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.     

Release of free fatty acids from Ehrlich ascites tumor cells

Ehrlich ascites tumor cells release free fatty acids (FFA) during in vitro incubation in media that contain albumin. The released FFA are derived by lipolysis from endogenous lipid esters. Addition of glucose to the incubation medium greatly decreases the quantity of fatty acid released by the cells. Cyanide, which inhibits endogenous lipid oxidation but not lipolysis, increases the quantity of fatty acid released to media containing albumin and causes free fatty acid to accumulate in the cells in the absence of exogenous albumin. The release of fatty acid, either preformed or derived by lipolysis during prolonged incubations, occurs under conditions of net fatty acid uptake from the incubation medium. Net release of fatty acid from the cell occurs only when fatty acid-extracted albumin is present in the extracellular medium; extrapolation of the data suggests that net release will not occur under physiological conditions. It is postulated that free fatty acid uptake and release are independent processes, the direction of net fatty acid movement being determined by the relationship between cellular free fatty acid concentration (regulating efflux) and the molar ratio of free fatty acid to albumin in the extracellular medium (regulating uptake).     

Control of fatty-acid synthetase levels by exogeneous long-chain fatty acids in the yeasts Candida lipolytica and Saccharomyces cerevisiae.

Endogeneous fatty acid biosynthesis in the two yeast species, Saccharomyces cerevisiae and Candida lipolytica is completely repressed by the addition of long-chain fatty acids to the growth medium. In Candida lipolytica, this repression is accompanied by a corresponding loss of fatty acid synthetase activity in the cell homogenate, when the cells were grown on fatty acids as the sole carbon source. The activity of the Saccharomyces cerevisiae fatty acid synthetase, however, remains unaffected by the addition of fatty acids to a glucose-containing growth medium. From fatty-acid-grown Candida lipolytica cells no fatty acid synthetase complex can be isolated, nor is there any immunologically cross-reacting fatty acid synthetase protein detectable in the crude cell extract. From this it is concluded that Candida lipolytica, but not Saccharomyces cerevisiae, is able to adapt to the growth on fatty acids either by repression of fatty acid synthetase biosynthesis or by a fatty-acid-induced proteolytic degradation of the multienzyme complex. Similarly, the fatty acid synthetase complex disappears rapidly from stationary phase Candida lipolytica cells even after growth in fatty-acid-free medium. Finally, it was found that the fatty acid synthetase complexes from Saccharomyces cerevisiae and Candida lipolytica, though very similar in size and subunit composition, were immunologically different and had no common antigenic determinants.     

Fatty acyl-AMP as an intermediate in fatty acid reduction to aldehyde in luminescent bacteria

The acyl protein synthetase component (50K) of the fatty acid reductase complex from the luminescent system of Photobacterium phosphoreum has been found to catalyze the activation of fatty acid via formation of an enzyme bound acyl-AMP (carboxyphosphate mixed anhydride) immediately prior to the acylation of the enzyme. PPi-ATP exchange and nucleotide binding experiments are dependent on fatty acid and indicate that the fatty acyl-AMP is directly formed and that an adenylated enzyme intermediate is not part of the mechanism. The formation of acyl-AMP from fatty acid and ATP is reversible with a standard free energy of -2 kcal/mol, and is dependent on Mg2+. The fatty acyl-AMP intermediate has been isolated and shown to be part of the pathway of fatty acid reduction. The 34K component of the complex, which strongly stimulates the acylation of the 50K protein by fatty acyl-AMP or fatty acid and ATP, is not required for the formation of acyl-AMP showing that it differentially affects the fatty acid activation and acylation steps catalyzed by the 50K protein.     

Silencing of hepatic fatty acid transporter protein 5 in vivo reverses diet-induced non-alcoholic fatty liver disease and improves hyperglycemia

Non-alcoholic fatty liver disease is a serious health problem linked to obesity and type 2 diabetes. To investigate the biological outcome and therapeutic potential of hepatic fatty acid uptake inhibition, we utilized an adeno-associated virus-mediated RNA interference technique to knock down the expression of hepatic fatty acid transport protein 5 in vivo prior to or after establishing non-alcoholic fatty liver disease in mice. Using this approach, we demonstrate here the ability to achieve specific, non-toxic, and persistent knockdown of fatty acid transport protein 5 in mouse livers from a single adeno-associated virus injection, resulting in a marked reduction of hepatic dietary fatty acid uptake, reduced caloric uptake, and concomitant protection from diet-induced non-alcoholic fatty liver disease. Importantly, knockdown of fatty acid transport protein 5 was also able to reverse already established non-alcoholic fatty liver disease, resulting in significantly improved whole-body glucose homeostasis. Thus, continued activity of hepatic fatty acid transport protein 5 is required to sustain caloric uptake and fatty acid flux into the liver during high fat feeding and may present a novel avenue for the treatment of non-alcoholic fatty liver disease.     

Palmityl-CoA and palmitylcarnitine interactions in mitochondrial fatty acid elongation

We have investigated interactions of palmityl-CoA and l-palmitylcarnitine as substrates for mitochondrial fatty acid elongation. l-Palmitylcarnitine is a more effective substrate primer for fatty acid elongation by intact mitochondria than is palmityl-CoA. Exogenous l-carnitine inhibited l-palmitylcarnitine-supported mitochondrial fatty acid elongation by both sonically disrupted and intact heart mitochondria, probably by shifting the equilibrium between palmitylcarnitine and palmityl-CoA toward palmitylcarnitine, thus removing palmityl-CoA from the reaction. d-Carnitine was without effect. d-Palmitylcarnitine inhibition of palmitylcarnitine transferase activity decreased palmitylcarnitine-stimulated mitochondrial fatty acid elongation but increased palmityl-CoA supported fatty acid elongation, presumably by increasing the effective concentration of palmityl-CoA in the assay medium. The data indicate that, although l-palmitylcarnitine is an effective substrate primer for mitochondrial fatty acid elongation, palmityl-CoA rather than palmitylcarnitine is the immediate precursor for fatty acid chain elongation.     

Thematic Review Series: Living History of Lipids: Discovery of essential fatty acids

Dietary fat was recognized as a good source of energy and fat-soluble vitamins by the first part of the 20th century, but fatty acids were not considered to be essential nutrients because they could be synthesized from dietary carbohydrate. This well-established view was challenged in 1929 by George and Mildred Burr who reported that dietary fatty acid was required to prevent a deficiency disease that occurred in rats fed a fat-free diet. They concluded that fatty acids were essential nutrients and showed that linoleic acid prevented the disease and is an essential fatty acid. The Burrs surmised that other unsaturated fatty acids were essential and subsequently demonstrated that linolenic acid, the omega-3 fatty acid analog of linoleic acid, is also an essential fatty acid. The discovery of essential fatty acids was a paradigm-changing finding, and it is now considered to be one of the landmark discoveries in lipid research.     

The essential fatty acid status in phenylketonuria patients under treatment

Phenylketonuric patients are on a special diet that lacks certain essential fatty acids. This study evaluates the essential fatty acid status of a group of phenylketonuric patients in the Netherlands undergoing dietary treatment. To this end, the essential fatty acid status of nine phenylketonuria patients was studied. On the basis of age and gender, two control subjects were selected for each patient. The essential fatty acid composition of duplicate food portions and the essential fatty acid status of plasma and erythrocytes were analyzed. Phenylketonuria subjects had a different essential fatty acid profile from their peers, especially concerning the n-3 fatty acids. N-6 and n-3 fatty long-chain polyenes were hardly consumed by phenylketonuria subjects, in contrast to the control subjects. Linoleic acid, on the other hand, was consumed in significantly higher amounts by phenylketonuria subjects and made up about 40% of their daily fat consumption. The essential fatty acid consumption pattern of the phenylketonuria subjects is mirrored by the essential fatty acid concentrations in blood. The essential fatty acid status of the phenylketonuric diet should be improved in order to prevent deficiency in n-3 fatty acids.     

Utilization of free fatty acids complexed to human plasma lipoproteins by mammalian cell suspensions

The purpose of this study was to determine whether lipoprotein-bound free fatty acid could be utilized by isolated mammalian cells. Ehrlich ascites tumor cells were incubated in vitro with radioactive free fatty acids that were bound to human plasma lipoproteins. Under these conditions, lipoprotein-bound free fatty acids were readily taken up by the cells. After 2 min of incubation with free fatty acids bound to low density lipoproteins, most of the radioactivity that was associated with the cells was in the form of free fatty acids. As the incubation continued, increasing amounts of radioactivity were incorporated into CO(2) and cell lipids, particularly phospholipids. Most of the free fatty acid uptake was the result of fatty acid transfer from low density lipoproteins to the cell, not from irreversible incorporation of the intact free fatty acid-low density lipoprotein complex. Fatty acid uptake increased as the ratio of free fatty acid to low density lipoprotein was raised. When albumin was added to the medium, free fatty acid uptake decreased. A large percentage of the newly incorporated cellular radioactivity was released into the medium if the cells were exposed subsequently to a solution containing albumin. Most of the released radioactivity was in the form of free fatty acid. The results with this experimental model suggest that lipoprotein-bound free fatty acid, like albumin-bound free fatty acid, is readily available for uptake by isolated cells. The mechanism of free fatty acid utilization by the Ehrlich cell is similar when either low density lipoprotein or serum albumin serves as the fatty acid carrier.     

Synthesis and utilization of fatty acids by wild-type and fatty acid auxotrophs of Caulobacter crescentus.

The fatty acid composition of the dimorphic bacterium Caulobacter crescentus was found to consist primarily of 16- and 18-carbon fatty acids, both saturated and monounsaturated, in agreement with the findings of Chow and Schmidt (J. Gen. Microbiol. 83:359-373, 1974). In addition, two minor but as yet unidentified fatty acids were detected. Chromatographic mobilities suggested that these fatty acids may be a cyclopropane and a branched-chain fatty acid. In addition, we demonstrated that the fatty acid composition of wild-type C. crescentus can be altered by growing the cells in medium supplemented with any one of a variety of unsaturated fatty acids. Linoleic acid, a diunsaturated fatty acid which is not synthesized by C. crescentus, was incorporated into phospholipids without apparent modification. In addition, we found that C. crescentus, like Escherichia coli, synthesizes vaccenic acid (18:1 delta 11,cis) rather than oleic acid (18:1 delta 9,cis). This result allowed us to deduce that the mechanism of fatty acid desaturation in C. crescentus is anaerobic, as it is in E. coli. Finally, we examined the fatty acid biosynthesis and composition of two unsaturated fatty acid auxotrophs of C. crescentus. Neither of these mutants resembled the E. coli unsaturated fatty acid auxotrophs, which have defined enzymatic lesions in fatty acid biosynthesis. Rather, the mutants appeared to have defects relating to the complex coordination of membrane biogenesis and cell cycle events in C. crescentus.     

Metabolic challenges reveal impaired fatty acid metabolism and translocation of FAT/CD36 but not FABPpm in obese Zucker rat muscle

We examined, in muscle of lean and obese Zucker rats, basal, insulin-induced, and contraction-induced fatty acid transporter translocation and fatty acid uptake, esterification, and oxidation. In lean rats, insulin and contraction induced the translocation of the fatty acid transporter FAT/CD36 (43 and 41%, respectively) and plasma membrane-associated fatty acid binding protein (FABPpm; 19 and 60%) and increased fatty acid uptake (63 and 40%, respectively). Insulin and contraction increased lean muscle palmitate esterification and oxidation 72 and 61%, respectively. In obese rat muscle, basal levels of sarcolemmal FAT/CD36 (+33%) and FABPpm (+14%) and fatty acid uptake (+30%) and esterification (+32%) were increased, whereas fatty acid oxidation was reduced (-28%). Insulin stimulation of obese rat muscle increased plasmalemmal FABPpm (+15%) but not plasmalemmal FAT/CD36, blunted fatty acid uptake and esterification, and failed to reduce fatty acid oxidation. In contracting obese rat muscle, the increases in fatty acid uptake and esterification and FABPpm translocation were normal, but FAT/CD36 translocation was impaired and fatty acid oxidation was blunted. There was no relationship between plasmalemmal fatty acid transporters and palmitate partitioning. In conclusion, fatty acid metabolism is impaired at several levels in muscles of obese Zucker rats; specifically, they are 1) insulin resistant with respect to FAT/CD36 translocation and fatty acid uptake, esterification, and oxidation and 2) contraction resistant with respect to fatty acid oxidation and FAT/CD36 translocation, but, conversely, 3) obese muscles are neither insulin nor contraction resistant at the level of FABPpm. Finally, 4) there is no evidence that plasmalemmal fatty acid transporters contribute to the channeling of fatty acids to specific metabolic destinations within the muscle.     

脂肪酸标准物质的研究进展

脂肪酸是一类具有重要功能的生物分子,广泛存在于动植物体内,是人体的重要组成成分,与人体很多疾病的发生都息息相关。脂肪酸标准物质是脂肪酸检测结果准确与否的依据,世界各国都在积极研制,目前我国脂肪酸标准物质研制单位主要有国家粮食局科学研究院和中国计量科学研究院等。综述了国内外脂肪酸标准物质的现状,对比分析了国内外脂肪酸标准物质的种类、特性量值组成和定值方法等。国外脂肪酸标准物质一般含有多种特性组分,特性量值较多,国内脂肪酸标准物质一般仅针对脂肪酸测量研制,基体比较单一,特性组分较少,量值较少。     

Fatty acid synthesis is a target for antibacterial activity of unsaturated fatty acids

Long-chain unsaturated fatty acids, such as linoleic acid, show antibacterial activity and are the key ingredients of antimicrobial food additives and some antibacterial herbs. However, the precise mechanism for this antimicrobial activity remains unclear. We found that linoleic acid inhibited bacterial enoyl-acyl carrier protein reductase (FabI), an essential component of bacterial fatty acid synthesis, which has served as a promising target for antibacterial drugs. Additional unsaturated fatty acids including palmitoleic acid, oleic acid, linolenic acid, and arachidonic acid also exhibited the inhibition of FabI. However, neither the saturated form (stearic acid) nor the methyl ester of linoleic acid inhibited FabI. These FabI-inhibitory activities of various fatty acids and their derivatives very well correlated with the inhibition of fatty acid biosynthesis using [(14)C] acetate incorporation assay, and importantly, also correlated with antibacterial activity. Furthermore, the supplementation with exogenous fatty acids reversed the antibacterial effect of linoleic acid, which showing that it target fatty acid synthesis. Our data demonstrate for the first time that the antibacterial action of unsaturated fatty acids is mediated by the inhibition of fatty acid synthesis.     

The Manipulation of the Fatty Acid Composition of Glycerolipids in Cyanobacteria Using Exogenous Fatty Acids

The fatty acid composition of the major diacylglycerolipidsof Anacystis nidulans R2 cells was modified by the careful additionof exogenous fatty acids to the growth medium of the cyanobacteria.In this way it is possible to incorporate significant levelsof different fatty acids into the membranes of the cells andchange their physical properties without changing other environmentalconditions. The exogenous fatty acid reduces or inhibits thebiosynthesis of native fatty acids and substitutes for themin all the major diacylglycerolipids. Although modifying thefatty acid composition in this way is lethal above certain concentrations,the data demonstrate a remarkable ability of the cells to growand develop over a wide range of fatty acid compositions. Similardata show that the fatty acid composition of Synechocystis sp.6803 cells also undergo significant changes in fatty acid compositionon the addition of exogenous fatty acid to the growth mediumand can tolerate similar changes in the degree of unsaturationof the fatty acids of the diacylglycerolipids of the cell membranes. (Received September 4, 1989; Accepted March 13, 1990)     

Modification of the fatty acid composition of individual phospholipids and neutral lipids after infection of the simian erythrocyte by Plasmodium knowlesi

Using capillary gas-liquid chromatography, we have analyzed the alteration in the total fatty acid, phospholipid and neutral lipid compositions of the monkey erythrocyte, after infection by the malarial parasite Plasmodium knowlesi. Data based on fatty acid quantitation show that the phospholipid composition is altered, with particularly large increases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the most abundant phospholipids in normal and P. knowlesi-schizont-infected cells. Unesterified fatty acids were found to be less abundant in infected cells. The total fatty acid content of the cell is increased 6-fold during infection, and total fatty acid composition is also changed: the infected cells are richer in palmitate (+23%), oleate (+29%) and linoleate (+89%), but contained less stearate (-27%) and arachidonate (-40%). The determination of the fatty acid composition of individual phospholipids, neutral lipids and unesterified fatty acids showed that choline-containing phospholipids (PC and sphingomyelin) were not as altered in their fatty acid pattern as anionic phospholipids (PE, phosphatidylserine (PS) and phosphatidylinositol (PI) and lysophosphatidylcholine (lysoPC). Specific alterations in the fatty acid compositions of individual phospholipids were detected, whereas the rise in linoleic acid was the only change during infection that was recovered in each phospholipid (except PC), neutral lipid and unesterified fatty acids. The fatty acid composition of the neutral lipids and unesterified fatty acids was particularly modified: the only rise in arachidonic acid level was observed in these lipid classes after infection. The total plasmalogen level of the erythrocyte is decreased in infected cells (-60%), but their level is increased in PI.     

Utilization of exogenous linolenic and oleic acids for plasma membrane phosphoglyceride systhesis in L-Fibroblasts

The inability of the strain L-fibroblast to synthesize quatitatively significant amounts of polyenoic fatty acid and the apparent lack of turnover of their phosphoglyceride acyl groups under the usual conditions of cell culture makes them especially well suited for studies concerning the effect of fatty acid unsaturation on biological membranes. Such cells grown in the absence of exogenous lipid sources have in their phosphoglycerides only traces of polyenoic fatty acid.By infusing fatty acid supplements into suspension cultures of logarithmically growing cultures of L-fibroblasts it is possible to increase singnificantly their phosphoglyceride polyenoic fatty content to as much as 50% of the total lipid phosphoglyceride fatty acids. The infusion of fatty acid supplements at a constant rate over a 48 h time period diminishes the toxic effects which may accompany single doses of unesterified fatty acid and reduces considerably the accumulation of cytoplasmic lipid droplets. Cultures supplemented in this way have virtually the same generation times as non-supplemented control cultures. The data show that alterations in surface membrane and homogenate polyenoic fatty acid composition are minimal when oleic acid is supplied to the culture. During exposure to large amounts of polyenoic fatty acid, however, the unsaturation of plasma membrane total phosphoglyceride fraction is less than that of the cell homogenate. This effect is more pronounced in the phosphatidylethanolamine than in the phosphatidylcholine fraction.     

Interaction of fatty acids with recombinant rat intestinal and liver fatty acid-binding proteins

Intestinal enterocytes contain two homologous fatty acid-binding proteins, intestinal fatty acid-binding protein (I-FABP)2 and liver fatty acid-binding protein (L-FABP). Since the functional basis for this multiplicity is not known, the fatty acid-binding specificity of recombinant forms of both rat I-FABP and rat L-FABP was examined. A systematic comparative analysis of the 18 carbon chain length fatty acid binding parameters, using both radiolabeled (stearic, oleic, and linoleic) and fluorescent (trans-parinaric and cis-parinaric) fatty acids, was undertaken. Results obtained with a classical Lipidex-1000 binding assay, which requires separation of bound from free fatty acid, were confirmed with a fluorescent fatty acid-binding assay not requiring separation of bound and unbound ligand. Depending on the nature of the fatty acid ligand, I-FABP bound fatty acid had dissociation constants between 0.2 and 3.1 microM and a consistent 1:1 molar ratio. The dissociation constants for L-FABP bound fatty acids ranged between 0.9 and 2.6 microM and the protein bound up to 2 mol fatty acid per mole of protein. Both fatty acid-binding proteins exhibited relatively higher affinity for unsaturated fatty acids as compared to saturated fatty acids of the same chain length. cis-Parinaric acid or trans-parinaric acid (each containing four double bonds) bound to L-FABP and I-FABP were displaced in a competitive manner by non-fluorescent fatty acid. Hill plots of the binding of cis- and trans- parinaric acid to L-FABP showed that the binding affinities of the two sites were very similar and did not exhibit cooperativity. The lack of fluorescence self-quenching upon binding 2 mol of either trans- or cis-parinaric acid/mol L-FABP is consistent with the presence of two binding sites with dissimilar orientation in the L-FABP. Thus, the difference in binding capacity between I-FABP and L-FABP predicts a structurally different binding site or sites.     

Molecular cloning of gene sequences for avian fatty acid synthase and evidence for nutritional regulation of fatty acid synthase mRNA concentration

A double-stranded cDNA library was constructed using total poly(A)+ RNA from the goose uropygial gland. Clones containing sequences complementary to fatty acid synthase mRNA were initially identified by colony hybridization with a 32P-labeled cDNA transcribed from RNA enriched for fatty acid synthase mRNA. Identity of the fatty acid synthase clones was confirmed by hybrid-selected translation. Mature fatty acid synthase mRNA is approximately 16 kilobases in length. When unfed neonatal goslings were fed for 24 hr, relative synthesis of hepatic fatty acid synthase increased more than 42-fold. Concomitantly, hepatic fatty acid synthase mRNA levels increased 70-fold. Thus, nutritional regulation of the synthesis of hepatic fatty acid synthase probably occurs at the pretranslational level. The availability of a specific probe for fatty acid synthase mRNA should allow us to analyze the regulation of expression of this gene during development, by nutrition and by hormones in both liver and uropygial gland.