Long-chain Fatty Acid Analogue Research Articles

Evidence for a role of SNMP2 and antennal support cells in sensillum lymph clearance processes of moth pheromone-responsive sensilla

In insect antenna, following the activation of olfactory sensory neurons, odorant molecules are inactivated by enzymes in the sensillum lymph. How the inactivation products are cleared from the sensillum lymph is presently unknown. Here we studied the role of support cells (SCs) and the so-called sensory neuron membrane protein 2 (SNMP2), a member of the CD36 family of lipid transporters abundantly expressed in SCs, in sensillum lymph clearance processes in the moths Heliothis virescens and Bombyx mori. In these species, the sex pheromone components are inactivated to long-chain fatty acids. To approach a role of SNMP2 in the removal of such inactivation products, we analyzed the uptake of a fluorescent long-chain fatty acid analog into a newly generated HvirSNMP2-expressing cell line. We found an increased uptake of the analog into SNMP2-cells compared to control cells, which could be blocked by the CD36 protein inhibitor, SSO. Furthermore, analyses of sensilla from antenna treated with the fatty acid analog indicated that SNMP2-expressing SCs are able to take up fatty acids from the sensillum lymph. In addition, sensilla from SSO-pretreated antenna of B. mori showed reduced removal of the fluorescent analog from the sensillum lymph. Finally, we revealed that SSO pretreatment of male silkmoth antenna significantly prolonged the duration of the female pheromone-induced wing-fluttering behavior, possibly as a result of impaired lymph clearance processes. Together our findings in H. virescens and B. mori support a pivotal role of olfactory SCs in sensillum lymph maintenance processes and suggest an integral role of SNMP2 in the removal of lipophilic “waste products” such as fatty acids resulting from sex pheromone inactivation.

Suppression of multiple myeloma by mitochondrial targeting

Treatment of multiple myeloma (MM) aims at inducing cell apoptosis by surpassing the limited capacity of MM cells to cope with oxidative stress. MM cell survival may further be suppressed by limiting cellular cholesterol. Long-chain fatty acid analogs of the MEDICA series promote mitochondrial stress and inhibit cholesterol biosynthesis, thus prompting us to verify their efficacy and mode-of-action in suppressing MM cell survival, in comparison to bortezomib. MEDICA analog is shown here to effectively suppress survival of MM cells, and to inhibit growth of MM xenograft. Suppression of MM cell survival by MEDICA is accompanied by inhibition of the STAT3, MAPK and the mTORC1 transduction pathways due to mitochondrial oxidative stress. MEDICA-induced oxidative stress is abrogated by added exogenous cholesterol. Suppression of MM cell survival by bortezomib is similarly driven by bortezomib-induced oxidative stress, being abrogated by added cholesterol. In line with that, the time-to-best-response of MM patients to bortezomib-based treatment protocols is shown to be positively correlated with their plasma cholesterol level. MEDICA profile may indicate novel therapeutic potential in the management of MM.

Pharmacological ACC inhibition decreases thrombin-induced platelet aggregation by a mechanism independent of lipid content

Acetyl-CoA carboxylase (ACC) is the first committed enzyme regulating lipids synthesis. We have recently demonstrated that the overexpression of a constitutively active form of ACC in platelets leads to an increase in the phospholipid content which stimulates thrombus formation. Based on these data, we believe that ACC inhibition might be a counter-regulatory mechanism limiting endogenous lipogenesis and platelet reactivity in certain pathological circumstances. Our study aims to assess the impact of pharmacological ACC1 inhibition on platelet functions, de novo lipogenesis and lipid content. Platelets were treated with the long-chain fatty acid (FA) analog TOFA (30 μM) or the active site-directed CP640.186 (CP, 60 μM), two ACC inhibitors, for 2H before thrombin stimulation. Lipogenesis was measured via 14C-acetate incorporation into FA. Platelet functions were assessed by aggregometry. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Both ACC inhibitors decreased lipogenesis and thrombin-induced platelet aggregation. TOFA, but not CP, drastically decreased mitochondrial oxygen consumption rate, an effect associated with an increased reactive oxygen species production and AMPK activation. Surprisingly, the quantitative lipidomics analyses showed that a preincubation with CP did not affect global platelet lipid content. However, the short-term ACC inhibition (and potential acetyl-CoA accumulation) was sufficient to modify the level of acetylated proteins such as tubulin, a key player in the regulation of platelet shape change and aggregation. Given the effect of TOFA on mitochondrial respiration, oxidative stress and AMPK activation, CP seems more appropriate to investigate the impact of ACC inhibition in platelets. A short-term acute treatment with CP inhibits thrombin-induced aggregation by a mechanism which doesn’t depend on lipid content but might involve changes in protein acetylation.

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.

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..

Longitudinal Evaluation of Myocardial Fatty Acid and Glucose Metabolism in Fasted and Nonfasted Spontaneously Hypertensive Rats Using MicroPET/CT.

Using longitudinal micro positron emission tomography (microPET)/computed tomography (CT) studies, we quantified changes in myocardial metabolism and perfusion in spontaneously hypertensive rats (SHRs), a model of left ventricular hypertrophy (LVH). Fatty acid and glucose metabolism were quantified in the hearts of SHRs and Wistar-Kyoto (WKY) normotensive rats using long-chain fatty acid analog 18F-fluoro-6-thia heptadecanoic acid (18F-FTHA) and glucose analog 18F-fluorodeoxyglucose (18F-FDG) under normal or fasting conditions. We also used 18F-fluorodihydrorotenol (18F-FDHROL) to investigate perfusion in their hearts without fasting. Rats were imaged at 4 or 5 times over their life cycle. Compartment modeling was used to estimate the rate constants for the radiotracers. Blood samples were obtained and analyzed for glucose and free fatty acid concentrations. SHRs demonstrated no significant difference in 18F-FDHROL wash-in rate constant (P = .1) and distribution volume (P = .1), significantly higher 18F-FDG myocardial influx rate constant (P = 4×10−8), and significantly lower 18F-FTHA myocardial influx rate constant (P = .007) than WKYs during the 2009-2010 study without fasting. SHRs demonstrated a significantly higher 18F-FDHROL wash-in rate constant (P = 5×10−6) and distribution volume (P = 3×10−8), significantly higher 18F-FDG myocardial influx rate constant (P = 3×10−8), and a higher trend of 18F-FTHA myocardial influx rate constant (not significant, P = .1) than WKYs during the 2011–2012 study with fasting. Changes in glucose plasma concentrations were generally negatively correlated with corresponding radiotracer influx rate constant changes. The study indicates a switch from preferred fatty acid metabolism to increased glucose metabolism with hypertrophy. Increased perfusion during the 2011-2012 study may be indicative of increased aerobic metabolism in the SHR model of LVH.

Real-Time Tracking of BODIPY-C12 Long-Chain Fatty Acid in Human Term Placenta Reveals Unique Lipid Dynamics in Cytotrophoblast Cells.

While the human placenta must provide selected long-chain fatty acids to support the developing fetal brain, little is known about the mechanisms underlying the transport process. We tracked the movement of the fluorescently labeled long-chain fatty acid analogue, BODIPY-C12, across the cell layers of living explants of human term placenta. Although all layers took up the fatty acid, rapid esterification of long-chain fatty acids and incorporation into lipid droplets was exclusive to the inner layer cytotrophoblast cells rather than the expected outer syncytiotrophoblast layer. Cytotrophoblast is a progenitor cell layer previously relegated to a repair role. As isolated cytotrophoblasts differentiated into syncytialized cells in culture, they weakened their lipid processing capacity. Syncytializing cells suppress previously active genes that regulate fatty-acid uptake (SLC27A2/FATP2, FABP4, ACSL5) and lipid metabolism (GPAT3, LPCAT3). We speculate that cytotrophoblast performs a previously unrecognized role in regulating placental fatty acid uptake and metabolism.

Suppression of B-Raf(V600E) cancers by MAPK hyper-activation.

B-Raf(V600E) activates MEK/MAPK signalling and acts as oncogenic driver of a variety of cancers, including melanoma, colorectal and papillary thyroid carcinoma. Specific B-Raf(V600E) kinase inhibitors (e.g., Vemurafenib) prove initial efficacy in melanoma followed shortly by acquired resistance, while failing in most other B-Raf(V600E) cancers due to primary resistance. Resistance is due to acquired mutations in the Ras/Raf/MEK/MAPK pathway and/or other oncogenic drivers that bypass B-Raf(V600E). Surprisingly, hyper-activation of MAPK by inhibiting its protein phosphatase 2A by a synthetic long-chain fatty acid analogue (MEDICA), results in oncogene-induced growth arrest and apoptosis of B-Raf(V600E) cancer cells. Growth arrest is accompanied by MAPK-mediated serine/threonine phosphorylation and suppression of a variety of oncogenic drivers that resist treatment by B-Raf(V600E) kinase inhibitors, including ErbB members, c-Met, IGFR, IRS, STAT3 and Akt. The combined activities of mutated B-Raf and MEDICA are required for generating hyper-activated MAPK, growth arrest and apoptosis, implying strict specificity for mutated B-Raf cancer cells.

Thyroid Hormone, Thyromimetics, and Metabolic Efficiency

Thyroid hormone (TH) has long been recognized as a major modulator of metabolic efficiency, energy expenditure, and thermogenesis. TH effects in regulating metabolic efficiency are transduced by controlling the coupling of mitochondrial oxidative phosphorylation and the cycling of extramitochondrial substrate/futile cycles. However, despite our present understanding of the genomic and nongenomic modes of action of TH, its control of mitochondrial coupling still remains elusive. This review summarizes historical and up-to-date findings concerned with TH regulation of metabolic energetics, while integrating its genomic and mitochondrial activities. It underscores the role played by TH-induced gating of the mitochondrial permeability transition pore (PTP) in controlling metabolic efficiency. PTP gating may offer a unified target for some TH pleiotropic activities and may serve as a novel target for synthetic functional thyromimetics designed to modulate metabolic efficiency. PTP gating by long-chain fatty acid analogs may serve as a model for such strategy.

Suppression of adipose lipolysis by long-chain fatty acid analogs

Agonist-induced lipolysis of adipose fat is robustly inhibited by insulin or by feedback inhibition by the long-chain fatty acids (LCFA) produced during lipolysis. However, the mode of action of LCFA in suppressing adipose lipolysis is not clear. β,β'-Tetramethyl hexadecanedioic acid (Mββ/ EDICA16) is a synthetic LCFA that is neither esterified into lipids nor β-oxidized, and therefore, it was exploited for suppressing agonist-induced lipolysis in analogy to natural LCFA. Mββ is shown here to suppress isoproterenol-induced lipolysis in the rat in vivo as well as in 3T3-L1 adipocytes. Inhibition of isoproterenol-induced lipolysis is due to decrease in isoproterenol-induced cAMP with concomitant inhibition of the phosphorylation of hormone-sensitive lipase and perilipin by protein kinase A. Suppression of cellular cAMP levels is accounted for by inhibition of the adenylate cyclase due to suppression of Raf1 expression by Mββ-activated AMPK. Suppression of Raf1 is further complemented by induction of components of the unfolded-protein-response by Mββ. Our findings imply genuine inhibition of agonist-induced adipose lipolysis by LCFA, independent of their β-oxidation or reesterification. Mββ suppression of agonist-induced lipolysis and cellular cAMP levels independent of the insulin transduction pathway may indicate that synthetic LCFA could serve as insulin mimetics in the lipolysis context under conditions of insulin resistance.

Arabidopsis LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1), LACS2, and LACS3 facilitate fatty acid uptake in yeast

The plant cuticle is a lipid-based barrier on the aerial surfaces of plants that plays a variety of protective roles. The cuticle is comprised largely of long-chain and very-long-chain fatty acids and their derivatives. In Arabidopsis, LONG-CHAIN ACYL-COA SYNTHETASE1 (LACS1), LACS2, and LACS3 are known or suspected cuticle biosynthetic genes. Very-long-chain acyl-coenzyme A (CoA) synthetase activity has been demonstrated for LACS1 and LACS2, although the role for such an activity in cuticle biosynthesis is currently unclear. In yeast and mammalian systems, some very-long-chain acyl-CoA synthetases are also called fatty acid transport proteins (FATPs) due to a second function of mediating transmembrane movement of fatty acids. We sought to determine if LACS1-3 also have this dual functionality. A yeast fat1Δ mutant is deficient in both very-long-chain acyl-CoA synthetase activity and exogenous fatty acid uptake. We demonstrate that heterologous expression of LACS1, 2, or 3 is able to complement both of these deficiencies. Furthermore, expression of each LACS enzyme in yeast resulted in uptake of the long-chain fatty acid analogue, C(1)-BODIPY-C(12). Only expression of LACS1 resulted in uptake of the very-long-chain fatty acid analogue, BODIPY-C(16). These results demonstrate that LACS1, LACS2, and LACS3 have the dual functionality of yeast and mammalian FATP enzymes. These findings have implications in the transmembrane transport and intracellular trafficking of plant lipids destined for export to the cuticle.

Reduced 123I-BMIPP uptake implies decreased myocardial flow reserve in patients with chronic stable angina

Long-chain fatty acid (LCFA) is the main energy source for normal myocardium at rest, but in ischemic myocardium, the main energy substrate shifts from LCFA to glucose. 123I-BMIPP is a radiolabeled LCFA analog. In chronic stable angina without previous infarction, we suppose that reduced 123I-BMIPP uptake is related to the substrate shift in myocardium with decreased myocardial flow reserve (MFR). The purpose of this study was to relate 123I-BMIPP uptake to rest myocardial blood flow (MBF), hyperemic MBF, and MFR assessed with 15O-water positron emission tomography (PET). We enrolled 21 patients with chronic stable angina without previous infarction, all of whom underwent 123I-BMIPP single-photon emission computed tomography (SPECT) and 15O-water PET. The left ventricle was divided into 13 segments. In each segment, rest MBF and hyperemic MBF were measured by PET. 123I-BMIPP uptake was evaluated as follows: score 0=normal, 1=slightly decreased uptake, 2=moderately decreased uptake, 3=severely decreased uptake, and 4=complete defect. 123I-BMIPP uptake was compared with rest MBF, hyperemic MBF, and MFR. The numbers of segments with 123I-BMIPP scores 0, 1, 2, 3, and 4 were 178, 40, 25, 24, and 0, respectively. The rest MBFs for scores 0, 1, 2, and 3 were 0.93+/-0.25, 0.86+/-0.21, 0.97+/-0.30, and 0.99+/-0.37 ml/min/g, respectively. The hyperemic MBFs for scores 0, 1, 2, and 3 were 2.76+/-1.29, 1.84+/-0.74, 1.37+/-0.39, and 1.08+/-0.40 ml/min/g, respectively. The MFRs for scores 0, 1, 2, and 3 were 3.01+/-1.38, 2.20+/-0.95, 1.44+/-0.22, and 1.10+/-0.26, respectively. As 123I-BMIPP uptake declined, hyperemic MBF and MFR decreased. In chronic stable angina without previous infarction, reduced 123I-BMIPP uptake implies decreased MFR.

The Acyl-CoA Synthetases Encoded within FAA1 andFAA4 in Saccharomyces cerevisiaeFunction as Components of the Fatty Acid Transport System Linking Import, Activation, and Intracellular Utilization

Exogenous long-chain fatty acids are activated to coenzyme A derivatives prior to metabolic utilization. In the yeast Saccharomyces cerevisiae, the activation of these compounds prior to metabolic utilization proceeds through the fatty acyl-CoA synthetases Faa1p and Faa4p. Faa1p or Faa4p are essential for long-chain fatty acid import, suggesting that one or both of these enzymes are components of the fatty acid transport system, which also includes Fat1p. By monitoring the intracellular accumulation of the fluorescent long-chain fatty acid analogue 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid, long-chain fatty acid transport was shown to be severely restricted in a faa1 Delta faa4 Delta strain. These data established for the first time a mechanistic linkage between the import and activation of exogenous fatty acids in yeast. To investigate this linkage further, oleoyl CoA levels were defined following incubation of wild type and mutant cells with limiting concentrations of exogenous oleate. These studies demonstrated oleoyl CoA levels were reduced to less than 10% wild-type levels in faa1 Delta and faa1 Delta faa4 Delta strains. Defects in metabolic utilization and intracellular trafficking were also found in the fatty acyl-CoA synthetase-deficient strains. The faa1 Delta faa4 Delta strain had a marked reduction in endogenous acyl-CoA pools, suggesting these enzymes play a role in maintenance of endogenous acyl-CoA pools, metabolism and trafficking. In addition, this strain had levels of in vivo beta-oxidation of exogenous oleate reduced 3-fold when compared with the isogenic parent. Northern analyses demonstrated an additional defect in fatty acid trafficking as FAA1 or FAA4 were required for the transcriptional regulation of the genes encoding the peroxisomal enzymes acyl-CoA oxidase (POX1) and medium-chain acyl-CoA synthetase (FAA2). These data support the hypothesis that fatty acyl-CoA synthetase (Faa1p or Faa4p) functions as a component of the fatty acid import system by linking import and activation of exogenous fatty acids to intracellular utilization and signaling.

Tetradecylthioacetic acid reduces stenosis development after balloon angioplasty injury of rabbit iliac arteries

Background: tetradecylthioacetic acid (TTA) is a synthetic long-chain fatty acid analogue that inhibits the oxidative modification of low-density lipoprotein particles in vitro. We examined the influence of TTA on the arterial wall response after balloon angioplasty injury in a rabbit iliac model. Methods and results: 14 rabbits were randomized to receiving either TTA fatty acids 800 mg daily perorally (weight 3.6±0.1 kg) or to normal diet (weight 3.5±0.5 kg, P=NS). Angioplasty was performed via right carotidotomy on both iliac arteries using an oversized balloon catheter, the TTA group being pretreated for 3 weeks. After angioplasty, the lumen diameter was 2.37±0.18 versus 2.36±0.13 mm for the TTA and control groups, respectively ( P=NS). At 10 weeks follow-up angiography, minimal luminal diameter was 1.64±0.27 versus 1.13±0.52 mm for the TTA and control groups respectively ( P<0.05). Histomorphometry did not show significant differences in intimal hyperplasia between the two groups (maximal intimal thickness 0.22±0.04 versus 0.19±0.10 mm, P=NS and intimal area 0.32±0.12 versus 0.36±0.23 mm 2, P=NS for the TTA and the control groups, respectively). In the heart, the sum of the n-3 fatty acids was 8.9±2.7 in the TTA group versus 4.3±0.2 mol% in the control group ( P<0.05). The anti-inflammatory fatty acid index, calculated as (22:5 n-3+22:6 n-3+20:3 n-6)/20:4 n-6, was 0.76±0.10 vs. 0.25±0.03 for the TTA and control groups, respectively ( P<0.05). In vitro TTA (100 μM) reduced the proliferation of human smooth muscle cell by more than 50%. Conclusion: treatment with TTA is associated with positive arterial remodeling after angioplasty injury. The significance of the in vitro inhibition of human smooth muscle cell proliferation needs to be further elucidated.

Can serum carnitine levels distinguish hypertrophic cardiomyopathy from hypertensive hearts?

Although echocardiography is a useful diagnostic tool in hypertrophic cardiomyopathy (HCM), it is sometimes difficult to differentiate it from hypertensive heart disease (HHD): some patients with HCM show symmetrical hypertrophy, whereas patients with HHD sometimes show asymmetrical septal hypertrophy. We used a radioiodinated long-chain fatty acid tracer to visualize the altered myocardial fatty acid metabolism of HCM and HHD. Carnitine is the essential substance for the beta-oxidation of long-chain fatty acids. We recently reported that serum free carnitine levels in HCM were elevated and that they were significantly correlated with the severity of myocardial fatty acid metabolic disorder. Therefore, we investigated serum carnitine levels in patients with HCM and HHD, which can contribute to the differentiation of each other. We studied 56 patients with HCM and 20 patients with essential hypertension. Serum free carnitine levels were significantly higher in patients with HCM than those with HHD (HCM 52.5+/-9.5 nmol/mL, HHD 46.6+/-6.4 nmol/mL, P<0.01), but they showed no statistical difference between patients with HHD and normal subjects. Serum acylcarnitine levels were significantly lower in patients with HCM than those with HHD (HCM 10.1+/-4.0 nmol/mL, HHD 14.5+/-4.9 nmol/mL, P<0.0005), although they did not differ between patients with HHD and normal subjects. Scintigraphic analyses with a long-chain fatty acid analog revealed that myocardial tracer uptake was much reduced in patients with HCM compared with that in patients with HHD (quantitative analysis: HCM 2.11+/-0.12, HHD 2.22+/-0.17, P<0.05; semiquantitative analysis: HCM 13.6+/-6.3, HHD 2.0+/-1.5, P<0.0001). In conclusion, the differences in serum carnitine levels between HCM and HHD reflect altered myocardial fatty acid metabolic impairment, and the levels can help to distinguish these 2 diseases.

Disruption of the Saccharomyces cerevisiae Homologue to the Murine Fatty Acid Transport Protein Impairs Uptake and Growth on Long-chain Fatty Acids

The yeast Saccharomyces cerevisiae is able to utilize exogenous fatty acids for a variety of cellular processes including beta-oxidation, phospholipid biosynthesis, and protein modification. The molecular mechanisms that govern the uptake of these compounds in S. cerevisiae have not been described. We report the characterization of FAT1, a gene that encodes a putative membrane-bound long-chain fatty acid transport protein (Fat1p). Fat1p contains 623 amino acid residues that are 33% identical and 54% with similar chemical properties as compared with the fatty acid transport protein FATP described in 3T3-L1 adipocytes (Schaffer and Lodish (1994) Cell 79, 427-436), suggesting a similar function. Disruption of FAT1 results in 1) an impaired growth in YPD medium containing 25 microM cerulenin and 500 microM fatty acid (myristate (C14:0), palmitate (C16:0), or oleate (C18:1)); 2) a marked decrease in the uptake of the fluorescent long-chain fatty acid analogue boron dipyrromethene difluoride dodecanoic acid (BODIPY-3823); 3) a reduced rate of exogenous oleate incorporation into phospholipids; and 4) a 2-3-fold decrease in the rates of oleate uptake. These data support the hypothesis that Fat1p is involved in long-chain fatty acid uptake and may represent a long-chain fatty acid transport protein.

Syntheses of Long-chain Fatty Acids Containing Various Functional Groups and Their Myristoyl CoA : Protein N-myristoyltransferase (NMT) Activities

Numerous eukaryotic cellular and viral proteins can be modified by the covalent attachment of fatty acyl groups. Protein acylation is classified into three types, viz., N-myristoylation, thioester (or ester) -linked acylation, and glycosylphosphatidylinositol-linked acylation. ProteinN-myristoylation involves the co-translational attachment of myristic acid (tetradecanoic acid, C14 : 0) to N-terminal glycine residues of various proteins. Myristoyl-CoA : proteinN-myristoyltransferase (NMT) is the enzyme by which the myristoyl group is transferred from myristoyl-CoA thioester toN-terminal glycine residues of nascent acylproteins.With consideration to the above, the syntheses of long-chain fatty acid analogs, especially myristic acid analogs, containing oxygen, sulfur, double bonds, triple bonds, aromatic residue, carbonyl, ester, and amide are reviewed. The structure-NMT activity relationship is also eluci-dated by using myristic acid analogs, N-terminal glycine-attached octapeptides, and Saccharomyces cervisiae NMT in vitro. The inhibition of the replication of human immunodeficiency virus-1 by these myristic analogs is briefly discussed.

Non-β-oxidizable ω-[ 18F]fluoro long chain fatty acid analogs show cytochrome P-450-mediated defluorination: Implications for the design of PET tracers of myocardial fatty acid utilization

The nature of the in vivo defluorination of non-β-oxidizable no-carrier-added ω-[ 18F]fluoro long chain fatty acid (LCFA) analogs was studied with the aim of developing PET tracers of LCFA utilization. Extensive defluorination of 15-[ 18F]fluoro-3-thia-pentadecanoic acid (FTPA) in mouse was evidenced by radioactivity uptake by bone. [ 18F]Fluoride in the blood was verified analytically. Incubations of FTPA in rat-liver homogenates and subcellular fractions thereof showed a strong defluorination process in microsomes which was O 2- and NADPH-dependent. In contrast, defluorination of FTPA was relatively slow in Langendorff perfused rat heart. High bone uptake in mouse was also observed with 14-[ 18F]fluoro-13, 13-dimethyl-3-thia-tetradecanoic acid, where gem-dimethyl substitution precludes direct elimination of H 18F. These data indicate that the defluorination of non-β-oxidizable ω-[ 18F]fluoro LCFA analogs is primarily governed by cytochrome P-450-mediated ω-oxidation. Therefore, labeling at the (ω-3) carbon was proposed to provide a more stabile 18F-label. Defluorination of the (ω-3)-labeled 13 ( R,S)-[ 18F]fluoro-3-thia-hexadecanoic acid was lower than that of FTPA in mouse and was independent of O 2 and NADPH in vitro. Thus, (ω-3) labeling with 18F is preferable to ω labeling of non-β-oxidizable LCFA analogs.

3-[ p-(6-Phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (PA-DPH): characterization as a fluorescent membrane probe and binding to fatty acid binding proteins

The negatively charged fluorophore 3-[ p-(6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (PA-DPH) was characterized by comparison with its parent compound DPH, and with cationic trimethylammonium-DPH (TMA-DPH). The molar absorption coefficient of PA-DPH (60 000 cm −1 · mol −1) as well as its quantum yield (0.7) and fluorescence lifetime (5 ns) in fluid phase membranes are intermediate between DPH and TMA-DPH. Steady-state fluorescence polarization studies show that PA-DPH detects the phase transition of both neutral and anionic bilayers. In fluid phase membranes the absolute values of PA-DPH polarization are considerably higher than DPH and somewhat lower than TMA-DPH. The results suggest that like TMA-DPH, PA-DPH is anchored to the surface of the membrane by its charge, but that it is probing a region somewhat deeper along the bilayer normal. PA-DPH binds to rat hepatic fatty acid binding protein (hFABP) and bovine serum albumin at PA-DPH/protein molar ratios of 1.5:1 and at least 6:1, respectively. Native oleic acid competes with PA-DPH for binding to both proteins, suggesting that the two ligands compete for similar binding sites. The affinity of PA-DPH for hFABP is similar to that of oleic acid. Thus, PA-DPH should be useful both as an anionic fluorescent membrane probe and a long-chain free fatty acid analogue.