Regulation of fatty acid transport and membrane transporters in health and disease

Long chain fatty acid uptake across the plasma membrane occurs, in part, via a protein-mediated process involving a number of fatty acid binding proteins known as fatty acid transporters. A critical step in furthering the understandings of fatty acid transport was the discovery that giant vesicles, prepared from tissues such as muscle and heart, provided a suitable system for measuring fatty acid uptake. These vesicles are large (10–15 m diameter), are oriented fully right side out, and contain cytosolic FABP in the lumen, which acts as a fatty acid sink, while none of the fatty acid taken up is metabolized or associated with the plasma membrane. The key fatty acid transporters FAT/CD36 and FABPpm are expressed in muscle and heart and their plasma membrane content is positively correlated with rates of fatty acid transport. These transporters are regulated acutely (within minutes) and chronically (days). For instance, both muscle contraction and insulin can translocate FAT/CD36 from an intracellular pool to the plasma membrane, thereby increasing fatty acid transport. With obesity, fatty acid transport is increased along with a concomitant increase in plasmalemmal FAT/CD36 (heart, muscle) and FABPpm (heart only), but without change in the expression of these transporters. This latter observation suggests that some of the fatty acid transporters are permanently relocated to the plasma membrane. In other studies it also appears that fatty acid transport rates are altered in a reciprocal manner to glucose transport. Since disorders in lipid metabolism appear to be an important factor contributing to the etiology of a number of common human diseases such as diabetes and obesity, our evidence that protein-mediated fatty acid transport is a key step in lipid metabolism allows the speculation that malfunctioning of the fatty acid transport process could be a common critical factor in the pathogenesis of these diseases.     

Free fatty acid transport across adipocytes is mediated by an unknown membrane protein pump

The role of cell membranes in regulating the flux of long chain free fatty acids (FFA) into and out of adipocytes is intensely debated. Four different membrane proteins including, FABPpm, CD36/FAT, caveolin-1, and FATP have been identified as facilitating FFA transport. Moreover, CD36 and caveolin-1 are also reported to mediate transport in conjunction with lipid rafts. The principal evidence for these findings is a correlation of the level of FFA uptake with the expression level of these proteins and with the integrity of lipid rafts. The 3T3-L1 and 3T3-F442A cell lines in their preadipocyte states reveal little or no expression of these proteins and correspondingly low levels of uptake. Here we have microinjected the adipocyte and preadipocyte cell lines with ADIFAB, the fluorescent indicator of FFA. The ADIFAB fluorescence allowed us to monitor the intracellular unbound FFA concentration during FFA influx and efflux. We show that these measurements of transport, in contrast to FFA uptake measurements, correlate neither with expression of these proteins nor with lipid raft integrity in preadipocytes and adipocytes. Transport characteristics, including the generation of an ATP-dependent FFA concentration gradient, are virtually identical in adipocytes and preadipocytes. We suggest that the origin of the discrepancy between uptake and our measurements is that most of the FFA transported into the cells is lost during the uptake but not in the transport protocols. We conclude that long chain fatty acid transport in adipocytes is very likely mediated by an as-yet-unidentified membrane protein pump.     

Regulation of fatty acid transport

PURPOSE OF REVIEW: The aim of the present review is to summarize recent developments in the area of regulation of fatty acid transport. RECENT FINDINGS: While controversy still exists regarding the contribution of passive diffusion versus protein-mediated fatty acid transport, both processes are now widely accepted. With the recent identification of an increasing number of putative fatty acid transporters, emphasis has been placed on regulation including fatty acid transport function of the protein, and also possible associated functions (acylCoA synthase activity and vectorial channelling to intracellular processing). Deciphering these issues has been facilitated through the use of loss-of-function (such as knockout) and gain-of-function (cell transfectants and transgenic mice) models. SUMMARY: It is likely that our concept of fatty acid transport will continue to converge, incorporating the individual functions of the wide variety of fatty acid transporters into an integrated physiologic framework with relevance to a number of diseases.     

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.     

Fatty acid transport in adipocytes monitored by imaging intracellular free fatty acid levels

Transport of free fatty acids (FFA) across the adipocyte plasma membrane is critical for maintaining homeostasis. To determine the membrane's role in regulating transport we describe here the first measurements of the intracellular (unbound) FFA concentration ([FFA(i)]) and their use in monitoring transport of FFA across 3T3F442A adipocytes. [FFA(i)] was measured by microinjecting cells with ADIFAB, a fluorescently labeled fatty acid-binding protein that is used to measure unbound FFA levels. We used ratio fluorescence microscopy of intracellular ADIFAB to image unbound FFA levels and determined the time course of [FFA(i)] in response to changing the extracellular unbound FFA concentration ([FFA(o)]). [FFA(o)] was clamped at defined levels using complexes of FFA and bovine serum albumin. We show that FFA influx is slow, requiring about 300 s to reach steady state (rate constant approximately 0.02 s(-1)) and saturable (K(o) approximately 200 nm). Efflux is twice as fast as influx, for zero [FFA(o)], but decreases with increasing [FFA(o)]. Surprisingly, at steady state [FFA(i)] is 2-5-fold (average 2-fold) greater than [FFA(o)] and this [FFA(i)]/[FFA(o)] gradient is abolished by depleting cellular ATP. Our results indicate that FFA transport across adipocyte membranes is highly regulated, involving an ATP-driven pump and a mechanism for gating efflux that is sensitive to [FFA(o)]. These characteristics are well described by a membrane carrier model but are not consistent with FFA transport across the membrane's lipid phase. We suggest that these characteristics are important in regulating circulating FFA levels by the adipocyte.     

Fatty acid transport into the brain: of fatty acid fables and lipid tails

The blood-brain barrier formed by the brain capillary endothelial cells provides a protective barrier between the systemic blood and the extracellular environment of the central nervous system. Brain capillaries are a continuous layer of endothelial cells with highly developed tight junctional complexes and a lack of fenestrations. The presence of these tight junctions in the cerebral microvessel endothelial cells aids in the restriction of movement of molecules and solutes into the brain. Fatty acids are important components of biological membranes, are precursors for the biosynthesis of phospholipids and sphingolipids and are utilized for mitochondrial β-oxidation. The brain is capable of synthesizing only a few fatty acids. Hence, most fatty acids must enter into the brain from the blood. Here we review current mechanisms of transport of free fatty acids into cells and describe how free fatty acids move from the blood into the brain. We discuss both diffusional as well as protein-mediated movement of fatty acids across biological membranes.     

Studies on lithium transport across the red cell membrane

Summary Binding of³H-saxitoxin to Na⁺ channels was studied in subcellular fractions prepared from rat brain homogenates. Saxitoxin binding to synaptosomes was saturable with an apparent dissociation constant of about 1nm; about 1 pmol/mg protein was bound at saturating saxitoxin concentrations. A linear, nonsaturable component of saxitoxin binding accounted for less than 3% of the total binding at 30nm. Saxitoxin binding to synaptosomes was unaffected by depolarization with elevated K⁺ concentrations, or by activation of the Na⁺ channels with batrachotoxin plus a purified polypeptide toxin from the scorpionLeiurus quinquestriatus. A procedure is described for preparing a membrane fraction that contains 70–80% of the total saxitoxin binding activity of the crude homogenate. The specific activity of this fraction was about 4 to 6 pmol/mg protein. About 60–70% of the saxitoxin binding sites were solubilized by incubating these membranes with the nonionic detergent Triton X-100; the detergent-solubilized binding sites eluted at a position corresponding to a mol wt of about 700,000 on gel filtration chromatography. Both membrane-bound and solubilized saxitoxin binding were assayed by a new cation exchange column method. The binding of saxitoxin to both membrane-bound and detergent-solubilized binding sites was saturable with an apparent dissociation constant of about 2nm. Dissociation of the saxitoxin-receptor complex followed a single exponential decay with a rate constant at 0° of 0.1 min^–1 for membrane bound and 0.2 min^–1 for detergent-solubilized binding sites. The measured association rate constant was 6×10⁸ m ^–1 min^–1 at 0° for membrane-bound saxitoxin binding sites.     

Anion transport across the red blood cell membrane mediated by dielectric pores

Summary The anion transport across the red blood cell membrane is assumed to occur by ionic diffusion through dielectric pores which are formed by protein molecules spanning the red blood cell membrane. The access of anions to the dielectric pores is regulated by anion adsorption sites positioned at the entrances of the pores. The adsorption of small inorganic anions to the adsorption sites is facilitated by ionizing cationic groups setting up a surface potential at the respective membrane surfaces. Applying the transition state theory of rate processes, flux equations for the unidirectional flux were derived expressing the unidirectional flux as a function of the fractional occupancies of anion adsorption sites at both membrane surfaces.The basic properties of the transport model were investigated. The concentration-dependence and the pH-dependence of the unidirectional fluxes were shown to depend upon the surface charge density and upon the affinity of the transported anion species to the anion binding sites. The concentration-response and the pH-response of the unidirectional fluxes of different anion species may differ substantially even if the anion species are transported by the same anion transport system. The model predicts a characteristic behavior of the Lineweaver-Burk plot and of the Dixon plot.A comparison between computer simulated and experimentally determined flux curves was made. By choosing a suitable set of parameters, the anion transport model is capable of simulating the concentration-dependencies and the pH-dependencies of the unidirectional sulfate and chloride flux. It is sufficient to change one single constant in order to convert the sulfate transport system into a chloride transport system. Furthermore, the model is capable of predicting the inhibitory action of chloride on the sulfate transport system. No attempts were made to fit the experimental data to the model. The behavior of the model was qualitatively in accordance with the experimental results.     

Studies on lithium transport across the red cell membrane

Summary Ouabain-resistant Na⁺–Li⁺ countertransport was studied on erythrocytes of man, sheep, rabbit, and beef. A transport system, exchanging Li⁺ for Na⁺ in a ratio of 11, was present in all four species. Li⁺ uptake by the exchange system increased 30-fold in the order man +–Na⁺ exchange in these species, but bears no relation to the Na⁺–K⁺ pump activity. The activity of the Na⁺–Li⁺ exchange system varied up to 7 and 16-fold among individual red cell specimens from man and beef, the variability being much smaller in sheep and rabbit erythrocytes. The affinities of the system for Li⁺ and Na⁺ were similar among the species and individuals (half saturation of the external site at about 1mm Li⁺ and 50mm Na⁺, respectively).50–60% of Na⁺–Li⁺ exchange was blocked by N-ethylmaleimide in all species.p-Chloromercuribenzene sulfonate inhibited the exchange only in beef and sheep erythrocytes (60–80%). The two SH-reagents act by decreasing the maximum activity of the system, whilst leaving its affinity for Li⁺ unaltered. Phloretin was a potent inhibitor in all species. 1mm each of furosemide, ethacrynic acid, and quinidine induced only a slight inhibition. The Na⁺–Li⁺ exchange of human and beef erythrocytes increased 3.5-fold upon elevation of the extracellular pH from 6 to 8.5, the pH-dependence arising from a change in affinity of the system for the cations and being similar to that reported for ouabain-resistant Na⁺–Na⁺ exchange in beef erythrocytes.It is concluded that a transport system exists in the red cell membranes of the four species which can mediate ouabain-resistant exchange of either Na⁺ for Na⁺, Na⁺ for Li⁺, or Li⁺ for Li⁺. The exchange system exhibits essentially identical transport characteristics in the four species, but shows a marked inter- and intra-species variability in maximum transport capacity and some differences in susceptibility towards inhibitors. A similar transport system is probably present also in other tissues. The exchange system seems to be distinct from the conventional Na⁺–K⁺ pump and shows no clear relation to one of the furosemide-sensitive, ouabain-resistant Na⁺ transport systems described in the literature.     

Direct determination of free fatty acid transport across the adipocyte plasma membrane using quantitative fluorescence microscopy.

Movement of free fatty acids (FFA) across the plasma membrane has been directly measured for the first time, using fluorescent FFA analogs and quantitative fluorescence microscopy. The rate of short chain FFA (less than or equal to 12 carbons) transport from the extracellular medium into intracellular lipid droplets of 3T3F442A adipocytes was more than 40-fold faster than long chain FFA (16 and 18 carbons). The membrane-impermeable amino reagent 4,4'-diisothiocyanostilbene-2,2'-disulfonate, inhibited greater than or equal to 50% of the long chain FFA transport but had no effect on short chain FFA transport. Oleic acid (2 microM) inhibited 90% of the fluorescent oleate transport but had no effect on the 11-carbon analog. These results indicate that a large fraction of long chain FFA uptake is mediated by a plasma membrane protein (s).     

Peptide transport across the animal cell plasma membrane: recent developments.

Transfer of intact peptides across the plasma membrane of animal cells, especially in the small intestine and the kidney, is a well established phenomenon. This process plays an important role in the maintenance of protein nutrition. Evidence is accumulating which suggests that the process may also have a great potential for pharmacological and clinical applications. It is therefore important to understand various aspects of peptide transport such as its function, chemical nature of the transport protein and its gene, the operational mechanisms and their regulation, and the relevance of the transport system to health and disease. Recent years have witnessed considerable progress in the field. The driving force for the transport system has been identified as the proton motive force which makes the system unique and distinct from the majority of solute transport systems in animal cells which are driven by a sodium motive force. A great deal is now known on the chemical nature of the active site. The protein responsible for the transport process in the small intestine has been purified and characterized. The system has been successfully expressed in its functional form in Xenopus laevis oocytes by microinjection into the oocytes of poly(A)+ mRNA isolated from intestinal mucosal cells. There is no doubt that the coming years will bring even more exciting information on the transport system, especially in areas such as hormonal regulation, clinical applicability and cloning, and characterization of the gene encoding the transport system.     

Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane

New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis are urgently needed. We report on the identification of an adamantyl urea compound that shows potent bactericidal activity against M. tuberculosis and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm, where they are synthesized to the periplasmic side of the plasma membrane and are in turn transferred onto cell wall arabinogalactan or used in the formation of virulence-associated, outer membrane, trehalose-containing glycolipids. Whole-genome sequencing of spontaneous-resistant mutants of M. tuberculosis selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane.