Radioactive Fatty Acids Research Articles

Correction: N-terminal palmitoylation within the appropriate amino acid environment conveys on NOS2 the ability to progress along the intracellular sorting pathways (doi:10.1242/jcs.02878).

This Correction updates and replaces the Expression of Concern (doi:10.1242/jcs.209999) relating to J. Cell Sci. (2006) 119, 1558-1569.Journal of Cell Science was made aware of issues with this paper by a reader. Fig. 1C contains data duplicated from a blot shown in fig. 4D of a paper the authors had previously published (Navarro-Lérida et al., 2004). Fig. 3A also contains data previously published in fig. 5A,B of Navarro-Lérida et al. (2004). After discussion with the corresponding author, Ignacio Rodríguez-Crespo, the journal referred this matter to Universidad Complutense de Madrid (UCM). The UCM investigating committee reviewed replicate experiments provided to determine whether they supported the scientific results and conclusions. They concluded that: “…despite the inadmissible manipulation in the production of the figures, the results and conclusions of the paper seem ultimately supported by the original experiments…”.The editorial policies of Journal of Cell Science state that: “Should an error appear in a published article that affects scientific meaning or author credibility but does not affect the overall results and conclusions of the paper, our policy is to publish a Correction…” and that a Retraction should be published when “…a published paper contain[s] one or more significant errors or inaccuracies that change the overall results and conclusions of the paper…”. We follow the guidelines of the Committee on Publication Ethics (COPE), which state: “Retraction should usually be reserved for publications that are so seriously flawed (for whatever reason) that their findings or conclusions should not be relied upon”. The standards of figure assembly and data presentation in this paper fall short of good scientific practice. However, given that the investigating committee at UCM decided that the conclusions of the paper were not affected by the errors, the appropriate course of action – according to COPE guidelines – is to publish a Correction, which the journal has made as detailed as possible.In Fig. 1C, replicate data supporting the results were available from numerous separate experiments. The authors state that because no comparisons between protein concentrations were made and as the data in this panel are ‘all-or-none’ results (meaning that proteins either incorporate radioactive fatty acids or they do not), separate blots can be used to represent the individual experiments. Replicate data for the experiment shown in Fig. 3A were available. Both corrected figure panels are shown here. The authors apologise to the journal and readers for these errors.Journal of Cell Science refers readers to other Corrections related to the UCM investigation:doi:10.1242/jcs.219634doi:10.1242/jcs.219667doi:10.1242/jcs.219683

Cellular growth and tube formation of HTR8/SVneo trophoblast: effects of exogenously added fatty acid-binding protein-4 and its inhibitor.

Adequate placental angiogenesis is critical for the establishment of the placental circulation and thus for normal feto-placental growth and development. Fatty acid-binding protein-4 (FABP4) plays a pro-angiogenic role in endothelial cells; however, very little information is available in placental first trimester trophoblast cells. Here we report that exogenously added FABP4 (exo-FABP4) stimulated tube formation (as a measure of in vitro angiogenesis) in HTR8/SVneo trophoblastic cells. HTR-8/SVneo cells were incubated in the presence of exogenously added FABP4 at different concentrations and time points. Cellular growth, proliferation, in vitro tube formation, expression of growth stimulatory-, fatty acid transporters, and angiogenic genes were investigated. Internalization of exo-FABP4 was carried out using immunocytochemistry. Radioactive fatty acid uptake was determined in the presence and absence of FABP4 metabolic inhibitor. Exo-FABP4 (10-100ng/ml) stimulated proliferation of HTR8/SVneo cells as compared to control. Exo-FABP4 dose dependently increased growth and viability of the cells to the similar extent as done by 50µM of arachidonic acid. Exo-FABP4-induced tube formation and proliferation were significantly inhibited by FABP4 (BMS309403) inhibitor. Exo-FABP4 stimulated the expression of growth stimulatory genes such as tissue inhibitor of matrix metalloproteinases-1 (TIMP1), insulin-like growth factor 1 (IGF1), and also prokineticin 2 (PROK2), the pro-angiogenic mediators in these cells. In addition, expressions of genes associated with proliferation and differentiation such as sonic hedgehog (SHH) and WNT1 inducible signalling pathway protein 1 (WISP1) were significantly expressed when cells were exposed to exo-FABP4. Our findings reveal a pro-angiogenic role of FABP4 in first trimester placental trophoblast cells and its regulation may have impact in placental physiology.

Fate of orally administered radioactive fatty acids in the late-pregnant rat.

To investigate the biodisponibility of placental transfer of fatty acids, rats pregnant for 20 days were given tracer amounts of [(14)C]palmitic (PA), oleic (OA), linoleic (LA), α-linolenic (LNA), or docosahexaenoic acid (DHA) orally and euthanized at 0.5, 1.0, 2.0, or 8.0 h thereafter. Maternal plasma radioactivity in lipids initially increased only to decline at later times. Most of the label appeared first as triacylglycerols (TAG); later, the proportion in phospholipids (PhL) increased. The percentage of label in placental lipids was also always highest shortly after administration and declined later; again, PhL increased with time. Fetal plasma radioactivity increased with time, with its highest value at 8.0 h after DHA or LNA administration. DHA initially appeared primarily in the nonesterified fatty acids (NEFA) and PA, OA, LA, and LNA as TAG followed by NEFA; in all cases, there was an increase in PhL at later times. Measurement of fatty acid concentrations allowed calculation of specific (radio)activities, and the ratio (fetal/maternal) of these in the plasmas gave an index of placental transfer activity, which was LNA > LA > DHA = OA > PA. It is proposed that a considerable proportion of most fatty acids transferred through the placenta are released into the fetal circulation in the form of TAG.

Association of Iodine-123 BMIPP and MIBG Cardiac Uptakes with Left Ventricular Functional Parameters Assessed by Gated Myocardial Perfusion SPECT

We investigated how the degree of cardiac uptakes of radioactive fatty acid (FA) analog ( 123 I-BMIPP) or norepinephrine (NE) analog ( 123 I-MIBG) was related to left ventricular (LV) functional parameters in patients with heart disease, which might be a predictor of cardiac events. Patients with ejection fraction (EF) of 16.2%-82.2% underwent 123 I-BMIPP or 123 I-MIBG myocardial scintigraphy, and were followed up over 1177±486 days. LV systolic functional parameter EF, diastolic parameters 1/3FR (first third filling rate), PFR (peak filling rate), and TPF (time to PFR) were calculated using quantitative gated SPECT. 123 I-BMIPP uptake (n=30) was significantly correlated with EF, 1/3FR, and PFR but not TPF. 123 I-MIBG uptake (n=37) was also correlated with EF, 1/3FR, and PFR but not TPF. In contrast, TPF was positively correlated with age, and was significantly longer in women than in men. Such findings were also observed in normal EF patients (≥50%, n=100). Kaplan–Meier analysis showed the occurrence of earlier cardiac events in the case of patients with decreased 123 I-BMIPP and 123 I-MIBG uptake, decreased PFR, and prolonged TPF. The degree of decreased cardiac uptakes of FA and NE may be associated with the grade of systolic and diastolic dysfunctions assessed by EF, PFR, and 1/3FR. In contrast, prolonged TPF, another diastolic parameter, may rather be caused by aging and female gender, independent of EF. Despite these differences, myocardial FA metabolism impairment, cardiac sympathetic nerve inactivity, decreased PFR, and prolonged TPF may all indicate poor cardiac prognosis in patients with heart disease. Keywords123 I-BMIPP; 123 I-MIBG; Cardiac Function; Heart Failure

Fully automated synthesis of 11C-acetate as tumor PET tracer by simple modified solid-phase extraction purification

IntroductionAutomated synthesis of 11C-acetate (11C-AC) as the most commonly used radioactive fatty acid tracer is performed by a simple, rapid, and modified solid-phase extraction (SPE) purification. MethodsAutomated synthesis of 11C-AC was implemented by carboxylation reaction of MeMgBr on a polyethylene Teflon loop ring with 11C-CO2, followed by acidic hydrolysis with acid and SCX cartridge, and purification on SCX, AG11A8 and C18 SPE cartridges using a commercially available 11C-tracer synthesizer. Quality control test and animals positron emission tomography (PET) imaging were also carried out. ResultsA high and reproducible decay-uncorrected radiochemical yield of (41.0±4.6)% (n=10) was obtained from 11C-CO2 within the whole synthesis time about 8min. The radiochemical purity of 11C-AC was over 95% by high-performance liquid chromatography (HPLC) analysis. Quality control test and PET imaging showed that 11C-AC injection produced by the simple SPE procedure was safe and efficient, and was in agreement with the current Chinese radiopharmaceutical quality control guidelines. ConclusionThe novel, simple, and rapid method is readily adapted to the fully automated synthesis of 11C-AC on several existing commercial synthesis module. The method can be used routinely to produce 11C-AC for preclinical and clinical studies with PET imaging.

Comparative analysis of S-fatty acylation of gel-separated proteins by stable isotope–coded fatty acid transmethylation and mass spectrometry

Covalent attachment of palmitic acid or other fatty acids to the thiol groups of cysteine residues of proteins through reversible thioester bonds has an important role in the regulation of diverse biological processes. We describe here the development of a mass spectrometry protocol based on stable isotope-coded fatty acid transmethylation (iFAT) for qualitative and comparative analysis of protein S-fatty acylation under different experimental conditions. In this approach, cellular proteins extracted from different cell states are separated by SDS-PAGE and then the gel is stained with either Coomassie blue or Nile red for improved sensitivity. Protein bands are excised and then an in-gel stable iFAT procedure is performed. The fatty acid methyl esters resulting from derivatization with d0- and d3-methanol are identified by mass spectrometry. By measuring the intensities of labeled and unlabeled fragment ion pairs of fatty acid methyl esters, the levels of S-fatty acylation in different cells or tissues can be compared. This approach has been applied to monitor the changes of S-fatty acylation of zebrafish liver proteome in response to environmental dichlorodiphenyltrichloroethane exposure. Compared with the approach using metabolic incorporation of radioactive fatty acid analogs, it is not only simple and effective but also eliminates the hazards of handling radioactive isotopes.

An automated SPE-based high-yield synthesis of [11C]acetate and [11C]palmitate: no liquid–liquid extraction, solvent evaporation or distillation required

An automated method is described for the rapid and high-yield synthesis of two of the most commonly used radioactive fatty acids: [(11)C]acetate and [(11)C]palmitate. Reaction of [(11)C]CO(2) with the respective Grignard reagents in diethyl ether solution proceeded for 2 min at 40°C. Quenching of the reaction and liberation of nonreacted [(11)C]CO(2) occurred upon addition of a fourfold molar excess of aqueous 0.1 M HCl (acetate) or nonaqueous HCl/Et(2)O (palmitate). Labeled products were then purified by adsorption to an Alumina-N Sep-Pak Plus cartridge and eluted with either aqueous NaH(2)PO(4) solution (acetate) or 100% ethanol (palmitate). High-performance liquid chromatography analysis confirmed that the radiochemical purity of each product was >98%, and decay-corrected radiochemical yields averaged 33% for [(11)C]palmitate and 40% for [(11)C]acetate. The method requires no liquid-liquid extraction, solvent evaporation or distillation capabilities and can be readily adapted to existing radiosynthesis modules.

Clinical Features of Myocardial Triglyceride in Different Types of Cardiomyopathy Assessed by Proton Magnetic Resonance Spectroscopy: Comparison With Myocardial Creatine

Background Myocardial lipid overstorage may produce cardiomyopathy, leading to dysfunction, but advanced heart failure may cause lipolysis via sympathetic nerve activation. In the failing heart, the creatine kinase system may also be impaired. The aims of this study were to assess myocardial triglyceride (TG) and creatine (CR) in different types of cardiomyopathy and to investigate whether they are related to the severity of cardiac dysfunction. Methods and Results In patients with hypertrophic cardiomyopathy (HCM, n = 8), dilated cardiomyopathy (DCM, n = 12) or ischemic cardiomyopathy (ICM, n = 10), and normal subjects (NML, n = 22), myocardial TG and CR were evaluated using proton magnetic resonance spectroscopy. To assess cardiac sympathetic nerve activity, myocardial MIBG (a radioactive guanethidine analog) uptake was measured in DCM. Myocardial TG was significantly lower in hypertrophic cardiomyopathy (HCM) (1.92 ± 0.99 μmol/g), but higher in ICM (7.59 ± 4.36 μmol/g) than in NML hearts (4.05 ± 1.94 μmol/g). There was no significant difference in TG between DCM (4.84 ± 6.45 μmol/g) and NML. Myocardial CR in HCM (20.4 ± 8.4 μmol/g), DCM (14.8 ± 4.8 μmol/g), and ICM (19.4 ± 6.3 μmol/g) was significantly lower than that in NML hearts (27.1 ± 4.3 μmol/g). Overall, myocardial CR correlated positively with the severity of heart failure estimated by ejection fraction or myocardial BMIPP (a radioactive fatty acid analog) uptake, but TG did not. In DCM, myocardial TG correlated with body mass index, but not with MIBG uptake. Conclusions Myocardial TG may be related to the specific cause of disease rather than the severity of cardiac dysfunction. In contrast, myocardial CR reflects the severity of heart failure despite different pathoetiologic mechanisms of dysfunction. In DCM, myocardial TG may be affected by an overweight state rather than cardiac sympathetic nerve dysfunction. Thus, myocardial CR has a closer relationship to heart failure severity than does myocardial TG.

Metabolism of exogenous fatty acids, fatty acid-mediated cholesterol efflux, PKA and PKC pathways in boar sperm acrosome reaction.

For understanding the roles of fatty acids on the induction of acrosome reaction which occurs under association of cholesterol efflux and PKA or PKC pathways in boar spermatozoa, metabolic fate of alone and combined radiolabeled 14C-oleic acid and 3H-linoleic acid incorporated in the sperm was compared, and behavior of cholesterol and effects of PKA and PKC inhibitors upon fatty acid-induced acrosome reaction were examined. Semen was collected from a Duroc boar, and the metabolic activities of fatty acids in the spermatozoa were measured using radioactive compounds and thin layer chromatography. Cholesterol efflux was measured with a cholesterol determination assay kit. Participation of fatty acids on the AR through PKA and PKC pathways was evaluated using a specific inhibitor of these enzymes. Incorporation rate of 14C-oleic acid into the sperm lipids was significantly higher than that of 3H-linoleic acid (P < 0.05). The oxidation of 14C-oleic acid was higher in combined radiolabeling rather than in one. The highest amounts of 3H-linoleic acid and 14C-oleic acid were recovered mainly in the triglycerides and phospholipids fraction, and 14C-oleic acid distribution was higher than the 3H-linoleic acid in both labeled (P < 0.05) sperm lipids. In the 3H-linoleic and 14C-oleic acid combined radiolabeling, the incorporation rate of the radioactive fatty acids in all the lipid fractions increased 15 times more than the alone radiolabeling. Boar sperm utilize oleic acid to generate energy for hyperactivation (P < 0.05). Supplementation of arachidonic acid significantly increased (P < 0.05) cholesterol efflux in sperm. When spermatozoa were incubated with PKA or PKC inhibitors, there was a significant reduction of arachidonic acid-induced acrosome reaction (AR) (P < 0.05), and inhibition by PKA inhibitor is stronger than that by PKC inhibitor. Incorporation of unsaturated fatty acids, especially oleic acid, into triglycerides and phospholipids provides prerequisite energy for AR. Cholesterol efflux by arachidonic acid triggers AR. Arachidonic acid activated PKA and PKC pathway participate in induction of the AR.

Characterization of Both Polyunsaturated Fatty Acid Biosynthetic Pathways in Schizochytrium sp.

Schizochytrium produces long chain polyunsaturated fatty acids (PUFAs) via a PUFA synthase. Targeted mutagenesis of one gene of this synthase was conducted to confirm PUFA synthase function and determine its metabolic necessity. The resulting mutants were auxotrophic and required supplementation with PUFAs. In vivo labeling experiments with radioactive fatty acids demonstrated the presence of several elongase and desaturase activities associated with the standard pathway of PUFA synthesis. However, this system was missing a critical Delta12 desaturase activity and was therefore not capable of synthesizing PUFAs from the 16- or 18-carbon saturated fatty acid products of the fatty acid synthase. Because Schizochytrium uses a PUFA synthase system for the production of PUFAs, the existence of a partial desaturase-elongase system (if not a simple vestige) is suggested to be either a scavenging mechanism for intermediate fatty acids prematurely released by the PUFA synthase or for PUFAs found in the organism's native environment.

Hydrolysis of tumor cell lipids after CTL-mediated death

Contributions of lipases to CTL function have been debated, including if T cell lipases damage target cells. Expression of the lipase pancreatic lipase-related protein 2 (PLRP2) was previously found in IL-4 cultured lymphocyte cell lines but absent from IL-2 cultured lymphocytes. Here, we evaluated IL-2 and IL-4 induced CTLs for hydrolysis of target cell lipids and killing. Using anti-CD3 redirected lysis of [(3)H]-oleic acid-labeled P815 tumor cells, we detected the release of the radioactive fatty acid (FA). When PLRP2(+/+) and PLRP2(-/-) CTLs were compared, there was more killing by the PLRP2(+/+) CTLs. However, [(3)H]-oleic acid release was similar per dead P815, suggesting that lipid hydrolysis was produced by the dead P815s rather than by PLRP2. The FA release and death were completely dependent on perforin and also occurred when P815s were killed by perforin-containing T cell granule extracts that lacked lipase activity. Death by the cytotoxic granules extracts was unaffected by the addition of lipases. A lipase inhibitor, tetrahydrolipstatin, blocked FA release without affecting CTL-mediated cytotoxicity. Also, CTL-mediated death caused as much FA release as death by disruption of cells by freeze-thawing. The released oleic acid may be sufficient to promote secondary apoptotic responses after CTL-induced trauma.

10.1007/s11827-008-1008-5

It is generally recognized nowadays that active lipid metabolism takes place in the nucleus of a mammalian cell. Experimental data testify to the biosynthesis of polyphosphoinositides and phosphatidylcholine and reveal corresponding enzymes within nuclei of mammalian cells. These findings suggest that lipidmediated signaling pathways in nuclei operate independently of lipid-mediated regulatory mechanisms functioning in membranes and cytosol. To explore the pathways of intranuclear lipid biosynthesis, we studied incorporation of 2-14C-acetate into lipids of cytosol and isolated nuclei of rat thymus cells after separate and combined incubation with the labeled precursor. The most efficient incorporation of 2-14C-acetate into lipids (cholesterol, free fatty acids, and phospholipids) was observed in a reaction mixture containing cytosol. When the reaction mixture contained only nuclei, incorporation of the radioactive precursor into lipids also took place, but specific radioactivity of the lipids was essentially lower than in the cytosol. In both cases, 2-14C-acetate incorporated into phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol, and cardiolipin. Phosphatidylcholine, the most abundant membrane phospholipid, demonstrated the lowest radioactivity, which was significantly lower than that of phosphatidylethanolamine. Incorporation of newly synthesized free fatty acids in nuclear phospholipids was inhibited, if nuclei were incubated with cytosol. As a result, radioactive free fatty acids were accumulated in nuclei, while in cytosol they were efficiently incorporated into phospholipids. The levels of phospholipids and cholesterol remained constant regardless of incubation protocol, while the overall yield of free fatty acids decreased after combined incubation of nuclear and cytosolic fractions or after incubation of cytosol without nuclei. Putative mechanisms underlying the appearance of radioactive lipids in isolated nuclei of thymus cells are discussed.

Creatine depletion and altered fatty acid metabolism in diseased human hearts: clinical investigation using 1h magnetic resonance spectroscopy and 123I BMIPP myocardial scintigraphy

In the heart, the creatine kinase system plays an important role in energy reserves, and myocardial energy production essentially depends upon fatty acid metabolism. To examine myocardial creatine (CR) concentration and altered cardiac fatty acid metabolism in various forms of heart disease. Myocardial CR concentration of the septum was measured by gated 1H magnetic resonance spectroscopy (MRS), applying a point-resolved spectroscopy (PRESS) sequence in 34 patients with heart disease. Of these patients, 14 underwent 123I BMIPP (radioactive fatty acid analogue) myocardial scintigraphy to evaluate myocardial fatty acid metabolism. Cardiac 123I BMIPP uptake was calculated as the heart-to-mediastinum count ratio. Myocardial CR concentration correlated positively with the left ventricular ejection fraction (LVEF) by echocardiography (R = 0.61, P<0.001, n = 34), suggesting that the degree of reduced CR is related to the severity of contractile dysfunction. Cardiac 123I BMIPP uptake also correlated positively with LVEF (initial image, R = 0.60, P<0.05; delayed image, R = 0.63, P<0.05; n = 14). There was a significant correlation between myocardial CR and cardiac 123I BMIPP uptake (initial image, R = 0.77, P<0.01; delayed image, R = 0.82, P<0.001; n = 14). Our study suggests an association between CR depletion and impaired fatty acid metabolism in various forms of heart diseases.

Fatty acid metabolism in adipocytes: functional analysis of fatty acid transport proteins 1 and 4

The role of fatty acid transport protein 1 (FATP1) and FATP4 in facilitating adipocyte fatty acid metabolism was investigated using stable FATP1 or FATP4 knockdown (kd) 3T3-L1 cell lines derived from retrovirus-delivered short hairpin RNA (shRNA). Decreased expression of FATP1 or FATP4 did not affect preadipocyte differentiation or the expression of FATP1 (in FATP4 kd), FATP4 (in FATP1 kd), fatty acid translocase, acyl-coenzyme A synthetase 1, and adipocyte fatty acid binding protein but did lead to increased levels of peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha. Both FATP1 and FATP4 kd adipocytes exhibited reduced triacylglycerol deposition and corresponding reductions in diacylglycerol and monoacylglycerol levels compared with control cells. FATP1 kd adipocytes displayed an approximately 25% reduction in basal (3)H-labeled fatty acid uptake and a complete loss of insulin-stimulated (3)H-labeled fatty acid uptake compared with control adipocytes. In contrast, FATP4 kd adipocytes as well as HEK-293 cells overexpressing FATP4 did not display any changes in fatty acid influx. FATP4 kd cells exhibited increased basal lipolysis, whereas FATP1 kd cells exhibited no change in lipolytic capacity. Consistent with reduced triacylglycerol accumulation, FATP1 and FATP4 kd adipocytes exhibited enhanced 2-deoxyglucose uptake compared with control adipocytes. These findings define unique and distinct roles for FATP1 and FATP4 in adipose fatty acid metabolism.

Lysophospholipid Acylation in RBC.

The formation, distribution and utilization of acyl-CoA plays a crucial role in plasma membrane phospholipid turnover in red blood cells (RBC). Upon de-acylation of glycero-phospholipids (PL) via the action of phospholipase, re-acylation of the lysophospholipids (LPL) requires activity of two enzymes of the Lands pathway. Long-chain acyl-CoA synthetases (ACSL) activate fatty acids to acyl-CoA which are subsequently ligated to LPL by LysoPhosphoLipid Acyl Transferase (LPLAT) a family of enzymes with exclusive specificity for the polar group of LPL (phosphatidic acid, choline, serine and ethanolamine). We recently identified ACSL6 as the enzyme responsible for the activation of fatty acid in RBC. None of the family members of LPLAT have been identified in RBC to date. LPC, either generated in the RBC or taken up from plasma, is rapidly acylated by RBC suggesting an important role for Lysohosphatidylcholine-acyl transferase (LPCAT) in RBC. We report the identification and characterization of LPCAT, the enzyme that generates PC from LPC and acylCoA. We identified the RNA expression of LPCAT, an approximately 60kD protein, in reticulocytes, confirming proteomic studies suggesting the presence of this protein in adult RBC membranes.). It is a modular protein containing an acyltransferase domain at the amino-terminus, three predicted membrane spanning domains, and a putative calcium binding site at the C-terminus, distinguishing it from the lysophosphatidic acid acyltransferease (LPAAT). The putative LPCAT was expressed in E. coli. It was found in the E. coli membrane fraction, and was able to use oleoyl-CoA and LPC as substrates to generate PC. Lysophosphatidic acid (LPA) was not acylated by this protein. In contrast the previously identified LPAAT (1) expressed in E. coli, utilized LPA but not LPC, indicating LPL specificity of these enzymes. Radioactive fatty acid added to RBC is also incorporated in phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS). Sequence analysis suggests that two other proteins present in the genome of mammals are homologues of LPCAT. We hypothesize that these putative acyltransferases are responsible for the acylation of lysophosphatidyl ethanolamine (LPE) and lysophosphatidyl serine (LPS). These proteins are essential to maintain a proper glycerophospholipid composition of the RBC membrane and thereby viability of the cells. A dysfunction of this system may underlie the observed differences in phospholipid molecular species composition in subpopulations of sickle cells contributing to sickle cell pathology. A complete description of these proteins involved in the maintenance of glycerophospholipid composition of RBC will aid to better understand the maintenance of plasma membrane lipid composition of all mammalian cells.

Iodine-123 BMIPP scintigraphy in the evaluation of patients with heart failure

To investigate whether cardiac parameters obtained by I-123 15-(p-iodophenyl)-3-methyl pentadecanoic acid (BMIPP, a radioactive fatty acid analogue) myocardial scintigraphy are useful as indicators of disease severity and predictors of cardiac events in congestive heart failure (CHF). Thirty-two CHF patients (functional class: 17 in NYHA II and 15 in NYHA III at the time of this study) were compared with 18 normal control subjects. Myocardial scintigraphy was performed 15 min and 3 hours after I-123 BMIPP injection. The heart-to-mediastinum count ratio of I-123 BMIPP on the initial (H/Mi) and delayed (H/Md) images and the washout rate (WR) were calculated. Both H/Mi and H/Md were lower in CHF than in controls (H/Mi 1.96+/-0.18 vs. 2.30+/-0.29; H/Md 1.72+/-0.15 vs. 1.97+/-0.21; both P<0.001), but WR was higher in CHF than in controls (WR (%) 23.7+/-5.7 vs. 18.2+/-6.0, P<0.01). Both H/Mi (R = 0.42, P<0.05) and H/Md (R = 0.45, P<0.05) correlated positively with the left ventricular (LV) ejection fraction (EF), estimated by echocardiography. The WR correlated positively with the plasma B-type natriuretic peptide (BNP) level (R = 0.47, P<0.01). Kaplan-Meier analysis showed an earlier CHF progression for patients with a lower H/Mi (<1.94). Myocardial metabolic abnormality evaluated by I-123 BMIPP scintigraphy is related to the severity of CHF. Furthermore, it may be useful as a predictor for cardiac events.

Low liver conversion rate of α-linolenic to docosahexaenoic acid in awake rats on a high-docosahexaenoate-containing diet

We quantified the rates of incorporation of alpha-linolenic acid (alpha-LNA; 18:3n-3) into "stable" lipids (triacylglycerol, phospholipid, cholesteryl ester) and the rate of conversion of alpha-LNA to docosahexaenoic acid (DHA; 22: 6n-3) in the liver of awake male rats on a high-DHA-containing diet after a 5-min intravenous infusion of [1-(14)C]alpha-LNA. At 5 min, 72.7% of liver radioactivity (excluding unesterified fatty acid radioactivity) was in stable lipids, with the remainder in the aqueous compartment. Using our measured specific activity of liver alpha-LNA-CoA, in the form of the dilution coefficient lambda(alpha-LNA-CoA), we calculated incorporation rates of unesterified alpha-LNA into liver triacylglycerol, phospholipid, and cholesteryl ester as 2,401, 749, and 9.6 nmol/s/g x 10(-4), respectively, corresponding to turnover rates of 3.2, 8.7, and 2.9%/min and half-lives of 8-24 min. A lower limit for the DHA synthesis rate from alpha-LNA equaled 15.8 nmol/s/g x 10(-4) (0.5% of the net in corporation rate). Thus, in rats on a high-DHA-containing diet, rates of beta-oxidation and esterification of alpha-LNA into stable liver lipids are high, whereas its conversion to DHA is comparatively low and insufficient to supply significant DHA to the brain. High incorporation and turnover rates likely reflect a high secretion rate by liver of stable lipids within very low density lipoproteins.

Triacylglycerol Is Synthesized in a Specific Subclass of Caveolae in Primary Adipocytes

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.

Palmitoylation of Inducible Nitric-oxide Synthase at Cys-3 Is Required for Proper Intracellular Traffic and Nitric Oxide Synthesis

A number of cell types express inducible nitric-oxide synthase (NOS2) in response to exogenous insults such as bacterial lipopolysaccharide or proinflammatory cytokines. Although it has been known for some time that the N-terminal end of NOS2 suffers a post-translational modification, its exact identification has remained elusive. Using radioactive fatty acids, we show herein that NOS2 becomes thioacylated at Cys-3 with palmitic acid. Site-directed mutagenesis of this single residue results in the absence of the radiolabel incorporation. Acylation of NOS2 is completely indispensable for intracellular sorting and .NO synthesis. In fact, a C3S mutant of NOS2 is completely inactive and accumulates to intracellular membranes that almost totally co-localize with the Golgi marker beta-cop. Likewise, low concentrations of the palmitoylation blocking agents 2-Br-palmitate or 8-Br-palmitate severely affected the .NO synthesis of both NOS2 induced in muscular myotubes and transfected NOS2. However, unlike endothelial NOS, palmitoylation of inducible NOS is not involved in its targeting to caveolae. We have created 16 NOS2-GFP chimeras to inspect the effect of the neighboring residues of Cys-3 on the degree of palmitoylation. In this regard, the hydrophobic residue Pro-4 and the basic residue Lys-6 seem to be indispensable for palmitoylation. In addition, agents that block the endoplasmic reticulum to Golgi transit such as brefeldin A and monensin drastically reduced NOS2 activity leading to its accumulation in perinuclear areas. In summary, palmitoylation of NOS2 at Cys-3 is required for both its activity and proper intracellular localization.

The N-terminal SH4 Region of the Src Family Kinase Fyn Is Modified by Methylation and Heterogeneous Fatty Acylation: ROLE IN MEMBRANE TARGETING, CELL ADHESION, AND SPREADING

The N-terminal SH4 domain of Src family kinases is responsible for promoting membrane binding and plasma membrane targeting. Most Src family kinases contain an N-terminal Met-Gly-Cys consensus sequence that undergoes dual acylation with myristate and palmitate after removal of methionine. Previous studies of Src family kinase fatty acylation have relied on radiolabeling of cells with radioactive fatty acids. Although this method is useful for verifying that a given fatty acid is attached to a protein, it does not reveal whether other fatty acids or other modifying groups are attached to the protein. Here we use matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to identify fatty acylated species of the Src family kinase Fyn. Our results reveal that Fyn is efficiently myristoylated and that some of the myristoylated proteins are also heterogeneously S-acylated with palmitate, palmitoleate, stearate, or oleate. Furthermore, we show for the first time that Fyn is trimethylated at lysine residues 7 and/or 9 within its N-terminal region. Both myristoylation and palmitoylation were required for methylation of Fyn. However, a general methylation inhibitor had no inhibitory effect on myristoylation and palmitoylation of Fyn, suggesting that methylation occurs after myristoylation and palmitoylation. Lysine mutants of Fyn that could not be methylated failed to promote cell adhesion and spreading, suggesting that methylation is important for Fyn function.