Fatty acid-binding proteins in the heart

Long-chain fatty acids are important fuel molecules for the heart, their oxidation in mitochondria providing the bulk of energy required for cardiac functioning. The low solubility of fatty acids in aqueous solutions impairs their cellular transport. However, cardiac tissue contains several proteins capable of binding fatty acids non-covalently. These fatty acid-binding proteins (FABPs) are thought to facilitate both cellular uptake and intracellular transport of fatty acids. The majority of fatty acids taken up by the heart seems to pass the sarcolemma through a carrier-mediated translocation mechanism consisting of one or more membrane-associated FABPs. Intracellular transport of fatty acids towards sites of metabolic conversion is most likely accomplished by cytoplasmic FABPs. In this review, the roles of membrane-associated and cytoplasmic FABPs in cardiac fatty acid metabolism under (patho)physiological circumstances are discussed.     

AMPK-mediated increase in myocardial long-chain fatty acid uptake critically depends on sarcolemmal CD36

CD36, also named fatty acid translocase, has been identified as a putative membrane transporter for long-chain fatty acids (LCFA). In the heart, contraction-induced 5′ AMP-activated protein kinase (AMPK) signaling regulates cellular LCFA uptake through translocation of CD36 and possibly of other LCFA transporters from intracellular storage compartments to the sarcolemma. In this study, isolated cardiomyocytes from CD36^+/+- and CD36^−/− mice were used to investigate to what extent basal and AMPK-mediated LCFA uptake are CD36-dependent. Basal LCFA uptake was not altered in CD36^−/− cardiomyocytes, most likely resulting from a (1.8-fold) compensatory upregulation of fatty acid-transport protein-1. The stimulatory effect of contraction-mimetic stimuli, oligomycin (2.5-fold) and dipyridamole (1.6-fold), on LCFA uptake into CD36^+/+ cardiomyocytes was almost completely lost in CD36^−/− cardiomyocytes, despite that AMPK signaling was fully intact. CD36 is almost entirely responsible for AMPK-mediated stimulation of LCFA uptake in cardiomyocytes, indicating a pivotal role for CD36 in mediating changes in cardiac LCFA fluxes.     

Insulin causes fatty acid transport protein translocation and enhanced fatty acid uptake in adipocytes

Fatty acid uptake into 3T3 L1 adipocytes is predominantly transporter mediated. Here we show that, during 3T3 L1 adipocyte differentiation, expression of fatty acid transport proteins (FATPs) 1 and 4 is induced. Using subcellular membrane fractionation and immunofluorescence microscopy, we demonstrate that, in adipocytes, insulin induces plasma membrane translocation of FATPs from an intracellular perinuclear compartment to the plasma membrane. This translocation was observed within minutes of insulin treatment and was paralleled by an increase in long chain fatty acid (LCFA) uptake. In contrast, treatment with TNF-alpha inhibited basal and insulin-induced LCFA uptake and reduced FATP1 and -4 levels. Thus, hormonal regulation of FATP activity may play an important role in energy homeostasis and metabolic disorders such as type 2 diabetes.     

Cellular lipid binding proteins as facilitators and regulators of lipid metabolism

Evidence is accumulating that cellular lipid binding proteins are playing central roles in cellular lipid uptake and metabolism. Membrane-associated fatty acid-binding proteins putatively function in protein-mediated transmembrane transport of fatty acids, likely coexisting with passive diffusional uptake. The intracellular trafficking of fatty acids, bile acids, and other lipid ligands, may involve their interaction with specific membrane or protein targets, which are unique properties of some but not of all cytoplasmic lipid binding proteins. Recent studies indicate that these proteins not only facilitate but also regulate cellular lipid utilization. For instance, muscle fatty acid uptake is subject to short-term regulation by translocation of fatty acid translocase (FAT)/CD36 from intracellular storage sites to the plasma membrane, and liver-type cytoplasmic fatty acid-binding protein (L-FABP_c) functions in long-term, ligand-induced regulation of gene expression by directly interacting with nuclear receptors. Therefore, the properties of the lipid-protein complex, rather than those of the lipid ligand itself, determine the fate of the ligand in the cell. Finally, there are an increasing number of reports that deficiencies or altered functioning of both membrane-associated and cytoplasmic lipid binding proteins are associated with disease states, such as obesity, diabetes and atherosclerosis. In conclusion, because of their central role in the regulation of lipid metabolism, cellular lipid binding proteins are promising targets for the treatment of diseases resulting from or characterised by disturbances in lipid metabolism, such as atherosclerosis, hyperlipidemia, and insulin resistance.     

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.     

Ablation of Iqgap2 protects from diet-induced hepatic steatosis due to impaired fatty acid uptake

Long-chain fatty acids (LCFA) serve as structural components for membrane biogenesis and as primary energy sources during mitochondrial β-oxidation reactions. Hepatic LCFA uptake is complex, with characteristics suggestive of a dual-kinetic model manifested by rapid (carrier-assisted/facilitated) and delayed (passive diffusional) phases. Our previous work using mice deficient of the Iqgap2 gene established a highly novel link between IQGAP2, a putative GTPase-activating protein, and hepatocarcinogenesis. Now we report that Iqgap2 deficiency also results in selective loss of the facilitated phase of hepatocyte LCFA uptake with preservation of the diffusional component. This molecular defect was seen in Iqgap2(-/-) hepatocytes of all ages studied (1-, 4-, 8-months). The loss of facilitated LCFA uptake protected against development of hepatic triglyceride accumulation in Iqgap2-deficient mice fed high-fat diet, consistent with a fundamental role in physiological fat partitioning. These phenotypic changes could not be explained by genetic loss of fatty acid processing proteins known to regulate lipid uptake or metabolic processing pathways. Iqgap2-deficient livers also displayed enhanced insulin sensitivity. Conclusion: These observations identify a novel property of the putative GTPase-activating protein IQGAP2 in LCFA uptake in vitro and in vivo, and implicate IQGAP2 in an intracellular signaling pathway necessary for functional fatty acid uptake, lipid processing, and, possibly, glucose homeostasis.     

Sulfo-N-succinimidyl esters of long chain fatty acids specifically inhibit fatty acid translocase (FAT/CD36)-mediated cellular fatty acid uptake

Sulfo-N-succinimidyl esters of LCFAs are a powerful tool to investigate the functional significance of plasmalemmal proteins in the LCFA uptake process. This notion is based on the following observations. First, sulfo-N-succinimidyl oleate (SSO) was found to inhibit the bulk of LCFA uptake into various cell types, i.e. rat adipocytes, type II pneumocytes and cardiac myocytes. Second, using cardiac giant membrane vesicles, in which LCFA uptake can be investigated in the absence of mitochondrial -oxidation, SSO retained the ability to largely inhibit LCFA uptake, indicating that inhibition of LCFA transsarcolemmal transport is its primary action. Third, SSO has no inhibitory effect on glucose and octanoate uptake into giant membrane vesicles derived from heart and skeletal muscle, indicating that its action is specific for LCFA uptake. Finally, SSO specifically binds to the 88 kDa plasmalemmal fatty acid transporter FAT, a rat homologue of human CD36, resulting in an arrest of the transport function of this protein.In addition to its inhibitory action at the plasma membrane level, evidence is presented for the lack of a direct inhibitory effect on subsequent LCFA metabolism. First, the relative contribution of oxidation and esterification to LCFA uptake is not altered in the presence of SSO. Second, isoproterenol-mediated channeling of LCFAs into oxidative pathways is not affected by sulfo-N-succinimidyl palmitate (SSP). As an example of its application we used SSP to study the role of FAT/CD36 in contraction- and insulin-stimulated LCFA uptake by cardiac myocytes , showing that this transporter is a primary site of regulation of cellular LCFA utilization.     

FATP1 is an insulin-sensitive fatty acid transporter involved in diet-induced obesity

Fatty acid transport protein 1 (FATP1), a member of the FATP/Slc27 protein family, enhances the cellular uptake of long-chain fatty acids (LCFAs) and is expressed in several insulin-sensitive tissues. In adipocytes and skeletal muscle, FATP1 translocates from an intracellular compartment to the plasma membrane in response to insulin. Here we show that insulin-stimulated fatty acid uptake is completely abolished in FATP1-null adipocytes and greatly reduced in skeletal muscle of FATP1-knockout animals while basal LCFA uptake by both tissues was unaffected. Moreover, loss of FATP1 function altered regulation of postprandial serum LCFA, causing a redistribution of lipids from adipocyte tissue and muscle to the liver, and led to a complete protection from diet-induced obesity and insulin desensitization. This is the first in vivo evidence that insulin can regulate the uptake of LCFA by tissues via FATP1 activation and that FATPs determine the tissue distribution of dietary lipids. The strong protection against diet-induced obesity and insulin desensitization observed in FATP1-null animals suggests FATP1 as a novel antidiabetic target.     

Localization of adipocyte long-chain fatty acyl-CoA synthetase at the plasma membrane

Long-chain fatty acyl-CoA synthetase (FACS) catalyzes esterification of long-chain fatty acids (LCFAs) with coenzyme A (CoA), the first step in fatty acid metabolism. FACS has been shown to play a role in LCFA import into bacteria and implicated to function in mammalian cell LCFA import. In the present study, we demonstrate that FACS overexpression in fibroblasts increases LCFA uptake, and overexpression of both FACS and the fatty acid transport protein (FATP) have synergistic effects on LCFA uptake. To explore how FACS contributes to LCFA import, we examined the subcellular location of this enzyme in 3T3-L1 adipocytes which natively express this protein and which efficiently take up LCFAs. We demonstrate for the first time that FACS is an integral membrane protein. Subcellular fractionation of adipocytes by differential density centrifugation reveals immunoreactive and enzymatically active FACS in several membrane fractions, including the plasma membrane. Immunofluorescence studies on adipocyte plasma membrane lawns confirm that FACS resides at the plasma membrane of adipocytes, where it co-distributes with FATP. Taken together, our data support a model in which imported LCFAs are immediately esterified at the plasma membrane upon uptake, and in which FATP and FACS function coordinately to facilitate LCFA movement across the plasma membrane of mammalian cells.     

Transport of long-chain fatty acids in Escherichia coli. Evidence for role of fadL gene product as long-chain fatty acid receptor

Transport of long-chain fatty acids (LCFA) across the cytoplasmic membrane of Escherichia coli requires functional fadL and fadD genes. The fadD gene codes for an acyl-CoA synthetase (fatty acid: CoA ligase (AMP forming] which has broad chain length specificity and is loosely bound to the cytoplasmic membrane. The fadL gene codes for a 43,000-dalton cytoplasmic membrane protein which, acting by an unknown mechanism, is needed specifically for LCFA transport. As a first step to define the role of the fadL gene product, studies were performed to determine if it functions as a LCFA receptor. The LCFA-binding activity was quantitated in intact cells in the absence of LCFA transport by comparing the binding of LCFA in fadD fadL and fadD fadL+ strains. These studies revealed that (i) fadD fadL+ strains bind 6-fold more LCFA than fadD fadL strains; (ii) fadD fadL strains harboring a plasmid containing the fadL gene bind 16-fold more LCFA than fadD fadL strains harboring only the plasmid vector; and (iii) the fadL-specific LCFA-binding activity is regulated by the fadR gene and catabolite repression. Studies with fadL strains harboring fadL plasmids containing in vitro constructed deletions indicate that mutations which alter the physical properties of the 43,000-dalton fadL gene product also affect fadL gene product-specific LCFA-binding activity. Overall, these studies suggest that one role of the fadL gene product in the LCFA transport process is to sequester LCFA at sites in the cell membrane for transport.     

Induction of hepatic cytosolic fatty acid binding protein with clofibrate accelerates both membrane and cytoplasmic transport of palmitate

The role of liver cytosolic fatty acid binding protein (L-FABP) in fatty acid transport and metabolism is unclear. Female liver contains substantially more L-FABP than male liver. Female liver also has a different fatty acid transport phenotype, including more rapid uptake, efflux and cytoplasmic transport. However, it is not known if the greater levels of L-FABP are responsible for these differences. We therefore determined whether increasing L-FABP using clofibrate causes male liver to acquire a female transport phenotype. The multiple indicator dilution (MID) method was used to estimate the rate constants for influx, efflux and cytoplasmic diffusion of palmitate in isolated perfused rat livers. Clofibrate treatment increased cytosolic concentrations of L-FABP 4.2+/-0.8-fold, the rate of cytoplasmic diffusion of palmitate 4.3+/-1.7-fold, and the steady-state palmitate extraction 1.5+/-0.3-fold (mean+/-S.E.). Influx and efflux constants were both increased (by 44% and 79%, respectively) to levels typical of female livers. These data suggest that clofibrate-induced elevation of cytosolic L-FABP not only stimulates intracellular diffusion but also influx and efflux of fatty acids. Possible mechanisms include reducing fatty acid binding to cytoplasmic membranes, induction of membrane fatty acid carriers, and catalyzing fatty acid exchange between aqueous cytoplasm and the plasma membrane.     

FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires plasma membrane rafts

We previously reported that lipid rafts are involved in long-chain fatty acid (LCFA) uptake in 3T3-L1 adipocytes. The present data show that LCFA uptake does not depend on caveolae endocytosis because expression of a dominant negative mutant of dynamin had no effect on uptake of [3H]oleic acid, whereas it effectively prevented endocytosis of cholera toxin. Isolation of detergent-resistant membranes (DRMs) from 3T3-L1 cell homogenates revealed that FAT/CD36 was expressed in both DRMs and detergent-soluble membranes (DSMs), whereas FATP1 and FATP4 were present only in DSMs but not DRMs. Disruption of lipid rafts by cyclodextrin and specific inhibition of FAT/CD36 by sulfo-N-succinimidyl oleate (SSO) significantly decreased uptake of [3H]oleic acid, but simultaneous treatment had no additional or synergistic effects, suggesting that both treatments target the same mechanism. Indeed, subcellular fractionation demonstrated that plasma membrane fatty acid translocase (FAT/CD36) is exclusively located in lipid rafts, whereas intracellular FAT/CD36 cofractionated with DSMs. Binding assays confirmed that [3H]SSO predominantly binds to FAT/CD36 within plasma membrane DRMs. In conclusion, our data strongly suggest that FAT/CD36 mediates raft-dependent LCFA uptake. Plasma membrane lipid rafts might control LCFA uptake by regulating surface availability of FAT/CD36.     

Cytosolic fatty acid binding proteins catalyze two distinct steps in intracellular transport of their ligands

Cytosolic long-chain fatty acid binding proteins (FABPs) are found in tissues that metabolize fatty acids. Like most lipid binding proteins, their specific functions remain unclear. Two classes have been described. Membrane-active FABPs interact directly with membranes during exchange of fatty acids between the protein binding site and the membrane, while membrane-inactive FABPs bind only to fatty acids that are already in aqueous solution. Despite these binding proteins, most fatty acids in cell cytoplasm appear to be bound to membranes. This paper reviews data suggesting that FABPs catalyze transfer of fatty acids between intracellular membranes, often across considerable intracellular distances. This process occurs in three distinct steps: dissociation of the fatty acid from a donor membrane, diffusion of the fatty acid across the intervening water layer, and binding to an acceptor membrane. Membrane-active FABPs catalyze dissociation of the fatty acid from the donor membrane and binding to the acceptor membrane, while membrane-inactive FABPs catalyze diffusion of fatty acids across the aqueous cytosol. Thus, FABPs catalyze all three steps in intracellular transport. A simple quantitative model has been developed that predicts the rate of intracellular transport as a function of the concentration, affinity and diffusional mobility of the binding protein. Different FABPs may have evolved to match the specific transport requirements of the cell type within which they are found.     

Insulin and Chromium Picolinate Induce Translocation of CD36 to the Plasma Membrane Through Different Signaling Pathways in 3T3-L1 Adipocytes,and with a Differential Functionality of the CD36

Chromium picolinate (CrPic) has been indicated to activate glucose transporter 4 (GLUT4) trafficking to the plasma membrane (PM) to enhance glucose uptake in 3T3-L1 adipocytes. In skeletal and heart muscle cells, insulin directs the intracellular trafficking of the fatty acid translocase/CD36 to induce the uptake of cellular long-chain fatty acid (LCFA). The current study describes the effects of CrPic and insulin on the translocation of CD36 from intracellular storage pools to the PM in 3T3-L1 adipocytes in comparison with that of GLUT4. Immunofluorescence microscopy and immunoblotting revealed that both CD36 and GLUT4 were expressed and primarily located intracellularly in 3T3-L1 adipocytes. Upon insulin or CrPic stimulation, PM expression of CD36 increased in a similar manner as that for GLUT4; the CrPic-stimulated PM expression was less strong than that of insulin. The increase in PM localization for these two proteins by insulin paralleled LCFA ([1-¹⁴C]palmitate) or [³H]deoxyglucose uptake in 3T3-L1 adipocytes. The induction of the PM expression of GLUT4, but not CD36, or substrate uptake by insulin and CrPic appears to be additive in adipocytes. Furthermore, wortmannin completely inhibited the insulin-stimulated translocation of GLUT4 or CD36 and prevented the increased uptake of glucose or LCFA in these cells. Taken together, for the first time, these findings suggest that both insulin and CrPic induce CD36 translocation to the PM in 3T3-L1 adipocytes and that their translocation-inducing effects are not additive. The signaling pathway inducing the translocations is different, apparently resulting in a differential activity of CD36.     

Towards an Understanding of Mesocestoides vogae Fatty Acid Binding Proteins’ Roles

Two fatty acid binding proteins, MvFABPa and MvFABPb were identified in the parasite Mesocestoides vogae (Platyhelmithes, Cestoda). Fatty acid binding proteins are small intracellular proteins whose members exhibit great diversity. Proteins of this family have been identified in many organisms, of which Platyhelminthes are among the most primitive. These proteins have particular relevance in flatworms since de novo synthesis of fatty acids is absent. Fatty acids should be captured from the media needing an efficient transport system to uptake and distribute these molecules. While HLBPs could be involved in the shuttle of fatty acids to the surrounding host tissues and convey them into the parasite, FABPs could be responsible for the intracellular trafficking. In an effort to understand the role of MvFABPs in fatty acid transport of M. vogae larvae, we analysed the intracellular localization of both MvFABPs and the co-localization with in vivo uptake of fatty acid analogue BODIPY FL C₁₆. Immunohistochemical studies on larvae sections using specific antibodies, showed a diffuse cytoplasmic distribution of each protein with some expression in nuclei and mitochondria. MvFABPs distribution was confirmed by mass spectrometry identification from 2D-electrophoresis of larvae subcellular fractions. This work is the first report showing intracellular distribution of MvFABPs as well as the co-localization of these proteins with the BODIPY FL C₁₆ incorporated from the media. Our results suggest that fatty acid binding proteins could target fatty acids to cellular compartments including nuclei. In this sense, M. vogae FABPs could participate in several cellular processes fulfilling most of the functions attributed to vertebrate’s counterparts.     

Evidence for vesicles that mediate long-chain fatty acid uptake by human microvascular endothelial cells

This study analyzes the mechanisms of long-chain fatty acid (LCFA) uptake by human microvascular endothelial cells (HMEC). The time course revealed the presence of an early, carrier-mediated uptake component and a later component mediated by clathrin-coated vesicles (CCV) and caveolae, as evidenced by three different experimental approaches: 1) significant reduction of [3H]oleate uptake over 5 min by either inhibition of CCV formation by potassium depletion or hypertonic medium, or disruption of caveolae by filipin III or cyclodextrin. 2) Co-localization of intracellular 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]octadecanoic acid with CCV and caveolae using confocal laser scanning microscopy. 3) Enrichment of [3H]oleate in a subcellular fraction containing CCV and caveolae. Within 10 min, more than 75% of intracellular [3H]oleate remained unmetabolized, suggesting that HMEC preferentially shuttle LCFA through the cell using CCV and caveolae as carriers. The uptake of albumin paralleled that of oleate within the first 10 min, suggesting internalization of at least some LCFA bound to albumin. Compared to oleate and albumin, the uptake of sucrose and dextran was low, indicating a potential minor contribution of fluid-phase endocytosis to the total vesicular LCFA uptake. The data indicate a previously unrecognized role of both CCV and caveolae for the uptake of LCFA by HMEC.     

Role of fatty acid uptake and fatty acid β-oxidation in mediating insulin resistance in heart and skeletal muscle

Fatty acids are a major fuel source used to sustain contractile function in heart and oxidative skeletal muscle. To meet the energy demands of these muscles, the uptake and β-oxidation of fatty acids must be coordinately regulated in order to ensure an adequate, but not excessive, supply for mitochondrial β-oxidation. However, imbalance between fatty acid uptake and β-oxidation has the potential to contribute to muscle insulin resistance. The action of insulin is initiated by binding to its receptor and activation of the intrinsic protein tyrosine kinase activity of the receptor, resulting in the initiation of an intracellular signaling cascade that eventually leads to insulin-mediated alterations in a number of cellular processes, including an increase in glucose transport. Accumulation of fatty acids and lipid metabolites (such as long chain acyl CoA, diacylglycerol, triacylglycerol, and/or ceramide) can lead to alterations in this insulin signaling pathway. An imbalance between fatty acid uptake and oxidation is believed to be responsible for this lipid accumulation, and is thought to be a major cause of insulin resistance in obesity and diabetes, due to lipid accumulation and inhibition of one or more steps in the insulin-signaling cascade. As a result, decreasing muscle fatty acid uptake can improve insulin sensitivity. However, the potential role of increasing fatty acid β-oxidation in the heart or skeletal muscle in order to prevent cytoplasmic lipid accumulation and decrease insulin resistance is controversial. While increased fatty acid β-oxidation may lower cytoplasmic lipid accumulation, increasing fatty acid β-oxidation can decrease muscle glucose metabolism, and incomplete fatty acid oxidation has the potential to also contribute to insulin resistance. In this review, we discuss the proposed mechanisms by which alterations in fatty acid uptake and oxidation contribute to insulin resistance, and how targeting fatty acid uptake and oxidation is a potential therapeutic approach to treat insulin resistance.     

Long-chain fatty acid uptake into adipocytes depends on lipid raft function

This study investigates the role of lipid rafts and caveolae, a subclass of lipid raft microdomains, in the binding and uptake of long-chain fatty acids (LCFA) by 3T3-L1 cells during differentiation. Disruption of lipid rafts by beta-cyclodextrin (betaCD) or selective inhibition of caveolae by overexpression of a dominant-negative mutant of caveolin-3 (Cav(DGV)) resulted in disassembly of caveolae structures at the cell surface, as assessed by electron microscopy. While in 3T3-L1 fibroblasts, which express few caveolae, Cav(DGV) or betaCD had no effect on LCFA uptake, in 3T3-L1 adipocytes the same treatments decreased the level of [(3)H]oleic acid uptake by up to 55 +/- 8 and 49 +/- 7%, respectively. In contrast, cholesterol loading of 3T3-L1 adipocytes resulted in a 4-fold increase in the extent of caveolin-1 expression and a 1.7-fold increase in the level of LCFA uptake. Both the inhibitory and enhancing effects of these treatments were constantly increasing with the [(3)H]oleic acid incubation time up to 5 min. Incubation of 3T3-L1 adipocytes with [(3)H]stearate followed by isolation of a caveolin-1 positive detergent-resistant membrane (DRM) fraction revealed that [(3)H]stearate binds to caveolae. Fatty acid translocase (FAT/CD36) was found to be present in this DRM fraction as well. Our data thus strongly indicate a critical involvement of lipid rafts in the binding and uptake of LCFA into 3T3-L1 adipocytes. Furthermore, our findings suggest that caveolae play a pivotal role in lipid raft-dependent LCFA uptake. This transport mechanism is induced in conjunction with cell differentiation and might be mediated by FAT/CD36.     

The ligand-mediated affinity of brain-type fatty acid-binding protein for membranes determines the directionality of lipophilic cargo transport

The intracellular transport of lipophilic cargoes is a highly dynamic process. In eukaryotic cells, the uptake and release of long-chain fatty acids (LCFAs) are executed by fatty-acid binding proteins. However, how these carriers control the directionality of cargo trafficking remains unclear. Here, we revealed that the unliganded archetypal Drosophila brain-type fatty acid-binding protein (dFABP) possesses a stronger binding affinity than its liganded counterpart for empty nanodiscs (ND). Titrating unliganded dFABP and nanodiscs with LCFAs rescued the broadening of FABP cross-peak intensities in HSQC spectra from a weakened protein–membrane interaction. Two out of the 3 strongest LCFA contacting residues in dFABP identified by NMR HSQC chemical shift perturbation (CSP) are also part of the 30 ND-contacting residues (out of the total 130 residues in dFABP), revealed by attenuated TROSY signal in the presence of lipid ND to apo-like dFABP. Our crystallographic temperature factor data suggest enhanced αII helix dynamics upon LCFA binding, compensating for the entropic loss in the βC-D/βE-F loops. The aliphatic tail of bound LCFA impedes the charge-charge interaction between dFABP and the head groups of the membrane, and dFABP is prone to dissociate from the membrane upon ligand binding. We therefore conclude that lipophilic ligands participate directly in the control of the functionally required membrane association and dissociation of FABPs.     

Uptake of long-chain fatty acids in HepG2 cells involves caveolae: analysis of a novel pathway

We investigated the role of caveolae in uptake and intracellular trafficking of long chain fatty acids (LCFA) in HepG2 human hepatoma cells. The uptake of [(3)H]oleic acid and [(3)H]stearic acid into HepG2 cells was measured by radioactive assays and internalization of the non-metabolizable fluorescent fatty acid 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] (12-NBD) stearate into single HepG2 cells was semi-quantitatively assessed by laser scanning microscopy. The initial rate of [(3)H]oleic acid uptake (V(0)) in HepG2 cells exhibited saturable transport kinetics with increasing concentrations of free oleic acid (V(max) 854 +/- 46 pmol mg protein(-1) min(-1), K(m) 100 +/- 14 nmol/l). While inhibition of clathrin coated pits did not influence LCFA uptake in HepG2, inhibition of caveolae formation by filipin III, cyclodextrin, and caveolin-1 antisense oligonucleotides resulted in reduction of [(3)H]oleic acid uptake by 54%, 45%, and 23%, respectively. Furthermore, filipin III inhibited the uptake of [(3)H]stearic acid and its fluorescent derivative 12-NBD stearate by 44% and 50%, respectively. Transfection studies with alpha-caveolin-1/cyanofluorescent protein chimeras showed significant colocalization of caveolae and internalized 12-NBD stearate. In conclusion, these data suggest a significant role for caveolae mediated uptake and intracellular trafficking of LCFA in HepG2 cells.