Trafficking of exogenous fatty acids within Caco-2 cells.

Dietary fatty acids (FAs) crossing the apical plasma membrane of small intestinal enterocytes are targeted to different metabolic pathways than serum FAs crossing the basolateral membrane. This apparent compartmentalization of FA metabolism in enterocytes was further investigated using a model human enterocyte-like intestinal cell line. [3H]Oleic acid bound to bovine serum albumin (BSA) was added to the apical or basolateral surfaces of confluent monolayers of Caco-2 cells growing on uncoated polycarbonate filters. In other experiments, [3H]oleic acid incorporated into micelles with taurocholate (+/- 2-monoacylglycerol) was added apically. Caco-2 cells absorbed oleic acid bound to BSA from both the apical and basolateral surfaces at the same rate. Oleic acid in micellar solution was absorbed more efficiently than oleic acid bound to BSA. Regardless of its site or mode of presentation, the majority of the incorporated oleic acid was found in triglycerides. Only a small fraction was subjected to beta-oxidation or esterification into phospholipids. Most of the incorporated oleic acid was still retained intracellularly at 24 h. The polarity of triglyceride secretion was influenced by the experimental conditions. Triglyceride secretion was not significantly polarized when oleic acid-BSA was presented apically. However, the ratio of basolateral to apical secretion at 24 h was 9:1 for oleic acid-BSA presented basolaterally. For oleic acid in taurocholate micelles there was a trend toward polarity of secretion to the apical media (apical to basolateral ratio = 2:1). The inclusion of 2-monoacylglycerol in oleic acid-taurocholate micelles did not augment triglyceride synthesis or secretion. These differences indicate that compartmentation of FA metabolism in Caco-2 cells is influenced by the site of FA presentation. Northern and Western blot hybridization studies indicated that the liver fatty acid-binding protein but not the intestinal fatty acid-binding protein gene is expressed in these cells. The absence of this latter 15 kDa protein indicates that it is not required by Caco-2 cells for the synthesis of triglycerides or for the polarized export of triglyceride. These studies indicate that the Caco-2 cell line will be a useful model system for studying the polarization of FA trafficking/metabolism in enterocytes and defining the role of intracellular fatty acid binding proteins in these processes.     

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.     

Role of caveolin-1 and cholesterol in transmembrane fatty acid movement

We have created by transfection a series of HEK 293 cell lines that express varying amounts of caveolin-1 to test the possible effect of this protein on the transport and metabolism of long chain fatty acids (FA) in cells with this gain of function. We used an extracellular fluorescent probe (ADIFAB) to monitor binding of exogenous FA to the plasma membrane and an intracellular pH probe to monitor FA equilibration across the plasma membrane. Real-time fluorescence measurements showed rapid binding of oleic acid to the extracellular side of the plasma membrane and a rapid translocation across the lipid bilayer by the flip-flop mechanism (<5 s). Two cell lines expressing levels of caveolin-1 roughly comparable to that of adipocytes, which have a very high level of endogenous expression of caveolin-1, showed a relatively slow change in intracellular pH (t(1/2) < 100 s) in addition to the fast changes in fluorescence. We interpret this additional second phase to represent translocation of additional FA from the outer to inner leaflet of the plasma membrane. The slower kinetics could represent either slower flip-flop of FA across highly organized, rigid regions of the plasma membrane or binding of FA to caveolin-1 in the intracellular leaflet of the plasma membrane. The kinetics of palmitate and elaidate (a trans FA) transmembrane movement were identical to that for oleate. These results were observed in the absence of the putative FA transport protein, CD36, and in the absence of any changes in expression of fatty acid transport proteins (FATP) 2 and 4, and are in direct correlation with increased cellular free cholesterol content. FA metabolism was slow in all cell lines and was not enhanced by caveolin-1 expression. We conclude that transport of FA across the plasma membrane is modulated by caveolin-1 and cholesterol and is not dependent on the putative FA transport proteins CD36 and FATP.     

Fatty acids affect micellar properties and modulate vitamin D uptake and basolateral efflux in Caco-2 cells

We have recently shown that vitamin D₃ (cholecalciferol) absorption is not a simple passive diffusion but involves cholesterol transporters. As free fatty acids (FAs) modulate cholesterol intestinal absorption and metabolism, we hypothesized that FAs may also interact with vitamin D absorption. Effects of FAs were evaluated at different levels of cholecalciferol intestinal absorption. First, the physicochemical properties of micelles formed with different FAs were analyzed. The micelles were then administered to human Caco-2 cells in culture to evaluate FA effects on (i) cholecalciferol uptake and basolateral efflux and (ii) the regulation of genes coding proteins involved in lipid absorption process. Micellar electric charge was correlated with both FA chain length and degree of unsaturation. Long-chain FAs at 500 μM in mixed micelles decreased cholecalciferol uptake in Caco-2 cells. This decrease was annihilated as soon as the long-chain FAs were mixed with other FAs. Oleic acid significantly improved cholecalciferol basolateral efflux compared to other FAs. These results were partly explained by a modulation of genes coding for lipid transport proteins such as Niemann-pick C1-like 1 and scavenger receptor class B type I. The data reported here show for the first time that FAs can interact with cholecalciferol intestinal absorption at different key steps of the absorption process. Cholecalciferol intestinal absorption may thus be optimized according to oil FA composition.     

Rapid flip-flop of oleic acid across the plasma membrane of adipocytes

Nonesterified long-chain fatty acids may enter cells by free diffusion or by membrane protein transporters. A requirement for proteins to transport fatty acids across the plasma membrane would imply low partitioning of fatty acids into the membrane lipids, and/or a slower rate of diffusion (flip-flop) through the lipid domains compared to the rates of intracellular metabolism of fatty acids. We used both vesicles of the plasma membrane of adipocytes and intact adipocytes to study transmembrane fluxes of externally added oleic acid at concentrations below its solubility limit at pH 7.4. Binding of oleic acid to the plasma membrane was determined by measuring the fluorescent fatty acid-binding protein ADIFAB added to the external medium. Changes in internal pH caused by flip-flop and metabolism were measured by trapping a fluorescent pH indicator in the cells. The metabolic end products of oleic acid were evaluated over the time interval required for the return of intracellular pH to its initial value. The primary findings were that (i) oleic acid rapidly binds with high avidity in the lipid domains of the plasma membrane with an apparent partition coefficient similar to that of protein-free phospholipid bilayers; (ii) oleic acid rapidly crosses the plasma membrane by the flip-flop mechanism (both events occur within 5 s); and (iii) the kinetics of esterification of oleic acid closely follow the time dependence of the recovery of intracellular pH. Any postulated transport mechanism for facilitating translocation of fatty acid across the plasma membrane of adipocytes, including a protein transporter, would have to compete with the highly effective flip-flop mechanism.     

Fatty acid transport across the cell membrane: Regulation by fatty acid transporters

Transport of long-chain fatty acids across the cell membrane has long been thought to occur by passive diffusion. However, in recent years there has been a fundamental shift in understanding, and it is now generally recognized that fatty acids cross the cell membrane via a protein-mediated mechanism. Membrane-associated fatty acid-binding proteins (‘fatty acid transporters’) not only facilitate but also regulate cellular fatty acid uptake, for instance through their inducible rapid (and reversible) translocation from intracellular storage pools to the cell membrane. A number of fatty acid transporters have been identified, including CD36, plasma membrane-associated fatty acid-binding protein (FABP_pm), and a family of fatty acid transport proteins (FATP1–6). Fatty acid transporters are also implicated in metabolic disease, such as insulin resistance and type-2 diabetes. In this report we briefly review current understanding of the mechanism of transmembrane fatty acid transport, and the function of fatty acid transporters in healthy cardiac and skeletal muscle, and in insulin resistance/type-2 diabetes. Fatty acid transporters hold promise as a future target to rectify lipid fluxes in the body and regain metabolic homeostasis.     

A biophysical study on molecular physiology of the uncoupling proteins of the central nervous system

Mitochondrial inner membrane uncoupling proteins (UCPs) facilitate transmembrane (TM) proton flux and consequently reduce the membrane potential and ATP production. It has been proposed that the three neuronal human UCPs (UCP2, UCP4 and UCP5) in the central nervous system (CNS) play significant roles in reducing cellular oxidative stress. However, the structure and ion transport mechanism of these proteins remain relatively unexplored. Recently, we reported a novel expression system for obtaining functionally folded UCP1 in bacterial membranes and applied this system to obtain highly pure neuronal UCPs in high yields. In the present study, we report on the structure and function of the three neuronal UCP homologues. Reconstituted neuronal UCPs were dominantly helical in lipid membranes and transported protons in the presence of physiologically-relevant fatty acid (FA) activators. Under similar conditions, all neuronal UCPs also exhibited chloride transport activities that were partially inhibited by FAs. CD, fluorescence and MS measurements and semi-native gel electrophoresis collectively suggest that the reconstituted proteins self-associate in the lipid membranes. Based on SDS titration experiments and other evidence, a general molecular model for the monomeric, dimeric and tetrameric functional forms of UCPs in lipid membranes is proposed. In addition to their shared structural and ion transport features, neuronal UCPs differ in their conformations and proton transport activities (and possibly mechanism) in the presence of different FA activators. The differences in FA-activated UCP-mediated proton transport could serve as an essential factor in understanding and differentiating the physiological roles of UCP homologues in the CNS.     

Channel character of uncoupling protein-mediated transport

Mitochondrial uncoupling proteins (UCPs) are pure anion uniporters, which mediate fatty acid (FA) uniport leading to FA cycling. Protonated FAs then flip-flop back across the lipid bilayer. An existence of pure proton channel in UCPs is excluded by the equivalent flux-voltage dependencies for uniport of FAs and halide anions, which are best described by the Eyring barrier variant with a single energy well in the middle of two peaks. Experiments with FAs unable to flip and alkylsulfonates also support this view. Phylogenetically, UCPs took advantage of the common FA-uncoupling function of SLC25 family carriers and dropped their solute transport function.     

Interaction of mitochondrial phosphate carrier with fatty acids and hydrophobic phosphate analogs

Mitochondrial transporters, in particular uncoupling proteins and the ADP/ATP carrier, are known to mediate uniport of anionic fatty acids (FAs), allowing FA cycling which is completed by the passive movement of FAs across the membrane in their protonated form. This study investigated the ability of the mitochondrial phosphate carrier to catalyze such a mechanism and, furthermore, how this putative activity is related to the previously observed HgCl(2)-induced uniport mode. The yeast mitochondrial phosphate carrier was expressed in Escherichia coli and then reconstituted into lipid vesicles. The FA-induced H(+) uniport or Cl(-) uniport were monitored fluorometrically after HgCl(2) addition. These transport activities were further characterized by testing various inhibitors of the two different transport modes. The phosphate carrier was found to mediate FA cycling, which led to H(+) efflux in proteoliposomes. This activity was insensitive to ATP, mersalyl or N-ethylmaleimide and was inhibited by methylenediphosphonate and iminodi(methylenephosphonate), which are new inhibitors of mitochondrial phosphate transport. Also, the HgCl(2) induced Cl(-) uniport mediated by the reconstituted yeast PIC, was found to be inhibited by these reagents. Both methylenediphosphonate and iminodi(methylenephosphonate) blocked unidirectional Cl(-) uptake, whereas Cl(-) efflux was inhibited by iminodi(methylenephosphonate) and phosphonoformic acid only. These results suggest that a hydrophobic domain, interacting with FAs, exists in the mitochondrial phosphate carrier, which is distinct from the phosphate transport pathway. This domain allows for FA anion uniport via the phosphate carrier and consequently, FA cycling that should lead to uncoupling in mitochondria. This might be considered as a side function of this carrier.     

Fats for thoughts: An update on brain fatty acid metabolism

Brain fatty acid (FA) metabolism deserves a close attention not only for its energetic aspects but also because FAs and their metabolites/derivatives are able to influence many neural functions, contributing to brain pathologies or representing potential targets for pharmacological and/or nutritional interventions.Glucose is the preferred energy substrate for the brain, whereas the role of FAs is more marginal. In conditions of decreased glucose supply, ketone bodies, mainly formed by FA oxidation, are the alternative main energy source. Ketogenic diets or medium-chain fatty acid supplementations were shown to produce therapeutic effects in several brain pathologies.Moreover, the positive effects exerted on brain functions by short-chain FAs and the consideration that they can be produced by intestinal flora metabolism contributed to the better understanding of the link between “gut-health” and “brain-health”.Finally, attention was paid also to the regulatory role of essential polyunsaturated FAs and their derivatives on brain homeostasis.     

Cellular fatty acid uptake is acutely regulated by membrane-associated fatty acid-binding proteins

Cellular long-chain fatty acid uptake is believed to occur largely by protein-mediated transmembrane transport of fatty acids, and also by passive diffusional uptake. It is postulated that the membrane proteins function in trapping of fatty acids from extracellular sources, whereafter their transmembrane translocation occurs by passive diffusion through the lipid bilayer. The key membrane-associated proteins involved are plasma membrane fatty acid-binding protein (FABP(pm)) and fatty acid translocase (FAT/CD36). Their plasma membrane contents are positively correlated with rates of fatty acid uptake. In studies with heart and skeletal muscle we observed that FAT/CD36 is regulated acutely, in that both contraction and insulin can translocate FAT/CD36 from an intracellular depot to the sarcolemma, thereby increasing the rate of fatty acid uptake. In addition, from studies with obese Zucker rats, an established rodent model of obesity and insulin resistance, evidence has been obtained that in heart, muscle and adipose tissue FAT/CD36 is permanently relocated from an intracellular pool to the plasma membrane, resulting in increased fatty acid uptake rates in this condition. These combined observations indicate that protein-mediated fatty acid uptake is a key step in cellular fatty acid utilization, and suggest that malfunctioning of the uptake process could be a critical factor in the pathogenesis of insulin resistance.     

Similar mechanisms of fatty acid transfer from human anal rodent fatty acid-binding proteins to membranes: Liver,intestine, heart muscle,and adipose tissue FABPs

The mammalian fatty acid-binding proteins (FABPs) are thought to be important for the transport and metabolism of fatty acids in numerous cell types. The transfer of FA from different members of the FABP family to membranes has been shown to occur by two distinct mechanisms, an aqueous diffusion-based mechanism and a collisional mechanism, wherein the FABP interacts directly with membrane acceptors. Much of the work that underlies this concept comes from efforts using rodent FABPs. Given the increasing awareness of links between FABPs and several chronic diseases in humans, it was important to establish the mechanisms of FA transfer for human FABPs. In the present studies, we examined the rate and mechanism of fatty acid transfer from four pairs of human and rodent (rat or mouse, as specified) FABPs: hLFABP and rLFABP, hIFABP and rIFABP, hHFABP and rHFABP, and hAFABP and mAFABP. In the case of human IFABP, both the Ala⁵⁴ and Thr⁵⁴ forms were examined. The results show clearly that for all FABPs examined, the mechanisms of ligand transfer observed for rodent proteins hold true for their human counterparts. Moreover, it appears that the Ala to Thr substitution at residue 54 of the human IFABP does not alter the fundamental mechanism of ligand transfer to membranes, but nevertheless causes a consistent decrease in the rate of transfer.     

Identification of the major intestinal fatty acid transport protein.

While intestinal transport systems for metabolites such as carbohydrates have been well characterized, the molecular mechanisms of fatty acid (FA) transport across the apical plasmalemma of enterocytes have remained largely unclear. Here, we show that FATP4, a member of a large family of FA transport proteins (FATPs), is expressed at high levels on the apical side of mature enterocytes in the small intestine. Further, overexpression of FATP4 in 293 cells facilitates uptake of long chain FAs with the same specificity as enterocytes, while reduction of FATP4 expression in primary enterocytes by antisense oligonucleotides inhibits FA uptake by 50%. This suggests that FATP4 is the principal fatty acid transporter in enterocytes and may constitute a novel target for antiobesity therapy.     

LPS decreases fatty acid oxidation and nuclear hormone receptors in the kidney

Inflammation produces marked changes in lipid metabolism, including increased serum fatty acids (FAs) and triglycerides (TGs), increased hepatic TG production and VLDL secretion, increased adipose tissue lipolysis, and decreased FA oxidation in liver and heart. Lipopolysaccharide (LPS) also increases TG and cholesteryl ester levels in kidneys. Here we confirm these findings and define potential mechanisms. LPS decreases renal FA oxidation by 40% and the expression of key proteins required for oxidation of FAs, including FA transport protein-2, fatty acyl-CoA synthase, carnitine palmitoyltransferase-1, medium-chain acyl-CoA dehydrogenase, and acyl-CoA oxidase. Similar decreases were observed in peroxisome proliferator-activated receptor alpha (PPARalpha)-deficient mice. LPS also caused a reduction in renal mRNA levels of PPARalpha (75% decrease), thyroid hormone receptor alpha (TRalpha) (92% decrease), and TRbeta (84% decrease), whereas PPARbeta/delta and gamma were not altered. Expression of PGC1 alpha and beta, coactivators required for PPARs and TR, was also decreased in kidneys of LPS-treated mice, as were mitochondrial genes regulated by PGC1 (Atp5g1, COX5a, Idh3a, and Ndufs8). Decreased renal FA oxidation could be a by-product of the systemic coordinated host response to increase FAs and TGs available for host defense and/or tissue repair. However, the kidney requires energy to support its transport functions, and the inability to generate energy via FA oxidation might contribute to the renal failure seen in severe sepsis.     

A null mutation in H-FABP only partially inhibits skeletal muscle fatty acid metabolism

The low-molecular-mass, cytosolic heart-type fatty acid-binding protein (H-FABP) is thought to be required for shuttling FA through the cytosol. Therefore, we examined the effects of an H-FABP-null mutation on FA and carbohydrate metabolism in isolated soleus muscle at rest and during a period of increased metabolic demand (30-min contraction). There were lower concentrations of creatine phosphate (-41%), ATP (-22%), glycogen (-34%), and lactate (-31%) (P < 0.05) in H-FABP-null soleus muscles, but no differences in citrate synthase and beta-3-hydroxyacyl-CoA dehydrogenase activities or in the intramuscular triacylglycerol (TAG) depots. There was a 43% increase in subsarcolemmal mitochondria in H-FABP-null solei. FA transport was reduced by 30% despite normal content of sarcolemmal long-chain fatty acid transporters fatty acid translocase/CD36 and plasma membrane-associated FABP transport proteins. Compared with wild-type soleus muscles, the H-FABP-null muscles at rest hydrolyzed less TAG (-22%), esterified less TAG (-49%), and oxidized less palmitate (-71%). The H-FABP-null soleus muscles retained a substantial capacity to increase FA metabolism during contraction (TAG esterification by +72%, CO2 production by +120%), although these rates remained lower (TAG esterification -26% and CO2 production -64%) than in contracting wild-type soleus muscles. Glycogen utilization during 30 min of contraction did not differ, whereas glucose oxidation was lower at rest (-24%) and during contraction (-32%) in H-FABP-null solei. Although these studies demonstrate that the absence of H-FABP alters rates of FA metabolism, it is also apparent that glucose oxidation is downregulated. The substantial increase in FA metabolism in contracting H-FABP-null muscle may indicate that other FABPs are also present, a possibility that we were not able to completely eliminate.     

Characterization of a fatty acid binding protein from the swimming crab Portunus trituberculatus and its effects on the composition of fatty acids in different tissues

Fatty acid binding proteins (FABPs) belonging to the lipocalin protein family, have an affinity for fatty acids (FAs) and participate in both the transport of FAs and lipid metabolism. In this study, a 909-bp full-length pt-FABP cDNA containing a 411-bp open reading frame (ORF) was obtained from the swimming crab Portunus trituberculatus. Quantitative real-time PCR revealed that during larval development, pt-FABP expression increased gradually from the zoea to the megalopa, and decreased in the juvenile crab. pt-FABP mRNA was detected at the highest level in the testis, followed by the muscles, with the lowest level in the hepatopancreas. The composition of fatty acids in three different tissues from crabs of different sex was detected by gas chromatography–mass spectrometry (GC-MS). The results indicated that the FA profile was in accordance with the expression of pt-FABP, which suggested that pt-FABP participates in the unsaturated fatty acid transportation and storage system of P. trituberculatus.     

Measuring the adsorption of Fatty acids to phospholipid vesicles by multiple fluorescence probes

Fatty acids (FA) are important nutrients that the body uses to regulate the storage and use of energy resources. The predominant mechanism by which long-chain fatty acids enter cells is still debated widely as it is unclear whether long-chain fatty acids require protein transporters to catalyze their transmembrane movement. We use stopped-flow fluorescence (millisecond time resolution) with three fluorescent probes to monitor different aspects of FA binding to phospholipid vesicles. In addition to acrylodan-labeled fatty acid binding protein, a probe that detects unbound FA in equilibrium with the lipid bilayer, and cis-parinaric acid, which detects the insertion of the FA acyl chain into the membrane, we introduce fluorescein-labeled phosphatidylethanolamine as a new probe to measure the binding of FA anions to the outer membrane leaflet. We combined these three approaches with measurement of intravesicular pH to show very fast FA binding and translocation in the same experiment. We validated quantitative predictions of our flip-flop model by measuring the number of H⁺ delivered across the membrane by a single dose of FA with the probe 6-methoxy-N-(3-sulfopropyl) quinolinium. These studies provide a framework and basis for evaluation of the potential roles of proteins in binding and transport of FA in biological membranes.     

Kinetic model of protein-mediated ligand transport: influence of soluble binding proteins on the intermembrane diffusion of a fluorescent fatty acid

The mechanism (or mechanisms) whereby fatty acids and other amphipathic compounds are transported from the plasma membrane to intracellular sites of biotransformation remains poorly defined. In an attempt to better characterize the role of cytosolic binding proteins in this process, a kinetic model of intermembrane ligand transport was developed in which diffusional transfer of ligand between membrane and protein is assumed. The model was tested by utilizing stopped-flow techniques to monitor the transfer of the fluorescent fatty acid analogue, 12-anthroyloxy stearate (12-AS), between model membrane vesicles. Studies were conducted in the presence or absence of bovine serum albumin (BSA), liver fatty acid-binding protein (L-FABP), and intestinal fatty acid-binding protein (I-FABP) in order to determine the effect of soluble proteins on the rate of intermembrane ligand transfer. As predicted by the model, the initial velocity of 12-AS arrival at the acceptor membrane increases in an asymptotic manner with the acceptor concentration. Furthermore, probe transfer velocity was found to decline asymptotically with increasing concentrations of BSA or L-FABP, proteins that exhibit diffusional transfer kinetics. This observation was found to hold true independent of whether donor or acceptor vesicles were preequilibrated with the protein. In contrast, 12-AS transfer velocity exhibited a linear correlation with the concentration of I-FABP, a protein that is thought to transport fatty acids, at least in part, via a collisional mechanism. Taken together, these findings validate the derived kinetic model of protein-mediated ligand transport and further suggest that the mechanism of ligand-protein interaction is a key determinant of the effect of cytosolic proteins on intracellular ligand diffusion.     

Long, saturated chains: tasty domains for kinases of insulin resistance

The mechanistic basis of how cells respond to increased fatty acids (FAs) is murky but potentially involves receptor-mediated activation or inhibition by different FA classes. Holzer et?al. (2011) recently propose in Cell that expansion of intracellular membrane microdomains induced by saturated FA recruit and activate c-Src for JNK activation.