Fatty Acid Analogue Research Articles

Detection of Heterogeneous Protein S-Acylation in Cells.

The use of synthetically synthesized azide and alkyne fatty acid analogs coupled with bioorthogonal Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction-based detection methods to study protein S-acylation reactions has replaced the traditional method of using in vivo metabolic radiolabeling with tritiated palmitic acid and has greatly facilitated our understanding of this essential cellular process. Here, we describe the chemical synthesis of myristic (C:14), palmitic (C16:0), and stearic (C18:0) acid-azide probes and detail how they may be utilized as chemical reporters for the analysis of S-acylation of exogenously expressed proteins in cells.

Aryl-urea fatty acids that activate the p38 MAP kinase and down-regulate multiple cyclins decrease the viability of MDA-MB-231 breast cancer cells

We recently developed a novel aryl-urea fatty acid (CTU; 16({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)hexadecanoic acid) that impaired the viability of MDA-MB-231 breast cancer cells in vitro and in mouse xenograft models in vivo. At present there is a deficiency of information on the structural requirements for the activity of CTU. Our initial study suggested that electron withdrawing groups were required on the aryl ring, and in this study we further evaluated the influence of the electronic properties of aromatic substitution on the capacity of CTU analogues to decrease MDA-MB-231 breast cancer cell viability. Analogues that contained strong electron-withdrawing groups in the meta- and para-positions of the aryl ring exhibited improved activity over CTU. Effective analogues down-regulated the cyclins D1, E1 and B1, and the cyclin-dependent kinases (CDKs) 4 and 6, that form complexes to coordinate cell cycle progression. Active CTU analogues also stimulated the phosphorylation and activation of the p38 MAP kinase signalling pathway in cells and both decreased proliferation (5-bromo-2′-deoxyuridine (brdU) incorporation) and activated apoptosis (executioner caspase-3/7 activity). These agents offer a new approach to target the cell cycle at multiple phases in order to efficiently prevent cancer cell expansion. Inclusion of the present structural information in drug design approaches could enhance the development of optimal analogues of aryl-urea fatty acids as potential anti-cancer agents.

Alloantigen-Activated Human T-Cells Increase Extracellular Fatty Acid Uptake and Intracellular Lipid Metabolism during Xenogeneic Gvhd

BackgroundFollowing activation by recognition of foreign antigens, human T-cells alter their metabolic pathways to meet the increasing energetic demands for efficient immune response. Like cancer cells, alloreactive T-cells show a preference for aerobic glycolysis rather than oxidative phosphorylation, which is referred to as “Warburg effect”. Until recently, it has been thought that extracellular fatty acid (FA) uptake and β-oxidation are severely reduced in alloreactive T-cells; however, some studies have indicated that lipid metabolism is rather increased in alloreactive mouse T-cells, and that metabolic pathway of FA can be a promising target for GVHD. To determine the role of lipid metabolism in human alloreactive T-cells after hematopoietic stem cell transplantation, we investigated the metabolic changes in human T-cells in vivo using human-into-mouse xenogeneic GVHD models.MethodsNOG mice received 250cGy of total body irradiation (TBI) and were subsequently injected intravenously with human pan T-cells. All mice developed severe GVHD and died within 2 weeks, while mice that received TBI only survived without any symptoms of GVHD. Cells were harvested from GVHD target organs of mice at day 9 after transplantation. For the measurement of glucose and fatty acid (FA) uptake by flow cytometry, cells were stained with fluorescent-labeled deoxyglucose analogue (2-NBDG) and long-chain fatty acid analogue (BODIPY 500/510 C12), respectively. PCR array and extracellular flux analysis were performed according to manufacturer's instructions.ResultsGlucose uptake, determined by flow cytometry, was significantly increased in human T-cells obtained from GVHD mice. Extracellular FA uptake was also increased in human T-cells in GVHD mice, and was associated with cell proliferation rate. Effector memory T-cells followed by central memory T-cells showed a higher FA uptake than did naive T-cells. These findings were similarly observed in both human CD4+ and CD8+ T-cells. Robust T-cell proliferation was observed even in MHC class I/II deficient (MHC−/−) NOG mice after transplantation, although to a lesser extent than MHC+/+ NOG mice, in a process known as homeostatic proliferation. Extracellular uptake of FA as well as glucose in T-cells was significantly decreased in MHC−/− NOG mice. Of note, even when compared among only fully proliferated T-cells between MHC+/+ and MHC−/− NOG mice, FA uptake was still significantly decreased in MHC−/− NOG mice, suggesting that the recognition of host MHC molecules by allogeneic T-cells accelerate this process.To compare the ability of human naive and memory T-cells to incorporate extracellular FA, we isolated human naive (CD45RA high) and memory (CD45RA low) T-cells and separately injected into NOG mice. Although it has been shown that memory T-cells exhibit different effector functions, the FA uptake in memory T-cells was comparable to that in naive T-cells. This suggests that memory T-cells can also alter their lipid metabolism following encounter with alloantigens.Finally, we assessed the expression of genes associated with lipid metabolism in human T-cells obtained from GVHD mice. Quantitative real-time PCR analysis detected up-regulation of mRNAs encoding the enzymes involved in FA transport including carnitine palmitoyltransferase (CPT1B), fatty acid binding protein (FABP1-4, FABP6, and FABP7), and β-oxidation pathway including acyl-CoA synthase (ACSBG2) and acyl-CoA dehydrogenase (ACAD9-11, ACADS, and ACADL) when compared with T-cells in MHC−/− NOG mice. Similarly, the expression of genes encoding the enzymes in triacylglycerol metabolism such as glycerol kinase (GK, GK2) and lipoprotein lipase (LPL) was up-regulated in GVHD mice. Furthermore, the expression of genes associated with mevalonate pathways such as HMG-CoA synthase (HMGCS1, HMGCS2), was also upregulated. These observations suggest that T-cells activated by alloantigens in vivo promote lipid hydrolysis, mitochondrial FA transport, and β-oxidation, resulting in greater utilization of free FA.ConclusionHuman alloreactive T-cells increased extracellular uptake of FA as well as glucose, and intracellular lipid metabolism in response to alloantigens (summarized in the graphical abstract). Therapeutic effects of specific inhibition of lipid metabolic pathways by pharmacological inhibitors including etomoxir are now being investigated in this model. [Display omitted] DisclosuresFujiwara:Shire: Consultancy; Pfizer: Consultancy; Chugai: Consultancy; Kirin: Consultancy; Kyowa-Hakko: Consultancy; Astellas: Consultancy. Ohmine:Kyowa Hakko Kirin: Speakers Bureau; Takara Bio: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Celgene Corporation: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Alexion Pharmaceuticals: Speakers Bureau; Ono Pharmaceutical: Consultancy. Muroi:Japanese Red Cross Society: Speakers Bureau; Dickinson and Company: Speakers Bureau; Becton: Speakers Bureau; JCR: Speakers Bureau. Kanda:Astellas: Consultancy, Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Taiho: Research Funding; Nippon-Shinyaku: Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Dainippon-Sumitomo: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Otsuka: Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; MSD: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Asahi-Kasei: Research Funding; Ono: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Taisho-Toyama: Research Funding; CSL Behring: Research Funding; Tanabe-Mitsubishi: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Takara-bio: Consultancy, Honoraria.

Mechanisms Underlying the Dual Effect of Polyunsaturated Fatty Acid Analogs on Kv7.1

Polyunsaturated fatty acid (PUFA) analogs represent a new class of potential anti-arrhythmic KV7.1 and KV7.1+KCNE1 channel activators. In this study, we describe dual independent activating effects of negatively charged PUFA analogs on KV7.1 and KV7.1+KCNE1 that are dependent on discrete channel motifs. PUFA analogs are critically dependent on K326 in S6 of KV7.1 to increase the maximum conductance and critically dependent on specific S4 arginines in KV7.1 to shift the voltage dependence of channel opening toward negative voltages. Our findings provide insights into how KV7.1+KCNE1 activators may interact electrostatically both with the pore domain and the voltage-sensing domain to augment channel activity. We believe that the molecular understanding of how PUFA analogs induce dual independent activating effects is an important step toward the development of effective anti-arrhythmic drugs that target KV7.1 channels.

Real-time microscopic assessment of fatty acid uptake kinetics in the human term placenta

IntroductionThe placenta employs an efficient and selective fatty acid transport system to supply lipids for fetal development. Disruptions in placental fatty acid transport lead to restricted fetal growth along with cardiovascular and neurologic deficits. Nevertheless, little is known about the molecular mechanisms involved in human placental fatty acid trafficking during the initial steps of uptake, or the importance of fatty acid chain length in determining uptake rates. MethodsWe employed BODIPY fluorophore conjugated fatty acid analogues of three chain lengths, medium (BODIPY-C5), long (BODIPY-C12), and very-long (BODIPY-C16), to study fatty acid uptake in isolated human trophoblast and explants using confocal microscopy. The three BODIPY-labeled fatty acids were added to freshly isolated explants and tracked for up to 30 min. Fatty acid uptake kinetics were quantified in trophoblast (cytotrophoblast and syncytiotrophoblast together) and the fetal capillary lumen. ResultsLong- (BODIPY-C12) and Very long-chain (BODIPY-C16) fatty acids accumulated more rapidly in the trophoblast layer than did medium-chain (BODIPY-C5) whereas BODIPY-C5 accumulated more rapidly in the fetal capillary than did the longer chain length fatty acids. The long-chain fatty acids, BODIPY-C12 and BODIPY-C16, are esterified and stored in lipid droplets in the cytotrophoblast layer, but medium-chain fatty acid, BODIPY-C5, is not. DiscussionFatty acids accumulate in trophoblast and fetal capillaries inversely according to their chain length. BODIPY-C5 accumulates in the fetal capillary in concentrations far greater than in the trophoblast, suggesting that medium-chain length BODIPY-labeled fatty acids are capable of being transported against a concentration gradient.

Abstract 4109: Multi-modality imaging to interrogate lipidome changes during melanoma progression in zebrafish

Abstract Lipids play diverse roles in the body, including cell structure, cell signalling, and energy production; all of which are altered in cancer. Lipid metabolism is therefore a potential therapeutic target, and here has been investigated using a transgenic zebrafish model. This study combines transcriptome and lipidomic analyses for the first time, with in-vivo positron emission tomography (PET) and ex-vivo desorption electrospray ionisation-mass spectrometry (DESI-MS), to investigate lipid metabolism changes in melanoma. Most lipids are made of fatty acids, and the methods by which tumours acquire fatty acids can be altered compared to normal tissue. Here, the PET tracer [18F]-FTHA (a fatty acid analogue) has been used to demonstrate free fatty acid uptake in melanoma tumours in-vivo. Additionally, the excellent spatial resolution of mass spectrometry imaging has been utilised by imaging the FTHA ex-vivo by DESI-MS, allowing more precise spatial correlation with the histology, and revealing the heterogeneous nature of this fatty acid uptake. These imaging data complement the findings of the transcriptome analyses which show upregulation of genes associated with fatty acid uptake. Integrating transcriptome and lipidome data analyses revealed dysregulation of glycerophospholipid pathways, and was supported by DESI-MS data, which revealed heterogeneous changes in lipid metabolism. Citation Format: Fiona Henderson, Hannah Johnston, Emrys Jones, Duncan Foster, Raghavendar T. Nagaraju, Michael Green, Michael Fairclough, Irene Barinaga-Rementeria Ramirez, Shuning He, B.Ewa Snaar-Jagalska, Katherine A. Hollywood, Warwick Dunn, Herman P. Spaink, Paul Lorigan, Emmanuelle Claude, Kaye Williams, Adam Hurlstone, Adam McMahon. Multi-modality imaging to interrogate lipidome changes during melanoma progression in zebrafish [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4109.

Early Effects of Sleeve Gastrectomy (SG) on Systemic and Organ-Specific Dietary Fatty Acid Uptake, Postprandial Free Fatty Acids (FFA), and Glucose Metabolism in Type 2 Diabetes (T2D)

Postprandial dietary fatty acid (FA) biodistribution is impaired in subjects with glucose intolerance, with decreased FA storage in adipose tissues (AT) and increased FA uptake by the myocardium. These abnormalities are improved after 1 year of lifestyle-induced weight loss. Bariatric surgery leads to early metabolic improvements before weight loss, but the early effects on dietary FA metabolism and biodistribution have never been studied. 9 subjects with T2D underwent a 6-hour postprandial metabolic protocol before and 12 days after SG. A liquid meal containing [U-13C]-palmitate and [2H]-glucose was ingested at time 0, with an IV perfusion of [3H]-glucose and [7,7,8,8-2H]-palmitate, allowing quantification of glucose absorption, endogenous glucose production (EGP), lipolysis and dietary FA spillover. A capsule containing a positron-emitting long-chain FA analog (18FTHA) was taken with the meal for the quantification of organ-specific dietary FA uptake using PET/CT (n=6). Twelve days after SG, whole body and hepatic insulin sensitivity increased significantly whereas fasting and postprandial EGP decreased. Postprandial GLP-1, GIP, PYY and glucagon were higher after vs. before SG. Postprandial AUC of FFA (175 vs. 228 mmol/Lx min; p< 0.05) and total FA oxidation were higher (p< 0.02 from 180 min to 360 min), but dietary FA oxidation was reduced after SG (p< 0.002 from 180 min to 360 min). After SG, uptake of dietary FA 6 hours after the meal was reduced in the myocardium (mean SUV 2.07± 0.66 vs. 1.36± 0.44; p=0.06) and was increased in visceral AT (mean SUV 0.24± 0.2 vs. 0.42± 0.2; p=0.03), without changes in other organs. In conclusion, there is reduced myocardial and increased visceral AT uptake of dietary FA associated with reduced dietary FA oxidation and increased FFA mobilization and oxidation, concomitant with reduced EGP and insulin sensitization in patients with T2D early after SG. Disclosure A. Carreau: None. C. Noll: None. B. Guerin: None. L. Biertho: Advisory Panel; Self; Novo Nordisk Inc.. Consultant; Self; Johnson & Johnson Services, Inc., Valeant Pharmaceuticals International, Inc.. E.E. Turcotte: None. A. Tchernof: Research Support; Self; Johnson & Johnson Services, Inc., Medtronic. A. Carpentier: Consultant; Self; UniQure, Janssen Pharmaceuticals, Inc., Siemens. Research Support; Self; Caprion, Merck Sharp & Dohme Corp., GlaxoSmithKline plc., Novartis Pharmaceuticals Corporation, AstraZeneca, Bristol-Myers Squibb Company, Sanofi-Aventis, Pfizer Inc., Novo Nordisk Inc., Exelixis, Amgen Inc..

Cloning and functional characterization of long-chain acyl-CoA synthetase 1 from the mesocarp of African oil palm (Elaeis guineensis Jacq.)

African oil palm (Elaeis guineensis Jacq.) is the highest-yielding oil crop in the world. Recent studies have shown the importance of long-chain acyl-CoA synthetases (LACSs) in lipid biosynthesis and degradation, but few studies have focused on oil palm. In this work, we found that transcription of EgLACS1was positively correlated with lipid accumulation in the oil palm mesocarp. In order to clarify the role of EgLACS1 on lipid metabolism, three variants of the EgLACS1 gene were cloned from oil palm and designated EgLACS1-V1, EgLACS1-V2, and EgLACS1-V3. Sequence analysis revealed that the three variant proteins contained several highly conserved motifs, shared important sequence similarities with other known LACS1s, and demonstrated that EgLACS1-V1, EgLACS1-V2 and EgLACS1-V3 were 666-, 664- and 601-aminoacid proteins, respectively. All of the three variant genes encodingEgLACS1 were expressed and tested for their functionality in an acyl-coenzyme-A synthetase- (ACS-) deficient mutant yeast strain YB525. Only EgLACS1-V1 and EgLACS1-V2 could rescue the growth deficiency (an inability to grow on a fatty acid auxotrophic medium) of YB525 in complementation tests. Using a fluorescent fatty acid analogue confirmed that EgLACS1-V1 and EgLACS1-V2 facilitated exogenous fatty acid uptake. EgLACS1 overexpression in yeast reduced fatty acid content. All these results suggested that not only did both the EgLACS1-V1 and EgLACS1-V2 enzymes exhibit acyl-coenzyme-A activities, but that they were also involved in the transfer of fatty acid-coenzyme A. They may also take part in the storage of lipid during fruit development in oil palm. The characterization of EgLACS1 will provide a molecular basis for the study of acyl-coenzyme A synthetase-mediated lipid synthesis and degradation in oil palm.

A novel system to quantify intestinal lipid digestion and transport

The zebrafish larva is a powerful tool for the study of dietary triglyceride (TG) digestion and how fatty acids (FA) derived from dietary lipids are absorbed, metabolized and distributed to the body. While fluorescent FA analogues have enabled visualization of FA metabolism, methods for specifically assaying TG digestion are badly needed. Here we present a novel High Performance Liquid Chromatography (HPLC) method that quantitatively differentiates TG and phospholipid (PL) molecules with one or two fluorescent FA analogues. We show how this tool may be used to discriminate between undigested and digested TG or phosphatidylcholine (PC), and also the products of TG or PC that have been digested, absorbed and re-synthesized into new lipid molecules. Using this approach, we explored the dietary requirement of zebrafish larvae for phospholipids. Here we demonstrate that dietary TG is digested and absorbed in the intestinal epithelium, but without dietary PC, TG accumulates and is not transported out of the enterocytes. Consequently, intestinal ER stress increases and the ingested lipid is not available support the energy and metabolic needs of other tissues. In TG diets with PC, TG is readily transported from the intestine and subsequently metabolized.

Carboxylate Analogues of Aryl-Urea-Substituted Fatty Acids That Target the Mitochondria in MDA-MB-231 Breast Cancer Cells to Promote Cell Death.

Selective targeting of the tumor cell mitochondrion is a viable approach for the development of anticancer agents because the organelle is functionally different from the mitochondria of normal cells. We recently developed a novel aryl-urea fatty acid, 16({[4-chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)hexadecanoic acid (1) that was found to disrupt mitochondria and to activate apoptosis in MDA-MB-231 breast cancer cells. However, there is currently little information on the structural requirements for the activity of compound 1 analogues. The present study evaluated the role of the carboxylic acid group on the anticancer activity of 1. Bioisosteric replacement of the carboxylate in 1 maintained activity. Thus, like 1, the sulfonic acid analogue 1-SA and the oxo-thiadiazole analogue 1-OT were also found to target the mitochondrion and to activate cell killing capacity. The hydroxamic acid analogue 1-HA also killed MDA-MB-231 cells, but its onset of action was slower than that of 1-SA and 1-OT. In contrast, replacement of the carboxylate with non-bioisosteric amido and methylamido groups produced analogues that minimally altered mitochondrial function and showed little capacity to decrease tumor cell viability. These findings suggest that the carboxylate moiety in the novel mitochondrially targeted agent 1 is an important determinant of the kinetics and efficacy of anticancer cell activities of compound 1 analogues. Further development of carboxylate-modified analogues of aryl-urea fatty acids as potential anticancer agents could now be warranted.

C. elegans Avoids Toxin‐Producing Streptomyces

Species of the non‐motile bacterium Streptomyces protect themselves from predation by the bacteriovore Caenorhabditis elegans through the secretion of nematicides. To evade death by these agents, C. elegans has evolved the ability to detect and avoid the bacterium. We characterize fatty acid analogs, including dodecanoic acid, which elicit a fast avoidance response by C. elegans. We also detect dodecanoic acid that has been secreted by Streptomyces by derivatization and detection with high resolution mass spectrometry. By sensing these signals, C. elegans can escape the nematicidal bacteria that would otherwise be a good food source.Support or Funding InformationLaura Miller Conrad: National Institutes of Health (5SC3GM118199); Angelina Tang and Sarah Matthews: National Science Foundation (HRD‐1302873); Miri VanHoven: National Institutes of Health (NS087544, GM089595), National Science Foundation (1355202).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mutation in MON2, Which Encodes a Protein Implicated in Vesicular Transport, Affects Response to Exogenous Fatty Acids in Yeast

Metabolism of exogenous fatty acids requires cells to take up, traffic, and molecularly respond to fed fatty acids. Our lab has determined that a mutation in mon2 results in defective growth on the fatty acid 10‐undecenoic acid. Like wild type cells, mon2Δ mutants are capable of normal response to the feeding of long chain unsaturated fatty acids (UFAs) by reducing transcription of OLE1, (which encodes the Δ9 desaturase), and by reducing the accumulation of endogenously produced UFAs. However, wild type cells when fed 10‐undecenoic acid will continue to express OLE1 and increase their content of endogenously produced UFAs. In contrast, mon2Δ mutants do not differentially respond to the two fatty acids. Staining with a BODIPY‐labeled fluorescent fatty acid analog indicates that there is a difference in localization/amount of the supplied fatty acid in mon2Δ cells compared to wild type cells. Thus, it appears that the mon2 mutation decreases the cell's ability to differentiate between fatty acids, perhaps due to mislocalization of the fed fatty acid or regulatory proteins.Support or Funding InformationThis work was supported by a Jacob Nyenhuis Faculty Award (VM), NSF‐REU (AG, MS, BG) and a Summer Scholar award from the Dean for Natural and Applied Science (RD).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A fatty acid analogue targeting mitochondria exerts a plasma triacylglycerol lowering effect in rats with impaired carnitine biosynthesis.

L-carnitine is important for the catabolism of long-chain fatty acids in the mitochondria. We investigated how the triacylglycerol (TAG)-lowering drug 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA) influenced lipid metabolism in carnitine-depleted, 3-(2,2,2-trimethylhydrazinium)propionate dehydrate (Mildronate; meldonium)-treated male Wistar rats. As indicated, carnitine biosynthesis was impaired by Mildronate. However, TAG levels of both plasma and liver were decreased by 1-triple TTA in Mildronate-treated animals. This was accompanied by increased gene expression of proteins involved in mitochondrial activity and proliferation and reduced mRNA levels of Dgat2, ApoB and ApoCIII in liver. The hepatic energy state was reduced in the group of Mildronate and 1-triple TTA as reflected by increased AMP/ATP ratio, reduced energy charge and induced gene expression of uncoupling proteins 2 and 3. The increase in mitochondrial fatty acid oxidation was observed despite low plasma carnitine levels, and was linked to strongly induced gene expression of carnitine acetyltransferase, translocase and carnitine transporter, suggesting an efficient carnitine turnover. The present data suggest that the plasma TAG-lowering effect of 1-triple TTA in Mildronate-treated rats is not only due to increased mitochondrial fatty acid oxidation reflected by increased mitochondrial biogenesis, but also to changes in plasma clearance and reduced TAG biosynthesis.

Molecular Mechanism of Polyunsaturated Fatty Acid Analogues as KV7.1-Channel Modulators

Negatively charged analogues of polyunsaturated fatty acids (PUFAs) have been shown to activate the human KV7.1+KCNE1 channel, which generates the repolarizing cardiac IKs current. Specific PUFA analogues activate both wild type KV7.1+KCNE1 and KV7.1+KCNE1 containing Long QT syndrome-associated mutations. Therefore, PUFA analogues represent a new class of KV7.1+KCNE1 channel activators that may aid in development of novel anti-arrhythmic drugs. To determine the molecular mechanism of how PUFA analogues activate KV7.1+KCNE1, we here combine electrophysiology recordings and site-directed mutagenesis with a panel of PUFA analogues with distinct properties. We identify two main effects of PUFA analogues on KV7.1 that are dependent on discrete channel motifs. The first effect is a shift in the voltage dependence of channel opening that is critically dependent on specific S4 arginines in the voltage sensor domain of KV7.1. Intriguingly, different S4 arginines are important in KV7.1 and KV7.1+KCNE1. The second effect is alteration in the maximum conductance that is critically dependent on the S6 residue K326 in the pore domain of KV7.1. Both effects appear to be primarily electrostatic. Our findings provide novel insights into how KV7.1+KCNE1 channel activators, such as PUFA analogues, simultaneously may interact with the voltage sensor domain and the pore domain to alter channel activity. Our findings also contribute pharmacological support to the hypothesis that KV7.1 and KV7.1+KCNE1 open from different conformational states.

Synthesis and Cytotoxicity Evaluation of Unusual Mixture Acid Acetate-protected Monogalactosyldiacylglycerols

In the present study, acetate-protected monogalactosyldiacylglycerol and its fatty acid and aromatic acid analogues were synthesized using trichloroacetimidate methodology. All compounds synthesized were evaluated for cytotoxicity against four cancer cell lines and one normal cell line. Among the compounds tested, mixed acid compound 7b exhibited good activity with IC50 value of 27.5 μM. In a similar manner, the combination of unsaturated oleic acid and cinnamic acid compound 7d also exhibited significant activity with IC50 value 34.9 μM. Based on these results, it could be concluded that the combination of oleic acid with cinnamic acid type acid and other saturated fatty acids enhance the cytotoxicity of glycoglycerolipids.

Effect of Kampo Extract Formulation (Japanese Traditional Medicine) on Pro- and Anti-angiogenic Factors Secreted from Primary Endothelial Cells and Placental Cells

The placenta employs an efficient and selective fatty acid transport system to supply lipids for fetal development. Disruptions in placental fatty acid transport lead to restricted fetal growth along with cardiovascular and neurologic deficits. Nevertheless, little is known about the molecular mechanisms involved in human placental fatty acid trafficking during the initial steps of uptake, or the importance of fatty acid chain length in determining uptake rates.We employed BODIPY fluorophore conjugated fatty acid analogues of three chain lengths, medium (BODIPY-C5), long (BODIPY-C12), and very-long (BODIPY-C16), to study fatty acid uptake in isolated human trophoblast and explants using confocal microscopy. The three BODIPY-labeled fatty acids were added to freshly isolated explants and tracked for up to 30 min. Fatty acid uptake kinetics were quantified in trophoblast (cytotrophoblast and syncytiotrophoblast together) and the fetal capillary lumen.Long- (BODIPY-C12) and Very long-chain (BODIPY-C16) fatty acids accumulated more rapidly in the trophoblast layer than did medium-chain (BODIPY-C5) whereas BODIPY-C5 accumulated more rapidly in the fetal capillary than did the longer chain length fatty acids. The long-chain fatty acids, BODIPY-C12 and BODIPY-C16, are esterified and stored in lipid droplets in the cytotrophoblast layer, but medium-chain fatty acid, BODIPY-C5, is not.Fatty acids accumulate in trophoblast and fetal capillaries inversely according to their chain length. BODIPY-C5 accumulates in the fetal capillary in concentrations far greater than in the trophoblast, suggesting that medium-chain length BODIPY-labeled fatty acids are capable of being transported against a concentration gradient.

Use of confocal laser endomicroscopy with a fluorescently labeled fatty acid to diagnose colorectal neoplasms.

Endoscopic treatment for early colorectal cancer closely correlates with patient prognosis. However, endoscopic differentiation between carcinomas and non-neoplastic lesions remains difficult. Here, we topically stained colorectal neoplasms with a fatty acid analogue (BODIPY-FA) and quantified the fluorescent signals using confocal laser endomicroscopy (CLE) and fluorescence microscopy. We also analyzed protein expression in colorectal cancer tissues. We found that expression of fatty acid synthase was elevated, while the expression of fatty acid transporters was reduced in colorectal cancer. In colorectal cancer mouse models and patients, the BODIPY-FA signals were higher in normal epithelia than in carcinomas or colonic intraepithelial neoplasias. BODIPY-FA staining revealed both the arrangement of intestinal glands and the intracellular structures under CLE screening. In a double-blind trial, CLE images stained with BODIPY-FA exhibited greater consistency (κ = 0.68) and overall validity (74.65%) than those stained using intravenous fluorescein sodium (κ = 0.43, 55.88%) when the results were compared with histological diagnoses. These findings suggest that topical use of BODIPY-FA with CLE is a promising imaging approach for early colorectal neoplasm screening.

Synthesis and biological evaluation of fatty acid derivatives for myocardial imaging containing [99mTc(CO)3

Radiolabeled fatty acids as myocardial metabolic agent are used for detecting ischemic heart disease, however, no 99mTc-labeled fatty acids have potential use in clinical diagnosis. In this work, five fatty acid analogues labeled with 99mTc were firstly synthesized and characterized, their biological behaviors were investigated. All Radiotracers had good stability when incubated in rat serum for 3 h at 37 °C. 99mTc -CpT-12-ODPPA (8b) showed higher initial myocardial uptake (8.17% ID/g at 1 min postinjection) and good heart/blood ratio (2.58 at 30 min postinjection). 99mTc-11-dpa-OUFA (2b) as positively charged lipophilic compounds had reasonable heart uptake (5.59% ID/g at 1 min postinjection) and good retention (1.89% ID/g at 60 min postinjection). Unfortunately, no great improvement was obtained by the five 99mTc-labeled fatty acid analogues for the short myocardial retention and poor heart/liver ratios.

Initial evaluation of 99m Tc-tricarbonyl-cyclopentadienyl fatty acids derivatives as SPECT tracers for myocardium.

Fatty acids are myocardial metabolic agent for detecting myocardial ischemia and infraction. However, no 99m Tc-labeled fatty acids had potential use in clinical practice. In this study, 99m Tc-CpTT-10-oxo-para-PPA (1d), 99m Tc-CpTT-11-oxo-para-PPA (2d), 99m Tc-CpTT-12-oxo-para-PPA (3d), 99m Tc-CpTT-11-oxo-ortho-PPA (4d), and 99m Tc-CpTT-11-oxo-meta-PPA (5d) were synthesized by a double ligand transfer reaction, and their biological behaviors were investigated. Compound 2d achieved good heart to blood ratio (3.39 at 5min after intravenous), and 2d showed high-heart uptake of 6.20%ID/g at 5minutes after injection. Compound 3d displayed a prolonged retention in the myocardium (1.43%ID/g at 60min after injection). Radioactivity accumulation in the lungs, spleen, and blood was eliminated rapidly. In vivo, metabolite analysis presented that compound 6d may be metabolite of 2d through β-oxidation in tissue. Unfortunately, the biodistribution studies of 1d, 2d, 3d, 4d, and 5d showed fast heart clearance and poor heart to liver ratios, which suggested that the 5 99m Tc-labeled fatty acid analogues cannot be used for diagnosis.

Saturated Fatty Acid Analogues of Cell-Penetrating Peptide PepFect14: Role of Fatty Acid Modification in Complexation and Delivery of Splice-Correcting Oligonucleotides

Modifying cell-penetrating peptides (CPPs) with fatty acids has long been used to improve peptide-mediated nucleic acid delivery. In this study we have revisited this phenomenon with a systematic approach where we developed a structure-activity relationship to describe the role of the acyl chain length in the transfection process. For that we took a well-studied CPP, PepFect14, as the basis and varied its N-terminal acyl chain length from 2 to 22 carbons. To evaluate the delivery efficiency, the peptides were noncovalently complexed with a splice-correcting oligonucleotide (SCO) and tested in HeLa pLuc705 reporter cell line. Our results demonstrate that biological splice-correction activity emerges from acyl chain of 12 carbons and increases linearly with each additional carbon. To assess the underlying factors regarding how the transfection efficacy of these complexes is dependent on hydrophobicity, we used an array of different methods. For the functionally active peptides (C12-22) there was no apparent difference in their physicochemical properties, including complex formation efficiency, hydrodynamic size, and zeta potential. Moreover, membrane activity studies with peptides and their complexes with SCOs confirmed that the toxicity of the complexes at higher molar ratios is mainly caused by the free fraction of the peptide which is not incorporated into the peptide/oligonucleotide complexes. Finally, we show that the increase in splice-correcting activity correlates with the ability of the complexes to associate with the cells. Collectively these studies lay the ground work for how to design highly efficient CPPs and how to optimize their oligonucleotide complexes for lowest toxicity without losing efficiency.