Metabolic challenges reveal impaired fatty acid metabolism and translocation of FAT/CD36 but not FABPpm in obese Zucker rat muscle

We examined, in muscle of lean and obese Zucker rats, basal, insulin-induced, and contraction-induced fatty acid transporter translocation and fatty acid uptake, esterification, and oxidation. In lean rats, insulin and contraction induced the translocation of the fatty acid transporter FAT/CD36 (43 and 41%, respectively) and plasma membrane-associated fatty acid binding protein (FABPpm; 19 and 60%) and increased fatty acid uptake (63 and 40%, respectively). Insulin and contraction increased lean muscle palmitate esterification and oxidation 72 and 61%, respectively. In obese rat muscle, basal levels of sarcolemmal FAT/CD36 (+33%) and FABPpm (+14%) and fatty acid uptake (+30%) and esterification (+32%) were increased, whereas fatty acid oxidation was reduced (-28%). Insulin stimulation of obese rat muscle increased plasmalemmal FABPpm (+15%) but not plasmalemmal FAT/CD36, blunted fatty acid uptake and esterification, and failed to reduce fatty acid oxidation. In contracting obese rat muscle, the increases in fatty acid uptake and esterification and FABPpm translocation were normal, but FAT/CD36 translocation was impaired and fatty acid oxidation was blunted. There was no relationship between plasmalemmal fatty acid transporters and palmitate partitioning. In conclusion, fatty acid metabolism is impaired at several levels in muscles of obese Zucker rats; specifically, they are 1) insulin resistant with respect to FAT/CD36 translocation and fatty acid uptake, esterification, and oxidation and 2) contraction resistant with respect to fatty acid oxidation and FAT/CD36 translocation, but, conversely, 3) obese muscles are neither insulin nor contraction resistant at the level of FABPpm. Finally, 4) there is no evidence that plasmalemmal fatty acid transporters contribute to the channeling of fatty acids to specific metabolic destinations within the muscle.     

The subcellular compartmentation of fatty acid transporters is regulated differently by insulin and by AICAR

Cellular fatty acid uptake is facilitated by a number of fatty acid transporters, FAT/CD36, FABPpm and FATP1. It had been presumed that FABPpm, was confined to the plasma membrane and was not regulated. Here, we demonstrate for the first time that FABPpm and FATP1 are also present in intracellular depots in cardiac myocytes. While we confirmed previous work that insulin and AICAR each induced the translocation of FAT/CD36 from an intracellular depot to the PM, only AICAR, but not insulin, induced the translocation of FABPpm. Moreover, neither insulin nor AICAR induced the translocation of FATP1. Importantly, the increased plasmalemmal content of these LCFA transporters was associated with a concomitant increase in the initial rate of palmitate uptake into cardiac myocytes. Specifically, the insulin-stimulated increase in the rate of palmitate uptake (+60%) paralleled the insulin-stimulated increase in plasmalemmal FAT/CD36 (+34%). Similarly, the greater AICAR-stimulated increase in the rate of palmitate uptake (+90%) paralleled the AICAR-induced increase in both plasmalemmal proteins (FAT/CD36 (+40%)+FABPpm (+36%)). Inhibition of palmitate uptake with the specific FAT/CD36 inhibitor SSO indicated that FABPpm interacts with FAT/CD36 at the plasma membrane to facilitate the uptake of palmitate. In conclusion, (1) there appears to be tissue-specific sensitivity to insulin-induced FATP1 translocation, as it has been shown elsewhere that insulin induces FATP1 translocation in 3T3-L1 adipocytes, and (2) clearly, the subcellular distribution of FABPpm, as well as FAT/CD36, is acutely regulated in cardiac myocytes, although FABPpm and FAT/CD36 do not necessarily respond identically to the same stimuli.     

Different mechanisms can alter fatty acid transport when muscle contractile activity is chronically altered

We examined whether skeletal muscle transport rates of long-chain fatty acids (LCFAs) were altered when muscle activity was eliminated (denervation) or increased (chronic stimulation). After 7 days of chronically stimulating the hindlimb muscles of female Sprague-Dawley rats, the LCFA transporter proteins fatty acid translocase (FAT)/CD36 (+43%) and plasma membrane-associated fatty acid-binding protein (FABPpm; +30%) were increased (P < 0.05), which resulted in the increased plasmalemmal content of these proteins (FAT/CD36, +42%; FABPpm +13%, P < 0.05) and a concomitant increase in the LCFA transport rate into giant sarcolemmal vesicles (+44%, P < 0.05). Although the total muscle contents of FAT/CD36 and FABPpm were not altered (P > 0.05) after 7 days of denervation, the LCFA transport rate was markedly decreased (-39%). This was associated with reductions in plasmalemmal FAT/CD36 (-24%) and FABPpm (-28%; P < 0.05). These data suggest that these LCFA transporters were resequestered to their intracellular depot(s) within the muscle. Combining the results from these experiments indicated that changes in rates of LCFA transport were correlated with concomitant changes in plasmalemmal FAT/CD36 and FABPpm, but not necessarily with their total muscle content. Thus chronic alterations in muscle activity can alter the rates of LCFA transport via different mechanisms, either 1) by increasing the total muscle content of FAT/CD36 and FABPpm, resulting in a concomitant increase at the sarcolemma, or 2) by reducing the plasma membrane content of these proteins in the absence of any changes in their total muscle content.     

Evidence for concerted action of FAT/CD36 and FABPpm to increase fatty acid transport across the plasma membrane

There is substantial molecular, biochemical and physiologic evidence that long-chain fatty acid transport involves a protein-mediated process. A number of fatty acid transport proteins have been identified, and for unknown reasons, some of these are coexpressed in the same tissues. In muscle and heart FAT/CD36 and FABPpm appear to be key transporters. Both proteins are regulated acutely (within minutes) and chronically (hours to days) by selected physiologic stimuli (insulin, AMP kinase activation). Acute regulation involves the translocation of FAT/CD36 by insulin, muscle contraction and AMP kinase activation, while FABPpm is induced to translocate by muscle contraction and AMP kinase activation, but not by insulin. Protein expression ofFAT/CD36 and FABPpm is regulated by prolonged AMP kinase activation (heart) or increased muscle contraction. Prolonged insulin exposure increases the expression of FAT/CD36 but not FABPpm. Trafficking of fatty acid transporters between an intracellular compartment(s) and the plasma membrane is altered in insulin-resistant skeletal muscle, as some FAT/CD36 is permanently relocated to plasma membrane, thereby contributing to insulin resistance due to the increased influx of fatty acids into muscle cells. Studies in FAT/CD36 null mice have revealed that this transporter is key to regulating the increase in the rate of fatty acid metabolism in heart and skeletal muscle. It appears based on a number of experiments that FAT/CD36 and FABPpm may collaborate to increase the rates of fatty acid transport, as these proteins co-immunoprecipitate.     

Munc18c provides stimulus-selective regulation of GLUT4 but not fatty acid transporter trafficking in skeletal muscle

Insulin-, and contraction-induced GLUT4 and fatty acid (FA) transporter translocation may share common trafficking mechanisms. Our objective was to examine the effects of partial Munc18c ablation on muscle glucose and FA transport, FA oxidation, GLUT4 and FA transporter (FAT/CD36, FABPpm, FATP1, FATP4) trafficking to the sarcolemma, and FAT/CD36 to mitochondria. In Munc18c(-/+) mice, insulin-stimulated glucose transport and GLUT4 sarcolemmal appearance were impaired, but were unaffected by contraction. Insulin- and contraction-stimulated FA transport, sarcolemmal FA transporter appearance, and contraction-mediated mitochondrial FAT/CD36 were increased normally in Munc18c(-/+) mice. Hence, Munc18c provides stimulus-specific regulation of GLUT4 trafficking, but not FA transporter trafficking.     

Folic acid rivals methylenetetrahydrofolate reductase (MTHFR) gene-silencing effect on MEPM cell proliferation and apoptosis

Rates of fatty acid oxidation increase rapidly in both rat heart and skeletal muscle in the early postnatal period. Therefore, we examined in heart and soleus muscle, (a) whether there were rapid changes in fatty acid transporter (FAT/CD36, FABPpm) mRNA and protein expression early in life (days 10 –36) and thereafter (days 84, 160, 365), and (b) whether the rates of fatty acid transport and the plasmalemmal content of FAT/CD36 and FABPpm were altered. Protein expression was altered rapidly from day 10–36 in both heart (FAT/CD36 only, +21%, P < 0.05)) and soleus muscle (FAT/CD36 + 100%, P < 0.05; FABPpm −20%, P < 0.05), with no further changes thereafter (P < 0.05). Rates of fatty acid transport (day 10 vs day 160) were increased in heart (+33%, P < 0.05) and muscle (+85%, P < 0.05), and were associated with concomitant increases in plasmalemmal FABPpm (+44%, P < 0.05) and FAT/CD36 (+16%, P < 0.05) in the heart, and only plasmalemmal FAT/CD36 in muscle (+90%, P < 0.05). Therefore, known changes in the rates of fatty acid oxidation in heart and muscle early in life appear to be accompanied by a concurrent upregulation in the rates of fatty acid transport and the expression of FAT/CD36 in heart and muscle, as well as an increase in plasmalemmal FAT/CD36 and FABPpm in the heart, and only plasmalemmal FAT/CD36 in soleus muscle. We speculate that the rapid upregulation of fatty acid transport rates in heart and muscle are needed to support the increased rates of fatty oxidation that have been previously observed in these tissues.     

Insulin stimulates fatty acid transport by regulating expression of FAT/CD36 but not FABPpm

Because insulin has been shown to stimulate long-chain fatty acid (LCFA) esterification in skeletal muscle and cardiac myocytes, we investigated whether insulin increased the rate of LCFA transport by altering the expression and the subcellular distribution of the fatty acid transporters FAT/CD36 and FABPpm. In cardiac myocytes, insulin very rapidly increased the expression of FAT/CD36 protein in a time- and dose-dependent manner. During a 2-h period, insulin (10 nM) increased cardiac myocyte FAT/CD36 protein by 25% after 60 min and attained a maximum after 90-120 min (+40-50%). There was a dose-dependent relationship between insulin (10(-12) to 10(-7) M) and FAT/CD36 expression. The half-maximal increase in FAT/CD36 protein occurred at 0.5 x 10(-9) M insulin, and the maximal increase occurred at 10(-9) to 10(-8) M insulin (+40-50%). There were similar insulin-induced increments in FAT/CD36 protein in cardiac myocytes (+43%) and in Langendorff-perfused hearts (+32%). In contrast to FAT/CD36, insulin did not alter the expression of FABPpm protein in either cardiac myocytes or the perfused heart. By use of specific inhibitors of insulin-signaling pathways, it was shown that insulin-induced expression of FAT/CD36 occurred via the PI 3-kinase/Akt insulin-signaling pathway. Subcellular fractionation of cardiac myocytes revealed that insulin not only increased the expression of FAT/CD36, but this hormone also targeted some of the FAT/CD36 to the plasma membrane while concomitantly lowering the intracellular depot of FAT/CD36. At the functional level, the insulin-induced increase in FAT/CD36 protein resulted in an increased rate of palmitate transport into giant vesicles (+34%), which paralleled the increase in plasmalemmal FAT/CD36 (+29%). The present studies have shown that insulin regulates protein expression of FAT/CD36, but not FABPpm, via the PI 3-kinase/Akt insulin-signaling pathway.     

Fatty acid transport and FAT/CD36 are increased in red but not in white skeletal muscle of ZDF rats

An increased rate of fatty acid transport into skeletal muscle has been has been linked to the accumulation of intramuscular lipids and insulin resistance, and red muscles are more susceptible than white muscles in developing fatty acid-mediated insulin resistance. Therefore, we examined in Zucker diabetic fatty (ZDF) rats, relative to lean rats, 1) whether rates of fatty acid transport and transporters (FAT/CD36 and FABPpm) were upregulated in skeletal muscle during the transition from insulin resistance (week 6) to type 2 diabetes (weeks 12 and 24), 2) whether such changes occurred primarily in red skeletal muscle, and 3) whether changes in FAT/CD36 and GLUT4 were correlated. In red muscles of ZDF compared with lean rats, the rates of fatty acid transport were upregulated (+66%) early in life (week 6). Compared with the increase in fatty acid transport in lean red muscle from weeks 12-24 (+57%), the increase in fatty acid transport rate in ZDF red muscle was 50% greater during this same period. In contrast, no differences in fatty acid transport rates were observed in the white muscles of lean and ZDF rats at any time (weeks 6-24). In red muscle only, there was an inverse relationship between FAT/CD36 and GLUT4 protein expression as well as their plasmalemmal content. These studies have shown that, 1) before the onset of diabetes, as well as during diabetes, fatty acid transport and FAT/CD36 expression and plasmalemmal content are upregulated in ZDF rats, but importantly, 2) these changes occurred only in red, not white, muscles of ZDF rats.     

Changes in fatty acid transport and transporters are related to the severity of insulin deficiency

We have examined the effects of streptozotocin (STZ)-induced diabetes (moderate and severe) on fatty acid transport and fatty acid transporter (FAT/CD36) and plasma membrane-bound fatty acid binding protein (FABPpm) expression, at the mRNA and protein level, as well as their plasmalemmal localization. These studies have shown that, with STZ-induced diabetes, 1) fatty acid transport across the plasma membrane is increased in heart, skeletal muscle, and adipose tissue and is reduced in liver; 2) changes in fatty acid transport are generally not associated with changes in fatty acid transporter mRNAs, except in the heart; 3) increases in fatty acid transport in heart and skeletal muscle occurred with concomitant increases in plasma membrane FAT/CD36, whereas in contrast, the increase and decrease in fatty acid transport in adipose tissue and liver, respectively, were accompanied by concomitant increments and reductions in plasma membrane FABPpm; and finally, 4) the increases in plasma membrane transporters (FAT/CD36 in heart and skeletal muscle; FABPpm in adipose tissue) were attributable to their increased expression, whereas in liver, the reduced plasma membrane FABPpm appeared to be due to its relocation within the cell in the face of slightly increased expression. Taken together, STZ-induced changes in fatty acid uptake demonstrate a complex and tissue-specific pattern, involving different fatty acid transporters in different tissues, in combination with different underlying mechanisms to alter their surface abundance.     

Hypoxia-induced fatty acid transporter translocation increases fatty acid transport and contributes to lipid accumulation in the heart

Protein-mediated LCFA transport across plasma membranes is highly regulated by the fatty acid transporters FAT/CD36 and FABPpm. Physiologic stimuli (insulin stimulation, AMP kinase activation) induce the translocation of one or both transporters to the plasma membrane and increase the rate of LCFA transport. In the hypoxic/ischemic heart, intramyocardial lipid accumulation has been attributed to a reduced rate of fatty acid oxidation. However, since acute hypoxia (15 min) activates AMPK, we examined whether an increased accumulation of intramyocardial lipid during hypoxia was also attributable to an increased rate of LCFA uptake as a result AMPK-induced translocation of FAT/CD36 and FABPpm. In cardiac myocytes, hypoxia (15 min) induced the redistribution of FAT/CD36 from an intracellular pool (LDM) (-25%, P<0.05) to the plasma membranes (PM) (+54%, P<0.05). Hypoxia also induced an increase in FABPpm at the PM (+56%, P<0.05) and a concomitant FABPpm reduction in the LDM (-24%, P<0.05). Similarly, in intact, Langendorff perfused hearts, hypoxia induced the translocation of a both FAT/CD36 and FABPpm to the PM (+66% and +61%, respectively, P<0.05), with a concomitant decline in FAT/CD36 and FABPpm in the LDM (-24% and -23%, respectively, P<0.05). Importantly, the increased plasmalemmal content of these transporters was associated with increases in the initial rates of palmitate uptake into cardiac myocytes (+40%, P<0.05). Acute hypoxia also redirected palmitate into intracellular lipid pools, mainly to PL and TG (+48% and +28%, respectively, P<0.05), while fatty acid oxidation was reduced (-35%, P<0.05). Thus, our data indicate that the increased intracellular lipid accumulation in hypoxic hearts is attributable to both: (a) a reduced rate of fatty acid oxidation and (b) an increased rate of fatty acid transport into the heart, the latter being attributable to a hypoxia-induced translocation of fatty acid transporters.     

Protein-mediated palmitate uptake and expression of fatty acid transport proteins in heart giant vesicles.

Giant sarcolemmal vesicles were isolated from rat heart and hindlimb muscles for a) characterization of long-chain fatty acid transport in the absence of metabolism and b) comparison of fatty acid transport protein expression with fatty acid transport. Giant vesicles contained cytosolic fatty acid binding protein. Palmitate uptake was completely divorced from its metabolism. All palmitate taken up was recovered in the intravesicular cytosol as unesterified FA. Palmitate uptake by heart vesicles exhibited a K m of 9.7 nm, similar to that of muscle (K m = 9.7 nm). Vmax (2.7 pmol/mg protein/s) in heart was 8-fold higher than in muscle (0.34 pmol/mg protein/s). Palmitate uptake was inhibited in heart (55-80%) and muscle (31-50%) by trypsin, phloretin, sulfo-N-succinimidyloleate (SSO), or a polyclonal antiserum against the 40 kDa plasma membrane fatty acid binding protein (FABPpm). Palmitate uptake by heart and by red and white muscle vesicles correlated well with the expression of fatty acid translocase (FAT/CD36) and fatty acid binding protein FABPpm, which may act in concert. The expression of fatty acid transport protein (FATP), was 10-fold lower in heart vesicles than in white muscle vesicles.It is concluded that long-chain fatty acid uptake by heart and muscle vesicles is largely protein-mediated, involving FAT/CD36 and FABPpm. The role of FATP in muscle and heart remains uncertain.     

Acute endurance exercise increases plasma membrane fatty acid transport proteins in rat and human skeletal muscle

Fatty acid transport proteins are present on the plasma membrane and are involved in the uptake of long-chain fatty acids into skeletal muscle. The present study determined whether acute endurance exercise increased the plasma membrane content of fatty acid transport proteins in rat and human skeletal muscle and whether the increase was accompanied by an increase in long-chain fatty acid transport in rat skeletal muscle. Sixteen subjects cycled for 120 min at ～60 ± 2% Vo(2) peak. Two skeletal muscle biopsies were taken at rest and again following cycling. In a parallel study, eight Sprague-Dawley rats ran for 120 min at 20 m/min, whereas eight rats acted as nonrunning controls. Giant sarcolemmal vesicles were prepared, and protein content of FAT/CD36 and FABPpm was measured in human and rat vesicles and whole muscle homogenate. Palmitate uptake was measured in the rat vesicles. In human muscle, plasma membrane FAT/CD36 and FABPpm protein contents increased 75 and 20%, respectively, following 120 min of exercise. In rat muscle, plasma membrane FAT/CD36 and FABPpm increased 20 and 30%, respectively, and correlated with a 30% increase in palmitate transport following 120 min of running. These data suggest that the translocation of FAT/CD36 and FABPpm to the plasma membrane in rat skeletal muscle is related to the increase in fatty acid transport and oxidation that occurs with endurance running. This study is also the first to demonstrate that endurance cycling induces an increase in plasma membrane FAT/CD36 and FABPpm content in human skeletal muscle, which is predicted to increase fatty acid transport.     

Hepatic fatty acid uptake is regulated by the sphingolipid acyl chain length

Ceramide synthase 2 (CerS2) null mice cannot synthesize very-long acyl chain (C22–C24) ceramides resulting in significant alterations in the acyl chain composition of sphingolipids. We now demonstrate that hepatic triacylglycerol (TG) levels are reduced in the liver but not in the adipose tissue or skeletal muscle of the CerS2 null mouse, both before and after feeding with a high fat diet (HFD), where no weight gain was observed and large hepatic nodules appeared. Uptake of both BODIPY-palmitate and [³H]-palmitate was also abrogated in the hepatocytes and liver. The role of a number of key proteins involved in fatty acid uptake was examined, including FATP5, CD36/FAT, FABPpm and cytoplasmic FABP1. Levels of FATP5 and FABP1 were decreased in the CerS2 null mouse liver, whereas CD36/FAT levels were significantly elevated and CD36/FAT was also mislocalized upon insulin treatment. Moreover, treatment of hepatocytes with C22–C24-ceramides down-regulated CD36/FAT levels. Infection of CerS2 null mice with recombinant adeno-associated virus (rAAV)-CerS2 restored normal TG levels and corrected the mislocalization of CD36/FAT, but had no effect on the intracellular localization or levels of FATP5 or FABP1. Together, these results demonstrate that hepatic fatty acid uptake via CD36/FAT can be regulated by altering the acyl chain composition of sphingolipids.     

Acute regulation of fatty acid uptake involves the cellular redistribution of fatty acid translocase

We used muscle contraction, which increases fatty acid oxidation, as a model to determine whether fatty acid transport is acutely regulated by fatty acid translocase (FAT/CD36). Palmitate uptake by giant vesicles, obtained from skeletal muscle, was increased by muscle contraction. Kinetic studies indicated that muscle contraction increased V(max), but K(m) remained unaltered. Sulfo-N-succinimidyl oleate, a specific inhibitor of FAT/CD36, fully blocked the contraction-induced increase in palmitate uptake. In giant vesicles from contracting muscles, plasma membrane FAT/CD36 was also increased in parallel with the increase in long chain fatty acid uptake. Further studies showed that like GLUT-4, FAT/CD36 is located in both the plasma membrane and intracellularly (endosomally). With muscle contraction, FAT/CD36 at the surface of the muscle was increased, while concomitantly, FAT/CD36 in the intracellular pool was reduced. Similar responses were observed for GLUT-4. We conclude that fatty acid uptake is subject to short term regulation by muscle contraction and involves the translocation of FAT/CD36 from intracellular stores to the sarcolemma, analogous to the regulation of glucose uptake by GLUT-4.     

Greater Transport Efficiencies of the Membrane Fatty Acid Transporters FAT/CD36 and FATP4 Compared with FABPpm and FATP1 and Differential Effects on Fatty Acid Esterification and Oxidation in Rat Skeletal Muscle

In selected mammalian tissues, long chain fatty acid transporters (FABPpm, FAT/CD36, FATP1, and FATP4) are co-expressed. There is controversy as to whether they all function as membrane-bound transporters and whether they channel fatty acids to oxidation and/or esterification. Among skeletal muscles, the protein expression of FABPpm, FAT/CD36, and FATP4, but not FATP1, correlated highly with the capacities for oxidative metabolism (r ≥ 0.94), fatty acid oxidation (r ≥ 0.88), and triacylglycerol esterification (r ≥ 0.87). We overexpressed independently FABPpm, FAT/CD36, FATP1, and FATP4, within a normal physiologic range, in rat skeletal muscle, to determine the effects on fatty acid transport and metabolism. Independent overexpression of each fatty acid transporter occurred without altering either the expression or plasmalemmal content of other fatty acid transporters. All transporters increased fatty acid transport, but FAT/CD36 and FATP4 were 2.3- and 1.7-fold more effective than FABPpm and FATP1, respectively. Fatty acid transporters failed to alter the rates of fatty acid esterification into triacylglycerols. In contrast, all transporters increased the rates of long chain fatty acid oxidation, but the effects of FABPpm and FAT/CD36 were 3-fold greater than for FATP1 and FATP4. Thus, fatty acid transporters exhibit different capacities for fatty acid transport and metabolism. In vivo, FAT/CD36 and FATP4 are the most effective fatty acid transporters, whereas FABPpm and FAT/CD36 are key for stimulating fatty acid oxidation.Uptake of long chain fatty acids across the plasma membrane had long been considered to occur via passive diffusion. However, in recent years, there has been a fundamental shift in our understanding, and it is now widely recognized that long chain fatty acids cross the plasma membrane via a protein-mediated mechanism (for reviews, see Refs. 1–3). A number of fatty acid transporters have been identified, including fatty acid translocase/CD36 (FAT/CD36), plasma membrane-associated fatty acid binding proteins (FABPpm), and a family of fatty acid transport proteins (FATP1–6)⁵ (for reviews, see Refs. 1 and 4). Selected stimuli (muscle contraction, insulin, and AICAR) induce the translocation of selected fatty acid transporters (FABPpm, FAT/CD36, and FATP1) from an intracellular depot to the plasma membrane, in both heart and skeletal muscle, resulting in concurrently increased rates of fatty acid transport (for a review, see Ref. 1). Some fatty acid transporters have now also been implicated in the dysregulation of fatty acid metabolism in heart and skeletal muscle in models of insulin resistance and type 1 and 2 diabetes, including FAT/CD36 (5–9), FATP1 (10, 11), and possibly FATP4 (11, 12) but not FABPpm (5–7). Thus, in recent years, it has become widely accepted that (a) long chain fatty acids traverse the plasma membrane via a protein-mediated mechanism and (b) some of the fatty acid transporters are central to the dysregulation in skeletal muscle fatty acid metabolism in obesity and type 2 diabetes.In vivo, many of the fatty acid transporters are frequently co-expressed in different tissues. FAT/CD36 and FABPpm are ubiquitously expressed (1), whereas FATP1–6 exhibit a somewhat tissue-specific distribution pattern (13, 14). The reason for the co-expression of different fatty acid transporters within the same tissue remains unclear. It has been speculated that selected fatty acid transporters may need to interact with each other (15, 16). Alternatively, it is also possible that (a) different fatty acid transporters have discrepant transport capacities, and (b) selected transporters may channel fatty acids differentially to fatty acid oxidation and esterification into triacylglycerols in mammalian tissue.Recent evidence has shown that the transport capacities among FATPs can differ substantially, as revealed by overexpression (14, 17, 18) or knockdown studies (19), but there is little agreement as to which FATP is most effective. Extensive studies by DiRusso et al. (17) in yeast revealed that when FATP1–6 were overexpressed to similar levels (qualitative assessment), FATP4 exhibited 1.7- and 3-fold greater fatty acid transport effectiveness compared with FATP1 and FATP2, respectively, whereas no fatty acid transport capacities were attributable to FATP3, -5, and -6 (17). In contrast, in HEK293 cells, the FATP6 transport capacity was 3- and 6.5-fold greater than FATP1 and FATP4, respectively (14), whereas in 3T3-L1 adipocytes, a fatty acid transport role was evident only for FATP1 and not FATP4 (19). Others have also questioned the transport role of FATP4 (20). These discrepant findings with respect to the transport effectiveness of FATPs may reflect, in part, the use of diverse cell types with ill defined metabolic needs and/or machinery for fatty acid uptake and metabolism. Indeed, several recent reports indicate that fatty acid transport cannot be adequately examined in some cells, because these appear to lack accessory proteins that may be involved in fatty acid transport (21, 22). In addition, extrapolation of results from cultured cells to metabolically important tissue in vivo may also be problematic, since cells and mammalian tissues probably have different requirements for fatty acid utilization, and their regulation of fatty acid uptake may also differ. For example, the mechanisms regulating the acute contraction-induced up-regulation of fatty acid transport and oxidation, such as occurs in heart and skeletal muscle, is probably absent in selected cell cultures.Assessment of fatty acid transporter effectiveness, in vivo, cannot be determined in knock-out animals, since compensatory responses in some fatty acid transporters (FATP1 and -4) occur when another fatty acid transporter (FAT/CD36) has been ablated (23, 24). Thus, the relative effectiveness of selected fatty acid transporters on fatty acid transport in vivo remains unknown. In addition, whether fatty acid transporters channel fatty acids to a particular metabolic fate, as has been suggested based on studies in cultured cells (18, 19, 25), may depend on the cell type being examined.It is desirable to discern the effectiveness of selected fatty acid transporters in mammalian tissues that have a well known system for transporting and utilizing fatty acids and in which fatty acid transporters can be independently up-regulated without disturbing the expression of other fatty acid transporters. These criteria can be satisfied in rat skeletal muscle in which genes can be up-regulated under controlled conditions within a physiologically meaningful range (26–28). Therefore, in the present study, we have compared the independent transport effectiveness of fatty acid transporters (FABPpm, FAT/CD36, FATP1, and FATP4) in skeletal muscle, without disturbing the expression and plasmalemmal content of other fatty acid transporters. In addition, we also examined the contributions of these transporters to fatty acid oxidation and esterification into triacylglycerols. These are the first studies to reveal that in vivo (a) the fatty acid transport effectiveness of fatty acid transporters differs considerably, and (b) in skeletal muscle, these transporters serve to channel fatty acids to oxidation, not esterification into triacylglycerols.     

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.     

Chronic leptin administration decreases fatty acid uptake and fatty acid transporters in rat skeletal muscle.

Chronic leptin administration reduces triacylglycerol content in skeletal muscle. We hypothesized that chronic leptin treatment, within physiologic limits, would reduce the fatty acid uptake capacity of red and white skeletal muscle due to a reduction in transport protein expression (fatty acid translocase (FAT/CD36) and plasma membrane-associated fatty acid-binding protein (FABPpm)) at the plasma membrane. Female Sprague-Dawley rats were infused for 2 weeks with leptin (0.5 mg/kg/day) using subcutaneously implanted miniosmotic pumps. Control and pair-fed animals received saline-filled implants. Leptin levels were significantly elevated (approximately 4-fold; p < 0.001) in treated animals, whereas pair-fed treated animals had reduced serum leptin levels (approximately -2-fold; p < 0.01) relative to controls. Palmitate transport rates into giant sarcolemmal vesicles were reduced following leptin treatment in both red (-45%) and white (-84%) skeletal muscle compared with control and pair-fed animals (p < 0.05). Leptin treatment reduced FAT mRNA (red, -70%, p < 0.001; white, -48%, p < 0.01) and FAT/CD36 protein expression (red, -32%; p < 0.05) in whole muscle homogenates, whereas FABPpm mRNA and protein expression were unaltered. However, in leptin-treated animals plasma membrane fractions of both FAT/CD36 and FABPpm protein expression were significantly reduced in red (-28 and -34%, respectively) and white (-44 and -56%, respectively) muscles (p < 0.05). Across all experimental treatments and muscles, palmitate uptake by giant sarcolemmal vesicles was highly correlated with the plasma membrane FAT/CD36 protein (r = 0.88, p < 0.01) and plasma membrane FABPpm protein (r = 0.94, p < 0.01). These studies provide the first evidence that protein-mediated long chain fatty acid transport is subject to long term regulation by leptin.     

Impact on fatty acid metabolism and differential localization of FATP1 and FAT/CD36 proteins delivered in cultured human muscle cells

We compared the intracellular distribution and regulatory role of fatty acid transporter protein (FATP1) and fatty acid translocase (FAT/CD36) on muscle cell fatty acid metabolism. With the use of adenoviruses, FATP1 and FAT genes were delivered to primary cultured human muscle cells. FATP1 and FAT moderately enhanced palmitate and oleate transport evenly at concentrations of 0.05, 0.5, and 1 mM. Long-term (16 h) consumption of palmitate and oleate from the media, and particularly incorporation into triacylglyceride (TAG), was stimulated equivalently by FATP1 and FAT at all fatty acid concentrations tested. In contrast, long-term CO₂ production was reduced by FATP1 and FAT at all doses of palmitate and at the lower concentrations of oleate. Neither FATP1 nor FAT markedly altered the production of acid-soluble metabolic intermediates from palmitate or oleate. The intracellular localization of fusion constructs of FATP1 and FAT with enhanced green fluorescent protein (EGFP) was examined. Independently of fatty acid treatment, FATPGFP was observed throughout the cytosol in a reticular pattern and concentrated in the perinuclear region, partly overlapping with the Golgi marker GM-130. FATGFP was found in the extracellular membrane and in cytosolic vesicles not coincident with GM-130. Neither FATP1 nor FAT proteins colocalized with lipid droplets in oleate-treated cells. We conclude that whereas FAT is localized on the extracellular membrane, FATP1 is active in the cytosol and imports fatty acids into myotubes. Overall, both FATP1 and FAT stimulated transport and consumption of palmitate and oleate, which they channeled away from complete oxidation and toward TAG synthesis. palmitate; oleate; fatty acid binding proteins; skeletal muscle