Fatty 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 Vesicles
For 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 Transport
In 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 Transport
We 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 Transporters
Different 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 Vitro
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).
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).
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 Vivo
The 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.
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 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).
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).
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).
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).
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).
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 Acid Transporters?
Evidence in vitro and in vivo has established clearly that a number of proteins facilitate the cellular entry of long-chain fatty acids. However, the need for so many different fatty acid transporters is not clear. This may reflect different fatty acid transport capacities, congruent with different capacities for fatty acid metabolism in different tissues. Alternatively, it has been suggested that different fatty acid transporters interact with each other to move fatty acids across the plasma membrane (50, 52, 102), since some fatty acid transporters co-immunoprepitate [FAT/CD36 and FATP6 in mouse heart (50), and FAT/CD36 and FABPpm in rat heart (Chabowski and Bonen, unpublished data)]. Functional studies have also shown that blocking either FAT/CD36 or FABPpm is sufficient to markedly inhibit fatty acid transport in heart and skeletal muscle (102, 139), suggesting a close collaboration between these two transporters at the plasma membrane. Since FABPpm is a peripheral membrane protein at the outer leaflet of the plasma membrane (134) and FAT/CD36 is an integral membrane protein (1), we (93, 102) have speculated that FABPpm acts as a receptor for long-chain fatty acids, facilitating the diffusion of the fatty acid-albumin complex through the unstirred fluid layer, and that it then interacts with FAT/CD36 to mediate the transmembrane passage of long-chain fatty acids, possibly by facilitating their flip-flop across the bilayer. In this respect, it should be mentioned that FAT/CD36 has also been reported to interact with FABPc (125). This interaction will facilitate the desorption of fatty acids from the inner plasma membrane leaflet or the intracellular site of the transporter to this intracellular carrier protein.
Another view has been proposed in a recent review by Stahl and Doege (40). They suggested a number of fatty acid transport models, all centered around a key role for FATP in mediating fatty acid transport (40). The proposed models either view FAT/CD36 as interacting with FATPs, possibly by transferring fatty acids from FAT/CD36 to FATPs at the plasma membrane, or alternatively as requiring only FATP (40), while apparently excluding any role for other known fatty acid transporters. As yet, there is no experimental evidence for any of these proposed models. Moreover, the models may not be correct. In the schemes presented, FABPpm has not been included, despite clear evidence that it has a role in fatty acid transport (31, 76, 102, 139). In addition, the possibility of an interaction of FAT/CD36 with FATPs (40, 126) has been challenged (43), largely because convincing experimental work has shown that FATPs and FAT/CD36 are present within distinct plasma membrane compartments (113). Thus, in contrast to one of the proposed schemes (40, 126), such discrete plasmalemmal compartmentation virtually excludes a direct interaction of FATP and FAT/CD36 proteins in mediating fatty acid transport (43). Finally, the proposed models (40, 126) ignore the physiological regulation of fatty acid transporter by fatty acid transporter translocation, particularly FAT/CD36 and FABPpm, as detailed below. Thus recent hypothetical models (40, 126) purporting to describe the regulation of fatty acid transport are far too FATP-centered, while largely ignoring a considerable body of work concerning other fatty acid transporters. Experimental work is needed to discern the interactions of the fatty acid transport proteins in promoting fatty acid transport. In our preliminary work, there is a substantial synergy among fatty acid transporters in stimulating fatty acid transport when several fatty acid transporters are overexpressed simultaneously in skeletal muscle (111).
Do Fatty Acid Transporters Channel Fatty Acids to a Particular Metabolic Fate?
It has been suggested, based on the overexpression of fatty acid transporters in different cell lines, that specific transport proteins interact with specific intracellular proteins to channel fatty acids to different metabolic fates within the cell (3, 47, 65, 116, 145). Some evidence was recently shown for this in 3T3-L1 adipocytes, in which FATP1 and long-chain acyl coenzyme A synthetase 1 (ACSL1) coimmunoprecipitate (116). Overexpression of ACSL1 increased the rate of fatty acid transport, leading to the conclusion that constitutive interaction between FATP1 and ACSL1 contributes to the efficient cellular uptake of fatty acids in adipocytes through vectorial acylation. This has led to the hypothesis “that fatty acid esterification might be required for fatty acid uptake into adipocytes” (116). Recently, FAT/CD36 was linked with triacylglycerol cycling in C2C12 cells (3). Others have shown a role for ACSL1, 4, and 6 in contributing to fatty acid transport by vectorial acylation (136). Aspects of vectorial acylation have recently been reviewed in detail by Black and DiRusso (9). Despite the attractiveness of fatty acid channeling hypothesis via specific fatty acid transporters, this may apply primarily to cell lines in which normal physiological regulation is absent.
In vivo, fatty acid channeling to a particular metabolic fate via specific fatty acid transporters would not seem to hold, particularly in metabolically dynamic tissues such as skeletal muscle and the heart. Such a scheme ignores the important role of physiological signals to direct fatty acids toward esterification by insulin or to oxidation by muscle contraction. There is already good evidence to indicate that channeling of long-chain fatty acids by FAT/CD36 to triacylglycerols, as observed in C2C12 myotubes (3), does not occur in heart and skeletal muscle. Muscle-specific overexpression of FAT/CD36 in mice failed to increase rates of palmitate esterification or oxidation in isolated skeletal muscle at rest (73). However, in muscle and heart, the contraction- and insulin-induced increases in the sarcolemmal pool of FAT/CD36 (see below) direct long-chain fatty acids to oxidation (13, 73, 97, 103) and esterification (98, 100), respectively. Thus, in vivo, the metabolic fate of long-chain fatty acids that have been transported into heart and muscle is dictated by intracellular signals fashioned by the energetic demands [i.e., contractile activity and AMPK activation (41, 97, 123, 124)] and/or the endocrine milieu [e.g., insulin and leptin (42, 98, 100, 109, 110, 130)].
Chronic Regulation of Fatty Acid Transport and Transporters
Given the fact that long-chain fatty acid uptake across the plasma membrane is largely protein mediated, the regulation of fatty acid transporters is expected to be a key determinant governing the rate of cellular long-chain fatty acid entry. Therefore, it has been important to establish whether fatty acid transport and transporters are regulated by selected physiological stimuli. A key aspect of this work in our laboratories has been correlating functional measures of fatty acid transport with the subcellular redistribution of fatty acid transporters. Unfortunately, until quite recently, suitable antibodies against rodent FATPs in metabolically important tissues had not been available. Thus, for the most part, we report our work over the past 7 years, in which we have examined the regulation of FABPpm and FAT/CD36 in heart and skeletal muscles by physiological stimuli (see below). Within the past year, we (28, 111) and others (143) have also begun to examine the physiological regulation of FATP1 in vivo.
Perturbations in muscle activity over weeks or days can alter glucose or lactate transport due to changes in the expression of their transporters (77, 104, 107). Similarly, when muscle activity was increased with chronic muscle stimulation (5–7 days), FAT/CD36 protein expression and plasmalemmal content were upregulated, and there was a concomitant increase in the rates of fatty acid transport into giant sarcolemmal vesicles (4, 11, 87) (FIGURE 8⇓). Exercise training also upregulated the fatty acid transport proteins FABPpm and FAT/CD36 (81, 117, 137, 140) and increased palmitate utilization (140). Muscle activity-induced alterations in fatty acid transporters may be mediated by the activation of AMP kinase, since in cardiac myocytes and the heart AMP kinase activation by AICAR upregulates the expression and plasmalemmal content of FAT/CD36 and FABPpm, resulting in an increased rate of fatty acid transport (29).
When muscle activity was eliminated (denervation, 7 days), the rate of fatty acid transport into giant vesicles was reduced (87). However, skeletal muscle FAT/CD36 protein expression was not altered. The reduction in fatty acid transport was therefore attributable to the permanent relocation of this transporter from the plasma membrane to its intracellular depot(s) (87) (FIGURE 8⇑). Indeed, changes in the rates of fatty acid transport into giant vesicles, in 5-to 7-day chronically stimulated and denervated muscles, correlated directly with the altered plasmalemmal FAT/CD36 (FIGURE 9A⇓) (87). This was also shown in AICAR-perfused hearts and AICAR-stimulated cardiac myocytes, as the AMPK activation by AICAR upregulated the expression of FAT/CD36 (~40%) and FABPpm proteins (+25%) after 90 min in both of these preparations. There was a concomitantly increased plasmalemmal content of FAT/CD36 (+49%) and FABPpm (+42%), which resulted in a 2.5-fold increase in the rate of fatty acid transport (29).
Endocrine regulation of fatty acid transport and transporters has also been shown. Prolonged leptin infusion (2 wk) repressed FAT/CD36 and FABPpm protein expression, which contributed to their reduced plasmalemmal content (129). The concomitant reductions in the rates of fatty acid transport into giant vesicles were highly correlated with the reduction in plasmalemmal FAT/CD36 (r = 0.88; FIGURE 9B⇑) and plasmalemmal FABPpm (r = 0.94) (129). In cardiac myocytes, insulin upregulated the expression of FAT/CD36 protein, but not FABPpm protein, in a dose-dependent manner (27). The resulting increase in FAT/CD36 protein expression (+32% in 2 h) and the concomitant increase in plasmalemmal FAT/CD36 (+29%) increased the rate of fatty acid transport by 34% (27).
Collectively, our studies have shown that, in heart an skeletal muscle, physiological stimuli alter the protein expression of fatty acid transporters (FAT/CD36 and FABPpm), and due to the concomitant changes in plasmalemmal FAT/CD36 and FABPpm the rates of fatty acid transport are altered accordingly. Importantly, fatty acid transport is not always regulated by changes in the expression of these proteins. At times, their subcellular distribution may be altered independent of changes in their protein expression. It is important to remember that it is the plasmalemmal pool of fatty acid transporters, not the total tissue pool, that regulates the rate of fatty acid transport into the cell.
Acute Regulation of Fatty Acid Transporters and Transport by Muscle Contraction, AMPK Activation and Insulin
It has long been known that glucose transport is upregulated by insulin and muscle contraction and that insulin stimulates glycogenesis, whereas muscle contraction promotes glucose oxidation (12, 15, 19–21), since these stimuli induce the translocation of the glucose transporter GLUT4 to the plasma membrane (cf. Ref. 53). Similar effects on fatty acid metabolism are provoked by insulin and muscle contraction; in both heart and skeletal muscle, insulin stimulates the rate of fatty acid esterfication (42, 98) and contraction increases the rate of fatty acid oxidation (41, 75). Therefore, we began to examine whether muscle contraction and insulin rapidly upregulated long-chain fatty acid transport by inducing the translocation of one or more fatty acid transporters in a manner analogous to that of the insulin- and contraction-induced GLUT4 translocation.
We have found that FAT/CD36, FABPpm, and FATP1 are located at the plasma membrane and in an intracellular depot in skeletal muscle and in the heart (13, 28, 34, 96–98, 100, 111). During 30 min of muscle contraction, fatty acid transport into giant vesicles was rapidly increased (within 5 min) and continued to increase for the remaining 25 min (FIGURE 10A⇓). On cessation of muscle contraction, the rates of fatty acid transport returned to basal levels during the next 60 min (FIGURE 10A⇓) (13). Blocking plasmalemmal FAT/CD36 with its specific inhibitor, SSO, completely inhibited the contraction-induced increase in palmitate transport into giant vesicles. Interestingly, the rates of palmitate transport into giant sarcolemmal vesicles were increased in direct proportion to the rate of muscle contraction (FIGURE 10B⇓), and kinetic studies showed that Vmax for palmitate transport was increased (FIGURE 10C⇓). Importantly, all these changes in the rates of palmitate transport into giant sarcolemmal vesicles occurred while the palmitate concentration was held constant in the assay. We also observed that the rapid increases and reductions in the rates of fatty acid transport were associated with concomitant increases and reductions in plasmalemmal FAT/CD36, respectively (FIGURE 10D⇓), while at the same time the intracellular FAT/CD36 was altered reciprocally (13). This then was the first study to demonstrate that fatty acid transport could be regulated acutely (i.e., within minutes) due to the translocation of FAT/CD36 from its intracellular depot to the plasma membrane. These observations have now been confirmed by others (138). In more recent work, we have also found that muscle contraction induces the translocation of FABPpm from an intracellular depot to the cell surface (61).
AMPK and ERK activation.
We have also demonstrated that AMPK activation induces the translocation of both FAT/CD36 and FABPpm, thereby upregulating the rate of fatty acid transport into skeletal muscle (62) and cardiac myocytes (28, 97, 103) (FIGURE 11A⇓). However, AMPK activation failed to induce the translocation of FATP1 in cardiac myocytes (FIGURE 11A⇓) (28). Recently, it has also been shown that activation of ERK1/2 signaling may also be involved in the contraction-induced translocation of FAT/CD36 to cell surface in skeletal muscle (138).
Since we had shown that it was possible to stimulate fatty acid transport by inducing the translocation of FAT/CD36 in contracting muscle (13), we also examined whether insulin stimulated fatty acid transport by a translocation mechanism involving one or more fatty acid transporters. Our results demonstrated clearly that, in muscle (61, 62, 98) and heart (28, 100), insulin also induced the translocation of FAT/CD36 to the plasma membrane, thereby stimulating fatty acid uptake into these tissues (28, 61, 62, 98, 100) and increasing fatty acid esterification (98). Insulin did not induce the translocation of FABPpm in cardiac myocytes (28) (FIGURE 11B⇑), but may do so in muscle (61). Blocking PI3-kinase with the inhibitor LY 29004 prevented the insulin-stimulated translocation of FAT/CD36 in cardiac myocytes (100) and skeletal muscle (98). This insulin-mediated signaling of FAT/CD36 translocation resembles that of the insulin-stimulated signaling of GLUT4 translocation, as has been reviewed by us in considerable detail elsewhere (93). It should be noted, however, that the signaling pathways for inducing GLUT4 and FAT/CD36 are not entirely similar, as we can recruit FAT/CD36 and GLUT4 to the plasma membrane by selected stimuli (94, 101). There is also some evidence that the cell surface of activity of FAT/CD36 can be altered, since 3-isobutyl-1-methylxanthine and milrinone increased the intrinsic activity of FAT/CD36, possibly through its covalent modification (95).
Shortly after our initial reports, Stahl et al. (127) reported that insulin induced the translocation of FATP1 in 3T3-L1 adipocytes (127). Unfortunately, others have had difficulty replicating these results in either 3T3-L1 adipocytes (116) or in cardiac myocytes (28), since insulin failed to induce the translocation of FATP1 in these studies, despite the fact that insulin did stimulate fatty acid uptake in both 3T3-L1 adipocytes (116) and cardiac myocytes (28) (FIGURE 11B⇑). From our work, it is has been clear for some time that the insulin-induced translocation of FAT/CD36 stimulates fatty acid uptake in muscle (61, 62, 98) and heart (28, 100), but in the studies in 3T3-L1 adipocytes (116, 127) the possibility of an insulin-induced translocation of FAT/CD36 was not examined. Given that FATP1 is not induced to translocate by insulin in 3T3-L1 adipocytes (116) and cardiac myocytes (28) and that AMPK activation in cardiac myocytes also fails to induce the translocation of FATP1 (28), the recent report indicating that insulin induces the translocation of FATP1 in skeletal muscle (143) needs to be viewed cautiously. It is at odds with some other studies (28, 116) that have failed to support the idea that FATP1 can be induced to translocate by insulin or by the activation of AMPK.
Signaling of Fatty Acid Transporters.
Ours were the first studies (13, 98, 100) to document the very rapid upregulation of fatty acid transport by the contraction-, AMPK-, and insulin-mediated translocation of selected fatty acid transporters. This regulation of fatty acid transport and fatty acid transporter translocation is remarkably parallel to contraction-, AMPK-, and insulin-induced stimulation of glucose transport into skeletal muscle (cf. Refs. 53, 118). We have recently reviewed these parallel mechanisms in detail elsewhere (cf. Ref. 93). Hence, the signaling pathways involved in mediating fatty acid transporter translocation have become of interest. In FIGURE 12⇓, we have shown schematically the mechanisms, as far as these are known, that induce the translocation of selected fatty acid transporters (FAT/CD36 and FABPpm) in muscle and heart.
Fatty Acid Transport and Plasmalemmal FAT/CD36 are Upregulated in Insulin Resistance and Type 2 Diabetes
Since it is well known that fatty acid metabolism in heart and skeletal muscle is dysregulated in obesity and Type 2 diabetes, we examined the possibility that a contributing mechanism to intramyocellular lipid accumulation was the increase in fatty transport. In an animal model of insulin resistance (obese Zucker rats), we observed a markedly upregulated rate of fatty acid transport in muscle and in the heart (FIGURE 12⇑) (35, 96). But this was not attributable to an increased expression of FAT/CD36 or FABPm (35, 96). Instead, the plasmalemmal content of FAT/CD36 accounted for the increase in fatty acid transport (FIGURE 12⇑) (96). Similarly, in insulin-resistant human skeletal muscle (obesity and Type 2 diabetes), we (18) observed that rates of fatty acid transport were increased as a result of the increased plasmalemmal FAT/CD36, but not plasmalemmal FABPpm, whereas the expression of these proteins was not altered. Importantly, these studies (18, 35, 96) indicate that, in the absence of any changes in FAT/CD36 protein expression, this transporter can be permanently relocated to the plasma membrane. This augments the rate of fatty acid uptake and contributes to the accumulation of intramuscular lipids, which in turn can cause insulin resistance by interfering with insulin signaling (84, 135, 144). Thus we (18, 35, 96) have established an apparent causal link between the upregulation of FAT/CD36-mediated fatty acid transport with insulin resistance in skeletal muscle and heart. It is important to note that this is observed under resting conditions when there is little need for an augmented rate of fatty acid transport into these tissues.
Unsuspected Role of FAT/CD36 in Mitochondrial Fatty Acid Oxidation
Recent work from our laboratory has also shown that FAT/CD36 is located at the mitochondrion, both in rodent (26) and human muscle (7, 71). The reason for its presence in mitochondria is not fully understood. It was observed that blocking FAT/CD36 in isolated mitochondria greatly reduces long-chain, but not short-chain, fatty acid oxidation (26), and during muscle contraction FAT/CD36 is translocated to the mitochondrion (26, 71). FAT/CD36 was found to interact with CPT1 to deliver fatty acyl moieties across the mitochondrial membrane (7). When FAT/CD36 and CPT1 were entered simultaneously in a multiple regression equation, a very strong prediction of fatty acid oxidation in mitochondria was obtained (r = 0.90) (7). This interaction of FAT/CD36 and FATP1 is further supported by their co-immunoprecipitation from skeletal muscle mitochondria (120). How these proteins interact at the mitochondrion to facilitate fatty acid entry into this organelle remains to be determined. Whether impaired mitochondrial FAT/CD36 availability would lead to impairments in fatty acid oxidation remains to be determined. Nevertheless, FAT/CD36 appears to be involved not only with transporting long-chain fatty acids into the muscle cell but also into the mitochondrion where this protein apparently recognizes long-chain fatty-acyl CoAs.
There is now considerable evidence that long-chain fatty acids are taken across the plasma membrane by binding to one or more membrane-associated proteins in heart and skeletal muscle and also in adipose tissue. A number of fatty acid transporters have been identified, many of which increase rates of fatty acid transport, including FAT/CD36, FABPpm, FATP1, 2, and 4, and possibly FATP6 in mouse heart but not in rat heart. There is some question whether FATP3 and 5 are fatty acid transporters. In metabolically active tissue (heart and muscle), specific transporters are unlikely to channel fatty acids to a particular metabolic fate, since this is known to be regulated by intracellular metabolic signals provoked by contraction or the endocrine milieu.
It is clear that, in vivo, fatty acid transport in muscle or heart is up- or downregulated in response to changes in plasmalemmal content of fatty acid transporters, particularly FAT/CD36 and/or FABPpm. These plasmalemmal protein changes may or may not reflect changes in their protein expression. In insulin-resistant heart and skeletal muscle, there is a permanent relocation of FAT/CD36 to the plasma membrane, whereas its expression is not altered. A temporary relocation (i.e., translocation) of FAT/CD36 to the plasma membrane, induced by classic physiological stimuli (contraction and insulin), can rapidly and transiently upregulate the rate of long-chain fatty acid transport. FABPpm is induced to translocate selectively by muscle contraction and AMPK activation but not by insulin. There is contradictory evidence as to whether FATP1 is induced to translocate by insulin. Finally, the involvement of FAT/CD36 in mitochondrial fatty acid oxidation is unexpected but further extends the multiple functions of this protein.
Clearly, there is now a wealth of genetic, molecular, biochemical, and physiological evidence to support the idea that fatty acids traverse the plasma membrane via a highly regulated, protein-mediated process, for which a number of proteins have now been identified. We cannot exclude the possibility that additional fatty acid transporters may yet be identified. We concur with Kampf and Kleinfeld (80) that the physical mechanisms of this protein-mediated process, at or within the plasma membrane, remain to be determined. Fatty acid transport and the proteins involved may well differ in tissues such as muscle and adipose tissue, which have very different metabolic roles. The limited understanding of the physical mechanisms of fatty acid per sé, does not invalidate the fact that fatty acid transport across the plasma membrane has repeatedly been shown to occur via a highly regulated protein-mediated process.
Studies in our laboratories are supported by grants from the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, The Heart and Stroke Foundation of Ontario, the Netherlands Heart Foundation (2002T049), the European Community (Integrated Project LSHM-CT-2004-005272, Exgenesis), and the Canada Research Chair program. J.J.F.P. Luiken is a recipient of a VIDI-Innovation Research Grant from the Netherlands Organization for Scientific Research (NWO-ZonMw Grant 016.036.305). J.F.C. Glatz is Netherlands Heart Foundation Professor of Cardiac Metabolism. A. Bonen is the Canada Research Chair in Metabolism and Health.
- © 2007 Int. Union Physiol. Sci./Am. Physiol. Soc.