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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.

Background: Lipid droplets have been implicated in HCV virion assembly. Results: Inhibitors of the synthesis of triacylglycerol and cholesterol ester, the main components of lipid droplets, impair virion assembly. Conclusion: Production of infectious HCV is linked integrally with the biosynthesis of the major components of lipid droplets. Significance: Inhibitors of enzymes that generate acylglycerols and cholesterol esters have antiviral activity., In hepatitis C virus infection, replication of the viral genome and virion assembly are linked to cellular metabolic processes. In particular, lipid droplets, which store principally triacylglycerides (TAGs) and cholesterol esters (CEs), have been implicated in production of infectious virus. Here, we examine the effect on productive infection of triacsin C and YIC-C8-434, which inhibit synthesis of TAGs and CEs by targeting long-chain acyl-CoA synthetase and acyl-CoA:cholesterol acyltransferase, respectively. Our results present high resolution data on the acylglycerol and cholesterol ester species that were affected by the compounds. Moreover, triacsin C, which blocks both triglyceride and cholesterol ester synthesis, cleared most of the lipid droplets in cells. By contrast, YIC-C8-434, which only abrogates production of cholesterol esters, induced an increase in size of droplets. Although both compounds slightly reduced viral RNA synthesis, they significantly impaired assembly of infectious virions in infected cells. In the case of triacsin C, reduced stability of the viral core protein, which forms the virion nucleocapsid and is targeted to the surface of lipid droplets, correlated with lower virion assembly. In addition, the virus particles that were released from cells had reduced specific infectivity. YIC-C8-434 did not alter the association of core with lipid droplets but appeared to decrease production of infectious virus particles, suggesting a block in virion assembly. Thus, the compounds have antiviral properties, indicating that targeting synthesis of lipids stored in lipid droplets might be an option for therapeutic intervention in treating chronic hepatitis C virus infection.

Hepatitis B virus X protein (HBx) plays crucial roles in the development of hepatocellular carcinoma (HCC). The abnormal lipid metabolism is involved in the hepatocarcinogenesis. We previously reported that HBx suppressed miR-205 in hepatoma cells. In this study, we supposed that HBx-decreased miR-205 might contribute to the abnormal lipid metabolism according to the bioinformatics analysis. Interestingly, we showed that the expression levels of acyl-CoA synthetase long-chain family member 4 (ACSL4) were negatively associated with those of miR-205 in clinical HCC tissues. Then, we validated that miR-205 was able to inhibit the expression of ACSL4 at the levels of mRNA and protein through targeting its 3'UTR. Strikingly, we found that HBx was able to increase the levels of cellular cholesterol, a metabolite of ACSL4, in hepatoma cells, which could be blocked by miR-205 (or Triacsin C, an inhibitor of ACSL4). However, anti-miR-205 could increase the levels of cholesterol in the cells. Moreover, we demonstrated that the levels of cholesterol were increased in the liver of HBx transgenic mice in a time course manner. Functionally, oil red O staining revealed that HBx promoted lipogenesis in HepG2 cells, which could be abolished by miR-205 (or Triacsin C). However, anti-miR-205 was able to accelerate lipogenesis in the cells. Interestingly, the treatment with Triacsin C could remarkably block the role of anti-miR-205 in the event. Thus, we conclude that miR-205 is able to target ACSL4 mRNA. The HBx-depressed miR-205 is responsible for the abnormal lipid metabolism through accumulating cholesterol in hepatoma cells.

Various triacsin C analogs, containing different alkenyl chains and carboxylic acid bioisoteres including 4-aminobenzoic acid, isothiazolidine dioxide, hydroxylamine, hydroxytriazene, and oxadiazolidine dione, were synthesized and their inhibitions of long chain fatty acyl-CoA synthetase (ACSL) were examined. Two methods, a cell-based assay of ACSL activity and an in situ [(14)C]-palmitate incorporation into extractable lipids were used to study the inhibition. Using an in vivo leukocyte recruitment inhibition protocol, the translocation of one or more cell adhesion molecules from the cytoplasm to the plasma membrane on either the endothelium or leukocyte or both was inhibited by inhibitors 1, 9, and triacsin C. The results suggest that inhibition of ACSL may attenuate the vascular inflammatory component associated with ischemia reperfusion injury and lead to a decrease of infarct expansion.

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

Sphingosine 1-phosphate (SIP) plays important roles both as a bioactive lipid molecule and an intermediate of the sphingolipid-to-glycerophospholipid metabolic pathway. To identify human acyl-CoA synthetases (ACSs) involved in SIP metabolism, we cloned all 26 human ACS genes and examined their abilities to restore deficient sphingolipid-to-glycerophospholipid metabolism in a yeast mutant lacking two ACS genes, FAA1 and FAA4. Here, in addition to the previously identified ACSL family members (ACSL1, 3, 4, 5, and 6), we found that ACSVL1, ACSVL4, and ACSBG1 also restored metabolism. All 8 ACSs were localized either exclusively or partly to the endoplasmic reticulum (ER), where SIP metabolism takes place. We previously proposed the entire SIP metabolic pathway from results obtained using yeast cells, i.e., SIP is metabolized to glycerophospholipids via trans-2-hexadecenal, trans-2-hexadecenoic acid, trans-2-hexadecenoyl-CoA, and palmitoyl-CoA. However, as SIP is not a naturally occurring long-chain base 1-phosphate in yeast, the validity of this pathway required further verification using mammalian cells. In the present study, we treated HeLa cells with the ACS inhibitor triacsin C and found that inhibition of ACSs resulted in accumulation of trans-2-hexadecenoic acid as in ACS mutant yeast. From these results, we conclude that SIP is metabolized by a common pathway in eukaryotes. (C) 2013 Elsevier Inc. All rights reserved.

Cryptosporidium parvum is a unicellular zoonotic pathogen that can cause severe watery diarrhea in both humans and animals [1]. It is emerging as 1 of the 4 top diarrheal pathogens in children

Fatty acids regulate angiogenesis although no such information is available in first trimester placental trophoblast cells despite the fact that angiogenesis is a critical step involving these cells in early placentation. We investigated effects of different fatty acids on angiogenesis, their uptake and metabolism and expression of lipid metabolic genes in first trimester placental trophoblast cells using HTR-8/SVneo cell line. Fatty acid uptake by these cells exhibited a saturable kinetics. Uptake of AA was consistently greater compared with that of EPA and DHA throughout the incubation period of 180 min. Use of triacsin C, an inhibitor of acyl-CoA synthetase, significantly inhibited fatty acid uptake as well as fatty acid induced cell proliferation in these cells. Angiogenic effect (as measured by tube formation) of these fatty acids was in the following order DHA> EPA> AA> OA. Angiogenic effect of these fatty acids (AA, EPA, OA) was significantly decreased in ANGPTL4 knocked down cells, indicating ANGPTL4 may be involved at least in part in fatty acid induced angiogenesis. In addition, these fatty acids altered expression of several lipid metabolic genes such as ADRP, FABP4, FABP3, and COX-2 those are involved in angiogenesis. All these data suggest that fatty acids regulate angiogenic processes in these cells via different mechanisms.

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.

Recently our group has demonstrated that cellular triglycerides (TG) levels play an important role in rotavirus replication. In this study, we further examined the roles of the key enzymes for TG synthesis (lipogenesis) in the replication of rotaviruses by using inhibitors of fatty acid synthase, long chain fatty acid acyl-CoA synthetase (ACSL), and diacylglycerol acyltransferase and acyl-CoA:cholesterol acyltransferase in association with lipid droplets of which TG is a major component. Triacsin C, a natural ACSL inhibitor from Streptomyces aureofaciens, was found to be highly effective against rotavirus replication. Thus, novel triacsin C analogs were synthesized and evaluated for their efficacies against the replication of rotaviruses in cells. Many of the analogs significantly reduced rotavirus replication, and one analog (1e) was highly effective at a nanomolar concentration range (ED50 0.1 μM) with a high therapeutic index in cell culture. Our results suggest a crucial role of lipid metabolism in rotavirus replication, and triacsin C and/or its analogs as potential therapeutic options for rotavirus infections.

Background: Micro-lipid droplets (mLDs) appear in adipocytes upon lipolytic stimulation. LDs may grow by spontaneous, homotypic fusion. Results: Scavenging of fatty acids prevents mLD formation. LDs grow by a slow transfer of lipids between LDs. Conclusion: mLDs form due to fatty acid overflow. LD growth is a controlled process. Significance: Novel mechanistic insights into LD remodeling are provided., Synthesis, storage, and turnover of triacylglycerols (TAGs) in adipocytes are critical cellular processes to maintain lipid and energy homeostasis in mammals. TAGs are stored in metabolically highly dynamic lipid droplets (LDs), which are believed to undergo fragmentation and fusion under lipolytic and lipogenic conditions, respectively. Time lapse fluorescence microscopy showed that stimulation of lipolysis in 3T3-L1 adipocytes causes progressive shrinkage and almost complete degradation of all cellular LDs but without any detectable fragmentation into micro-LDs (mLDs). However, mLDs were rapidly formed after induction of lipolysis in the absence of BSA in the culture medium that acts as a fatty acid scavenger. Moreover, mLD formation was blocked by the acyl-CoA synthetase inhibitor triacsin C, implicating that mLDs are synthesized de novo in response to cellular fatty acid overload. Using label-free coherent anti-Stokes Raman scattering microscopy, we demonstrate that LDs grow by transfer of lipids from one organelle to another. Notably, this lipid transfer between closely associated LDs is not a rapid and spontaneous process but rather occurs over several h and does not appear to require physical interaction over large LD surface areas. These data indicate that LD growth is a highly regulated process leading to the heterogeneous LD size distribution within and between individual cells. Our findings suggest that lipolysis and lipogenesis occur in parallel in a cell to prevent cellular fatty acid overflow. Furthermore, we propose that formation of large LDs requires a yet uncharacterized protein machinery mediating LD interaction and lipid transfer.

Triacsin C, an inhibitor of acyl-CoA synthetase, is known to have antiatherosclerotic and vasodilatory activities. The aims of this study were to evaluate the effects of triacsin C on endotoxin-induced (lipopolysaccharide, LPS) inflammation in 3T3-L1 adipocytes and also to evaluate its synergistic effect with triacsin C and resveratrol, a potent antiinflammatory agent. Exposure to LPS for 18 hr increased secretion of IL-6 into the culture medium and mRNA expression of IL-6, MCP-1, TLR and iNOS. Pretreatment of triacsin C for 2 hr suppressed IL-6 accumulation in the medium and the induction of IL-6 expression by LPS, which was more effective than resveratrol treatment. The synergistic effect of triacsin C and resveratrol was found to reduce the expression of iNOS by LPS. However, neither triacsin C nor resveratrol affected the LPS-induced expression of MCP-1, TLR or iNOS. These findings indicate that triacsin C may be a local regulator of inflammation in the adipocyte, although detailed mechanisms are needed to elucidate this through further research.

Long-chain polyunsaturated fatty acids (LCPUFAs) such as docosahexaenoic acid (DHA) and arachidonic acid (AA) are essential for proper development of fetal brain and retina. These LCPUFAs are selectively enriched in the fetal circulation compared with the maternal circulation. In the current study we investigated the transfer of LCPUFAs and a non-essential fatty acid (oleic acid, OA) in a transwell monolayer system of placental choriocarcinoma (BeWo) cells. We show that incubation with OA results in increased triglyceride accumulation and lipid droplet formation compared with that of DHA. The relative amount of transfer of DHA across the cell monolayer was approximately 4-fold greater compared with that of OA when these fatty acids were added individually at 100 muM. This reflects the different fates of these two fatty acids in their metabolism and subsequent transport across the placental trophoblasts to the fetus. When using a mixture of fatty acids mimicking the composition of plasma non-esterified fatty acids during the last trimester of pregnancy, the transfer of OA and the LCPUFAs (DHA and AA) into the basolateral reservoir was not significantly different, whereas the transfer of palmitic acid (PA) was approximately 3.5-fold higher than OA transfer. However, since the concentration of OA compared to LCPUFAs was 10-fold higher in the donor chamber, the relative transport of the LCPUFAs was higher compared with that of OA. In addition, we show that inhibiting esterification of fatty acids into acyl-CoA can modulate, in part, the degree of transport through the cells. In conclusion, the transwell model system closely mimics the mechanisms of differential fatty acid transport as observed in vivo. LCPUFAs were transported through the cells more efficiently than shorter fatty acids such as OA.

ATP-sensitive potassium channels (KATP channels) determine the excitability of pancreatic β-cells and importantly regulate glucose-stimulated insulin secretion (GSIS). Long-chain free fatty acids (FFAs) decrease GSIS after long-term exposure to β-cells, but the effects of exogenous FFAs on KATP channels are not yet well clarified. In this study, the effects of linoleic acid (LA) on membrane potential (MP) and KATP channels were observed in primary cultured rat pancreatic β-cells. LA (20 μM) induced hyperpolarization of MP and opening of KATP channels, which was totally reversed and inhibited by tolbutamide, a KATP channel blocker. Inhibition of LA metabolism by acyl-CoA synthetase inhibitor, triacsin C (10 μM), partially inhibited LA-induced opening of KATP channels by 64%. The non-FFA G protein-coupled receptor (GPR) 40 agonist, GW9508 (40 μM), induced an opening of KATP channels, which was similar to that induced by LA under triacsin C treatment. Blockade of protein kinases A and C did not influence the opening of KATP channels induced by LA and GW9508, indicating that these two protein kinase pathways are not involved in the action of LA on KATP channels. The present study demonstrates that LA induces hyperpolarization of MP by activating KATP channels via both intracellular metabolites and activation of GPR40. It indicates that not only intracellular metabolites of FFAs but also GPR40-mediated pathways take part in the inhibition of GSIS and β-cell dysfunction induced by FFAs.

AMP-activated protein kinase (AMPK) is activated in adipocytes during exercise and other states in which lipolysis is stimulated. However, the mechanism(s) responsible for this effect and its physiological relevance are unclear. To examine these questions, 3T3-L1 adipocytes were treated with cAMP-inducing agents (isoproterenol, forskolin, and isobutylmethylxanthine), which stimulate lipolysis and activate AMPK. When lipolysis was partially inhibited with the general lipase inhibitor orlistat, AMPK activation by these agents was also partially reduced, but the increases in cAMP levels and cAMP-dependent protein kinase (PKA) activity were unaffected. Likewise, small hairpin RNA-mediated silencing of adipose tissue triglyceride lipase inhibited both forskolin-stimulated lipolysis and AMPK activation but not that of PKA. Forskolin treatment increased the AMP:ATP ratio, and this too was reduced by orlistat. When acyl-CoA synthetase, which catalyzes the conversion of fatty acids to fatty acyl-CoA, was inhibited with triacsin C, the increases in both AMPK activity and AMP:ATP ratio were blunted. Isoproterenol-stimulated lipolysis was accompanied by an increase in oxidative stress, an effect that was quintupled in cells incubated with the AMPK inhibitor compound C. The isoproterenol-induced increase in the AMP:ATP ratio was also much greater in these cells. In conclusion, the results indicate that activation of AMPK in adipocytes by cAMP-inducing agents is a consequence of lipolysis and not of PKA activation. They suggest that AMPK activation in this setting is caused by an increase in the AMP:ATP ratio that appears to be due, at least in part, to the acylation of fatty acids. Finally, this AMPK activation appears to restrain the energy depletion and oxidative stress caused by lipolysis.

As previously reported, triacsin C, a selective inhibitor of acyl-CoA synthetase, inhibited the synthesis of cholesteryl ester and triacylglycerol in mouse peritoneal macrophages, leading to a reduction of lipid droplets. Therefore, the in vivo efficacy was studied. Low-density lipoprotein receptor-knockout (LDLR � /� ) mice were fed a high cholesterol diet (0.15%) for two months to measure the atherogenic areas of the hearts and aortas. When triacsin C was orally administered (10 mg/kg/day), the atherosclerotic areas were significantly reduced by 86% in aorta and 36% in hearts. The results strongly suggested that triacsin C shows anti-atherogenic activity by inhibiting acyl-CoA synthetase activity.

Previous studies have reported both positive and negative effects of culture of islets at high glucose concentrations on regulated insulin secretion. Here, we have re-examined this question in mouse islets and determined the role of changes in lipid synthesis in the effects of glucose. Glucose-stimulated insulin secretion (GSIS) and gene expression were examined in islets from C57BL/6 mice or littermates deleted for sterol-regulatory element binding protein-1 (SREBP1) after 4 days of culture at high glucose concentrations. Culture of control islets at 30 versus 8 mmol/1 glucose led to enhanced secretion at both basal (3 mmol/1) and stimulatory (17 mmol/1) glucose concentrations and to enhanced triacylglycerol accumulation. These changes were associated with increases in the expression of genes involved in glucose sensing (glucose transporter 2, glucokinase, sulfonylurea receptor 1, inwardly rectifying K + channel 6.2), differentiation (pancreatic duodenal homeobox 1), and lipogenesis (Srebp1, fatty acid synthase, acetyl-coenzyme A carboxylase 1, stearoyl-coenzyme A desaturase 1). When cultured at either 8 or 30 mmol/1 glucose, SREBP1-deficient (SREBP1 -/- ) islets displayed reduced GSIS and triacylglycerol content compared with normal islets. Correspondingly, glucose induction of the above genes in control islets was no longer observed in SREBP1 -/- mouse islets. We conclude that enhanced lipid synthesis mediated by SREBP1c-dependent genes is required for the adaptive changes in islet gene expression and insulin secretion at high glucose concentrations.

The mere presence of lipids in the intestine is known to reduce nutrient intake in both rodents and humans by activating the intestine–brain neural axis. More recently, studies have indicated that the brain senses blood lipids directly to inhibit glucose production and maintain glucose homeostasis in rodents through a brain–liver neural axis. Now comes the first demonstration that upper intestinal lipids rapidly inhibit glucose production through an intestine–brain–liver neurocircuit. Work in rats shows that the lipids or fats which enter the small intestine trigger the afferent neuronal signal to the brain, which then sends signals to the liver to lower glucose production and blood glucose levels in as little as fifteen minutes. But eating a high-fat diet for just three days can interfere with this signal, disabling it so that it does not signal the other organs to lower blood glucose levels. This holds out the prospect that novel sites and targets may be revealed to lower glucose or blood sugar levels in those who are obese or have diabetes. The presence of lipids in the intestine causes a reduction in nutrient intake, but it is now shown that lipids present in the intestine also regulate endogenous nutrient production. According to the data, putative intra-intestinal lipid sensors signal the presence of ingested lipids, via the brain, to the liver, which reduces the endogenous glucose production accordingly. Energy and glucose homeostasis are regulated by food intake and liver glucose production, respectively. The upper intestine has a critical role in nutrient digestion and absorption. However, studies indicate that upper intestinal lipids inhibit food intake as well in rodents and humans by the activation of an intestine–brain axis1,2,3,4. In parallel, a brain–liver axis has recently been proposed to detect blood lipids to inhibit glucose production in rodents5. Thus, we tested the hypothesis that upper intestinal lipids activate an intestine–brain–liver neural axis to regulate glucose homeostasis. Here we demonstrate that direct administration of lipids into the upper intestine increased upper intestinal long-chain fatty acyl-coenzyme A (LCFA-CoA) levels and suppressed glucose production. Co-infusion of the acyl-CoA synthase inhibitor triacsin C or the anaesthetic tetracaine with duodenal lipids abolished the inhibition of glucose production, indicating that upper intestinal LCFA-CoAs regulate glucose production in the preabsorptive state. Subdiaphragmatic vagotomy or gut vagal deafferentation interrupts the neural connection between the gut and the brain, and blocks the ability of upper intestinal lipids to inhibit glucose production. Direct administration of the N-methyl-d-aspartate ion channel blocker MK-801 into the fourth ventricle or the nucleus of the solitary tract where gut sensory fibres terminate abolished the upper-intestinal-lipid-induced inhibition of glucose production. Finally, hepatic vagotomy negated the inhibitory effects of upper intestinal lipids on glucose production. These findings indicate that upper intestinal lipids activate an intestine–brain–liver neural axis to inhibit glucose production, and thereby reveal a previously unappreciated pathway that regulates glucose homeostasis.

Lipid droplets (LDs) function as intracellular storage depots of neutral lipids. Recently, we identified long-chain acyl-coenzyme A synthetase 3 (ACSL3) as a major LD-associated protein in the human hepatocyte cell line HuH7. In this study, we investigated whether droplet-associated ACSL is involved in lipid metabolism in LDs. Addition of oleic acid (OA) to culture medium was shown to enhance the intracellular accumulation of LDs in the cells, which was accompanied by an increase of droplet ACSL3. When LD-enriched cells induced by OA were further incubated without OA for 3 days, approximately 80% of LDs were retained in the cells. Conversely, cellular LD content was greatly decreased after the addition of an ACSL inhibitor, triacsin C. This was accompanied by a concomitant decrease of the droplet ACSL3. Incubation of isolated LD fractions with (14)C-labeled OA or palmitic acid resulted in [(14)C]acyl-CoA generation in vitro, indicating the presence of ACSL activity in LDs. The droplet ACSL activity varied according to the quantity of LDs in their emergence and disappearance in cells. Incubation of the LD fraction with [(14)C]oleoyl-CoA resulted in radioactive triacylglycerol and cholesteryl esters. These results suggest that LD ACSL activity is involved in local synthesis of neutral lipids and LD formation.

The apicomplexan Cryptosporidium parvum possesses a unique 1500-kDa polyketide synthase (CpPKS1) comprised of 29 enzymes for synthesising a yet undetermined polyketide. This study focuses on the biochemical characterization of the 845-amino acid loading unit containing acyl-[ACP] ligase (AL) and acyl carrier protein (ACP). The CpPKS1-AL domain has a substrate preference for long chain fatty acids, particularly for the C20:0 arachidic acid. When using [3H]palmitic acid and CoA as co-substrates, the AL domain displayed allosteric kinetics towards palmitic acid (Hill coefficient, h=1.46, K50=0.751 microM, Vmax=2.236 micromol mg(-1) min(-1)) and CoA (h=0.704, K50=5.627 microM, Vmax=0.557 micromol mg(-1) min(-1)), and biphasic kinetics towards adenosine 5'-triphosphate (Km1=3.149 microM, Vmax1=373.3 nmol mg(-1) min(-1), Km2=121.0 microM, and Vmax2=563.7 nmol mg(-1) min(-1)). The AL domain is Mg2+-dependent and its activity could be inhibited by triacsin C (IC50=6.64 microM). Furthermore, the ACP domain within the loading unit could be activated by the C. parvum surfactin production element-type phosphopantetheinyl transferase. After attachment of the fatty acid substrate to the AL domain for conversion into the fatty-acyl intermediate, the AL domain is able to transfer palmitic acid to the activated holo-ACP in vitro. These observations ultimately validate the function of the CpPKS1-AL-ACP unit, and make it possible to further dissect the function of this megasynthase using recombinant proteins in a stepwise procedure.

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.

The expression and function of the GH receptor is critical for the actions of pituitary GH in the intact animal. The role of systemic factors in the reduced expression of the GH receptor and consequent GH insensitivity in pathological states such as sepsis, malnutrition, and poorly controlled diabetes mellitus is unclear. In the current study, we demonstrate that saturated (palmitic and myristic; 50 μm) fatty acids (FA) inhibit activity of the promoter of the major (L2) transcript of the GH receptor gene; unsaturated (oleic and linoleic) FA (200 μm) do not alter activity of the promoter. Comparable effects with palmitic acid and the nonmetabolizable analog bromo-palmitic acid, and failure of triacsin C to abrogate palmitic acids effects on GH receptor expression indicate that this effect is due to direct action(s) of FA. Palmitic acid, but not the unsaturated FA linoleic acid, decreased steady-state levels of endogenous L2 mRNA and GHR protein in 3T3-L1 preadipocytes. The effect of FA was localized to two cis elements located approximately 600 bp apart on the L2 promoter. EMSA and chromatin immunoprecipitation assays established that both these cis elements bind the Krüppel-type zinc finger transcription factor, ZBP-89. Ectopic expression of ZBP-89 amplified the inhibitory effect of FA on L2 promoter activity and on steady-state levels of endogenous L2 mRNA in 3T3-L1 preadipocytes. Mutational analyses of the two ZBP-89 binding sites revealed that both the sites are essential for palmitic acid’s inhibitory effect on the L2 promoter and for the enhancing effect of ZBP-89 on palmitic acid-induced inhibition of the L2 promoter. Our results establish a molecular basis for FA-induced inhibition of GH receptor gene expression in the pathogenesis of acquired GH insensitivity in pathological states such as poorly controlled diabetes mellitus and small for gestational age.

It is well documented that arachidonic acid (AA) and its metabolites are intimately linked to cancer biology. However, the downstream mechanism(s) that link AA levels to cancer cell proliferation remain to be elucidated. Initial experiments in the current study showed that exogenous AA and inhibitors of AA metabolism that lead to the accumulation of unesterified AA are cytotoxic to the colon cancer cell line, HCT-116. Additionally, exogenous AA and triacsin C, an inhibitor of AA acylation, induced apoptosis and related caspase-3 activity in a transcriptionally dependent manner. Gene array analysis revealed that both exogenous AA and triacsin C alter the expression of similar genes in HCT-116 cells. For example, both down-regulate several genes with well-documented roles in cell survival and apoptotic resistance. Conversely, both upregulate genes encoding activator protein-1 (AP-1) transcription factors, which have known roles in inducing apoptosis, and genes that counteract ras (Erk/MAPK) growth signaling pathways. Real-time polymerase chain reaction and immunoblotting demonstrated that mRNA and protein levels of one of the major AP-1 transcription factors, c-Jun, is markedly elevated by exogenous AA and triacsin C. Additionally, the cyclooxygenase inhibitor, sulindac sulfide, increases c-Jun mRNA levels. Together, these studies reveal that the generation of intracellular AA and its subsequent impact on gene expression probably represents a critical step that regulates colon cancer cell proliferation.

Arachidonic acid (AA) and its metabolites are intimately linked to carcinogenesis. Inhibitors of AA metabolic enzymes have demonstrated anti-carcinogenic effects in vivo and induce apoptosis of many cancer cell lines in vitro. The mechanism by which AA influences carcinogenesis, however, remains unresolved. The current study explores the growth inhibitory potential of Triacsin C, PLT-98625, and NS-398 which inhibit three distinct metabolic enzymes that control intracellular AA levels: fatty acid coenzyme-A ligase 4 (FACL-4), coenzyme-A independent transacylase (CoA-IT), and cyclooxygenase (COX), respectively. Results reveal the anti-proliferative effects of these inhibitors in a number of human cancer cell lines. Further studies in the SK-MES-1 cell line demonstrate that all three inhibitors induce accumulation of unesterified AA which correlates with induction of apoptosis. Addition of exogenous AA also induces apoptosis. Furthermore, in combination, these inhibitors act cooperatively to induce AA accumulation which correlates to a synergistic reduction in cell viability. Taken together, these results suggest that accumulation of unesterified AA is a common mechanism in the induction of cancer cell apoptosis by various inhibitors of AA metabolism, confirm that previously described AA remodeling pathways are valid in cancer cells, and indicate that combination treatment strategies utilizing these inhibitors may represent a novel approach to blocking cancer cell growth. Further study is required to determine the downstream pathway(s) whereby high cellular burdens of unesterified AA promote apoptosis.

Abnormal high density lipoprotein (HDL) metabolism among patients with diabetes and insulin resistance may contribute to their increased risk of atherosclerosis. ATP-binding cassette transporter ABCA1 mediates the transport of cholesterol and phospholipids from cells to HDL apolipoproteins and thus modulates HDL levels and atherogenesis. Unsaturated fatty acids, which are elevated in diabetes, impair the ABCA1 pathway in cultured cells by destabilizing ABCA1 protein. Here we examined the cellular pathway that mediates the ABCA1 destabilizing effects of fatty acids. The long-chain acyl-CoA synthetase inhibitor triacsin C completely reversed fatty acid-induced ABCA1 destabilization, indicating that fatty acids need to be activated to their CoA derivatives to enhance ABCA1 degradation. Unsaturated but not saturated fatty acids stimulated phospholipase D (PLD) activity, the PLD inhibitor 1-butanol prevented the unsaturated fatty acid-induced reduction in ABCA1 levels, and the PLD2 activator mastoparan markedly reduced ABCA1 protein levels, implicating a role for PLD2 in the ABCA1 destabilizing effects of fatty acids. Unsaturated fatty acids and mastoparan increased phosphorylation of ABCA1 serines. PLD2 small interfering RNA abolished the ability of unsaturated fatty acids to inhibit lipid transport activity, to reduce protein levels, and to increase serine phosphorylation of ABCA1. The diacylglycerol analog oleoylacetylglycerol also reduced ABCA1 protein levels and increased its serine phosphorylation, suggesting that PLD2-generated diacylglycerols promote the destabilizing phosphorylation of ABCA1. These data provide evidence that intracellular unsaturated acyl-CoA derivatives destabilize ABCA1 by activating a PLD2 signaling pathway.

We have described that, in adrenal and Leydig cells, the hormonal regulation of free arachidonic acid (AA) concentration is mediated by the concerted action of two enzymes: an acyl-CoA thioesterase (MTE-I or ARTISt) and an acyl-CoA synthetase (ACS4). In this study we analyzed the potential regulation of these proteins by hormonal action in steroidogenic cells. We demonstrated that ACS4 is rapidly induced by adrenocorticotropin (ACTH) and cAMP in Y1 adrenocortical cells. The hormone and its second messenger increased ACS4 protein levels in a time and concentration dependent way. Maximal concentration of ACTH (10 mIU/ml) produced a significant effect after 15 min of treatment and exerted the highest increase (3-fold) after 30 min. Moreover, 35S-methionine incorporation showed that the increase in ACS4 protein levels is due to an increase in the de novo synthesis of the protein. On the contrary MTE-I protein levels in Y1 and MA-10 cells did not change after steroidogenic stimuli. In contrast with the effect observed on protein levels, stimulation of both cell lines did not change ACS4 RNA levels during the first hour of treatment, indicating that the effect of both stimuli is exerted at the level of ACS4 protein synthesis. StAR protein induction has a key role on the activation of steroidogenesis since this protein increases the rate of the limiting step of the whole process. In agreement with the fact that the inhibition of ACS4 activity by triacsin C blocks cAMP-stimulated progesterone production by MA-10 Leydig cells, here we demonstrated that ACS4 inhibition also reduces StAR protein levels. Moreover, exogenous AA was able to overcome the effect of triacsin C on both events, StAR induction and steroidogenesis. These results were confirmed by experiments using ACS4-targeted siRNA which result in a reduction in both ACS4 and StAR protein levels. The concomitant decrease in steroid production was overcome by the addition of AA to the knocked-out cells. In summary, this study suggests that in adrenal and Leydig cells the hormonal action prompts the synthesis of a labile protein, ACS4, which activity is involved in the regulation of AA release and is essential for steroidogenesis and StAR protein induction.

Background: Although cancer cells appear to maintain the machinery for intrinsic apoptosis, defects in the pathway develop during malignant transformation, preventing apoptosis from occurring. How to specifically induce apoptosis in cancer cells remains unclear. Methods: We determined the apoptosome activity and p53 status of normal human cells and of lung, colon, stomach, brain, and breast cancer cells by measuring cytochrome c-dependent caspase activation and by DNA sequencing, respectively, and we used COMPARE analysis to identify apoptosome-specific agonists. We compared cell death, cytochrome c release, and caspase activation in NCI-H23 (lung cancer), HCT-15 (colon cancer), and SF268 (brain cancer) cells treated with Triacsin c, an inhibitor of acyl-CoA synthetase (ACS), or with vehicle. The cells were mock, transiently, or stably transfected with genes for Triacsin c-resistant ACSL5, dominant negative caspase-9, or apoptotic protease activating factor-1 knockdown. We measured ACS activity and levels of cardiolipin, a mitochondrial phospholipid, in mock and ACSL5-transduced SF268 cells. Nude mice carrying NCI-H23 xenograft tumors (n = 10) were treated with Triacsin c or vehicle, and xenograft tumor growth was assessed. Groups were compared using two-sided Student t tests. Results: Of 21 p53-defective tumor cell lines analyzed, 17 had higher apoptosome activity than did normal cells. Triacsin c selectively induced apoptosome-mediated death in tumor cells (caspase activity of Triacsin c-treated versus untreated SF268 cells; means = 1020% and 100%, respectively; difference = 920%, 95% CI = 900% to 940%; P

Fatty acid transport protein 4 (FATP4) is an integral membrane protein expressed in the plasma and internal membranes of the small intestine and adipocyte as well as in the brain, kidney, liver, skin, and heart. FATP4 has been hypothesized to be bifunctional, exhibiting both fatty acid transport and acyl-CoA synthetase activities that work in concert to mediate fatty acid influx across biological membranes. To determine whether FATP4 is an acyl-CoA synthetase, the murine protein was engineered to contain a C-terminal FLAG epitope tag, expressed in COS1 cells via adenovirus-mediated infection and purified to near homogeneity using alpha-FLAG affinity chromatography. Kinetic analysis of the enzyme was carried out for long chain (palmitic acid, C16:0) and very long chain (lignoceric acid, C24:0) fatty acids as well as for ATP and CoA. FATP4 exhibited substrate specificity for C16:0 and C24:0 fatty acids with a V(max)/K(m) (C16:0)/V(max)/K(m) (C24:0) of 1.5. Like purified FATP1, FATP4 was insensitive to inhibition by triacsin C but was sensitive to feedback inhibition by acyl-CoA. Although purified FATP4 exhibited high levels of palmitoyl-CoA and lignoceroyl-CoA synthetase activity, extracts from the skin and intestine of FATP4 null mice exhibited reduced esterification for C24:0, but not C16:0 or C18:1, suggesting that in vivo, defects in very long chain fatty acid uptake may underlie the skin disorder phenotype of null mice.

Background: Triacsin C, a fatty acid analog, inhibits endothelial nitric oxide synthetase (eNOS) palmitoylation, increases nitric oxide synthesis and enhances methacholine-induced relaxation of vascular rings. The experiments presented here tested the hypothesis that triacsin C increases the synthesis of PGI2 and/or endothelial-derived hyperpolarizing factor. Methods: Long-chain fatty acyl CoA synthetase activity (LCFACoAS), agonist-induced prostacyclin synthesis and agonist-induced release of radioactivity in endothelial cells labeled with [3H]arachidonic acid were measured in the presence and absence of triacsin C. Results: Inhibition by triacsin C of palmitoyl CoA formation was significantly greater than inhibition of arachidonoyl CoA formation in solubilized endothelial cell preparations. While 24-hour triacsin C treatment significantly reduced basal 6-keto synthesis, it had no effect on agonist-stimulated synthesis. The release of arachidonic acid metabolites was examined in [3H]arachidonate-labeled cells. Triacsin C treatment had no effect on basal or vasopressin-, angiotensin-II-, bradykinin- or ionomycin-induced release of radioactivity, but significantly reduced release in response to isoproterenol or phenylephrine. Expression of neither immunoreactive eNOS nor immunoreactive inducible nitric oxide synthetase (iNOS) was changed by triacsin C treatment, but the fraction of immunoreactive eNOS in the cytoplasm of treated cells was significantly greater as compared to vehicle control cells. Phorbol myristoyl acetate or fenofibrate significantly increased in vitro LCFACoAS activity, and significantly decreased the nitrite/eNOS ratio. Conclusions: These data indicate that, while triacsin C can inhibit arachidonoyl CoA synthetase in endothelial cells, it does not increase the availability of endogenous substrate for basal or agonist-induced PGI2 synthesis, nor does it enhance release of arachidonic acid or its metabolites.

Circulating interleukin-6 (IL-6), insulin, and free fatty acid (FFA) concentrations are associated with impaired insulin action in obese and type 2 diabetic individuals. However, a causal relationship between elevated plasma FFAs and IL-6 has not been shown. Because skeletal muscle represents a major target of impaired insulin action, we studied whether FFAs may affect IL-6 expression in human myotubes. We demonstrate that specifically saturated FFAs, e.g. palmitate (0.25 mm), induce IL-6 mRNA expression and protein secretion by a proteasome-dependent mechanism that leads to a rapid and chronic activation of nuclear factor-κB. Insulin, high glucose concentrations, or unsaturated FFAs did not activate IL-6 expression. In fact, the unsaturated FFA linoleate inhibited palmitate-induced IL-6 production. Because inhibition of palmitate metabolism by the acyl-CoA synthetase inhibitor triacsin C did not abolish IL-6 expression, it appears that the palmitate molecule per se exerts the observed effects. Furthermore, we show that in human myotubes, IL-6 activates the phosphorylation of signal transducer and activator of transcription 3 in concentrations similar to hepatocytes. However, no inhibitory effect of IL-6 on insulin action, determined as phosphatidylinositol 3-kinase association with insulin receptor substrate-1, Akt phosphorylation, and glycogen synthesis, was detected. We conclude that IL-6 expression may be modulated by the composition of circulating FFA, e.g. by diet, and that skeletal muscle cells could be target cells for IL-6.