Mechanisms and Regulation of Protein-Mediated Cellular Fatty Acid Uptake: Molecular, Biochemical, and Physiological Evidence

Abstract

POINT/COUNTERPOINTMechanisms and Regulation of Protein-Mediated Cellular Fatty Acid Uptake: Molecular, Biochemical, and Physiological EvidenceArend Bonen, Adrian Chabowski, Joost J. F. P Luiken, and Jan F. C. GlatzArend BonenDepartment of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada, Search for more papers by this authorEmail the corresponding author at [email protected], Adrian ChabowskiDepartment of Physiology, Medical University of Bialystok, Bialystok, Poland, andSearch for more papers by this author, Joost J. F. P LuikenDepartment of Molecular Genetics, Maastricht University, Maastricht, The Netherlands.Search for more papers by this author, and Jan F. C. GlatzDepartment of Molecular Genetics, Maastricht University, Maastricht, The Netherlands.Search for more papers by this authorPublished Online:01 Feb 2007https://doi.org/10.1152/physiologyonline.2007.22.1.15MoreSectionsPDF (824 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmail Arend Bonen Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada [email protected]Download figureDownload PowerPointAdrian Chabowski Department of Physiology, Medical University of Bialystok, Bialystok, PolandDownload figureDownload PowerPointJoost J. F. P Luiken Department of Molecular Genetics, Maastricht University, Maastricht, The NetherlandsDownload figureDownload PowerPointJan F. C. Glatz Department of Molecular Genetics, Maastricht University, Maastricht, The NetherlandsDownload figureDownload PowerPointFatty acids serve as substrates for diverse cellular processes, including membrane biosynthesis, protein modification, regulation of transcription, intracellular signaling, and energy provision for tissues such as liver, heart, and skeletal muscle. Although these processes are highly regulated, it has long been thought that entry of long-chain fatty acids into parenchymal cells occurs in an unregulated manner by diffusion (57–60). However, from a physiological perspective, it may be speculated that it would be highly desirable to regulate the entry of fatty acids into the cell, given their important roles in many key cellular processes. In addition, since fatty acids may exert harmful effects, related to their detergent-like action (114, 122), such detrimental effects could be better controlled if fatty acid entry into the cell was regulated.In recent years, there has been considerable debate as to whether fatty acid is transported into cells or diffuses rapidly into the cell. It now appears that this debate is less strident, as it has been acknowledged recently that evidence supporting passive diffusion as the main mechanism for fatty acid uptake is apparently in error, since “previous reports for rapid flip-flop were based on an incorrect interpretation of the measurements” (79), Because of this (79) and other experiments (78), it has been concluded that “the lipid bilayer portion of biological membranes may present a significant barrier to transport of FFA across cell membranes” (36) and that “flip-flop is the rate limiting step for FFA transport across lipid vesicles” (78). Furthermore, “this implies that at least certain biological membranes may require protein-mediated transporters to catalyze the flip-flop step” (78).Since we (13, 27–29, 73, 97, 98, 100) and others (32, 45, 54) have previously provided considerable support for the protein-mediated entry of long-chain fatty acids into the cell, especially in metabolically important tissues such as heart and skeletal muscle, we concur with these recent conclusions (36, 78, 79) that (membrane-associated) proteins are involved in cellular fatty acid uptake. Indeed, very solid evidence, based on physiological, biochemical, molecular, and genetic studies, supports the idea that fatty acid entry into many tissues occurs via a protein-mediated mechanism that is highly regulatable both acutely (within minutes) and chronically (hours to weeks). We (13, 27–29, 73, 97, 98, 100) and others (32, 45, 54) have identified in heart and skeletal muscle some of the proteins that facilitate the movement of fatty acids across the plasma membrane, their regulation by physiological signals, as well as the signaling pathways involved. In addition, we (18, 96) have also found that these proteins may mediate, in part, the development of fatty acid-induced insulin resistance.We agree with Kampf and Kleinfeld (80) that the exact mechanics of how fatty acids are transferred across the plasma membrane are not known, whether in adipose tissue, heart, or skeletal muscle. We do not dismiss the idea that fatty acids can also diffuse into the cell. However, under physiological conditions, diffusion of fatty acids appears to be quantitatively less important (40), as has also been shown for lactate (105, 106) and glucose (85). It would not be completely surprising if the proteins involved in transporting fatty acids and/or their regulation would differ in adipocytes and muscle tissue (heart and skeletal muscle) given the very different metabolic roles and physiological functions of these tissues. The purpose of the present paper is to examine 1) the key evidence in support of a highly regulated, protein-mediated system that is involved in delivering fatty acids across the plasma membrane and 2) how this transport process may contribute to regulating fatty acid metabolism. The emphases throughout is on studies in mammalian tissues, primarily heart and skeletal muscle, in which fatty acid transport appears to be a key component in regulating fatty acid metabolism.Evidence that Fatty Acids are Taken Up via Protein-Mediated System into Giant Sarcolemmal VesiclesFor some time, determinations of fatty acid movement across the plasma membrane had occurred in either in vitro (reviewed in the accompanying paper Ref. 80) or in more biological preparations in which entry of fatty acids into the cell was not clearly distinguishable from the concurrent fatty acid metabolism, although the corrections introduced provided a reasonable approximation of fatty acid transport across the plasma membrane (2, 5, 6). Nevertheless, these different approaches led to conflicting results. In an attempt to solve the conundrum and realizing that both of these foregoing approaches may limit proper conclusions concerning the fatty acid uptake and its regulation, we set out 1) to use a vesicle preparation in which only the rates of fatty acid movement across the plasma membrane can be examined (i.e., in the absence of their metabolism) and 2) to derive such vesicles from metabolically important mammalian tissues. Therefore, we began to use the giant vesicles prepared from skeletal muscle (14) and the heart (102).We have detailed previously how these vesicles are prepared and how fatty uptake measurements are made (14, 88, 102). Briefly, giant vesicles can be prepared from most tissues by simply removing the tissue from the animals and scissoring these tissues into narrow strips (1–3 mm). Thereafter, the strips are incubated (1 h, 34°C) in 140 mM KCl/10 mM MOPS (pH 7.4) containing a protease inhibitor (aprotinin) and an appropriate collagenase for the tissue in question. Thereafter, the vesicles are recovered from the incubating medium after centrifugation, i.e., at the interface of a 3 ml, 4% Nycodenz layer, and a 1 ml KCl/MOPS upper layer. Recovered vesicles are then resuspended in KCl/MOPS. At this point, vesicles can be maintained for some time at room temperature or overnight at 4°C, and possibly even longer. For transport studies, protein concentrations of the vesicles are normally determined immediately. Thereafter, protein (≥50 μg) is added to small tubes. Radiolabeled palmitate and unlabeled palmitate (previously complexed to fat-free bovine serum albumin), as well as radiolabeled mannitol, are then added (40-μl volume) to the vesicles and quickly mixed. Typically, the reaction proceeds for 15 s. At that point, an ice-cold HgCl2 stop solution is added. Thereafter, vesicles are pelleted, the supernatant fraction is removed, and the radioactivity is determined in the pellet cut from the tip of the tube. Standard calculations are performed to determine the molar quantity of palmitate taken up in 15 s.Key features of this giant vesicle preparation include the following: 1) giant sarcolemmal vesicles have a large volume (10- to 15-μm diameter), which permits measurement of fatty acid entry over a 2- to 30-s range, during which time the rate of palmitate uptake remains linear (i.e., backflux is not occurring), while the concentration of extra-vesicular palmitate in the assay is not altered; 2) giant sarcolemmal vesicles are oriented 100% right-side out, which may be important depending on how fatty acids cross the plasma membrane; and 3) in the lumen of giant sarcolemmal vesicles there are excess quantities of the 15-kDa cytoplasmic fatty acid binding protein (FABPc), which is required to act as a sink for fatty acids that have crossed the plasma membrane (14, 86, 88). Absence of FABPc in the lumen of giant vesicles compromises the uptake of fatty acids (99), thereby demonstrating the critical role of this protein in the desorption of fatty acids from the plasma membrane.Studies of the rates of fatty acid entry into heart- and skeletal muscle-derived giant vesicles have provided convincing evidence in support of the involvement of a protein-mediated system. In these giant vesicles, the rate of entry of long-chain fatty acids is saturable (FIGURE 6A), suggesting the presence of a limiting factor. Pretreating giant sarcolemmal vesicles with protein modifying agents (trypsin and phloretin), as well as with a reactive oleate ester [sulfo-N-succinimidy-loleate (SSO)] and antisera to a putative fatty acid transporter, inhibited fatty acid entry into the vesicles by 30–50% (14, 102) (FIGURE 6B). In addition, palmitate entry into giant vesicles was displaced by another long-chain fatty acid (oleate) but not by a short-chain fatty acid (octanoate) or by glucose (14, 102), illustrating that the transport system is specific for long-chain fatty acids (FIGURE 6B). Finally, the palmitate that was transported into giant sarcolemmal vesicles was fully recovered in the lumen of these vesicles and was not further metabolized (FIGURE 6C). Collectively, these studies demonstrated that long-chain fatty acids traverse the plasma membrane in heart and skeletal muscle largely via a protein-mediated system.Interestingly, the Kms for fatty acid transport in heart, and in red and white skeletal muscles, were similar (6–9 nM), whereas the Vmax differed markedly (heart >> red muscle > white muscle) (14, 102) (FIGURE 6A). These Vmax differences corresponded directly with the well-known differences in the capacities for fatty acid oxidation in heart and in red and white skeletal muscles. This suggests strongly that the rate of protein-mediated fatty acid movement across the plasma membrane is scaled with the need to metabolize fatty acids in these tissues. Indeed, there was an excellent correlation between the plasmalemmal fatty acid transporter FAT/CD36 and the rate of palmitate transport into giant sarcolemmal vesicles derived from heart and skeletal muscle (FIGURE 6D). Protein-mediated fatty acid transport into heart and muscle does not appear to be energy consuming, as in muscle tissues the direction of net fatty acid movement is determined by the transmembrane gradient of fatty acids. Because the intracellular fatty acid concentration is at least 17-fold lower in muscle compared with the arterial circulation (82, 141), the driving gradient is always from the circulation/interstitial space into the myocyte. However, such an inwardly directed gradient may not occur in adipocytes, given the very different metabolic role of adipose tissue (i.e., lipid accretion and release), and, therefore, the mechanisms regulating protein-mediated fatty acid transport into or out of adipocytes appear to differ (78, 79) from those observed in muscle and heart. Importantly, therefore, adipocytes and muscle tissues cannot serve as interchangeable model systems for examining the regulation of fatty acid transport (and metabolism). To do so would probably add considerable confusion to our understanding of how fatty acid transport is regulated in different metabolic tissues, which are well known to have markedly different functions, in vivo.Clearly, the giant sarcolemmal vesicles used in our laboratories offer many advantages, including the ability to conduct studies using metabolically important studies from rodents as well as humans (see below). There are, however, also some limitations. First, considerable quantities of fresh tissue are required to generate sufficient vesicles for transport studies (preferably 0.5–1 g). Second, it is possible that preparation of the vesicles isolates specific subplasmalemmal domains, as we have observed that vesicles generated from heart, muscle, liver, and adipocytes are more or less similar in size (88, 96). Another concern is that giant vesicles are only derived from the plasma membrane, whereas T-tubules are completely excluded. This may be important, since substrate transport proteins are present in T-tubules [GLUT4 (92) and MCT1 and 4 (17)], and we have also found FAT/CD36 and FABPpm in this location (unpublished observations). It is known that GLUT4 can be induced to translocate to the T-tubules (90, 92), and these invaginations represent an efficient means of distributing substrates into the muscle cell’s interior. Extensive use of giant vesicles has therefore precluded any assessment of the role of T-tubules in fatty acid transport into muscle tissues.Critique of Determining Fatty Acid TransportIn the accompanying paper (80), it is noted that the uncertainty about plasma membrane fatty acid transport arises because uptake measurements do not clearly distinguish the actual transport step. Certainly, we (13, 27–29, 73, 97, 98, 100) and others (32, 45, 54) have focused on establishing whether there is a transport-like process of transferring fatty acids across the plasma membrane and determining which proteins are involved in this process. We concur that these approaches shed no light on the actual transport steps. However, the converse also appears to be the case: namely that exclusive focus on the specific transport steps in the membrane has limited an appreciation (57–60) for a considerable body of work supporting the evidence for a fatty acid transport-like process, particularly in metabolically important tissues.Concerns that uptake measurements are not sensitive to unbound cytosolic fatty acids (80) are likely not a serious problem in giant vesicles. Cytosolic fatty acid content in skeletal muscle is extraordinary low (82, 141), and fatty uptake by giant vesicles proceeds in a linear fashion for at least 30 s (14), indicating that uptake is not being impaired by intravesicular fatty acid accumulation. Moreover, within these giant vesicles, there is excess FABPc that acts as a fatty acid sink (14). In addition, all the fatty acids that have been transported into the vesicles are recovered as fatty acids from the lumen (cytosol) of the giant vesicles (14) (see FIGURE 6C).Other critiques (80) are that determination of saturated fatty acid uptake relies on the subtraction of a linear component, assumed to be diffusion, and there may be errors in calculating unbound fatty acid concentrations. However, inhibition of plasmalemmal fatty acid transporters (14, 102, 139) or ablation of a fatty acid transporter markedly reduces the rate of adipocyte fatty acid transport (45). In addition, in these FAT/CD36-null mice, only a linear (non-protein mediated) rate of fatty acid uptake is observed in adipocytes (45). Collectively, these experimental observations suggest strongly that determination of saturated fatty acid transport as currently done is not an artifact.The final critique (80) states that uptake of exogenous fatty acids must not perturb cell-associated fatty acids already present, and hence their efflux must be prevented with specific reagents, something that was not achieved in 3T3F442A adipocytes (79). This critique would seem to relate specifically to adipocytes, since these may well contain a considerable amount of intracellular fatty acids. As noted above, in giant vesicles derived from heart and muscle, the intracellular fatty acids are likely very low, and the linearity of initial rates of exogenous fatty acid uptake indicates little or no efflux is occurring. Moreover, we have had no difficulty in blocking fatty acid uptake into giant vesicles. Apparently, the difficulty in inhibiting fatty acid uptake in adipocytes (80) appears to be unique to this model system and does not apply to our giant vesicle preparation. Thus concerns about methodological inadequacies in fatty acid transport determinations appear to be far less problematical in muscle- and heart-derived giant vesicles as opposed to adipocytes, which appear to provide the frame of reference for some of the methodological critique (80).Defining the Process of Plasmalemmal Fatty Acid TransportWe have opted to refer to the process of protein-mediated fatty acid movement across the plasma membrane as fatty acid transport. We recognize that this transport process may differ from that of other substrates, such as glucose and lactate. Moreover, the molecular mechanisms of the transmembrane translocation of fatty acids remain elusive, because the molecular structure and membrane topology of the various fatty acid transporters are not fully known. Importantly, the molecular mechanism of transmembrane translocation of fatty acids may include a flip-flop of the fatty acid from one leaflet to another (as explained below) (51). These caveats, however, should not impede usage of the term fatty acid transport, especially since the regulation of protein-mediated long chain fatty acid movement across the plasma membrane is very similar to that of glucose transport [see detailed review by Luiken et al. (93) concerning these similarities].Molecular Evidence for Membrane Fatty Acid TransportersDifferent groups have identified integral and peripheral membrane proteins that appeared to be involved in the transport of fatty acids into parenchymal cells. These proteins are commonly referred to as fatty acid transporters, despite the remaining uncertainty as to the exact mechanism by which any one of these proteins participates in the transport process. The known fatty acid transport proteins include 1) a family of ~70-kDa fatty acid transport proteins (FATP1–6) (50, 70, 119), 2) the 40-kDa plasma membrane associated fatty acid binding protein (FABPpm) (121, 133), and 3) the heavily glycosylated 88-kDa fatty acid translocase, also known as CD36 (FAT/CD36) (1).The murine FATPs appear to be expressed in somewhat of a tissue-specific manner (50, 70, 91), and species specificity in expression patterns also appears to be present (91). In contrast, FAT/CD36 and FABPpm (10) are ubiquitously expressed in almost all rodent tissues examined. In humans, fatty acid transporters are expressed in skeletal muscle (FATP1 and 4, FABPpm, and FAT/CD36) (16, 24, 25), heart (FAT/CD36) (69), and adipose tissue (FATP1 and 4, FAT/CD36, FABPpm) (8, 22, 48). Support for facilitating long-chain fatty acid transport by many of these differing transporters has been obtained from genetic studies in cell lines and animals, as well as from physiological studies in mammalian tissues.Studies In VitroFATP1–6.FATP1 was discovered by Schaffer and Lodish (119) using an expression cloning strategy and a cDNA library from 3T3-L1 adipocytes to identify a cDNA that, when expressed in cultured cells, augments the uptake of long-chain fatty acids. Subsequently, others identified a family of integral membrane FATPs (FATP2–6) (50, 70). Given the number of FATP isoforms, their transport capacities are likely to vary widely. Zou et al. (145) have examined the role of Fat1p, the FATP1 ortholog in Saccharomyces cerevisiae. This work has shown that Fat1p and a long-chain acyl-CoA synthetase form a functional complex at the plasma membrane, which then couples transport of exogenous fatty acids and their activation (145). Because FATPs share considerable sequence homology and domain organization with acyl-CoA synthetases, there had been concerns that the apparent fatty acid transport function of FATPs merely reflected the long-chain fatty acid activation being coupled to fatty acid metabolism, as opposed to facilitating fatty acid transport per se (33, 112, 115). Although deletion of the Fat1p gene (FAT1) in yeast did not impair the activities of long-chain acyl-CoA synthetases (44), others observed that FAT1 deletion in S. cerevisiae reduced the activity of very long-chain acyl-CoA synthetase activities (142). Purified murine FATP1 and 4 also have acyl-CoA synthetase activities (55, 56), with FATP1 exhibiting a preference for a broad range of fatty acids (55) and FATP4 exhibiting a preference for long- and very long-chain fatty acids (56). Collectively, these studies caused some confusion as to whether the acyl-CoA synthetase activity associated with FATPs provided the driving force for fatty acid transport. However, elegant studies by DiRusso et al. (38) appear to have resolved this matter. They have shown in a genetically defined yeast strain, which normally cannot transport fatty acids and has a reduced acyl-CoA synthetase activity, that FATP1, 2, and 4 are particularly effective in facilitating the rates of long-chain fatty acid transport by 8.2-, 4.5-, and 13.1-fold, respectively, whereas FATP3 and 5 provide only a modest twofold increase, and FATP6 provides virtually no increase in long-chain fatty acid transport (38) (FIGURE 7). Importantly, these FATP-induced increases in fatty acid transport were not attributable to the concurrent upregulation (~1.6-fold) in oleoyl-CoA synthetase activity (38) (FIGURE 7). Others have also cast doubt on a fatty acid transport function for FATP3, since it catalyzes fatty acid activation but not fatty acid transport in MA-10 cells (112). Despite the independent transport functions of FATP1, 2, and 4 (38), it has been found recently that the yeast orthologs of FATP1 and long-chain acyl coenzyme A synthetase 1 co-immunoprecipitated (145) and that FATP1-mediated fatty acid uptake is coupled to long-chain acyl coenzyme A synthetase 1 in 3T3 L1 adipocytes (116).FABPpm.Plasma membrane-associated fatty acid binding protein (FABPpm) was identified by Stremmel and coworkers (37, 131–133) in a series of studies in liver and the heart. FABPpm is a peripheral membrane protein at the outer leaflet of the plasma membrane (134). Analysis of its amino acid sequences showed FABPpm to be identical to mitochondrial aspartate aminotransferase (mAspAt) (134). Apparently, FABPpm/mAspAt is a protein with distinct functions at different subcellular sites. FABPpm and mAspAt are derived from the same gene while not requiring alternative splicing of the mRNA (23). In 3T3 fibroblasts transfected with mAspAt cDNA, FABPpm was localized to the plasma membrane, and rates of saturable LCFA uptake were increased (76).FAT/CD36.An 88-kDa adipocyte membrane protein was identified by covalent labeling with N-sulfosuccinimidyl esters of long-chain fatty acids, which irreversibly inhibited fatty acid transport by 75% (63, 64). Subsequently, Abumrad et al. (1) cloned a heavily glycosylated fatty acid translocase (FAT), the sequence of which which was 85% homologous with that of glycoprotein IV (CD36) that had been previously identified in human platelets and in lactating mammary epithelium. Hence, this 88-kDa integral membrane protein is known as FAT/CD36 (1). FAT/CD36 is a class B scavenger receptor protein with multiple functions (46). It is involved in angiogenesis, atherosclerosis, inflammation, as well as lipid metabolism (46). A key role for FAT/CD36 in fatty acid transport was shown when fibroblasts were transfected with FAT/CD36, which resulted in increased rates of fatty acid uptake (3, 74).Studies In VivoThe foregoing studies in vitro have confirmed that a number of distinct proteins facilitate the transport of fatty acids across the plasma membrane. The metabolic consequences of overexpressing or deleting fatty acid transporters in rodents have recently been documented, although the quantitative contributions of fatty acid transport per se to fatty acid metabolism in these models has not always been clarified.FATP1.In vivo, FATPs are expressed in somewhat of a tissue-specific manner (40, 70). Transgenic mice with cardiac-specific overexpression of FATP1 demonstrated a fourfold increase in fatty acid uptake (30). This was associated with a twofold increase in cardiomyocyte fatty acid accumulation and in fatty acid metabolism, as well as cardiac dysfunction with pathophysiological findings similar to those in diabetic cardiomyopathy (30). Ablation of FATP1 reduced muscle triacylglycerol content, prevented development of high fat-diet-induced insulin resistance (83), and blunted insulin-stimulated triacylglycerol synthesis (143). Surprisingly, basal rates of fatty acid uptake were not altered (143). We have found that overexpression of FATP1 in rat skeletal muscle increases the rate of long-chain fatty transport into giant sarcolemmal vesicles (111).FATP2.FATP2 is expressed mainly in liver and kidney. In these tissues of FATP2-null mice, there was a decreased peroxisomal very long-chain acyl CoA synthetase activity and decreased peroxisomal very long-chain fatty acid -oxidation, but very long-chain fatty acids did not accumulate in liver or kidney (66).FATP3.It appears that FATP3 has little or no fatty acid transport function (38, 112). Its effects in vivo have apparently not been explored.FATP4.Because it is apparently the only FATP located in the small intestine, FATP4 has been implicated in the absorption of dietary lipids (128). Depending on the means used to ablate FATP4, FATP4-null mice show either an early embryonic (49) or an early neonatal lethality (68, 108). FATP4 does appear to be essential for maintaining normal skin structure and function (67). Deletion of one allele of FATP4 reduced the FATP4 protein by 40–50%, and this was accompanied by a reduced BODIPY-fatty acid uptake in intestinal epithelial cells (49). Overexpression of FATP4 in rat skeletal muscle increases the rate of long-chain fatty transport into giant sarcolemmal vesicles (111).FATP5.This protein, FATP5, is present in liver (40, 70). Studies in FATP5-null mice revealed that FATP5 is in hepatic lipid metabolism (39) and to have bile acid CoA synthetase activities (72). In FATP5 knockout mice, fatty acid uptake was reduced 50% in primary hepatocytes, and hepatic triacylglycerol content was reduced. This diminished uptake of fatty acids reduces fatty acid esterification, despite the increased expression of fatty acid synthetase (39).FATP6.Recently, it was reported that FATP6 is expressed only in heart (50). Stable transfection of FATP6 into 293 cells enhanced uptake of LCFAs and indicated that the FATP6 isoform is more important than FATP1 for taking up long-chain fatty acids (50). However, there is some debate as to the whether FATP6 has a transport role in rat heart (91), and FATP6 minimally promoted fatty acid uptake when expressed in yeast (38).Collectively, these studies demonstrate important metabolic functions for many of the FATPs. However, some concerns remain, as in FATP1-null mice, basal rates of fatty acid uptake were not impaired (143), whereas FATP1 overexpression increased fatty acid transport (111). Moreover, the putative, significant transport role for FATP6 (50) has been questioned (38, 91).FABPpm.The specific effects of FABPpm on fatty acid transport into mammalian tissue have only been examined in several studies. We (31, 111) have shown that electrotransfecting FABPpm into a single muscle, in vivo, upregulates FABPpm protein within days. It is localized to the plasma membrane and increases the rate of long-chain fatty acid transport into giant sarcolemmal vesicles, as well as increasing fatty acid oxidation but not esterification (31, 111). However, the increase in fatty acid transport (+79%) was far more modest than the increase in plasmalemmal FABPpm (+173%). This may suggest that overexpressing FABPpm alone is not optimally effective for increasing long-chain fatty acid transport rates and that FABPpm may function in conjunction with other fatty acid transporters, particularly FAT/CD36, as some of our recent work has shown (111).FAT/CD36.Many studies have now shown that FAT/CD36 is a key, long-chain fatty acid transporter in metabolically important tissues. Under basal conditions, a null mutation in murine FAT/CD36 reduced the uptake of fatty acid analogs in vivo in heart (50–80%), skeletal muscle (40–75%), and adipose tissue (60–70%) (32), and saturable fatty acid transport was lost in adipocytes (45). This reduced rate of fatty acid uptake in FAT/CD36-null mice lowered the basal rates of fatty acid esterification and oxidation in muscle and heart (32, 62, 75, 89). In addition, the metabolic responses (esterification and oxidation) to metabolic challenges provided by insulin, AICAR, and muscle contraction are severely blunted in FAT/CD36-null mice, and their exercise capacity is also markedly reduced (62). Muscle-specific overexpression of FAT/CD36 has shown that fatty acid oxidation was increased in this tissue, but only during muscle contraction, not at rest (73). This suggested to us that long-chain fatty acid transport is not simply regulated by the expression level of FAT/CD36 but possibly also by trafficking this protein to the plasma membrane and/or by activating it at the plasma membrane (see below).Why so Many Membrane Fatty A