochondrial aldehyde dehydrogenase (ALDH2) activity in elderly AD patients. In the present study we investigated the effect of ALDH2 overexpression on cardiac function in APP/PS1 mouse model of AD. Global ALDH2 transgenic mice were crossed with APP/PS1 mutant mice to generate the ALDH2-APP/PS1 mutant mice. Cognitive function, cardiac contractile, and morphological properties were assessed. We showed that APP/PS1 mice displayed significant cognitive deficit in Morris water maze test, myocardial ultrastructural, geometric (cardiac atrophy, interstitial fibrosis) and functional (reduced fractional shortening and cardiomyocyte contraction) anomalies along with oxidative stress, apoptosis, and inflammation in myocardium. ALDH2 transgene significantly attenuated or mitigated these anomalies. We also noted the markedly elevated levels of lipid peroxidation, the essential lipid peroxidation enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4), the transcriptional regulator for ACLS4 special protein 1 (SP1) and ferroptosis, evidenced by elevated NCOA4, decreased GPx4, and SLC7A11 in myocardium of APP/PS1 mutant mice; these effects were nullified by ALDH2 transgene. In cardiomyocytes isolated from WT mice and in H9C2 myoblasts in vitro, application of Aβ (20 μM) decreased cell survival, compromised cardiomyocyte contractile function, and induced lipid peroxidation; ALDH2 transgene or activator Alda-1 rescued Aβ-induced deteriorating effects. ALDH2-induced protection against Aβ-induced lipid peroxidation was mimicked by the SP1 inhibitor tolfenamic acid (TA) or the ACSL4 inhibitor triacsin C (TC), and mitigated by the lipid peroxidation inducer 5-hydroxyeicosatetraenoic acid (5-HETE) or the ferroptosis inducer erastin. These results demonstrate an essential role for ALDH2 in AD-induced cardiac anomalies through regulation of lipid peroxidation and ferroptosis.

IntroductionDocosahexaenoic acid (DHA) supplementation has been reported to negatively correlate with cancer cell proliferation and tumour development in many cancer types. Although cumulative evidence has demonstrated the apoptotic effect and cytotoxicity of DHA against tumour development in many cell types, the precise cellular and biochemical mechanisms of DHA-induced apoptosis in human endometrial cancer cells have not been investigated.Material and methodsMTT assay was performed to confirm the degree of apoptosis by combining treatment with DHA and triacsin C in endometrial cancer cell line. The synergistic effects of triacsin C and DHA were identified by performing flowcytometry and immunoblotting analysis.ResultsCombined treatment with DHA and triacsin C significantly induced apoptosis in RL95-2 endometrial carcinoma cells. Combined treatment with 125 µM DHA and 5 µM triacsin C significantly increased the sub-G1 population and apoptotic fragments in endometrial carcinoma cells. It was also demonstrated that DHA and triacsin C induced apoptosis through mitochondrial pathways via caspases-9, -3, and -7 as well as through the extrinsic pathway by activation of caspase-8/BID.ConclusionsFurther elucidation of the apoptotic mechanisms involving DHA treatment with ACS ablation could shed light on possible new treatment strategies for endometrial cancer. In addition, further research into the mechanisms of DHA and triacsin C-induced apoptotic mechanisms may lead to the development of therapeutic strategies for endometrial cancer.

In this study we have developed a method based on Flux Balance Analysis to identify human metabolic enzymes which can be targeted for therapeutic intervention against COVID-19. A literature search was carried out in order to identify suitable inhibitors of these enzymes, which were confirmed by docking calculations. In total, 10 targets and 12 bioactive molecules have been predicted. Among the most promising molecules we identified Triacsin C, which inhibits ACSL3, and which has been shown to be very effective against different viruses, including positive-sense single-stranded RNA viruses. Similarly, we also identified the drug Celgosivir, which has been successfully tested in cells infected with different types of viruses such as Dengue, Zika, Hepatitis C and Influenza. Finally, other drugs targeting enzymes of lipid metabolism, carbohydrate metabolism or protein palmitoylation (such as Propylthiouracil, 2-Bromopalmitate, Lipofermata, Tunicamycin, Benzyl Isothiocyanate, Tipifarnib and Lonafarnib) are also proposed.

Lipid is a crucial energy resource for mammalian oocyte. Melatonin could benefit the maturation of porcine oocyte in vitro, but the related mechanism is not elucidated yet. In the current study, methods to monitor lipid metabolism in single live oocytes were firstly established using probes (Lipi-Blue and Lipi-Green). It was observed that both lipid biogenesis and lipolysis occurred in maturing oocyte, but the general level of lipids dropped. Then maturing oocytes stained with probes were treated with melatonin or lipid metabolic-related inhibitors (triacsin C, rotenone, or etomoxir). The results showed that the lipid metabolism and maturation of porcine oocytes were all disrupted and that melatonin rescued the oocytes treated with triacsin C or rotenone, but not those treated with etomoxir. Further investigation demonstrated that cumulus cells are able to transfer lipids to oocytes via gap junctions. It was also observed that melatonin receptors exist in cumulus cells and are required for oocytes to maintain lipid metabolism. Meanwhile, the global gene expressing in cumulus cells was also modulated by melatonin, especially the genes related to antioxidants (SOD1, GPX1, GPX3, GPX4, PRDX2, and PRDX5), lipid metabolism (FABP3, FABP5, ACACB, TECR, etc.), and mitochondrial respiration (GPD1, ETFB, CYC1, and the genes of ATP synthase). Altogether the current research demonstrates that melatonin modulates lipid metabolism in maturing oocytes through its receptors in cumulus cells and benefits the developmental competence of oocytes.

Granulocyte&ndash, macrophage colony-stimulating factor (GM-CSF) is a monomeric glycoprotein that has been implicated in the tumor growth and progression of different types of cancer. GM-CSF is produced by various non-immune cells including MDA-MB-231 in response to various stimuli. However, the role of lipopolysaccharide (LPS) in the regulation of GM-CSF in MDA-MB-231 breast cancer cells so far remains unclear. Herein, we asked whether LPS could induce GM-CSF production in MDA-MB-231 cells, and if so, which signaling pathway was involved. MDA-MB-231 cells were treated with LPS or tumor necrosis factor alpha (TNF-&alpha, positive control), and GM-CSF expression levels were determined by qRT-PCR, ELISA, and confocal microscopy. Phosphorylation of the mitogen-activated protein kinases (MAPKs) and nuclear factor-&kappa, B (NF-kB) signaling proteins were evaluated by flow cytometry. Our results show that LPS induces GM-CSF expression at both mRNA and protein levels in MDA-MBA-231 cells. Inhibition of acyl-CoA synthetase 1 (ACSL1) activity in the cells with triacsin C significantly reduces the secretion of GM-CSF. Furthermore, the inhibition of ACSL1 activity significantly blocks the LPS-mediated phosphorylation of p38 MAPK, MEK1/2, extracellular signal-regulated kinase (ERK)1/2, c-Jun NH2-terminal kinase (JNK), and nuclear factor-&kappa, B (NF-kB) in the cells. These findings provide the first evidence that LPS induces ACSL1-dependent GM-CSF gene expression in MDA-MB-231 breast cancer cells, which requires the activation of p38 MAPK, MEK1/2, ERK1/2, JNK, and NF-kB.

Saturated fatty acid (SFA) induces proinflammatory response through a Toll-like receptor (TLR)-mediated mechanism, which is associated with cardiometabolic diseases such as obesity, insulin resistance, and endothelial dysfunction. Consistent with this notion, TLR2 or TLR4 knockout mice are protected from obesity-induced proinflammatory response and endothelial dysfunction. Although SFA causes endothelial dysfunction through TLR-mediated signaling pathways, the mechanisms underlying SFA-stimulated inflammatory response are not completely understood. To understand the proinflammatory response in vascular endothelial cells in high-lipid conditions, we compared the proinflammatory responses stimulated by palmitic acid (PA) and other canonical TLR agonists [lipopolysaccharide (LPS), Pam3-Cys-Ser-Lys4 (Pam3CSK4), or macrophage-activating lipopeptide-2)] in human aortic endothelial cells. The expression profiles of E-selectin and the signal transduction pathways stimulated by PA were distinct from those stimulated by canonical TLR agonists. Inhibition of long-chain acyl-CoA synthetases (ACSL) by a pharmacological inhibitor or knockdown of ACSL1 blunted the PA-stimulated, but not the LPS- or Pam3CSK4-stimulated proinflammatory responses. Furthermore, triacsin C restored the insulin-stimulated vasodilation, which was impaired by PA. From the results, we concluded that PA stimulates the proinflammatory response in the vascular endothelium through an ACSL1-mediated mechanism, which is distinct from LPS- or Pam3CSK4-stimulated responses. The results suggest that endothelial dysfunction caused by PA may require to undergo intracellular metabolism. This expands the understanding of the mechanisms by which TLRs mediate inflammatory responses in endothelial dysfunction and cardiovascular disease.

Therapeutics targeting replication of SARS coronavirus 2 (SARS-CoV-2) are urgently needed. Coronaviruses rely on host membranes for entry, establishment of replication centers and egress. Compounds targeting cellular membrane biology and lipid biosynthetic pathways have previously shown promise as antivirals and are actively being pursued as treatments for other conditions. Here, we tested small molecule inhibitors that target membrane dynamics or lipid metabolism. Included were inhibitors of the PI3 kinase VPS34, which functions in autophagy, endocytosis and other processes; Orlistat, an inhibitor of lipases and fatty acid synthetase, is approved by the FDA as a treatment for obesity; and Triacsin C which inhibits long chain fatty acyl-CoA synthetases. VPS34 inhibitors, Orlistat and Triacsin C inhibited virus growth in Vero E6 cells and in the human airway epithelial cell line Calu-3, acting at a post-entry step in the virus replication cycle. Of these the VPS34 inhibitors exhibit the most potent activity.

The lipid metabolic enzymes are considered candidate therapeutic targets for breast cancer. Long-chain acyl-coenzyme A (CoA) synthase (ACSL) is one of lipid metabolic enzymes and converts free-fatty acid to fatty acid-CoA. Five ACSL isoforms including ACSL1, ACSL3, ACSL4, ACSL5 and ACSL6 are identified in human. High ACSL4 expression has been observed in aggressive breast cancer phenotype. However, the role of other isoforms is still little-known. We therefore, analyzed the expression of ACSL isoforms in each subtype of breast cancer within METABRIC dataset and cancer cell line encyclopedia dataset. The expression levels of ACSL1, ACSL4 and ACSL5 in estrogen receptor (ER)-negative group were higher than that in ER-positive group. Similar expression pattern was detected among breast cancer cell lines MCF-7 (ER-positive) and MDA-MB-231 (ER-negative). Treatment of ACSL inhibitor triacsin C which inhibited enzyme activity of ACSL 1, 3, 4 and 5 suppressed cell growth of MCF-7 and MDA-MB-231. Our results further showed that high ACSL5 expression was associated with good prognosis in patients with both ER-positive and ER-negative breast cancer through KM plotter analysis. These results suggest that ACSL1, ACSL4 and ACSL5 expression is regulated by ER signaling pathways and ACSL5 is a potential novel biomarker for predicting prognosis of breast cancer patients.

Multiple myeloma (MM) is defined by the clonal expansion of malignant plasma cells in the bone marrow (BM) and has a 5-year survival rate of 50% (Siegel el al. 2018, Cancer J. Clin.). MM remains incurable due to the development of resistance to current chemotherapies; therefore, it is paramount to investigate novel treatments and the mechanisms of drug resistance in MM cells. Interestingly, obesity correlates with increased incidence of MM and high body mass index correlates with a poor treatment response (Marinac et al. 2019, JNCI Cancer Spectr, Groß et al. 2017, Oncotarget). Obesity is a major risk factor for many cancers, however, given the complexity of obesity, there are an array of mechanisms by which obesity may support tumor cells. Studies of obesity and MM are mainly at the epidemiological level and have not extensively explored the mechanism of this relationship. Therefore, there is a critical need to understand how obesity contributes to support cancers such as MM. Possible mechanisms may be through the increased availability of free fatty acids or through other factors that are found in obese patients. We hypothesize that lipid metabolism contributes to obesity-linked cancers such as MM. Recently, changes in lipid metabolism have been shown to support the proliferation, migration and the development of drug resistance in other blood cancers such as acute myeloid leukemia (Tabe et al. 2017, Cancer Res, Tabe et al. 2018, Sci. Reports) and solid tumors such as breast (Wang et al. 2017, JCI Insights) and prostate (Mitra et al. 2017, BMC Cancer) cancer. However, the role of lipid metabolism in MM cells has been understudied. Therefore, we hypothesized that genes within the Hallmark Fatty Acid Metabolism gene set (https://www.gsea-msigdb.org) would be differentially expressed between healthy patients and those with MM. We mined the clinical data (GSE6477, Chng et al. 2007, Cancer Res.) and found that transcripts of an enzyme critical for lipid metabolism, acyl-CoA synthetase long chain member 1 (ACSL1), was significantly downregulated (Figure 1A, Log2(Fold Change)=-2.33, adjusted p value=1.64*10-5, false discovery rate) in patients with newly diagnosed MM relative to normal plasma cells. Therefore, we hypothesized that ACSL1 may act as a tumor suppressor in MM. In order to test the role of the ACSL family as tumor suppressors, we treated human (MM1.S, OPM2 and RPMI-8226) and mouse myeloma (5TGM1) cell lines with an inhibitor (Triacsin C, TriC) of four of the five human acyl-CoA synthetase long chain family members (ACSL1,3,4 and 5). Contrary to our hypothesis, TriC treatment significantly decreased MM cell proliferation (Figure 1B, p Taken together, our data demonstrate that TriC-mediated ACSL inhibition in MM cells decreases proliferation, induces G0 arrest, apoptosis and decreases FAO-dependent respiration and mitochondrial function. It is unclear what ACSL family member is responsible for the phenotype we report here. To address these questions, future studies will focus on genetically targeting individual ACSL family members and characterizing the lipidomic profile of MM ACSL mutants. Our data also suggests that fatty acids are used as an energy source, therefore we will explore how FAO contributes to MM cell proliferation and survival. Disclosures No relevant conflicts of interest to declare.

Overexpression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in different types of cancer is associated with tumor growth and progression. Tumor necrosis factor-&alpha, (TNF&alpha, ) is involved in the induction of GM-CSF in different cells, however, the underlying molecular mechanism in this production of GM-CSF has not been fully revealed. Recently, it was noted that TNF&alpha, mediates inflammatory responses through long-chain acyl-CoA synthetase 1 (ACSL1). Therefore, we investigated the role of ACSL1 in the TNF&alpha, mediated production of GM-CSF. Our results showed that MDA-MB-231 cells displayed increased GM-CSF mRNA expression and secretion after incubation with TNF&alpha, Blocking of ACSL1 activity in the cells with triacsin C markedly suppressed the secretion of GM-CSF. However, inhibition of &beta, oxidation and ceramide biosynthesis were not required for GM-CSF production. By small interfering RNA mediated knockdown, we further demonstrated that TNF&alpha, induced GM-CSF production was significantly diminished in ACSL1 deficient cells. TNF&alpha, mediated GM-CSF expression was significantly reduced by inhibition of p38 MAPK, ERK1/2 and NF-&kappa, B signaling pathways. TNF&alpha, induced phosphorylation of p38, ERK1/2, and NF-&kappa, B was observed during the secretion of GM-CSF. On the other hand, inhibition of ACSL1 activity attenuates TNF&alpha, mediated phosphorylation of p38 MAPK, ERK1/2, and NF-&kappa, B in the cells. Importantly, our findings suggest that ACSL1 plays an important role in the regulation of GM-CSF induced by TNF&alpha, in MDA-MB-231 cells. Therefore, ACSL1 may be considered as a potential novel therapeutic target for tumor growth.

The lipophilic fungal pathogen Malassezia spp. must acquire long-chain fatty acids (LCFAs) from outside the cell. To clarify the mechanism of LCFA acquisition, we investigated fatty acid uptake by this fungus and identified the long-chain acyl-CoA synthetase (ACS) gene FAA1 in three Malassezia spp.: M. globosa, M. pachydermatis, and M. sympodialis. These FAA1 genes could compensate for the double mutation of FAA1 and FAA4 in Saccharomyces cerevisiae, suggesting that Malassezia Faa1 protein recognizes exogenous LCFAs. MgFaa1p and MpFaa1p utilized a medium-chain fatty acid, lauric acid (C12:0). Interestingly, the ACS inhibitor, triacsin C, affected the activity of the Malassezia Faa1 proteins but not that of S. cerevisiae. Triacsin C also reduced the growth of M. globosa, M. pachydermatis, and M. sympodialis. These results suggest that triacsin C and its derivatives are potential compounds for the development of new anti-Malassezia drugs.

Long chain acyl-CoA synthetases (ACSLs) convert fatty acids (FA) to fatty acyl-CoAs that are utilized for various cellular processes, including triglyceride synthesis, activation of transcription factors, signal transduction, and providing energy sources via oxidation. Among 5 isotypes (ACSL1, 3, 4, 5, and 6), ACSL1 is the predominant isotype in adipocytes, and it is known to play important roles in adipocyte physiology, including FA uptake, triglyceride (TG) synthesis, thermogenesis, and FA oxidation. However, the roles of ACSL1 in brown adipocyte differentiation is incompletely understood. During the in vitro differentiation of brown adipocytes from stromal vascular cells (SVC), mRNA expression of ACSL1 was drastically increased. Pharmacological inhibition of ACSLs by triacsin C suppressed the differentiation of brown adipocytes. Interestingly, triacsin C inhibited the expression of uncoupled protein 1 (UCP1), which plays the major role in thermogenesis in mitochondria. Thus, we hypothesize that ACSL1 regulates thermogenesis and energy utilization by regulation of brown adipocyte differentiation. To examine the role of ACSL1 in brown adipose tissue (BAT), we generated BAT specific ACSL1 knockout mice by breeding UCP1-Cre with ACSL1 flox/flox mice (ACSL1BATKO). ACSL1 deficiency in BAT reduced the weight gain and glucose intolerance in high fat diet (HFD)-fed mice. ACSL1BATKO mice were severely cold intolerant. Furthermore, ACSL1BATKO mice were resistant to beta 3-agonist (CL-316243)-stimulated weight loss. Fat mass in HFD-fed ACSL1BATKO was lower than HFD-fed wild type mice. These data suggest that ACSL1 in BAT contributes to BAT differentiation, which causes the impairment of FA utilization. However, the mechanisms by which deficiency of ACSL1 in BAT reduces HFD-induced weight gain need to be further investigated. Disclosure G. Ren: None. J. Kim: None. Funding National Institutes of Health (R01HL128695R03AG058078)

Cryptosporidiosis is a significant cause of gastroenteritis in both humans and livestock in developing countries. The only FDA-approved drug available against the same is nitazoxanide, with questionable efficacy in malnourished children and immunocompromised patients. Recent in vitro studies have indicated the viability of Triacsin C as a potential drug candidate, which targets the parasite’s long-chain fatty acyl coenzyme A synthetase enzyme (LC-FACS), a critical component of the fatty acid metabolism pathway. We have used this molecule as a baseline to propose more potent versions thereof. We have applied a combined approach of substructure replacement, literature search, and database screening to come up with 514 analogs of Triacsin C. A virtual screening protocol was carried out which lead us to identify a potential hit compound. This was further subjected to a 100-ns molecular dynamics simulation in complex to determine its stability and binding characteristics. After which, the ADME/tox properties were predicted to assess its viability as a drug. The molecule R134 was identified as the best hit due to its highest average binding affinity, stability in complex when subjected to MD simulations, and reasonable predicted ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) properties comparable to those of the Triacsin C parent molecule. We have proposed R134 as a putative drug candidate against the Cryptosporidium parvum LC-FACS enzyme isoforms, following an in silico protocol. We hope the results will be helpful when planning future in vitro experiments for identifying drugs against Cryptosporidium.

The mechanisms for supplying octanoic acid for ghrelin acylation in X/A-like cells are incompletely understood. We found that long-chain fatty acids were incorporated at a higher rate in the ghrelin-producing cell line MGN3-1 than in MIN6 cells, in part due to higher expression level of long-chain fatty acyl-CoA synthetase family member 1 (Acsl1). Inhibition of ACSLs by triacsin C profoundly suppressed acylated ghrelin production. These results suggest that high incorporation of long-chain fatty acids into the ghrelin-producing cells plays a role in the supply of octanoic acid for ghrelin acylation.

RPE65 is the isomerase catalyzing conversion of all-trans-retinyl ester (atRE) into 11-cis-retinol in the retinal visual cycle. Crystal structures of RPE65 and site-directed mutagenesis reveal aspects of its catalytic mechanism, especially retinyl moiety isomerization, but other aspects remain to be determined. To investigate potential interactions between RPE65 and lipid metabolism enzymes, HEK293-F cells were transfected with expression vectors for visual cycle proteins and co-transfected with either fatty acyl:CoA ligases (ACSLs) 1, 3, or 6 or the SLC27A family fatty acyl-CoA synthase FATP2/SLCA27A2 to test their effect on isomerase activity. These experiments showed that RPE65 activity was reduced by co-expression of ACSLs or FATP2. Surprisingly, however, in attempting to relieve the ACSL-mediated inhibition, we discovered that triacsin C, an inhibitor of ACSLs, also potently inhibited RPE65 isomerase activity in cellulo. We found triacsin C to be a competitive inhibitor of RPE65 (IC50 = 500 nm). We confirmed that triacsin C competes directly with atRE by incubating membranes prepared from chicken RPE65-transfected cells with liposomes containing 0-1 μM atRE. Other inhibitors of ACSLs had modest inhibitory effects compared with triascin C. In conclusion, we have identified an inhibitor of ACSLs as a potent inhibitor of RPE65 that competes with the atRE substrate of RPE65 for binding. Triacsin C, with an alkenyl chain resembling but not identical to either acyl or retinyl chains, may compete with binding of the acyl moiety of atRE via the alkenyl moiety. Its inhibitory effect, however, may reside in its nitrosohydrazone/triazene moiety.

Background The homeostasis of lipid droplets (LDs) plays a crucial role in maintaining the physical metabolic processes in cells, and is regulated by many LD-associated proteins, including perilipin 5 (Plin5) in liver. As the putative sites of hepatitis C virus (HCV) virion assembly, LDs are vital to viral infection. In addition, the hepatic LD metabolism can be disturbed by non-structural HCV proteins, such as NS5A, but the details are still inexplicit. Methods HCV NS5A was overexpressed in the livers and hepatocytes of wild-type and Plin5-null mice. BODIPY 493/503 and oil red O staining were used to detect the lipid content in mouse livers and hepatocytes. The levels of lipids, lipid peroxidation and inflammation biomarkers were further determined. Immunofluorescence assay and co-immunoprecipitation assay were performed to investigate the relationship of Plin5 and NS5A. Results One week after adenovirus injection, livers expressing NS5A showed more inflammatory cell aggregation and more severe hepatic injuries in Plin5-null mice than in control mice, which was consistent with the increased serum levels of IL-2 and TNF-α (P

Coxiella burnetiiis an obligate intracellular bacterial pathogen and a causative agent of culture-negative endocarditis. WhileC. burnetiiinitially infects alveolar macrophages, it has also been found in lipid droplet (LD)-containing foamy macrophages in the cardiac valves of endocarditis patients. In addition, transcriptional studies ofC. burnetii-infected macrophages reported differential regulation of the LD coat protein-encoding gene perilipin 2(plin-2). To further investigate the relationship between LDs andC. burnetii, we compared LD numbers using fluorescence microscopy in mock-infected andC. burnetii-infected alveolar macrophages. On average,C. burnetii-infected macrophages contained twice as many LDs as mock-infected macrophages. LD numbers increased as early as 24 hours post-infection, an effect reversed by blockingC. burnetiiprotein synthesis. The observed LD accumulation was dependent on theC. burnetiiType 4B Secretion System (T4BSS), a major virulence factor that manipulates host cellular processes by secreting bacterial effector proteins into the host cell cytoplasm. To determine the importance of LDs duringC. burnetiiinfection, we manipulated LD homeostasis and assessedC. burnetiiintracellular growth. Surprisingly, blocking LD formation with the pharmacological inhibitors triacsin C or T863, or knocking out acyl-CoA transferase-1 (acat-1) in alveolar macrophages, increasedC. burnetiigrowth at least 2-fold. Conversely, preventing LD lipolysis by inhibiting adipose triglyceride lipase (ATGL) with atglistatin almost completely blocked bacterial growth, suggesting LD breakdown is essential forC. burnetii.Together these data suggest that maintenance of LD homeostasis, possibly via theC. burnetiiT4BSS, is critical for bacterial growth.IMPORTANCEHost neutral lipid storage organelles known as lipid droplets (LDs) serve as a source of energy, nutrients, and signaling lipids. LDs are associated with infection of the intracellular bacterial pathogenCoxiella burnetii, a significant cause of culture-negative endocarditis. WhileC. burnetiiwas found in LD-rich foamy macrophages in endocarditis patients, little is known about the host LD-C. burnetiirelationship. We demonstratedC. burnetiiType 4B Secretion System (T4BSS)-dependent LD accumulation in macrophages, suggesting a T4BSS-mediated regulation of host LD homeostasis. Further, manipulating LD homeostasis significantly affected bacterial growth, indicating LDs play an important role duringC. burnetiiinfection. AsC. burnetiiendocarditis has a 19% mortality rate even in treated patients, exploring the LD-C. burnetiiassociation might identify novel therapeutic targets.

Long chain acyl-CoA synthetases (ACSLs) are a family of enzymes that convert free long chain fatty acids into their acyl-CoA forms. Among ACSL enzymes, ACSL4 prefers arachidonic acid (AA) as a substrate and plays an important role in re-esterification of free AA. We previously reported that the suppression of ACSL4 activity by treatment with an ACSL inhibitor or a small interfering RNA markedly enhanced interleukin-1β (IL-1β)-dependent prostaglandin (PG) biosynthesis in rat fibroblastic 3Y1 cells. We show here that in addition to these prostanoids, cytokine-dependent production of 5,11-dihydroxyeicosatetraenoic acid (5,11-diHETE), a cyclooxygenase product of 5-hydroxyeicosatetraenoic acid (5-HETE), was enhanced by the inhibition of ACSL4 activity. Treatment of several types of cells with an ACSL inhibitor, triacsin C, markedly enhanced IL-1β-dependent production of 5,11-diHETE. siRNA-mediated knockdown of ACSL4 also enhanced IL-1β-dependent production of 5,11-diHETE from 3Y1 cells. The production of 5,11-diHETE was significantly decreased by a cyclooxygenase (COX)-2 selective inhibitor, NS-398, but not by a 5-lipoxygenase activating protein (FLAP) inhibitor, MK-886. The inhibition of ACSL enzymes significantly facilitated release of not only 5-HETE but also 8-HETE, 9-HETE, 11-HETE, 12-HETE, and 15-HETE, independently of IL-1β stimulation. In vitro analysis showed that a recombinant COX-2 enzyme more effectively metabolized 5(S)-HETE to 5-11-diHETE compared to COX-1 enzyme. From these results, we proposed the following mechanism of 5,11-diHETE biosynthesis in these cells: 1) inhibition of ACSL4 causes accumulation of free AA; 2) the accumulated AA is nonspecifically converted into various HETEs; and 3) among these HETEs, 5-HETE is metabolized into 5,11-diHETE by cytokine-induced COX-2.

De novo lipid synthesis and scavenging of fatty acids (FA) are processes essential for the formation of the membrane of the human pathogen Chlamydia trachomatis (C.t.). Host FA are assimilated via esterification by the bacterial acyl-acyl carrier protein (ACP) synthase AasC but inhibitors of the host acyl-CoA synthetase enymes ACSL also impaired growth of C.t. in human cells. In E. coli, activity of AasC was sensitive to triacsin C and rosiglitazone G. The absence of a triacsin C-insensitive pathway and the increased inhibition by rosiglitazone G confirmed the sensitivity of the bacterial acyl-ACP synthase to these drugs in infected human cells. We found no evidence that the human ACSL enzymes are required for lipid formation by C.t. The broad substrate specificity of acyltransferase CT775 provides C.t. with the capacity to incorporate straight-chain and bacterial specific branched-chain fatty acids. CT775 accepts both acyl-ACP and acyl-CoA as acyl donors and, 1- or 2-acyl isomers of lysophosphoplipids as acyl acceptors. The enzyme responsible for remodeling of human phosphatidylserine to bacterial phosphatidylethanolamine was identified as CT699. These findings provide evidence that the pathogen has the ability to extend the lipid diversity of its membrane.

Made available in DSpace on 2019-10-04T12:42:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-08-05 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) In this study, we evaluated the modulation effect of long-chain Acyl-CoA synthetase during early embryo development. Bovine embryos were cultured in four groups: positive modulation (ACS+) with GW3965 hydrochloride, negative modulation (ACS-) with Triacsin C, association of both modulators (ACS +/-), and control. Embryo development rates were not altered (P>0.05) by treatments. Embryonic cytoplasmic lipid content increased in ACS+ but reduced in ACS- compared to the control (P < 0.05), whereas the membrane phospholipids profile was not altered by treatments. The total number of blastomeres did not differ (P > 0.05) between groups; however, an increased apoptotic cells percentage was found in ACS- compared to control. Twenty-four hours after warming, ACS+ and control grade I embryos presented the best hatching rates, whereas the ACS+ group equaled the hatching rates between their embryos of grades I, II and III 48 hours after warming. The relative abundance of transcripts for genes associated with lipid metabolism (ACSL3, ACSL6, ACAT1, SCD, and AUH), heatshock (HSP90AA1 and HSF1), oxidative stress (GPX4), and angiogenesis (VEGF), among other important genes for embryo development were affected by at least one of the treatments. The treatments were effective in modulating the level of transcripts for ACSL3 and the cytoplasmic lipid content. The ACS- was not effective in increasing embryonic cryosurvival, whereas ACS+ restored survival rates after vitrification of embryos with low quality, making them equivalent to embryos of excellent quality. Fed Univ Pampa, Sch Vet Med, Uruguaiana, RS, Brazil Fed Univ ABC, Ctr Nat & Human Sci, Santo Andre, SP, Brazil Univ Sao Paulo, Dept Anim Reprod, Sao Paulo, SP, Brazil ABS Pecplan, In Vitro Brasil, Mogi Mirim, SP, Brazil Univ Fed Uberlandia, Dept Chem, Uberlandia, MG, Brazil Univ Prebiteriana Mackenzie, Sch Engn, Sao Paulo, SP, Brazil Sao Paulo State Univ, Dept Anim Reprod, Botucatu, SP, Brazil Sao Paulo State Univ, Dept Biol Sci, Assis, SP, Brazil Sao Paulo State Univ, Dept Anim Reprod, Botucatu, SP, Brazil Sao Paulo State Univ, Dept Biol Sci, Assis, SP, Brazil CAPES: 1 CAPES: 2012/50533-2 CAPES: 2019/16239-9

Lipid disequilibrium induced by inhibition of long-chain acyl-CoA synthetases impairs ERAD substrate glycan trimming and dislocation independently of its effects on lipid droplet biogenesis. The disruptions in ER proteostasis activate the IRE1 and PERK branches of the unfolded protein response and ultimately induce IRE1-dependent cell death., The endoplasmic reticulum (ER) mediates the folding, maturation, and deployment of the secretory proteome. Proteins that fail to achieve their native conformation are retained in the ER and targeted for clearance by ER-associated degradation (ERAD), a sophisticated process that mediates the ubiquitin-dependent delivery of substrates to the 26S proteasome for proteolysis. Recent findings indicate that inhibition of long-chain acyl-CoA synthetases with triacsin C, a fatty acid analogue, impairs lipid droplet (LD) biogenesis and ERAD, suggesting a role for LDs in ERAD. However, whether LDs are involved in the ERAD process remains an outstanding question. Using chemical and genetic approaches to disrupt diacylglycerol acyltransferase (DGAT)–dependent LD biogenesis, we provide evidence that LDs are dispensable for ERAD in mammalian cells. Instead, our results suggest that triacsin C causes global alterations in the cellular lipid landscape that disrupt ER proteostasis by interfering with the glycan trimming and dislocation steps of ERAD. Prolonged triacsin C treatment activates both the IRE1 and PERK branches of the unfolded protein response and ultimately leads to IRE1-dependent cell death. These findings identify an intimate relationship between fatty acid metabolism and ER proteostasis that influences cell viability.

Neutral lipids - involved in many cellular processes - are stored as lipid droplets (LD), those mainly cytosolic (cLD) along with a small nuclear population (nLD). nLD could be involved in nuclear-lipid homeostasis serving as an endonuclear buffering system that would provide or incorporate lipids and proteins involved in signalling pathways as transcription factors and as enzymes of lipid metabolism and nuclear processes. Our aim was to determine if nLD constituted a dynamic domain. Oleic-acid (OA) added to rat hepatocytes or HepG2 cells in culture produced cellular-phenotypic LD modifications: increases in TAG, CE, C, and PL content and in cLD and nLD numbers and sizes. LD increments were reversed on exclusion of OA and were prevented by inhibition of acyl-CoA synthetase (with Triacsin C) and thus lipid biosynthesis. Under all conditions, nLD corresponded to a small population (2-10%) of total cellular LD. The anabolism triggered by OA, involving morphologic and size changes within the cLD and nLD populations, was reversed by a net balance of catabolism, upon eliminating OA. These catabolic processes included lipolysis and the mobilization of hydrolyzed FA from the LD to cytosolic-oxidation sites. These results would imply that nLD are actively involved in nuclear processes that include lipids. In conclusion, nLD are a dynamic nuclear domain since they are modified by OA through a reversible mechanism in combination with cLD; this process involves acyl-CoA-synthetase activity; ongoing TAG, CE, and PL biosynthesis. Thus, liver nLD and cLD are both dynamic cellular organelles., Instituto de Investigaciones Bioquímicas de La Plata, Facultad de Ciencias Médicas, Facultad de Ciencias Exactas

Intracellular long-chain acyl-CoA synthetases (ACSL) activate fatty acids to produce acyl-CoA, which undergoes β-oxidation and participates in the synthesis of esterified lipids such as triacylglycerol (TAG). Imbalances in these metabolic routes are closely associated with the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Triacsin C is one of the few compounds that inhibit TAG accumulation into lipid droplets (LD) by suppressing ACSL activity. Here we report that treatment of primary rat hepatocytes with triacsin C at concentrations lower than the IC50 (4.1 μM) for LD formation: (i) diminished LD number in a concentration-dependent manner; (ii) increased mitochondrial amount; (iii) markedly improved mitochondrial metabolism by enhancing the β-oxidation efficiency, electron transport chain capacity, and degree of coupling - treatment of isolated rat liver mitochondria with the same triacsin C concentrations did not affect the last two parameters; (iv) decreased the GSH/GSSG ratio and elevated the protein carbonyl level, which suggested an increased reactive oxygen species production, as observed in isolated mitochondria. The hepatocyte mitochondrial improvements were not related to either the transcriptional levels of PGC-1α or the content of mTOR and phosphorylated AMPK. Triacsin C at 10 μM induced hepatocyte death by necrosis and/or apoptosis through mechanisms associated with mitochondrial permeability transition pore opening, as demonstrated by experiments using isolated mitochondria. Therefore, triacsin C at sub-IC50 concentrations modulates the lipid imbalance by shifting hepatocytes to a more oxidative state and enhancing the fatty acid consumption, which can in turn accelerate lipid oxidation and reverse NAFLD in long-term therapies.

Giardiasis caused by Giardia intestinalis (syn. G. lamblia, G. duodenalis) is one of the leading causes of diarrheal parasitic diseases worldwide. Although limited drugs to treat giardiasis are available, there are concerns regarding toxicity in some patients and the emerging drug resistance. By data-mining genome sequences, we observed that G. intestinalis is incapable of synthesizing fatty acids de novo. However, this parasite has five long-chain fatty acyl-CoA synthetases (GiACS1 to GiACS5) to activate fatty acids scavenged from the host. ACS is an essential enzyme because fatty acids need to be activated to form acyl-CoA thioesters before they can enter subsequent metabolism. In the present study, we performed experiments to explore whether some GiACS enzymes could serve as drug targets in Giardia. Based on the high-throughput datasets and protein modeling analyses, we initially studied the GiACS1 and GiACS2, because genes encoding these two enzymes were found to be more consistently expressed in varied parasite life cycle stages and when interacting with host cells based on previously reported transcriptome data. These two proteins were cloned and expressed as recombinant proteins. Biochemical analysis revealed that both had apparent substrate preference towards palmitic acid (C16:0) and myristic acid (C14:0), and allosteric or Michaelis-Menten kinetics on palmitic acid or ATP. The ACS inhibitor triacsin C inhibited the activity of both enzymes (IC50 = 1.56 µM, Ki = 0.18 µM for GiACS1 and IC50 = 2.28 µM, Ki = 0.23 µM for GiACS2, respectively) and the growth of G. intestinalis in vitro (IC50 = 0.8 µM). As expected from giardial evolutionary characteristics, both GiACSs displayed differences in overall folding structure as compared with their human counterparts. These observations support the notion that some of the GiACS enzymes may be explored as drug targets in this parasite.