Composition and metabolism of phospholipids of human leukocytes

Human mononuclear (MN) and polymorphonuclear (PMN) leukocytes were analyzed for their phospholipid, triglyceride, cholesterol and fatty acid content. The phospholipid/cholesterol ratio was 1.24 for both cels. MN cells contain more phosphatidylcholine (PC), but less phosphatidylserine (PS), phosphatidylethanolamine (PE) and sphingomyelin (SPH) than PMN cells when expressed as percent of total phospholipid. When expressed on the basis of lipid content per cell, MN cells contain less PS, PE and SPH but more triglyceride than PMN cells. PMN cells incorporate palmitic, stearic, linoleic and linolenic acids into their phospholipids, triglycerides or cholesterol esters. The incorporation into triglycerides was highest for all fatty acids. Of the phospholipids, the incorporation was highest into PC. Labeled fatty acids also were found in proteins which had been delipidized by exhaustive extraction with organic solvents. These represent tightly or covalently bound fatty acids. The incorporation of [³H]palmitic acid into this protein fraction is stimulated by insulin.     

Sterol carrier protein-2/sterol carrier protein-x expression differentially alters fatty acid metabolism in L cell fibroblasts

Sterol carrier protein-2 (SCP-2) and SCP-x are ubiquitous proteins found in all mammalian tissues. Although both proteins interact with fatty acids, their relative contributions to the uptake, oxidation, and esterification of straight-chain (palmitic) and branched-chain (phytanic) fatty acids in living cells has not been resolved. Therefore, the effects of each gene product on fatty acid metabolism was individually examined. Based on the following, SCP-2 and SCP-x did not enhance the uptake/translocation of fatty acids across the plasma membrane into the cell: i) a 2-fold increase in phytanic and palmitic acid uptake was observed at long incubation times in SCP-2- and SCP-x-expressing cells, but no differences were observed at initial time points; ii) uptake of 2-bromo-palmitate, a nonoxidizable, poorly metabolizable fatty acid analog, was unaffected by SCP-2 or SCP-x overexpression; and iii) SCP-2 and SCP-x expression did not increase targeting of radiolabeled phytanic and palmitic acid to the unesterified fatty acid pool. Moreover, SCP-2 and SCP-x expression enhanced fatty acid uptake by stimulating the intracellular metabolism via fatty acid oxidation and esterification. In summary, these data showed for the first time that SCP-2 and SCP-x stimulate oxidation and esterification of branched-chain as well as straight-chain fatty acids in intact cells.     

Cellular fatty acid uptake: the contribution of metabolism

PURPOSE OF REVIEW: The aim of this review is to highlight the importance of fatty acid metabolism as a major determinant in fatty acid uptake. In particular, we emphasize how the activation, intracellular transport and downstream metabolism of fatty acids influence their uptake into cells. RECENT FINDINGS: Studies examining fatty acid entry into cells have focused primarily on the roles of plasma membrane proteins or the question of passive diffusion. Recent studies, however, strongly suggest that a driving force governing fatty acid uptake is the metabolic demand for fatty acids. Both gain and loss-of-function experiments indicate that fatty acid uptake can be modulated by activation at both the plasma membrane and internal sites, by intracellular fatty acid binding proteins, and by enzymes in synthetic or degradative metabolic pathways. Although the mechanism is not known, it appears that converting fatty acids to acyl-CoAs and downstream metabolic intermediates increases cellular fatty acid uptake, probably by limiting efflux. SUMMARY: Altered fatty acid metabolism and the accumulation of triacylglycerol and lipid metabolites has been strongly associated with insulin resistance and diabetes, but we do not fully understand how the entry of fatty acids into cells is regulated. Future studies of cellular fatty acid uptake should consider the influence of fatty acid metabolism and the possible interactions between fatty acid metabolism or metabolites and fatty acid transport proteins.     

Fatty acids regulate pigmentation via proteasomal degradation of tyrosinase: a new aspect of ubiquitin-proteasome function

Fatty acids are common components of biological membranes that are known to play important roles in intracellular signaling. We report here a novel mechanism by which fatty acids regulate the degradation of tyrosinase, a critical enzyme associated with melanin biosynthesis in melanocytes and melanoma cells. Linoleic acid (unsaturated fatty acid, C18:2) accelerated the spontaneous degradation of tyrosinase, whereas palmitic acid (saturated fatty acid, C16:0) retarded the proteolysis. The linoleic acid-induced acceleration of tyrosinase degradation could be abrogated by inhibitors of proteasomes, the multicatalytic proteinase complexes that selectively degrade intracellular ubiquitinated proteins. Linoleic acid increased the ubiquitination of many cellular proteins, whereas palmitic acid decreased such ubiquitination, as compared with untreated controls, when a proteasome inhibitor was used to stabilize ubiquitinated proteins. Immunoprecipitation analysis also revealed that treatment with fatty acids modulated the ubiquitination of tyrosinase, i.e. linoleic acid increased the amount of ubiquitinated tyrosinase whereas, in contrast, palmitic acid decreased it. Furthermore, confocal immunomicroscopy showed that the colocalization of ubiquitin and tyrosinase was facilitated by linoleic acid and diminished by palmitic acid. Taken together, these data support the view that fatty acids regulate the ubiquitination of tyrosinase and are responsible for modulating the proteasomal degradation of tyrosinase. In broader terms, the function of the ubiquitin-proteasome pathway might be regulated physiologically, at least in part, by fatty acids within cellular membranes.     

Human Fatty Acid Transport Protein 2a/Very Long Chain Acyl-CoA Synthetase 1 (FATP2a/Acsvl1) Has a Preference in Mediating the Channeling of Exogenous n-3 Fatty Acids into Phosphatidylinositol

The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (M_r 70,000) and FATP2b (M_r 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14–C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6). Using the same cells expressing FATP2a or FATP2b, electrospray ionization/MS was used to follow the trafficking of stable isotopically labeled n-3 fatty acids into phosphatidylcholine and phosphatidylinositol. The expression of FATP2a resulted in the trafficking of C18:3-CoA and C22:6-CoA into both phosphatidylcholine and phosphatidylinositol but with a distinct preference for phosphatidylinositol. Collectively these data demonstrate FATP2a functions in fatty acid transport and activation and provides specificity toward n-3 fatty acids in which the corresponding n-3 acyl-CoAs are preferentially trafficked into acyl-CoA pools destined for phosphatidylinositol incorporation.     

Specificity of fatty acid acylation of cellular proteins

Labeling of the BC3H1 muscle cell line with [3H] palmitate and [3H]myristate results in the incorporation of these fatty acids into a broad spectrum of different proteins. The patterns of proteins which are labeled with palmitate and myristate are distinct, indicating a high degree of specificity of fatty acylation with respect to acyl chain length. The protein-linked [3H]palmitate is released by treatment with neutral hydroxylamine or by alkaline methanolysis consistent with a thioester linkage or a very reactive ester linkage. In contrast, only a small fraction of the [3H]myristate which is attached to proteins is released by treatment with hydroxylamine or alkaline methanolysis, suggesting that myristate is linked to proteins primarily through amide bonds. The specificity of fatty acid acylation has also been examined in 3T3 mouse fibroblasts and in PC12 cells, a rat pheochromacytoma cell line. In both cells, palmitate is primarily linked to proteins by a hydroxylamine-labile linkage while the major fraction of the myristic acid (60-70%) is linked to protein via amide linkage and the remainder via an ester linkage. Major differences were noted in the rate of fatty acid metabolism in these cells; in particular in 3T3 cells only 33% of the radioactivity incorporated from myristic acid into proteins is in the form of fatty acids. The remainder is presumably the result of conversion of label to amino acids. In BC3H1 cells, palmitate- and myristate-containing proteins also exhibit differences in subcellular localization. [3H]Palmitate-labeled proteins are found almost exclusively in membranes, whereas [3H]myristate-labeled proteins are distributed in both the soluble and membrane fractions. These results demonstrate that fatty acid acylation is a covalent modification common to a wide range of cellular proteins and is not restricted solely to membrane-associated proteins. The major acylated proteins in the various cell lines examined appear to be different, suggesting that the acylated proteins are concerned with specialized cell functions. The linkages through which fatty acids are attached to proteins also appear to be highly specific with respect to the fatty acid chain length.     

Acylation of cellular proteins with endogenously synthesized fatty acids

A number of cellular proteins contain covalently bound fatty acids. Previous studies have identified myristic acid and palmitic acid covalently linked to protein, the former usually attached to proteins by an amide linkage and the latter by ester or thio ester linkages. While in a few instances specific proteins have been isolated from cells and their fatty acid composition has been determined, the most frequent approach to the identification of protein-linked fatty acids is to biosynthetically label proteins with fatty acids added to intact cells. This procedure introduces possible bias in that only a selected fraction of proteins may be labeled, and it is not known whether the radioactive fatty acid linked to the protein is identical with that which is attached to the protein when the fatty acid is derived from endogenous sources. We have examined the distribution of protein-bound fatty acid following labeling with [3H]acetate, a general precursor of all fatty acids, using BC3H1 cells (a mouse muscle cell line) and A431 cells (a human epidermoid carcinoma). Myristate, palmitate, and stearate account for essentially all of the fatty acids linked to protein following labeling with [3H]acetate, but at least 30% of the protein-bound palmitate in these cells was present in amide linkage. In BC3H1 cells, exogenous palmitate becomes covalently bound to protein such that less than 10% of the fatty acid is present in amide linkage. These data are compatible with multiple protein acylating activities specific for acceptor protein fatty acid chain length and linkage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of fatty acids and lipid by a Candida sp. growing on a fraction of n-alkanes predominating in tridecane

A Candida sp. was grown on a fraction of n-alkanes (dodecane 22%, tridecane 48%, tetradecane 28%) as sole carbon source. The growth rate was increased most markedly by using high concentrations of n-alkanes (16.7% v/v). When grown in a 5 liter fermentor, the yeast reached its highest yield (60 g. of cell dry wt/l) with a concomitant high yield of fatty acids (21 g of fatty acids/l), by using a nitrogen-deficient medium. To achieve good growth, it was essential to use an inoculum (1 part into 10) of rapidly growing cells and beneficial to increase the agitation rate gradually once growth had begun. After 108 hr maximum conversions of substrate to product were: 71.5% (w/w) for alkanes into cells and 24.8% (w/w) for alkanes into fatty acids. Of the, total fatty acids at the end of the fat-accumulating phase of growth 54% were shorter in chain length than palmitic acid (C₁₆H₃₂O₂). When grown on glucose, as sole carbon source, less than 2% of the total fatty acids were shorter than palmitic acid. When n-alkanes were added to cells growing on glucose, short-chain fatty acids (C₁₀ to C₁₄) were synthesized immediately, indicating a derepressed enzyme system for hydrocarbon assimilation and the absence of diauxie. The production of these acids was at the apparent sacrifice of linoleic acid synthesis. In spite of the high conversion ratios, it is concluded that it would be uneconomical to produce fatty acids, even expensive ones such as lauric acid, by microbial transformation of n-alkanes.     

Characterization of a protein fatty acylesterase present in microsomal membranes of diverse origin

A microsomal activity of baby hamster kidney cells which cleaves ester-type bound fatty acids from acyl proteins in vitro has been characterized. This activity is also present in microsomal membranes from pig liver, calf kidney, and human mucous cells. Cell free deacylation is described for the Semliki Forest virus acyl proteins E1 and E2 and the precursor of E2 designated p62. Acyl chain cleavage operates with both exogenous and endogenous viral acyl protein substrates. The in vitro cleavage requires microsomes solubilized by detergents of which various kinds are equally effective (Nonidet P-40, Tween 20, sodium deoxycholate, Triton X-100, or octyl-beta-D-glucoside). If microsomes are boiled for 15 min prior to the incubation, deacylation is abolished completely and no radioactivity is released from the palmitoylated acyl proteins during incubation with either detergents or microsomes alone. No changes in the molecular structure of the deacylated Semliki Forest virus proteins were detected, and the cleavage product was identified as free fatty acid. Deacylation is time- and temperature-dependent and can be enhanced by increasing the concentration of microsomal protein in the incubation mixture. It is completely inhibited under acidic conditions (pH 5) and at low temperature (4 degrees C). Deacylation also occurs in the presence of EDTA and bivalent cations such as Mg2+, Mn2+, and Ca2+ which influence the reaction marginally. On the other hand, fatty acid release is drastically reduced with a mixture of Co2+, Zn2+, and Hg2+ ions. The activity is not identical with protein fatty acyltransferase operating in the reverse direction, since a partially purified preparation of this acyltransferase failed to cleave fatty acids from fatty acylated substrate proteins. Taken together, these data lead us to postulate an enzymatic activity which cleaves fatty acids from ester-type fatty acylated proteins, and we propose to designate this enzyme a protein fatty acylesterase.     

Metabolism of tween-Fatty Acid esters by cultured soybean cells : kinetics of incorporation into lipids, subsequent turnover, and associated changes in endogenous Fatty Acid synthesis

Uptake of Tween-fatty acid esters and incorporation of the fatty acids into lipids by soybean (Glycine max [L.] Merr.) suspension cultures was investigated, together with subsequent turnover of the incorporated fatty acids and associated changes in endogenous fatty acid synthesis. Tween uptake was saturable, and fatty acids were rapidly transferred from Tweens to all acylated lipids. Patterns of incorporation into glycerolipids were similar in cells treated with Tweens carrying [1-¹⁴C]-fatty acids and in cells treated with [1-¹⁴C]acetate, indicating that exogenous fatty acids were used for glycerolipid synthesis essentially as if they had been made by the cell. In Tween-treated cells neutral lipids (which include Tweens) initially accounted for the majority of lipid radioactivity. Radioactivity was then rapidly transferred to glycerolipids. A transient pool of free fatty acids accounting for up to 10% of lipid radioactivity was observed. This was consistent with the hypothesis that fatty acids are transferred from Tweens to lipids by deacylation of the Tweens, creating a pool of free fatty acids which are then used for lipid synthesis. Sterols were only slightly labeled in cells treated with Tweens, but accounted for nearly 50% of lipid radioactivity in cells treated with acetate. This suggested very little degradation and reutilization of the radioactive fatty acids in cells treated with Tweens. In cells treated with either [1-¹⁴C]acetate or Tween-[1-¹⁴C]-18:1, 70% of the initial fatty acid radioactivity remained in fatty acids after a 100 hour chase. By contrast, fatty acids not normally present disappeared more rapidly, suggesting differential treatment of such fatty acids compared with those normally present. Cells which had incorporated large amounts of exogenous fatty acids altered fatty acid synthesis in three distinct ways: (a) amounts of [1-¹⁴C]acetate incorporated into fatty acids were reduced; (b) cells incorporating exogenous unsaturated fatty acids increased the proportion of [1-¹⁴C]acetate partitioned into saturated fatty acids, while the converse was true of cells which had incorporated exogenous saturated fatty acids; (c) desaturation of 18:1 to 18:2 and 18:3 was reduced in cells which had incorporated unsaturated fatty acids. These results suggest that Tween-fatty acid esters will be useful for supplying fatty acids to cells for a variety of studies related to fatty acid or membrane metabolism.     

Effect of Polyunsaturated Fatty Acids on Fetal Mouse Brain Cells in Culture in a Chemically Defined Medium

Abstract: The biochemical and morphological effects of polyunsaturated fatty acids on fetal brain cells grown in a chemically defined medium were studied. Fetal brain cells were dissociated from mouse cerebral hemispheres taken on the 16th day of gestation. After cells had grown in chemically defined medium for 8 days, the proportion of polyunsaturated fatty acids of cultured cells was only one-half of that observed at day 0 and about 1.5 times less than that of cells grown in serum-supplemented medium. Fatty acid 20:3(n-9) was present in cultured cells grown in either chemically defined or serum-supple-mented medium. demonstrating the deficiency of essential fatty acids. The reduced amount of polyunsaturated fatty acids in cells grown in the chemically defined medium was balanced by an increase in monounsaturated fatty acids. The saturated fatty acids were not affected. When added at the seeding time, linoleic, linolenic, arachidonic, or docosahexaenoic acid stimulated the proliferation of small dense cells. Besides, we demonstrate that each of the four fatty acids studied was incorporated into phospholipids. Adding fatty acids of the n-6 series increased the content of n-6 fatty acids in the cells, but also provoked an increase in the n-3 fatty acids. Among several combinations of fatty acids, only 20:4 and 22:6, when added to the culture in a ratio of 2:1, restored a fatty acid profile similar to controls (i.e. in vivo tissue taken at post- natal dav 5).     

Metabolic engineering of Saccharomyces cerevisiae for production of novel lipid compounds

The yeast Saccharomyces cerevisiae has been modified successfully for production of numerous metabolites and therapeutic proteins through metabolic engineering, but has not been utilized to date for the production of lipid-derived compounds. We developed a lipid metabolic engineering strategy in S. cerevisiae based upon culturing techniques that are typically employed for studies of peroxisomal biogenesis; cells were grown in media containing fatty acids as a sole carbon source, which promotes peroxisomal proliferation and induction of enzymes associated with fatty acid beta-oxidation. Our results indicate that growth of yeast on fatty acids such as oleate results in extensive uptake of these fatty acids from the media and a subsequent increase in total cellular lipid content from 2% to 15% dry cell weight. We also show that co-expression of plant fatty acid desaturases 2 and 3 ( FAD2 and FAD3), using a fatty acid-inducible peroxisomal gene promoter, coupled the processes of fatty acid uptake with the induction of a new metabolic pathway leading from oleic acid (18:1) to linolenic acid (18:3). Finally, we show that cultivation of yeast cells in the presence of triacylglycerols and exogenously supplied lipase promotes extensive incorporation of triglyceride fatty acids into yeast cells. Collectively, these results provide a framework for bioconversion of low-cost oils into value-added lipid products.     

Influence of stringent and relaxed response on excretion of recombinant proteins and fatty acid composition in Escherichia coli

In contrast to stringent (relA⁺) cells of Escherichia coli, relaxed (relA) cells excreted recombinant proteins (-lactamase, interferon 1) into the culture medium during amino acid limitation. Comparative analyses of overall fatty acid composition in relA⁺ cells and relA cells were performed and revealed that, in wild-type cells, drastic alterations occurred during the stringent response. The portion of saturated fatty acids (C16:0) and the fractions of cyclopropane fatty acids (C17_cyc and C19_cyc) increased whereas the portions of unsaturated fatty acids (C16:1 and C18:1) decreased. In cells of the relaxed mutant, no significant changes in the overall composition of the fatty acids were observed after the onset amino acid limitation. These results indicate that a change in fatty acid composition of membrane lipids after starvation of cells may be responsible for the prevention of loss of cellular proteins into the culture medium in stringent controlled cells of Escherichia coli.     

Fatty acid interactions with proteins: what X-ray crystal and NMR solution structures tell us

The interactions of fatty acids with proteins have been studied by a variety of conventional approaches for decades. However, only limited aspects of fatty acid-protein interactions have been elucidated, even with the integration of information gleaned from the many techniques. Judgments must be made about what information is most reliable, particularly when derivatives of fatty acids are substituted for natural fatty acids. In recent years, the application of techniques of structural biology has brought about dramatic advances in this important area of lipid research. High-resolution crystallographic and NMR structures of several proteins with bound fatty acids reveal the complete tertiary structure of the protein and molecular details of fatty acid-protein interactions. The examples presented include most of the known structures of (non-enzymatic) proteins that bind fatty acids. The proteins are found in very different compartments of cells and organisms: the plasma compartment (human serum albumin); the cytosolic compartment of mammalian cells (fatty acid- binding proteins); the cytosol of plant cells (nonspecific lipid-transfer protein); the nucleus of mammalian cells (peroxisome proliferator-activated receptor and hepatic nuclear factor 4); and a bacterial membrane (halorhodopsin). This review discusses the structural features of these proteins and their binding pocket(s) and compares the specific modes of their interactions with fatty acids.     

Transport of fatty acids across membranes by the diffusion mechanism

In early research on fatty acid transport, passive diffusion seemed to provide an adequate explanation for movement of fatty acids through the membrane bilayer. This simple hypothesis was later challenged by the discovery of several proteins that appeared to be membrane-related fatty acid transporters. In addition, some biophysical studies suggested that fatty acids moved slowly through the simple model membranes (phospholipid bilayers), which would provide a rationale for protein-assisted transport. Furthermore, it was difficult to rationalize how fatty acids could diffuse passively across the bilayer as anions. Newer studies have shown that fatty acids are present in membranes in the un-ionized as well as the ionized form, and that the un-ionized form can cross a protein-free phospholipid bilayer quickly. This flip-flop mechanism has been validated in cells by intracellular pH measurements. The role of putative fatty acid transport proteins remains to be clarified.     

Effect of Substrate on the Fatty Acid Composition of Hydrocarbon-utilizing Microorganisms

The fatty acid pattern in three hydrocarbon-utilizing bacteria during growth on various substrates was examined. The predominant fatty acids in acetate-grown cells were C(16), C(16:1), C(18:1), and Br-C(19) and the major fatty acids in propane-grown cells were C(15), C(17), C(17:1), C(18:1), and Br-C(18). When one organism (Mycobacterium sp. strain OFS) was grown on the n-alkanes from C(13) to C(17), the major fatty acid in the cells was of the same chain length as the substrate. Studies on the incorporation of acetate into the cellular fatty acids of microorganisms growing on C(15) and C(17)n-alkanes suggest that the oxidative products of the substrate are incorporated into the cellular fatty acids without degradation to acetate.