Labeling of adipocyte membranes by sulfo-N-succinimidyl derivatives of long-chain fatty acids: Inhibition of fatty acid transport

Summary Sulfo-N-succinimidyl derivatives of the long-chain fatty acids, oleic and myristic, were synthesized and covalently reacted with isolated rat adipocytes. The plasma membrane proteins labeled by these compounds and the effect of labeling on the transport of long-chain fatty acids were investigated. Sulfo-N-succinimidyl oleate (SSO) and myristate (SSM) inhibited the transport of fatty acids (by about 70%). Inhibition of fatty acid transport was not a result of alterations in cell integrity, as intracellular water volume was not changed. It did not reflect effects on fatty acid metabolism, since it was observed under conditions where greater than 90% of the fatty acid taken up was recovered in the free form. The inhibitory effect was specific to the fatty acid transport system, as the transport of glucose and the permeation of retinoic acid, a substance with structural similarities to long-chain fatty acids, were unaffected. Sulfosuccinimidyl oleate reacted exclusively with a plasma membrane protein with an apparent size of 85 kDa while sulfosuccinimidyl myristate also labeled a 75-kDa while sulfosuccinimidyl myristate also labeled a 75-kDa protein. These proteins were among the ones labeled by diisothiocyanodisulfonic acid (DIDS) which also inhibits fatty acid transport irreversibly. The data suggest that the 85-kDa protein, which is the only one labeled by all three inhibitors is involved in facilitating membrane permeation of long-chain fatty acids.     

Functional characterization of ExFadLO,an outer membrane protein required for exporting oxygenated long-chain fatty acids in Pseudomonas aeruginosa

Bacterial proteins of the FadL family have frequently been associated to the uptake of exogenous hydrophobic substrates. However, their outer membrane location and involvement in substrate uptake have been inferred mainly from sequence similarity to Escherichia coli FadL, the first well-characterized outer membrane transporters of Long-Chain Fatty Acids (LCFAs) in bacteria. Here we report the functional characterization of a Pseudomonas aeruginosa outer membrane protein (ORF PA1288) showing similarities to the members of the FadL family, for which we propose the name ExFadLO. We demonstrate herein that this protein is required to export LCFAs 10-HOME and 7,10-DiHOME, derived from a diol synthase oxygenation activity on oleic acid, from the periplasm to the extracellular medium. Accumulation of 10-HOME and 7,10-DiHOME in the extracellular medium of P. aeruginosa was abolished by a transposon insertion mutation in exFadLO (ExFadLO¯ mutant). However, intact periplasm diol synthase activity was found in this mutant, indicating that ExFadLO participates in the export of these oxygenated LCFAs across the outer membrane. The capacity of ExFadLO¯ mutant to export 10-HOME and 7,10-DiHOME was recovered after complementation with a wild-type, plasmid-expressed ExFadLO protein. A western blot assay with a variant of ExFadLO tagged with a V5 epitope confirmed the location of ExFadLO in the bacterial outer membrane under the experimental conditions tested. Our results provide the first evidence that FadL family proteins, known to be involved in the uptake of hydrophobic substrates from the extracellular environment, also function as secretion elements for metabolites of biological relevance.     

Effects of arachidonic acid and the other long-chain fatty acids on the membrane currents in the squid giant axon

Summary The effects of arachidonic acid and some other long-chain fatty acids on the ionic currents of the voltage-clamped squid giant axon were investigated using intracellular application of the test substances. The effects of these acids, which are usually insoluble in solution, were examined by using -cyclodextrin as a solvent. -cyclodextrin itself had no effect on the excitable membrane. Arachidonic acid mainly suppresses the Na current but has little effect on the K current. These effects are completely reversed after washing with control solution. The concentration required to suppress the peak inward current by 50% (ED₅₀) was 0.18mm, which was 10 times larger than that of medium-chain fatty acids like 2-decenoic acid. The Hill number was 1.5 for arachidonic acid, which is almost the same value as for medium-chain fatty acids. This means that the mechanisms of the inhibition are similar in both long- and medium-chain fatty acids. When the long-chain fatty acids were compared, the efficacy of suppression of Na current was about the same value for arachidonic acid, docosatetraenoic acid and docosahexaenoic acid. The suppression effects of linoleic acid and linolenic acid on Na currents were one-third of that of arachidonic acid. Oleic acid had a small suppression effect and stearic acid had almost no effect on the Na current. The currents were fitted to equations similar to those proposed by Hodgkin and Huxley (Hodgkin, A.L., Huxley, A.F. (1952)J. Physiol (London) 117:500–544) and the change in the parameters of these equations in the presence of fatty acids were calculated. The curve of the steady-state activation parameter (m ) for the Na current against membrane potential and the time constant of activation ( _m ) were shifted 10 mV in a depolarizing direction by the application of fatty acids. The time constant for inactivation ( _h ) has almost unaffected by application of these fatty acids.     

Liver fatty acid binding protein enhances sterol transfer by membrane interaction

Among the large family of fatty acid binding proteins, the liver L-FABP is unique in that it not only binds fatty acids but also interacts with sterols to enhance sterol transfer between membranes. Nevertheless, the mechanism whereby L-FABP potentiates intermembrane sterol transfer is unknown. Both fluorescence and dialysis data indicate L-FABP mediated sterol transfer between L-cell fibroblast plasma membranes occurs by a direct membrane effect: First, dansylated-L-FABP (DNS-L-FABP) is bound to L-cell fibroblast plasma membranes as indicated by increased DNS-L-FABP steady state polarization and phase resolved limiting anisotropy. Second, coumarin-L-FABP (CPM-L-FABP) fluorescence lifetimes were significantly increased upon interaction with plasma membranes. Third, dialysis studies with³H-cholesterol loaded plasma membranes showed that L-FABP added to the donor compartment of the dialysis cell stimulated³H-cholesterol transfer whether or not the dialysis membrane was permeable to L-FABP. However, L-FABP mediated intermembrane sterol transfer did require a sterol binding site on L-FABP. Chemically blocking the ligand binding site also inhibited L-FABP activity in intermembrane sterol transfer. Finally, L-FABP did not act either as an aqueous carrier or in membrane fusion. The fact that L-FABP interacted with plasma membrane vesicles and required a sterol binding site was consistent with a mode of action whereby L-FABP binds to the membrane prior to releasing sterol from the bilayer.Abbreviations ³H-CHO [1,2-³H(N)]-cholesterol - ANTS 8-aminonaphthalene-1,3,6-trisulfonic acid - CF carboxyfluorescein - CHO cholesterol - CPM (coumarin maleimide) 7-diethylamino-3-(4-maleimidylphenyl)-4-methylcoumarin - cPNA cisparinaric acid - DHE (dehydroergosterol) ^5,7,9(11),22-ergostatetraen-3-ol - DMF dimethyl formamide - DMPOPOP 1,4-bis[4-methyl-5-phenyl-2-oxazolyl]benzene - DNS (dansyl chloride) 5-dimethylaminonaphthalene-1-sulfonylchloride - DPX p-xylene-bis-pyridinium bromide - FBS fetal bovine serum - fluorescamine 4-phenylspiro[furan-2(3H), 1 phthalan]-3,3-dione - L-FABP liver fatty acid binding protein - NPG p-nitrophenylglyoxal - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine - SUV small unilamellar vesicle(s) - TNM tetranitromethane This work was supported in part by the National Institutes of Health United States Public Health Service (GM31651 and DK41402) and the American Heart Association (Postdoctoral Fellowship to JKW). The helpful assistance of Dr. Scott M. Colles and Mr. Daniel R. Prows in isolating L-FABP was much appreciated.     

Selective transport of long-chain fatty acids by FAT/CD36 in skeletal muscle of broilers

Fatty acid translocase (FAT/CD36) is a membrane receptor that facilitates long-chain fatty acid uptake. To investigate its role in the regulation of long-chain fatty acid composition in muscle tissue, we studied and compared FAT/CD36 gene expression in muscle tissues of commercial broiler chickens and Chinese local Silky fowls. The results from gas chromatography–mass spectrometry analysis of muscle samples demonstrated that Chinese local Silky fowls had significantly higher (P < 0.05) proportions of linoleic acid (LA) and palmitic acid, lower proportions (P < 0.05) of arachidonic acid (AA) and oleic acid than the commercial broiler chickens. The mRNA expression levels of fatty acid (FA) transporters (FA transport protein-1, membrane FA-binding protein, FAT/CD36 and caveolin-1) in the m. ipsilateral pectoralis and biceps femoris were analyzed by Q-PCR, and FAT/CD36 expression levels showed significant differences between these types of chickens (P < 0.01). Interestingly, the levels of FAT/CD36 expression are positively correlated with LA content (r = 0.567, P < 0.01) but negatively correlated with palmitic acid content (r = −0.568, P < 0.01). Further experiments in the stably transfected Chinese hamster oocytes cells with chicken FAT/CD36 cDNA demonstrated that overexpression of FAT/CD36 improves total FA uptake with a significant increase in the proportion of LA and AA, and a decreased proportion of palmitic acid. These results suggest that chicken FAT/CD36 may selectively transport LA and AA, which may lead to the higher LA deposition in muscle tissue.     

Platelet membrane fatty acids in thrombocytosis due to myeloproliferative disorders

The fatty acid composition of platelet membranes has been analysed in patients with thrombocytosis due to myeloproliferative disorders, who had not taken any drugs. A significant increase in palmitic and oleic acid, together with a decrease in stearic, linoleic and arachidonic acids was observed. The fatty acid pattern of platelet membranes was also analysed in patients during treatment with ASA (acetylsalicylic acid). ASA ingestion completely normalizes the platelet content of palmitic acid and partially that of stearic and arachidonic acid, whereas it has no effect on the level of linoleic acid and raises that of oleic acid. The altered pattern of fatty acids observed in patients may interfere with platelet function by decreasing membrane fluidity. Treatment of patients with ASA seems to act on platelet membranes by partially normalizing the fatty acid composition.     

Regulation of glucose and protein metabolism in growing steers by long-chain n-3 fatty acids in muscle membrane phospholipids is dose-dependent

A previous study showed that long-chain n-3 polyunsaturated fatty acids (LCn-3PUFA; >18 carbons n-3) exert an anabolic effect on protein metabolism through the upregulation of insulin sensitivity and activation of the insulin signaling pathway. This study further delineates for the first time whether the anabolic effect of LCn-3PUFA on metabolism is dose responsive. Six steers were used to test three graded amounts of menhaden oil rich in LCn-3PUFA (0%, 2% and 4%; enteral infusions) according to a double 3 × 3 Latin square design. Treatment comparisons were made using iso-energetic substitutions of control oil for menhaden oil and using 6-week experimental periods. The LCn-3PUFA in muscle total membrane phospholipids increased from 8%, 14% to 20% as dietary menhaden oil increased. Feeding graded amounts of menhaden oil linearly decreased plasma insulin concentration (49, 35 and 25 μU/ml, P = 0.01). The insulin-stimulated amino acid disposal rates as assessed using hyperinsulinemic-euglycemic-euaminoacidemic clamps (20, 40 and 80 mU/kg per h) were linearly increased by the incremental administrations of menhaden oil from 169, 238 to 375 μmol/kg per h (P = 0.005) during the 40 mU/kg per h clamp, and from 295, 360 and 590 μmol/kg per h (P = 0.02) during the 80 mU/kg per h clamp. Glucose disposal rate responded according to a quadratic relationship with the incremental menhaden oil amounts (P < 0.05). A regression analysis showed that 47% of the amino acid disposal rates elicited during the hyperinsulinemic clamp was related to muscle membrane LCn-3PUFA content (P = 0.003). These results show for the first time that both protein and glucose metabolism respond in a dose-dependent manner to menhaden oil and to muscle membrane LCn-3PUFA.     

Role of fatty acid transport protein 4 in metabolic tissues: insights into obesity and fatty liver disease

Fatty acid (FA) metabolism is a series of processes that provide structural substances, signalling molecules and energy. Ample evidence has shown that FA uptake is mediated by plasma membrane transporters including FA transport proteins (FATPs), caveolin-1, fatty-acid translocase (FAT)/CD36, and fatty-acid binding proteins. Unlike other FA transporters, the functions of FATPs have been controversial because they contain both motifs of FA transport and fatty acyl-CoA synthetase (ACS). The widely distributed FATP4 is not a direct FA transporter but plays a predominant function as an ACS. FATP4 deficiency causes ichthyosis premature syndrome in mice and humans associated with suppression of polar lipids but an increase in neutral lipids including triglycerides (TGs). Such a shift has been extensively characterized in enterocyte-, hepatocyte-, and adipocyte-specific Fatp4-deficient mice. The mutants under obese and non-obese fatty livers induced by different diets persistently show an increase in blood non-esterified free fatty acids and glycerol indicating the lipolysis of TGs. This review also focuses on FATP4 role on regulatory networks and factors that modulate FATP4 expression in metabolic tissues including intestine, liver, muscle, and adipose tissues. Metabolic disorders especially regarding blood lipids by FATP4 deficiency in different cell types are herein discussed. Our results may be applicable to not only patients with FATP4 mutations but also represent a model of dysregulated lipid homeostasis, thus providing mechanistic insights into obesity and development of fatty liver disease.     

Structural and related functional changes in sarcoplasmic reticulum induced by long-chain fatty acids

The effect of palmitic and oleic acids on Ca²⁺-ATPase activity in coupled preparations of sarcoplasmic reticulum isolated from rabbit hind leg muscle have been compared with their effects on vesicles uncoupled with Ca²⁺ ionophore, A23187. Palmitate at 2 µM · mg protein^–1 has no significant effect on enzyme activity and does not uncouple catalytic activity from calcium accumulation within the vesicles. Oleic acid at 1 µM · mg protein^–1 uncouples the vesicles, whereas 2 µM · mg protein^–1 completely inhibits Ca²⁺-ATPase activity. Fluorescence anisotropy of diphenylhexatriene is not significantly altered by palmitate, but a large transient increase in motion of the probe is observed with addition of oleic acid. The effects of oleic acid on enzyme activity are not mediated via an effect on the bulk properties of the hydrophobic domain of the membrane lipids.     

Effects of fatty acids on membrane currents in the squid giant axon

Summary The effects of fatty acids on the ionic currents of the voltage-clamped squid giant axon were investigated using intracellular and extracellular application of the test substances. Fatty acids mainly suppress the Na current but have little effect on the K current. These effects are completely reversed after washing with control solution. The concentrations required to suppress the peak inward current by 50% and Hill number were determined for each fatty acid. ED₅₀ decreased about 1/3 for each increase of one carbon atom. The standard free energy was –3.05 kJ mole^–1 for CH₂. The Hill number was 1.58 for 2-decenoic acid. The suppression effect of the fatty acids depends on the number of carbon atoms in the compounds and their chemical structure. Suppression of the Na current was clearly observed when the number of carbon atoms exceeded eight. When fatty acids of the same chain length were compared, 2-decenoic acid had strong inhibitory activity, but sebacic acid had no effect at all on the Na channel. The currents were fitted to equations similar to those proposed by Hodgkin and Huxley (J. Physiol. (London) 117:500–544, 1952) and the changes in the parameters of these equations in the presence of fatty acids were calculated. The curve of the steady-state activation parameter (m ) for the Na current against membrane potential and the time constant of activation ({ie113-1}) were shifted 20 mV in a depolarizing direction by the application of fatty acids. The time constant for inactivation ({ie113-2}) was almost no change by application of the fatty acids. The time constant for activation ({ie113-3}) of K current was shifted 20 mV in a depolarizing direction by the application of the fatty acids.     

Quantitation of plasma membrane fatty acid-binding protein by enzyme dilution and monoclonal antibody based immunoassay

Summary A plasma membrane fatty acid-binding protein (h-FABPPm) has been isolated from rat hepatocytes. Analogous proteins have also been identified in adipocytes, jejunal enterocytes and cardiac myocytes, all cells with high transmembrane fluxes of fatty acids. These 43 kDa, highly basic (pl = 9.1) FABP_pm 's appear unrelated to the smaller, cytosolic FABP's (designated FABP's) identified previously in the same tissues. h-FABP_pm appears closely related to the mitochondrial isoform of glutamic-oxaloacetic transaminase (mGOT), and both the purified protein and liver cell plasma membranes (LPM) possess GOT enzymatic activity. From their relative GOT specific activities it is estimated that h-FABP_pm constitutes approximately 2% of LPM protein, or about 0.7 × 10⁷ sites per cell. A monoclonal antibody-based competitive inhibition enzyme immunoassay (CIEIA) for h-FABP_pm is described; it yields an estimate of 3.4 x 10⁷ h-FABP_pm sites per hepatocyte. Quantitated by either method, h-FABPPm appears to be a highly abundant protein constituent of LPM.     

Effect of hydralazine on myocardial plasma membrane fatty acid binding protein (PM-FABP) during diabetes mellitus

Irregularities in K⁺ currents form the basis of several cardiovascular dysfunctions, among which are arrhythmias and vasospasms. The developmental regulation of voltage-gated K⁺ channels, however, has been difficult to study. A novel approach was therefore employed to examine these channels in muscle tissue. Primers for a PCR-based analysis were designed using published nucleic acid sequences for voltage-gated K⁺ channels. Final selection of the primer pairs was based on the homology present in the S4 and H5 transmembrane domains. A specific band was amplified with these primers using RNA isolated from both rat A10 vascular smooth muscle cells and rat heart tissue.     

Interaction modes of long-chain fatty acids in dipalmitoylphosphatidylcholine bilayer membrane: contrast to mode of inhalation anesthetics

The effects of long-chain fatty acids (four saturated and two unsaturated fatty acids, one derivative) on phase transitions of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were examined in the low concentration region, and the results were compared with those for an inhalation anesthetic. The effects of all fatty acids on the pre- and main-transition temperatures of the DPPC bilayer membrane appeared in the concentration range of μM order while that of the anesthetic appeared in the mM order. The appearance modes of these ligand actions were significantly different from one another. The three differential partition coefficients of the ligands between two phases of the DPPC bilayer membrane were evaluated by applying the thermodynamic equation to the variation of the phase-transition temperatures. The DPPC bilayer membranes showed the different receptivity for the ligands; the saturated fatty acids had an affinity for gel phase whereas unsaturated fatty acids and an anesthetic had an affinity for liquid-crystalline phase to the contrary. In particular, the receptivity for the ligands in the gel phase markedly changed depending on kinds of ligands. The interaction modes between the DPPC and fatty acid molecules in the gel phase were considered from the hexagonal lattice model. The disappearance compositions of the pretransition by the fatty acids coincided with the compositions at which the membrane is all covered by the units in each of which two fatty acids molecules are regularly distributed in the hexagonal lattice in a different way, and the distribution depended on the chain length and existence of a double bond for the fatty acids. The interpretation did not hold for the case of the anesthetic at all, which proved that a number of anesthetic molecules act the surface region of the bilayer membrane nonspecifically. The present study clearly implies that DPPC bilayer membranes have high ability to recognize kinds of ligand molecules and can discriminate among them with specific interaction by the membrane states.     

The membrane fatty acid-binding protein is not identical to mitochondrial glutamic oxaloacetic transaminase (mGOT)

Summary For evaluation whether the membrane fatty acid-binding protein is related to mGOT, studies on the structure and function of both purified proteins were performed. Physicochemical characterization revealed that both proteins are different: the membrane fatty acid-binding protein has a molecular weight of 40 kD and a pI of 8.5–9.0, whereas rat mGOT has a molecular weight of 44 kD and a pI of 9.5–10.0. According to this distinct differences, they migrated separately on 2-dimensional electrophoresis. Furthermore, monospecific antibodies against the membrane fatty acid binding protein did not react with rat mGOT. In co-chromatography studies only the membrane fatty acid-binding protein showed affinity for long chain fatty acids, but not mGOT. Moreover, membrane binding studies were performed with the monospecific antibody to the membrane fatty acid binding protein. The inhibitory effect of this antibody on plasma membrane binding of oleate was reversed after preabsorption of the antibody with the membrane fatty acid binding protein, but was not affected after preabsorption with mGOT. These results indicate that the membrane fatty acid binding protein and mGOT are structurally and functionally not related. The data also support the significance of this membrane protein in the plasma membrane binding process of long chain fatty acids.     

Ceramides with 2-hydroxylated,very long-chain polyenoic fatty acids in rodents: From testis to fertilization-competent spermatozoa

Sphingolipids from rodent testis and spermatozoa are known to contain non-hydroxylated (N-) and 2-hydroxylated (2-OH) very-long-chain polyunsaturated fatty acids (VLCPUFA). In this study, the contribution of species with each type of fatty acids to the total ceramides (Cer) and sphingomyelins (SM) was investigated in rat and mouse testis and in rat spermatozoa. The major VLCPUFA in both lipids of testis were N- and 2-OH versions of 28:4n−6, 30:5n−6 and 32:5n−6 in the rat, and predominantly of 30:5n−6 in the mouse. Absent altogether from rat pre-puberal testes, SM and Cer with N-VLCPUFA appeared 10 days earlier than those with 2-OH VLCPUFA in postnatal development, in association with germ cell differentiation. Conversely, in adult fertile rats that were gradually deprived of germ cells in vivo after treatment with doxorubicin, SM and Cer with N-VLCPUFA decreased earlier than their 2-OH counterparts, and neither was present in aspermatogenic testes. In rat epididymal spermatozoa, the content of Cer prevailed over that of SM and 2-OH VLCPUFA prevailed over N-VLCPUFA in both lipids. In mature gametes, the acrosomal reaction resulted in an almost complete hydrolysis of the species of SM that contain both types of VLCPUFA to produce the corresponding Cer. Ceramides are biosynthetic precursors of SM in the testis, but themselves final products in spermatozoa. VLCPUFA-rich SM and Cer are thus produced in germ cells with the teleological objective of fulfilling their ultimate physiological role in spermatozoa that are apt and ready to fertilize an oocyte.     

亚麻籽粒中主要脂肪酸和蛋白质积累过程研究

以亚麻品种82(50)为材料,研究了亚麻籽粒中主要脂肪酸、蛋白质和糖积累的规律,结果表明:在亚麻籽粒发育成熟的过程中,棕榈酸、亚油酸含量持续下降,当籽粒完全成熟时降至最低;油酸和亚麻酸含量持续增加,当籽粒完全成熟时含量最高;硬脂酸在籽粒发育10～25d,其含量逐渐提高,以后逐渐下降。蛋白质随着籽粒发育成熟其含量持续提高,且蛋白质含量的提高主要发生在籽粒发育成熟的30d以前。总糖随籽粒发育成熟含量迅速下降,当种子完全成熟时降至最低。