Intracellular Fatty Acid Trafficking Research Articles

Microbial fatty acid transport proteins and their biotechnological potential.

Fatty acid metabolism has been widely studied in various organisms. However, fatty acid transport has received less attention, even though it plays vital physiological roles, such as export of toxic free fatty acids or uptake of exogenous fatty acids. Hence, there are important knowledge gaps in how fatty acids cross biological membranes, and many mechanisms and proteins involved in these processes still need to be determined. The lack of information is more predominant in microorganisms, even though the identification of fatty acids transporters in these cells could lead to establishing new drug targets or improvements in microbial cell factories. This review provides a thorough analysis of the current information on fatty acid transporters in microorganisms, including bacteria, yeasts and microalgae species. Most available information relates to the model organisms Escherichia coli and Saccharomyces cerevisiae, but transport systems of other species are also discussed. Intracellular trafficking of fatty acids and their transport through organelle membranes in eukaryotic organisms is described as well. Finally, applied studies and engineering efforts using fatty acids transporters are presented to show the applied potential of these transporters and to stress the need for further identification of new transporters and their engineering.

Fatty Acyl Synthetases and Thioesterases in Plant Lipid Metabolism: Diverse Functions and Biotechnological Applications

Plants use fatty acids to synthesize acyl lipids for many different cellular, physiological, and defensive roles. These roles include the synthesis of essential membrane, storage, or surface lipids, as well as the production of various fatty acid-derived metabolites used for signaling or defense. Fatty acids are activated for metabolic processing via a thioester linkage to either coenzyme A or acyl carrier protein. Acyl synthetases metabolically activate fatty acids to their thioester forms, and acyl thioesterases deactivate fatty acyl thioesters to free fatty acids by hydrolysis. These two enzyme classes therefore play critical roles in lipid metabolism. This review highlights the surprisingly complex and varying roles of fatty acyl synthetases in plant lipid metabolism, including roles in the intracellular trafficking of fatty acids. This review also surveys the many specialized fatty acyl thioesterases characterized to date in plants, which produce a great diversity of fatty acid products in a tissue-specific manner. While some acyl thioesterases produce fatty acids that clearly play roles in plant-insect or plant-microbial interactions, most plant acyl thioesterases have yet to be fully characterized both in terms of their substrate specificities and their functions. The biotechnological applications of plant acyl thioesterases and synthetases are also discussed, as there is significant interest in these enzymes as catalysts for the sustainable production of fatty acids and their derivatives for industrial uses.

“Passenger gene” problem in transgenic C57BL/6 mice used in hearing research

Despite recent advances in genome engineering technologies, traditional transgenic mice generated on a mixed genetic background of C57BL/6 and 129/Sv mice remain widely used in age-related hearing loss (AHL) research, since C57BL/6 mice exhibit early onset and progression of AHL due to a mutation in cadherin 23-encoding gene (Cdh23753G>A). In these transgenic mice, backcrossing for more than 10 generations results in replacement of the donor background (129/Sv) with that of the recipient (C57BL/6), so that approximately 99.9% of genes are C57BL/6-derived and are considered congenic. However, the regions flanking the target gene may still be of 129/Sv origin, creating a so-called “passenger gene problem” where the normal 129/Sv-derived Cdh23753G allele can travel with the target gene. In this study, we investigated the role of fatty acid-binding protein 7 (Fabp7), which is important for cellular uptake and intracellular trafficking of fatty acids in the cochlea, using traditional Fabp7 knockout (KO) mice on the C57BL/6 background. We found that Fabp7 KO mice showed delayed AHL progression and milder cochlear degeneration. However, the genotype of the Cdh23 region flanking Fabp7 was still that of 129/Sv origin (Cdh23753GG). Our findings reveal the potential risk of contamination for traditional transgenic mice generated on the C57BL/6 background.

Utilization of adipocyte-derived lipids and enhanced intracellular trafficking of fatty acids contribute to breast cancer progression

BackgroundTo determine whether adipocyte-derived lipids could be transferred into breast cancer cells and investigate the underlying mechanisms of subsequent lipolysis and fatty acid trafficking in breast cancer cells.MethodsA Transwell co-culture system was used in which human breast cancer cells were cultured in the absence or presence of differentiated murine 3 T3-L1 adipocytes. Migration/invasion and proliferation abilities were compared between breast cancer cells that were cultivated alone and those co-cultivated with mature adipocytes. The ability of lipolysis in breast cancer cells were measured, as well as the expression of the rate-limiting lipase ATGL and fatty acid transporter FABP5. ATGL and FABP5 were then ablated to investigate their impact on the aggressiveness of breast cancer cells that were surrounded by adipocytes. Further, immunohistochemistry was performed to detect differential expression of ATGL and FABP5 in breast cancer tissue sections.ResultsThe migration and invasion abilities of cancer cells were significantly enhanced after co-culture with adipocytes, accompanied by elevated lipolysis and expression of ATGL and FABP5. Abrogation of ATGL and FABP5 sharply attenuated the malignancy of co-cultivated breast cancer cells. However, this phenomenon was not observed if a lipid emulsion was added to the culture medium to substitute for adipocytes. Furthermore, epithelial-mesenchymal transaction was induced in co-cultivated breast cancer cells. That may partially due to the stimulation of PPARβ/δ and MAPK, which was resulted from upregulation of FABP5. As evidenced by immunohistochemistry, ATGL and FABP5 also had higher expression levels at the invasive front of the breast tumor, in where the adipocytes abound, compared to the central area in tissue specimens.ConclusionsLipid originating from tumor-surrounding adipocytes could be transferred into breast cancer cells. Adipocyte-cancer cell crosstalk rather than lipids alone induced upregulation of lipases and fatty acid transport protein in cancer cells to utilize stored lipids for tumor progression. The increased expression of the key lipase ATGL and intracellular fatty acid trafficking protein FABP5 played crucial roles in this process via fueling or signaling.

Adipose tissue fatty acid storage factors: effects of depot, sex and fat cell size.

Background/ObjectivesPatterns of postabsorptive adipose tissue fatty acid storage correlate with sex-specific body fat distribution. Some proteins and enzymes participating in this pathway include CD36 (facilitated transport), acyl-CoA synthetases (ACS; the first step in fat metabolism), and diacylglycerol acetyl-transferase (DGAT; the final step of triglyceride synthesis). Our goal was to better define CD36, ACS and DGAT in relation to sex, subcutaneous fat depots, and adipocyte size.Subjects/MethodsData was collected from studies conducted at Mayo Clinic between 2004 and 2012. Abdominal and femoral subcutaneous fat biopsy samples must have been collected in the postabsorptive state from healthy males and premenopausal females. Body composition was measured with DXA and abdominal CT scans. Adipocyte size (microscopy), CD36 protein content (ELISA), and ACS and DGAT enzyme activities were measured. Data are presented as medians; 25th:75th quartiles.ResultsMales (n=60) and females (n=78) did not differ by age (37;28:46 yr), BMI (28.4;24.6:32.1 kg/m2), or abdominal (0.60;0.45:0.83 μg/cell) and femoral adipocyte size (0.76;0.60:0.94 μg/cell). Femoral ACS and DGAT were greater in females than males when expressed per mg lipid (ACS: 73 vs. 55 pmol/mg lipid/min; DGAT: 5.5 vs. 4.0 pmol/ mg lipid/min; p<0.0001 for both) and per 1000 adipocytes (ACS: 59 vs. 39 pmol/1000adipocytes/min; DGAT: 4.3 vs. 3.1 pmol/1000adipocytes/min; p≤0.0003 for both). There were no differences in abdominal fat storage factors between sexes. ACS and DGAT decreased as a function of adipocyte size (p<0.0001 for both). The decrease in ACS was greater in males and abdominal subcutaneous fat. There were no sex differences in CD36 in either fat depot, nor did it vary across adipocyte size.ConclusionsFacilitated transport of fatty acids by CD36 under postabsorptive conditions would not be different in those with large vs. small adipocytes in either depot of both sexes. However, intracellular trafficking of fatty acids to triglyceride storage by ACS and DGAT may be less efficient in larger adipocytes.

Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of FATP2 resulted in increases in all four classes of phospholipid, indicating little selectivity. In the case of C22:6, there were significant increases of this exogenous fatty acids being trafficking into PC and PI. Collectively, these data support the conclusion that FATP2 has a dual function in the pathways linking the transport and activation of exogenous fatty acids. We discuss the differential roles of FATP2 and its role in both fatty acid transport and fatty acid activation in the context of lipid homeostasis.

Fatty acids induce leukotriene C4 synthesis in macrophages in a fatty acid binding protein-dependent manner

Obesity results in increased macrophage recruitment to adipose tissue that promotes a chronic low-grade inflammatory state linked to increased fatty acid efflux from adipocytes. Activated macrophages produce a variety of pro-inflammatory lipids such as leukotriene C4 (LTC4) and 5-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) suggesting the hypothesis that fatty acids may stimulate eicosanoid synthesis. To assess if eicosanoid production increases with obesity, adipose tissue of leptin deficient ob/ob mice was analyzed. In ob/ob mice, LTC4 and 12-HETE levels increased in the visceral (but not subcutaneous) adipose depot while the 5-HETE levels decreased and 15-HETE abundance was unchanged. Since macrophages produce the majority of inflammatory molecules in adipose tissue, treatment of RAW264.7 or primary peritoneal macrophages with free fatty acids led to increased secretion of LTC4 and 5-HETE, but not 12- or 15-HETE. Fatty acid binding proteins (FABPs) facilitate the intracellular trafficking of fatty acids and other hydrophobic ligands and in vitro stabilize the LTC4 precursor leukotriene A4 (LTA4) from non-enzymatic hydrolysis. Consistent with a role for FABPs in LTC4 synthesis, treatment of macrophages with HTS01037, a specific FABP inhibitor, resulted in a marked decrease in both basal and fatty acid-stimulated LTC4 secretion but no change in 5-HETE production or 5-lipoxygenase expression. These results indicate that the products of adipocyte lipolysis may stimulate the 5-lipoxygenase pathway leading to FABP-dependent production of LTC4 and contribute to the insulin resistant state.

Transcriptional regulation of lipid synthesis in bovine mammary epithelial cells by sterol regulatory element binding protein-1

The objective of this study was to investigate the transcriptional regulation of lipid synthesis by sterol regulatory element binding protein-1 (SREBP-1) in bovine mammary epithelial cells. In the current study, bovine mammary epithelial (MAC-T) cells cultured in insulin- and prolactin-containing medium were treated with a transfection reagent as control, a nontargeting small interfering (si)RNA sequence (100nM) as a negative control, or an SREBP-1–specific siRNA (100nM) for 48h. The mRNA expression of SREBP-1 was decreased more than 90% by siRNA. Precursor and mature forms of SREBP-1 protein were undetectable in cells treated with SREBP-1 siRNA. Fatty acid synthesis and fatty acid uptake, measured using isotope incorporation, were reduced significantly in cells treated with SREBP-1 siRNA compared with controls. Transcript abundance of acyl-CoA synthetase short-chain family member 2, acetyl-CoA carboxylase, fatty acid synthetase, and isocitrate dehydrogenase 1 (key enzymes of de novo lipogenesis) was decreased by 40 to 65% with SREBP-1 siRNA, in agreement with acetate incorporation data. The mRNA levels of fatty acid binding protein 3 and stearyl-CoA desaturase 1 (proteins responsible for intracellular fatty acid trafficking and long-chain fatty acid modification) were decreased 76 and 60%, respectively, by SREBP-1 siRNA treatment compared with controls. The mRNA expression of mitochondrial glycerol-3-phosphate acyltransferase and lipin 1 (involved in triglyceride synthesis) was significantly decreased in cells treated with SREBP-1 siRNA compared with control cells. However, the expression of milk fat globule membrane proteins measured did not differ among treatments. In conclusion, SREBP-1 plays an important role in integrated regulation of lipid synthesis in bovine mammary epithelial cells through regulation of key enzymes.

Gene networks driving bovine milk fat synthesis during the lactation cycle

BackgroundThe molecular events associated with regulation of milk fat synthesis in the bovine mammary gland remain largely unknown. Our objective was to study mammary tissue mRNA expression via quantitative PCR of 45 genes associated with lipid synthesis (triacylglycerol and phospholipids) and secretion from the late pre-partum/non-lactating period through the end of subsequent lactation. mRNA expression was coupled with milk fatty acid (FA) composition and calculated indexes of FA desaturation and de novo synthesis by the mammary gland.ResultsMarked up-regulation and/or % relative mRNA abundance during lactation were observed for genes associated with mammary FA uptake from blood (LPL, CD36), intracellular FA trafficking (FABP3), long-chain (ACSL1) and short-chain (ACSS2) intracellular FA activation, de novo FA synthesis (ACACA, FASN), desaturation (SCD, FADS1), triacylglycerol synthesis (AGPAT6, GPAM, LPIN1), lipid droplet formation (BTN1A1, XDH), ketone body utilization (BDH1), and transcription regulation (INSIG1, PPARG, PPARGC1A). Change in SREBF1 mRNA expression during lactation, thought to be central for milk fat synthesis regulation, was ≤2-fold in magnitude, while expression of INSIG1, which negatively regulates SREBP activation, was >12-fold and had a parallel pattern of expression to PPARGC1A. Genes involved in phospholipid synthesis had moderate up-regulation in expression and % relative mRNA abundance. The mRNA abundance and up-regulation in expression of ABCG2 during lactation was markedly high, suggesting a biological role of this gene in milk synthesis/secretion. Weak correlations were observed between both milk FA composition and desaturase indexes (i.e., apparent SCD activity) with mRNA expression pattern of genes measured.ConclusionA network of genes participates in coordinating milk fat synthesis and secretion. Results challenge the proposal that SREBF1 is central for milk fat synthesis regulation and highlight a pivotal role for a concerted action among PPARG, PPARGC1A, and INSIG1. Expression of SCD, the most abundant gene measured, appears to be key during milk fat synthesis. The lack of correlation between gene expression and calculated desaturase indexes does not support their use to infer mRNA expression or enzyme activity (e.g., SCD). Longitudinal mRNA expression allowed development of transcriptional regulation networks and an updated model of milk fat synthesis regulation.

Charge modification at conserved positively charged residues of fatty acid binding protein (FABP) from the giant liver fluke Fasciola gigantica: its effect on oligomerization and binding properties

Fatty acid binding proteins (FABPs) are capable of binding hydrophobic ligands with high affinity; thereby facilitating the cellular uptake and intracellular trafficking of fatty acids. In this study, functional characteristics of a cytoplasmic FABP from the giant liver fluke Fasciola gigantica (FgFABP) were determined. Binding of a fluorescent fatty acid analogue 11-[[5-dimethy aminonaphtalene-1-sulphonyl] amino] undecanoic acid (DAUDA) to FgFABP resulted in changes in the emission spectrum. The optimal excitation wavelength and maximum emission of fluorescence for binding activities with DAUDA were 350 nm and 550 nm, respectively. The binding activity for DAUDA was determined from titration experiments and revealed a Kd value of 2.95+/-0.54 microM. Furthermore, we found that cross-linking profile of FgFABP with dithiobis-(succinimidylpropionate) (DSP) in the presence of DAUDA resulted in increased formation of higher-ordered oligomers compared to that in the absence of DAUDA. We also replaced five highly conserved positively charged residues (K9, K58, K91, R107 and K131) with alanine and studied their oligomerization and binding properties of the modified FgFABPs. The obtained data demonstrate that these residues do not appear to be involved in oligomerization. However, the K58A and R107A substitutions exhibited a reduction in binding affinities. K91A and R107A revealed an increase in maximal specific binding.

Liver Fatty Acid-binding Protein Initiates Budding of Pre-chylomicron Transport Vesicles from Intestinal Endoplasmic Reticulum

The rate-limiting step in the transit of absorbed dietary fat across the enterocyte is the generation of the pre-chylomicron transport vesicle (PCTV) from the endoplasmic reticulum (ER). This vesicle does not require coatomer-II (COPII) proteins for budding from the ER membrane and contains vesicle-associated membrane protein 7, found in intestinal ER, which is a unique intracellular location for this SNARE protein. We wished to identify the protein(s) responsible for budding this vesicle from ER membranes in the absence of the requirement for COPII proteins. We chromatographed rat intestinal cytosol on Sephacryl S-100 and found that PCTV budding activity appeared in the low molecular weight fractions. Additional chromatographic steps produced a single major and several minor bands on SDS-PAGE. By tandem mass spectroscopy, the bands contained both liver and intestinal fatty acid-binding proteins (L- and I-FABP) as well as four other proteins. Recombinant proteins for each of the six proteins identified were tested for PCTV budding activity; only L-FABP and I-FABP (23% the activity of L-FABP) were active. The vesicles generated by L-FABP were sealed, contained apolipoproteins B48 and AIV, were of the same size as PCTV on Sepharose CL-6B, and by electron microscopy, excluded calnexin and calreticulin but did not fuse with cis-Golgi nor did L-FABP generate COPII-dependent vesicles. Gene-disrupted L-FABP mouse cytosol had 60% the activity of wild type mouse cytosol. We conclude that L-FABP can select cargo for and bud PCTV from intestinal ER membranes.

Zebrafish intestinal fatty acid binding protein (I-FABP) gene promoter drives gut-specific expression in stable transgenic fish.

Mammalian intestinal fatty acid-binding protein (I-FABP) is a small cytosolic protein and is thought to play a crucial role of intracellular fatty acid trafficking and metabolism in gut. To establish an in vivo system for investigating its tissue-specific regulation during zebrafish intestinal development, we isolated 5'-flanking sequences of the zebrafish L-FABP gene and used a transgenic strategy to generate gut-specific transgenic zebrafish with green/red fluorescent intestine. The 4.5-kb 5'-flanking sequence of zebrafish I-FABP gene was sufficient to direct fluorescent expression in intestinal tube, first observed in 3 dpf embryos and then continuously to the adult stage. This pattern of transgenic expression is consistent with the expression pattern of the endogenous gene. In all five transgenic lines 45-52% of the F2 inheritance rates were consistent with the ratio of Mendelian segregation. These fish can also provide a valuable resource of labeled adult intestinal cells for in vivo or in vitro studies. Finally, it is possible to establish an in vivo system using these fish for screening genes required for gut development. genesis 38:26-31, 2004.

In vivo studies of liver-type fatty acid binding protein (L-FABP) gene expression in liver of transgenic zebrafish (Danio rerio)

Mammalian liver fatty acid binding protein (L-FABP) is a small cytosolic protein in various tissues including liver, small intestine and kidney and is thought to play a crucial role in intracellular fatty acid trafficking and metabolism. To better understand its tissue-specific regulation during zebrafish hepatogenesis, we isolated 5′-flanking sequences of the zebrafish L-FABP gene and used a green fluorescent protein (GFP) transgenic strategy to generate liver-specific transgenic zebrafish. The 2.8-kb 5′-flanking sequence of zebrafish L-FABP gene was sufficient to direct GFP expression in liver primordia, first observed in 2 dpf embryos and then continuously to the adult stage. This pattern of transgenic expression is consistent with the expression pattern of the endogenous gene. F2 inheritance rates of 42–51% in all the seven transgenic lines were consistent with the ratio of Mendelian segregation. Further, hhex and zXbp-1 morphants displayed a visible liver defect, which suggests that it is possible to establish an in vivo system for screening genes required for liver development.

A new concept of cellular uptake and intracellular trafficking of long-chain fatty acids.

Fatty acids are the main structural and energy sources of the human body. Within the organism, they are presented to cells as fatty acid:albumin complexes. Dissociation from albumin represents the first step of the cellular uptake process, involving membrane proteins with high affinity for fatty acids, e.g., fatty acid translocase (FAT/CD 36) or the membrane fatty acid-binding protein (FABPpm). According to the thus created transmembrane concentration gradient, uncharged fatty acids can flip-flop from the outer leaflet across the phospholipid bilayer. At the cytosolic surface of the plasma membrane, fatty acids can associate with the cytosolic FABP (FABP(c)) or with caveolin-1. Caveolins are constituents of caveolae, which are proposed to serve as lipid delivery vehicles for subcellular organelles. It is not known whether protein (FABP(c))- and lipid (caveolae)-mediated intracellular trafficking of fatty acids operates in conjunction or in parallel. Channeling fatty acids to the different metabolic pathways requires activation to acyl-CoA. For this process, the family of fatty acid transport proteins (FATP 1-5/6) might be relevant because they have been shown to possess acyl-CoA synthetase activity. Their variable N-terminal signaling sequences suggest that they might be targeted to specific organelles by anchoring in the phospholipid bilayer of the different subcellular membranes. At the highly conserved cytosolic AMP-binding site of FATP, fatty acids are activated to acyl-CoA for subsequent metabolic disposition by specific organelles. Overall, fatty acid uptake represents a continuous flow involving the following: dissociation from albumin by membrane proteins with high affinity for fatty acids; passive flip-flop across the phospholipid bilayer; binding to FABP(C) and caveolin-1 at the cytosolic plasma membrane; and intracellular trafficking via FABP(c) and/or caveolae to sites of metabolic disposition. The uptake process is terminated after activation to acyl-CoA by the members of the FATP family targeted intracellularly to different organelles.

Sterol carrier protein-2 expression increases NBD-stearate uptake and cytoplasmic diffusion in L cells.

The effects of sterol carrier protein-2 (SCP-2) expression on fatty acid uptake and cytoplasmic diffusion were determined using L cell fibroblasts transfected with cDNA encoding either the 15- or 13. 2-kDa form of SCP-2. Cis-parinarate and 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate) were used as nonesterifiable fluorescent fatty acid probes. NBD-stearate and cis-parinarate uptake was rapid and saturable. In 15-kDa SCP-2-expressing cells, the extent of NBD-stearate and cis-parinarate uptake was increased 1.4- and 1. 2-fold, respectively, compared with control. In the 13.2-kDa SCP-2-expressing cells, the extent of NBD-stearate and cis-parinarate uptake was increased 1.3- and 1.1-fold, respectively, compared with control cells. NBD-stearate cytoplasmic diffusion was increased 1.5-fold in 15-kDa SCP-2-expressing cells, but not in 13. 2-kDa SCP-2-expressing cells, compared with control cells. After incubation with NBD-stearate for 30 min at 37 degrees C, fluorescence imaging indicated that NBD-stearate was localized primarily in lipid droplets in all cell lines. These results suggest that SCP-2 may be involved not only in fatty acid uptake but also in intracellular fatty acid trafficking.

Role of membrane-associated and cytoplasmic fatty acid-binding proteins in cellular fatty acid metabolism

A number of membrane-associated and cytoplasmic fatty acid-binding proteins (FABPs) are now being implicated in the cellular uptake and intracellular transport of long-chain fatty acids (FA). These proteins each have the capacity of non-covalent binding of FA, are present in tissues actively involved in FA metabolism, and are upregulated in conditions of increased cellular FA metabolism. To date, five distinct membrane FABPs have been described, ranging in mass from 22 to 88 kDa and each showing a characteristic tissue distribution. Evidence for involvement in cellular fatty acid uptake has been provided for several of them, because it was recently found that isolated cell lines transfected with 88-kDa putative fatty acid translocase (FAT; homologous to CD36) or with 63-kDa fatty acid-transport protein show an increased rate of FA uptake. The (at least nine) FABPs of cytoplasmic origin belong to a family of small (14–15 kDa) lipid binding proteins, all having a similar tertiairy structure but differing in binding properties and in tissue occurrence. The biological functions of the various FABPs, possibly exerted in a concerted action among them, comprise solubilization and compartmentalization of FA, facilitation of the cellular uptake and intracellular trafficking of FA, and modulation of mitosis, cell growth, and cell differentiation. In addition, the FABPs have been suggested to participate in and/or modulate FA-mediated signal transduction pathways and FA regulation of gene expression, and to prevent local high FA concentrations thereby contributing to the protection of cells against the toxic effects of FA. In conclusion, long-chain fatty acids are subject to continuous interaction with multiple proteins, which interplay influences their cellular metabolism.

Long-term effects of fatty acids on cell viability and gene expression of neonatal cardiac myocytes

Fatty acids are the most important source of energy for the adult heart. However, cardiac substrate preference changes during development and alters in pathophysiological states. Fatty acids have also been shown to be involved in signal transduction pathways, thereby affecting gene expression in various cell systems. In the present paper the significance of changes in substrate preference and the potential role of fatty acids in signal transduction in the cardiomyocyte are briefly reviewed. Furthermore, the development of a cellular model system, useful in exploring the long-term effects of fatty acids on the normal and hypertrophic cardiomyocyte, is described. Some aspects of this model system are illustrated by showing the effects of different fatty acid species on cell viability and the effects of fatty acids on the expression of heart type fatty acid-binding protein (H-FABP), a 15 kDa protein thought to be involved in intracellular trafficking of fatty acids. To this end primary cultures of rat neonatal ventricular myocytes were kept in defined medium containing various (combinations of) substrates for up to 48 h. First, the effects of prolonged exposure to different fatty acid species, complexed to BSA, on cell viability were investigated. Exposure of the cells to saturated fatty acids (C16:0 or C18:0), but not mono-unsaturated (C16:1 or C18:1) fatty acids, resulted in cell death, as evidenced by the release of intracellular proteins like lactate dehydrogenase. The detrimental effects of saturated fatty acids were nullified by the co-addition of mono-unsaturated fatty acids. Accordingly, the combination of C16:0 C18:1 was used to examine the effects of fatty acids on the expression of H-FABP. Therefore, the cells were incubated with either (i) glucose only, (ii) fatty acids only, or (iii) glucose plus fatty acids. Incubation with fatty acids (with or without glucose) resulted in a nearly four-fold increase of the H-FABP mRNA level. Similarly, at the protein level the cellular H- FABP LDH ratio increased almost two-fold. In hypertrophic cardiomyocytes (stimulated with the α 1-adrenergic agonist phenylephrine) the stimulatory effect of fatty acids on H-FABP expression was mitigated. These findings strongly suggest that fatty acids are able to modulate gene expression in the context of the cardiac muscle cell.

X-ray crystallographic structures of adipocyte lipid-binding protein complexed with palmitate and hexadecanesulfonic acid. Properties of cavity binding sites.

Adipocyte lipid-binding protein is a 14.6-kDa polypeptide that is responsible for the intracellular trafficking of fatty acids. Its structure previously has been solved in the apo and holo forms complexed with stearate and oleate. To examine the binding of lipids other than those with a carboxylate headgroup, we have determined the structure of ALBP in complex with a sulfonic acid, hexadecanesulfonic acid, and compared its structure with the natural fatty acid analog, palmitate. Crystallographic refinement led to similar models, both with R-factors of about 20% and a resolution of 1.6 A. results can be compared with earlier studies on C18 fatty acids, both saturated and unsaturated. The previously refined complexes with stearate and oleate in combination with the complexes of palmitate and hexadecanesulfonic acid demonstrate specific positions for water molecules bound in the internal cavity. Many of the water-binding sites are present in both the apo form and the holo forms of the protein. With ligand present, a network of 10 internalized water molecules appear to form a hydrophobic hydration region. In spite of the sp3 geometry of the sulfonic acid derivative, the headgroup occupies the same site as that of the planar carboxylate in natural fatty acids. These results demonstrate that intracellular lipid-binding proteins are capable of binding a wider variety of lipids than previously considered and reveal the importance of interior ordered water molecules in the binding cavity.

In situ binding of fatty acids to the liver fatty acid binding protein: analysis using 3-[ 125I]iodo-4-azido-n-hexadecylsalicylamide

A photoactivatable radioiodinated fatty acid analogue, 3-[125I]iodo-4-azido-N-hexadecylsalicylamide (125I-AHS) has been synthesized and used to investigate the involvement of cellular lipid carriers in hepatic fatty acid utilization. Photoactivation of Hep G2 internalized 125I-AHS revealed that several cellular proteins were crosslinked with the radiolabeled fatty acid analogue. Three predominant proteins in the membrane fraction of the cell with molecular masses 17, 50 and 127 kDa were crosslinked with the lipid analogue, as determined using autoradiography after SDS-PAGE. Three other proteins in the soluble fraction of the cell, with molecular masses 14, 24 and 35 kDa, were also labeled in situ. In contrast to the other labeled proteins, the fatty acid analogue accumulated on the cytoplasmic 14 kDa protein in a time and temperature dependent fashion. The in situ-labeled 14 kDa protein was identified from primary rat hepatocytes as the liver fatty acid binding protein by partial purification and its ability to be immunoprecipitated with immunospecific L-FABP antiserum. Collectively the results indicate that fatty acids traverse the plasma membrane and are bound cytoplasmically by the liver fatty acid binding protein, as well as other proteins in the cell. This represents the first demonstration in intact hepatocytes that the liver fatty acid binding protein participates in the process of intracellular fatty acid trafficking, and supports a model in which cytoplasmic lipid carriers solubilize fatty acids as a step in their metabolic utilization.