Inhibition Of Fatty Acid Synthesis Research Articles

Cellular pharmacokinetics and intracellular activity of the bacterial fatty acid synthesis inhibitor, afabicin desphosphono against different resistance phenotypes of Staphylococcus aureus in models of cultured phagocytic cells

Antibiotics with new modes of action that are active against intracellular forms of Staphylococcus aureus are sorely needed to fight recalcitrant infections caused by this bacterium. Afabicin desphosphono (Debio 1452, the active form of afabicin [Debio1450]) is an inhibitor of FabI enoyl-Acyl carrier protein reductase and has specific and extremely potent activity against Staphylococci, including strains resistant to current antistaphylococcal agents. Using mouse J774 macrophages and human THP-1 monocytes, we showed that afabicin desphosphono: (i) accumulates rapidly in cells, reaching stable cellular-to-extracellular concentration ratios of about 30; (ii) is recovered entirely and free in the cell-soluble fraction (no evidence of stable association with proteins or other macromolecules). Afabicin desphosphono caused a maximum cfu decrease of about 2.5 log10 after incubation in broth for 30h, including against strains resistant to vancomycin, daptomycin, and/or linezolid. Using a pharmacodynamic model of infected THP-1 monocytes (30h of incubation post-phagocytosis), we showed that afabicin desphosphono is bacteriostatic (maximum cfu decrease: 0.56 to 0.73 log10) towards all strains tested, a behaviour shared with the comparators (vancomycin, daptomycin, and linezolid) when tested against susceptible strains. We conclude that afabicin desphosphono has a similar potential as vancomycin, daptomycin or linezolid to control the intracellular growth and survival of phagocytized S.aureus and remains fully active against strains resistant to these comparators.

Leelamine Is a Novel Lipogenesis Inhibitor in Prostate Cancer Cells In Vitro and In Vivo.

Increased de novo synthesis of fatty acids is implicated in the pathogenesis of human prostate cancer, but a safe and effective clinical inhibitor of this metabolic pathway is still lacking. We have shown previously that leelamine (LLM) suppresses transcriptional activity of androgen receptor, which is known to regulate fatty acid synthesis. Therefore, the current study was designed to investigate the effect of LLM on fatty acid synthesis. Exposure of 22Rv1, LNCaP, and PC-3 prostate cancer cells, but not RWPE-1 normal prostate epithelial cell line, to LLM resulted in a decrease in intracellular levels of neutral lipids or total free fatty acids. LLM was superior to another fatty acid synthesis inhibitor (cerulenin) for suppression of total free fatty acid levels. LLM treatment downregulated protein and/or mRNA expression of key fatty acid synthesis enzymes, including ATP citrate lyase, acetyl-CoA carboxylase 1, fatty acid synthase, and sterol regulatory element-binding protein 1 (SREBP1) in each cell line. Consistent with these in vitro findings, we also observed a significant decrease in ATP citrate lyase and SREBP1 protein expression as well as number of neutral lipid droplets in vivo in 22Rv1 tumor sections of LLM-treated mice when compared with that of controls. LLM-mediated suppression of intracellular levels of total free fatty acids and neutral lipids was partly attenuated by overexpression of SREBP1. In conclusion, these results indicate that LLM is a novel inhibitor of SREBP1-regulated fatty acid/lipid synthesis in prostate cancer cells that is not affected by androgen receptor status.

Extending the Metabolite Diversity of the Endophyte Dimorphosporicola tragani.

Fungi are one of the most prolific sources of microbial secondary metabolites. The production of new metabolites can be achieved using multiple fermentation conditions and by adding small-molecule effectors, including epigenetic modifiers. In the framework of our Natural Product screening programme targeting the discovery of new antimicrobial compounds, we applied multiple fermentation conditions and adsorptive polymeric resins on a large collection of fungal endophytes, to increase and stimulate their fungal secondary metabolite production. During this work the endophytic fungus Dimorphosporicola tragani CF-090383 showed antimicrobial activity only when grown in presence of adsorptive polymeric resins. In addition, seven epigenetic modifiers were added to fermentations of this endophytic fungus, in an attempt to activate its cryptic pathways as well as to analyse the metabolites produced under these conditions. D. tragani was seen to produce three different mycotoxin dendrodolides when the epigenetic modifiers 5-azacytidine and valproic acid were added to the fermentations, and these compounds were further characterized. However, the fungus produced the fatty acid synthesis inhibitor cerulenin, a molecule not previously described to be produced by this fungal species, only when cultivated in presence of the XAD-16 resin. We have found that the addition of XAD-16 resin resulted in four-fold higher titers in the production of cerulenin when compared to the best production conditions described in literature for the original fungal producer strain, Cephalosporium caerulens KF-140 (=Sarocladium oryzae), in a zeolite-based fermentation, used as an ammonium ion-trapping agent. The production of cerulenin by this strain of D. tragani, represents an alternative source for the improved production of cerulenin with better yields.

The MYC Oncogene Cooperates with Sterol-Regulated Element-Binding Protein to Regulate Lipogenesis Essential for Neoplastic Growth

Lipid metabolism is frequently perturbed in cancers, but the underlying mechanism is unclear. We present comprehensive evidence that oncogene MYC, in collaboration with transcription factor sterol-regulated element-binding protein (SREBP1), regulates lipogenesis to promote tumorigenesis. We used human and mouse tumor-derived cell lines, tumor xenografts, and four conditional transgenic mouse models of MYC-induced tumors to show that MYC regulates lipogenesis genes, enzymes, and metabolites. We found that MYC induces SREBP1, and they collaborate to activate fatty acid (FA) synthesis and drive FA chain elongation from glucose and glutamine. Further, by employing desorption electrospray ionization mass spectrometry imaging (DESI-MSI), we observed invivo lipidomic changes upon MYC induction across different cancers, for example, aglobal increase in glycerophosphoglycerols. After inhibition of FA synthesis, tumorigenesis was blocked, and tumors regressed in both xenograft and primary transgenic mouse models, revealing the vulnerability of MYC-induced tumors to the inhibition of lipogenesis.

Abstract 5081: Leelamine is a novel inhibitor of fatty acid synthesis in prostate cancer

Abstract Increased beta-oxidation of fatty acids to meet energy demand is a rather unique characteristic of a subset of human prostate cancers. A safe and effective intervention for inhibition of fatty acid synthesis is still a clinically unmet need. We have shown previously that leelamine (LLM), a phytochemical derived from pine tree bark, suppresses expression and activity of full-length androgen receptor (AR) and its splice variants in vitro and in vivo in preclinical models of prostate cancer. Because AR is implicated in regulation of fatty acid metabolism, the present study was undertaken to determine the effect of LLM on this metabolic pathway. Treatment of a castration-resistant (22Rv1) as well as an androgen-responsive (LNCaP) human prostate cancer cell line with LLM (2.5 and 5 µmol/L) resulted in downregulation of key fatty acid synthesis enzyme proteins including ATP citrate lyase (ACLY), acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and sterol regulatory element-binding protein 1 (SREBP1). LLM treatment also decreased intracellular levels of total free fatty acids and neutral lipid droplets in LNCaP and 22Rv1 cells. Consistent with the in vitro results, we also observed a significant decrease in ACLY and SREBP1 protein expression and number of neutral lipid droplets in vivo in tumor tissue sections of 22Rv1 xenografts after intraperitoneal administration of LLM (9.1 mg/kg bw/ day, 5 times/week) compared to controls. Studies are in progress to determine if overexpression of AR and/or SREBP1 confers protection against fatty acid synthesis inhibition by LLM. In conclusion, it is reasonable to postulate that suppression of AR-SREBP1 regulated fatty acid metabolism is an important mechanism in prostate cancer inhibition by LLM. This study was supported by the grant RO1 CA101753 awarded by the National Cancer Institute. Citation Format: Krishna B. Singh, Shivendra V. Singh. Leelamine is a novel inhibitor of fatty acid synthesis in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5081.

Abstract 1846: Acid sensing G protein-coupled receptor OGR1 is required for acid induced adiposomogenesis in breast cancer cells

Abstract Malignant tumors exhibit altered metabolism and consume higher levels of glucose compared to surrounding normal tissue, resulting in acidic extracellular microenvironment. Acidity in the microenvironment is a critical stress factor and selection force for the evolution of aggressive tumor types. There are several acid sensing cell surface receptors and ion channels (ASICs) that can sense acidity in the microenvironment; among them proton sensing G protein-coupled receptors (GPCRs) such as OGR1 (GPR68), TDAG8 (GPR65), GPR4 and GPR132 form a major class of acid receptors. Our previous studies demonstrated that acid adaptation is associated with survival mechanisms like chronic activation of autophagy and redistribution of the lysosomal proteins to the plasma membrane. When grown under acidic pH, breast cancer cells accumulate lipids as revealed by staining with Nile Red and perilipin 2, a protein that coats adiposomes or lipid droplets. These are dynamic organelles that store neutral lipids surrounded by a shell of proteins and a phospholipid monolayer. The lipids stored in adiposomes are produced de novo, as acid-induced lipogenic phenotype is maintained, even if cells are grown with de-lipidated serum. Inhibition of fatty acid synthesis was selectively toxic under acidic conditions and attenuated adiposome accumulation. Among the acid sensing receptors, TDAG8 and OGR1 are highly expressed in a panel of breast cancer cell lines compared to non-malignant breast epithelial cells. Highly invasive and acid tolerant MDA-MB-231 cells express significantly higher levels of TDAG8 while MCF7 and T47D cells have high levels of OGR1. We investigated the role of these receptors in transducing the acid signal that results in the accumulation of lipid droplets. CRISPR/Cas9 mediated depletion of the major acid sensors in breast cancer cell lines MCF7 and T47D showed that depletion of OGR1, not TDAG8, specifically inhibited acid induced adiposome accumulation. Additionally, inducing the cells with ogerin, an allosteric activator of OGR1 resulted in adiposomogenesis. Further, OGR1 knock out cells showed inhibition in cell growth under acidic growth conditions compared to OGR1 expressing cells. OGR1 knockout cells were defective in acid induced autophagy. OGR1 is coupled with Gq/11 and, upon ligand (H+) binding, triggers activation of phospholipase C stimulating the formation of the second messenger IP3. Adiposome formation was inhibited in presence of PLC inhibitors, Edelfosine or U73122. These results indicate that OGR1 is the major acid sensing GPCR in these cells that mediate adiposomogenesis. Accumulation of adiposomes is a highly regulated process related to storing autophagic products, and appears to be important in cell survival in acid stress. Taken together, increased dependence on lipid metabolism by cancer cells under acidic conditions reveals novel therapeutic vulnerabilities. Citation Format: Smitha Pillai, Michael Langsen, Jonathan Nguyen, Jonathan Wojtkowiak, Marilyn Bui, Robert Gatenby, Robert Gillies. Acid sensing G protein-coupled receptor OGR1 is required for acid induced adiposomogenesis in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1846.

Autophagy-Mediated Regulation of Lipid Metabolism and Its Impact on the Growth in Algae and Seed Plants.

Under nutrient starvation conditions, algae and seed-plant cells accumulate carbon metabolites such as storage lipids, triacylglycerols (TAGs), and starches. Recent research has suggested the involvement of autophagy in the regulation of carbon metabolites under nutrient starvation. When algae are grown under carbon starvation conditions, such as growth in darkness or in the presence of a photosynthesis inhibitor, lipid droplets are surrounded by phagophores. Indeed, the amount of TAGs in an autophagy-deficient mutant has been found to be greater than that in wild type under nitrogen starvation, and cerulenin, which is one of the inhibitors of fatty acid synthesis, induces autophagy. In land plants, TAGs accumulate predominantly in seeds and etiolated seedlings. These TAGs are degraded in peroxisomes via β-oxidation during germination as a source of carbon for growth without photosynthesis. A global analysis of the role of autophagy in Arabidopsis seedlings under carbon starvation revealed that a lack of autophagy enhances the accumulation of TAGs and fatty acids. In Oryza sativa, autophagy-mediated degradation of TAGs and diacylglycerols has been suggested to be important for pollen development. In this review, we introduce and summarize research findings demonstrating that autophagy affects lipid metabolism and discuss the role of autophagy in membrane and storage-lipid homeostasis, each of which affects the growth and development of seed plants and algae.

Inhibition of fatty acid metabolism by etomoxir or TOFA suppresses murine dendritic cell activation without affecting viability

Objective: Dendritic cells (DCs) are important players in immunity against pathogens, but overactive DCs have been implicated in autoimmune diseases, like lupus, in which a paucity of targeted therapies remains. Recent research shows that DCs upregulate their immunometabolism when activating. We explored whether modulating fatty acid (FA) metabolism needed for oxidative phosphorylation can affect the activation of two main DC subsets.Material and methods: Sorted murine plasmacytoid DCs (pDCs) and conventional DCs (cDCs), generated in FLT3-L medium, were treated with etomoxir, an inhibitor of FA oxidation, or TOFA, an inhibitor of FA synthesis, then stimulated with TLR9 agonist CpGA. Surface activation markers and viability were analyzed by flow cytometry, cytokine, and chemokine production and were measured by ELISA.Results: Modulation of FA metabolism suppressed the upregulation of costimulatory molecules and the production of proinflammatory cytokine IL-6 and type I Interferon-dependent chemokine CXCL10 by both subsets of DCs, without affecting DC viability, neither of resting DCs or upon activation. Etomoxir inhibited pDCs at lower doses than cDCs, suggesting that pDCs may be more susceptible to FA metabolic modulation.Conclusions: Both cDCs, the primary antigen presenting cell, and pDCs, the primary type I IFN producer, exhibit a suppressed ability to activate but normal viability when their FA metabolism is inhibited by etomoxir or TOFA. Our findings indicate that FA metabolism plays an important role in the activation of both pDCs and cDCs and suggest that its modulation is an exploitable therapeutic target to suppress DC activation in inflammation or autoimmunity.

The Widely Used Antimicrobial Triclosan Induces High Levels of Antibiotic Tolerance In Vitro and Reduces Antibiotic Efficacy up to 100-Fold In Vivo.

The antimicrobial triclosan is used in a wide range of consumer products ranging from toothpaste, cleansers, socks, and baby toys. A bacteriostatic inhibitor of fatty acid synthesis, triclosan is extremely stable and accumulates in the environment. Approximately 75% of adults in the United States have detectable levels of the compound in their urine, with a sizeable fraction of individuals (>10%) having urine concentrations equal to or greater than the minimal inhibitory concentration for Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Previous work has identified connections between defects in fatty acid synthesis and accumulation of the alarmone guanosine tetraphosphate (ppGpp), which has been repeatedly associated with antibiotic tolerance and persistence. Based on these data, we hypothesized that triclosan exposure may inadvertently drive bacteria into a state in which they are able to tolerate normally lethal concentrations of antibiotics. Here we report that clinically relevant concentrations of triclosan increased E. coli and MRSA tolerance to bactericidal antibiotics as much as 10,000-fold in vitro and reduced antibiotic efficacy up to 100-fold in a mouse urinary tract infection model. Genetic analysis indicated that triclosan-mediated antibiotic tolerance requires ppGpp synthesis but is independent of growth. These data highlight an unexpected and certainly unintended consequence of adding high concentrations of antimicrobials in consumer products, supporting an urgent need to reevaluate the costs and benefits of the prophylactic use of triclosan and other bacteriostatic compounds.

Inhibition of fatty acid synthesis arrests colorectal neoplasm growth and metastasis: Anti-cancer therapeutical effects of natural cyclopeptide RA-XII

Emerging evidence has shown that metabolism, in particular the synthesis of fatty acids, has great significance for growth and metastasis of colorectal neoplasm. The previous results showed that RA-XII, a natural cyclopeptide isolated from Rubia yunnanensis, inhibits tumor growth and metastasis by AMPK/mTOR/P70S6K pathway and PI3K/AKT/NF-κB pathway. But if or not lipid metabolism involves the antitumor mechanism of RA-XII is not clear. Herein the results indicated that RA-XII reduced the cell motility by decreasing the expressions of β-catenin and β-catenin dependent proteins CD44 and MMP7 in HCT116 cells. Then RA-XII effectively reduced fatty acids levels by decreasing the expression of SREBP-1 and inhibiting the expressions of de novo fatty acid synthesis proteins FASN and SCD. Moreover the decreased cell motility caused by RA-XII was attenuated with the SREBP-1 knockdown. In addition, the in vivo experiments also demonstrated that RA-XII inhibited tumor growth and metastasis via restraining lipogenesis in colorectal neoplasm mouse models. Taken together, these results indicated that RA-XII suppressed the colorectal neoplasm growth and metastasis by inhibition of lipogenesis depended on SREBP-1 suppression.

Modulation of the central carbon metabolism of Corynebacterium glutamicum improves malonyl-CoA availability and increases plant polyphenol synthesis.

In recent years microorganisms have been engineered towards synthesizing interesting plant polyphenols such as flavonoids and stilbenes from glucose. Currently, the low endogenous supply of malonyl-CoA, indispensable for plant polyphenol synthesis, impedes high product titers. Usually, limited malonyl-CoA availability during plant polyphenol production is avoided by supplementing fatty acid synthesis-inhibiting antibiotics such as cerulenin, which are known to increase the intracellular malonyl-CoA pool as a side effect. Motivated by the goal of microbial polyphenol synthesis being independent of such expensive additives, we used rational metabolic engineering approaches to modulate regulation of fatty acid synthesis and flux into the tricarboxylic acid cycle (TCA cycle) in Corynebacterium glutamicum strains capable of flavonoid and stilbene synthesis. Initial experiments showed that sole overexpression of genes coding for the native malonyl-CoA-forming acetyl-CoA carboxylase is not sufficient for increasing polyphenol production in C. glutamicum. Hence, the intracellular acetyl-CoA availability was also increased by reducing the flux into the TCA cycle through reduction of citrate synthase activity. In defined cultivation medium, the constructed C. glutamicum strains accumulated 24 mg·L -1 (0.088 mM) naringenin or 112 mg·L -1 (0.49 mM) resveratrol from glucose without supplementation of phenylpropanoid precursor molecules or any inhibitors of fatty acid synthesis.

N-Benzylanilines as Fatty Acid Synthesis Inhibitors against Biofilm-related Methicillin-resistant Staphylococcus aureus.

Bacterial fatty acid synthase system is a well validated target for the development of novel antimicrobial agents. This study reports the synthesis of Schiff bases and their reductive N-benzylanilines. Most N-benzylanilines were active against Gram-positive bacteria, among which compound 4k performed best against both S. aureus and MRSA with the MIC value at 0.5 mg/L. Moreover, we identified the strong antibacterial activity for compound 4k against 19 clinical MRSA strains isolated from different specimen, which indicated its potential in clinical application. In vitro biofilm inhibition and microscopy assay revealed compound 4k inhibits biofilm formation and eradicates preformed biofilm effectively. The size-exclusion chromatography and docking study indicated that compound 4k mimics the binding mode of triclosan with saFabI. The efficiency of the protein-inhibitor interaction was evaluated by measuring NADPH reduction using trans-2-octenoyl-CoA as substrate. Overall, our data demonstrate that N-benzylaniline is a promising scaffold for anti-staphylococcal drug development.

Characterization of very long chain fatty acid synthesis inhibition by ipfencarbazone.

Ipfencarbazone exhibits excellent herbicidal activity against Echinochloa spp. and is safe for rice. The effects of ipfencarbazone on very long chain fatty acid (VLCFA) elongation in rice and late watergrass and its inhibitory mechanism were investigated in this study. Although ipfencarbazone inhibited VLCFA elongation in the microsomes prepared from late watergrass and rice at low concentrations, the inhibitory effect was higher in late watergrass than in rice. These results suggested that the primary site of action of ipfencarbazone is VLCFA elongase (VLCFAE) and ipfencarbazone has a differential affinity between the VLCFAEs of the plants. The inhibitory activity of ipfencarbazone became higher in proportion to pre-incubation period with the VLCFAE. The degree of inhibition did not decrease by dilution of the VLCFAE-ipfencarbazone complex. These results suggested that ipfencarbazone binds to the VLCFAE irreversibly.

Regulation of Stearoyl-Coenzyme A Desaturase 1 by trans-10,cis-12 Conjugated Linoleic Acid via SREBP1 in Primary Goat Mammary Epithelial Cells

trans-10, cis-12 Conjugated linoleic acid (t10c12-CLA) is a biohydrogenation intermediate in the rumen that inhibits mammary fatty acid de novo synthesis in lactating dairy goats. However, the underlying molecular pathways in milk-lipid metabolism affected by t10c12-CLA are not completely understood. The present study investigated the lipid-regulation mechanisms in goat mammary epithelial cells (GMECs) in response to t10c12-CLA. Gene-expression analysis indicated sterol-regulatory-element-binding transcription factor1 ( SREBF1) and its putative target gene stearoyl-CoA desaturase ( SCD1) were down-regulated (fold changes of 0.33 ± 0.04, P < 0.05, and 0.19 ± 0.01, P < 0.01, respectively). Concentrations of cellular palmitoleic acid (C16:1) and oleic acid (C18:1) were decreased (1.12 ± 0.05 vs 1.69 ± 0.11% and 15.70 ± 0.44 vs 24.97 ± 0.82%, respectively, P < 0.01), whereas those of linoleic acid (C18:2) were increased (5.00 ± 0.14 vs 3.81 ± 0.25%, P < 0.05); the desaturation indices of C16 and C18 were decreased in response to t10c12-CLA treatment (6.90 ± 0.05 vs 8.00 ± 0.30% and 61.41 ± 0.65 vs 67.73 ± 1.33%, respectively, P < 0.05). A luciferase-activity assay indicated that deletion of the sterol-response-element (SRE) site and the nuclear-factor (NF-Y) site in the SCD1-promoter region (-511/+65 bp) suppressed the regulatory effect of t10c12-CLA. Overexpression of SREBF1 partly counteracted the inhibitory effect of t10c12-CLA on de novo fatty acid synthesis. Overall, t10c12-CLA causes an inhibition of fatty acid synthesis and desaturation and regulates SCD1 expression by affecting the binding of SREBP1 protein to the SRE and NF-Y sites.

A method for the gross analysis of global protein acylation by gas-liquid chromatography.

Protein acylation is a posttranslational modification in which an amino acid residue of a protein is acylated by a fatty acid. This process plays a key role in regulating proteomic function. Studies of protein acylation have relied on the development and application of extremely complicated molecular methods. However, global protein acylation can be profiled following hydrolysis of fatty acyl groups from cellular proteins. The present study aimed to develop a method for analysis of global protein acylation using gas-liquid chromatography (GLC). The total protein was extracted from the human hepatocellular carcinoma (HepG2) cell line. Protein sedimentation and extensive wash were combined with differential O-, S-, or N-acyl hydrolysis using sodium hydroxide (NaOH), hydroxylamine (NH2 OH), or hydrochloric acid (HCl), respectively. GLC with a flame ionization detector system was used to analyze changes in the fatty acid composition of the released lipids. The effect of selective inhibition of monounsaturated fatty acid (MUFA) synthesis on global protein acylation and the expression of reprogramming markers were determined to further validate the proposed profiling approach. In all hydrolysis conditions, the amount of myristate released was significantly higher than of other fatty acids. Notable differences were observed in the release of individual fatty acids among the hydrolyzing agents. Only NH2 OH could release significant amounts of palmitoleate (>2.5-fold vs. NaOH and HCl). The acylation assay indicates that treatment with a chemical inhibitor of monounsaturated fatty acid synthesis led to an overall increase in saturated fatty acid O- and N-acylation, and a decrease in palmitoleate O- and S-acylation of cellular proteins (<-15%). This was accompanied by significant reductions in the gene expression of the reprogramming markers Oct4 (-26%, P < 0.01) and Sox2 (-40%, P < 0.01). GLC-based analysis of global protein acylation affords a semi-quantitative method that can be used to assess the gross changes in the protein acylation profile during cell differentiation and reprogramming. © 2018 IUBMB Life, 71(3):340-346, 2019.

20(S)-Protopanaxatriol Promotes Fatty Acid-Induced ER Stress and Apoptosis in Multiple Myeloma By Down-Regulating SCD1 Expression

Multiple myeloma (MM), is a hematological malignancy characterized by the accumulation of clonal malignant plasma cells. Nowadays more and more studies concern that alert metabolism including glycolysis, glutaminolysis and lipid metabolism has potent in vivo anticancer activity in multiple myeloma. While glycolysis and glutaminolysis was well established, lipid metabolism of MM is poorly understood and there is a need for a new low-toxic therapy that selectively target MM.Lipid metabolism studies such as on the use of inhibitors of fatty acid synthesis and their effects on MM cell survival have been reported. Our study showed the increase of the expression of stearoyl CoA desaturase 1 (SCD1) and the elevated fatty acid biosynthesis in MM cells (Fig.1A, p<0.001). We found that SCD1 is overexpression in MM patients' samples and associated with clinical stage of myeloma (Fig 1B, p<0.05). Then we examined the level of lipid droplets(LDs) in MM cells, and the high level of LDs detected in MM cells demonstrated the lipid accumulation in MM (Fig.1C). Stable depletion of SCD1 inhibited fatty acid biosynthesis and decreased LDs levels and this reduction of LDs remained at low levels in MM cells (Fig.1D, E). These results suggest that MM cell growth party relies on SCD1-mediated fatty acid metabolism.The finding that 20(S)-protopanaxatriol(PPT) has significant effect on inhibiting the transcription of lipogenic genes have reported. Western blotting analysis shows that PPT decreased SCD1 protein levels in RPMI-8226, ARH-77 cell lines. In addition, PPT treatment decreased fatty acid biosynthesis and blocked lipid storage in lipid droplets(LDs) (Fig.1F). The proportion of saturated and monounsaturated was also decreased after treatment (Fig. 1G). Given that 20(S)-protopanaxatriol(PPT) has lipid-lowering effect in MM, we hypothesized that PPT exerts anti-myeloma effects by disrupting lipogenesis. In vitro experiments demonstrate the significant effect of PPT on decreasing proliferation and inducing apoptosis in multiple myeloma (Fig.1H). Supplementation with the SCD1 enzymatic product, oleic acid, rescued MM cells from PPT cell killing and SCD1 silencing, decreasing levels of SCD1 inhibition induced apoptosis and proliferation inhibition (Fig.1I). The results of Western Blot Analysis show a positive correlation between SCD1 inhibition and endoplasmic reticulum stress (ER stress) (Fig.1J). In addition, PPT can obviously induce ER stress after inhibiting SCD1, while ER-stress inhibitor TUDCA can significantly reverse the induced apoptosis of PPT treatment in MM cells (Fig.1K, p<0.05). These results suggest that excessive endoplasmic reticulum stress is the main cause of PPT induced apoptosis.In summary, our studies reveal that regulation of fatty acid metabolism in MM cells is an essential target. We show that the redeployed drug PPT killed MM cells by decreasing SCD1 protein levels and promoting fatty acid-induced ER stress. This study is relevant to the wider context of multiple myeloma therapeutics that developing therapeutics which can disrupt fatty acid biosynthesis. To our knowledge, this is the first study to describe the aglycone of ginsenosides 20(S)-protopanaxatriol with demonstrable anti-myeloma activity that target fatty acid biosynthesis [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Acetyl-CoA Carboxylase Inhibition Reverses NAFLD and Hepatic Insulin Resistance but Promotes Hypertriglyceridemia in Rodents.

Pharmacologic inhibition of acetyl-CoA carboxylase (ACC) enzymes, ACC1 and ACC2, offers an attractive therapeutic strategy for nonalcoholic fatty liver disease (NAFLD) through simultaneous inhibition of fatty acid synthesis and stimulation of fatty acid oxidation. However, the effects of ACC inhibition on hepatic mitochondrial oxidation, anaplerosis, and ketogenesis in vivo are unknown. Here, we evaluated the effect of a liver-directed allosteric inhibitor of ACC1 and ACC2 (Compound 1) on these parameters, as well as glucose and lipid metabolism, in control and diet-induced rodent models of NAFLD. Oral administration of Compound 1 preferentially inhibited ACC enzymatic activity in the liver, reduced hepatic malonyl-CoA levels, and enhanced hepatic ketogenesis by 50%. Furthermore, administration for 6 days to high-fructose-fed rats resulted in a 20% reduction in hepatic de novo lipogenesis. Importantly, long-term treatment (21 days) significantly reduced high-fat sucrose diet-induced hepatic steatosis, protein kinase C epsilon activation, and hepatic insulin resistance. ACCi treatment was associated with a significant increase in plasma triglycerides (approximately 30% to 130%, depending on the length of fasting). ACCi-mediated hypertriglyceridemia could be attributed to approximately a 15% increase in hepatic very low-density lipoprotein production and approximately a 20% reduction in triglyceride clearance by lipoprotein lipase (P ≤ 0.05). At the molecular level, these changes were associated with increases in liver X receptor/sterol response element-binding protein-1 and decreases in peroxisome proliferator-activated receptor-α target activation and could be reversed with fenofibrate co-treatment in a high-fat diet mouse model. Conclusion: Collectively, these studies warrant further investigation into the therapeutic utility of liver-directed ACC inhibition for the treatment of NAFLD and hepatic insulin resistance.

Chloroplast Damage Induced by the Inhibition of Fatty Acid Synthesis Triggers Autophagy in Chlamydomonas.

Fatty acids are synthesized in the stroma of plant and algal chloroplasts by the fatty acid synthase complex. Newly synthesized fatty acids are then used to generate plastidial lipids that are essential for chloroplast structure and function. Here, we show that inhibition of fatty acid synthesis in the model alga Chlamydomonas reinhardtii activates autophagy, a highly conserved catabolic process by which cells degrade intracellular material under adverse conditions to maintain cell homeostasis. Treatment of Chlamydomonas cells with cerulenin, a specific fatty acid synthase inhibitor, stimulated lipidation of the autophagosome protein ATG8 and enhanced autophagic flux. We found that inhibition of fatty acid synthesis decreased monogalactosyldiacylglycerol abundance, increased lutein content, down-regulated photosynthesis, and increased the production of reactive oxygen species. Electron microscopy revealed a high degree of thylakoid membrane stacking in cerulenin-treated cells. Moreover, global transcriptomic analysis of these cells showed an up-regulation of genes encoding chloroplast proteins involved in protein folding and oxidative stress and the induction of major catabolic processes, including autophagy and proteasome pathways. Thus, our results uncovered a link between lipid metabolism, chloroplast integrity, and autophagy through a mechanism that involves the activation of a chloroplast quality control system.

Abstract A10: Fatty acid synthesis inhibitor, orlistat, potentiates cisplatin-induced toxicity in ovarian cancer cells

Abstract Background: Fatty acid synthase (FAS) is a key enzyme in the synthesis of fatty acids and has been shown to be upregulated in many cancers. Additional studies found that high levels of FAS correlated with worse prognosis and with chemoresistance in ovarian tumors. As such, FAS has become a promising target for antitumor therapies. The aim of this study was to gain mechanistic insights into the cytotoxic effects of fatty acid inhibition with orlistat treatment alone or in combination with cisplatin in the ovarian cancer cell line A2780. Methods: A2780 cells were treated with cisplatin (0.4-12 μM) for 4 hours followed by orlistat (25-100 μM, 24 hr). Cell proliferation was measured via CyQuant assay; cell apoptotic activity was measured by both caspase 3/7 assay and flow cytometry with annexin V and propidium iodide. Fatty acid β-oxidation was then characterized using a Seahorse XF24 Extracellular Flux Analyzer. ATP levels were measured with a luminescent ATP detection kit. Finally, Western blot analyses for capsase 3 and phospho-ATM were performed. Results: Cisplatin treatment (4 and 10 μM) of A2780 cells induced an increase in OXPHOS and steady-state levels of ATP 24 hr after damage. Orlistat (25 and 50 μM, 24 hr) decreased basal oxygen consumption and decreased steady-state ATP levels in cisplatin-treated cells. A2780 cells showed decreased proliferation and increased apoptotic activity (Caspase 3/7 and Annexin V) when treated with cisplatin followed by orlistat, as compared to either drug alone. Using the Chou Talaly method the combination index was found to be 0.73 indicating a synergistic relationship. Cisplatin increased the level of phospho-ATM that was reduced by orlistat. Conclusions: Orlistat in combination with cisplatin treatment of A2780 ovarian tumor cells increased apoptosis compared to either treatment alone. Orlistat caused decreased oxidative phosphorylation and prevented a cisplatin-induced increase in OXPHOS and ATP. The combination of cisplatin and orlistat decreases p-ATM, suggesting decreased activation of the DNA repair pathway after cisplatin treatment. This may represent the mechanism of synergistic action of the combination treatment. Citation Format: Bennett Van Houten, Molly Heft-Neal, Wei Qian, Vera Roginskaya. Fatty acid synthesis inhibitor, orlistat, potentiates cisplatin-induced toxicity in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A10.

Isolation of colonization-defective Escherichia coli mutants reveals critical requirement for fatty acids in bacterial colony formation.

Most bacterial cells in nature exhibit extremely low colony-forming activity, despite showing various signs of viability, impeding the isolation and utilization of many bacterial resources. However, the general causes responsible for this state of low colony formation are largely unknown. Because liquid cultivation typically yields more bacterial cell cultures than traditional solid cultivation, we hypothesized that colony formation requires one or more specific gene functions that are dispensable or less important for growth in liquid media. To verify our hypothesis and reveal the genetic background limiting colony formation among bacteria in nature, we isolated Escherichia coli mutants that had decreased frequencies of colony formation but could grow in liquid medium from a temperature-sensitive mutant collection. Mutations were identified in fabB, which is essential for the synthesis of long unsaturated fatty acids. We then constructed a fabB deletion mutant in a wild-type background. Detailed behavioural analysis of the mutant revealed that under fatty acid-limited conditions, colony formation on solid media was more sensitively and seriously impaired than growth in liquid media. Furthermore, growth under partial inhibition of fatty acid synthesis with cerulenin or triclosan brought about similar phenotypes, not only in E. coli but also in Bacillus subtilis and Corynebacterium glutamicum. These results indicate that fatty acids have a critical importance in colony formation and that depletion of fatty acids in the environment partly accounts for the low frequency of bacterial colony formation.