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.     

Defect in human myocardial long-chain fatty acid uptake is caused by FAT/CD36 mutations

Because of the importance of long-chain fatty acids (LCFAs) as a myocardial energy substrate, myocardial LCFA metabolism has been of particular interest for the understanding of cardiac pathophysiology. Recently, by using radiolabeled LCFA analogues, myocardial LCFA metabolism has been clinically evaluated, which revealed a total defect of myocardial LCFA accumulation in a small number of subjects. The mechanism for the cellular LCFA uptake process is still disputable, but recent results suggest that fatty acid translocase (FAT)/CD36 is a transporter in the heart. In the present study, we analyzed mutations and protein expression of the FAT/CD36 gene in 47 patients who showed total lack of the accumulation of a radiolabeled LCFA analogue in the heart. All the patients carried two mutations in the FAT/CD36 gene, and expression of the FAT/CD36 protein was not detected on either platelet or monocyte membranes. Our results showed the link between mutations of the FAT/CD36 gene and a defect in the accumulation of LCFAs in the human heart.     

Succinimidyl oleate, established inhibitor of CD36/FAT translocase inhibits complex III of mitochondrial respiratory chain

The functional role of CD36 protein detected in mitochondrial fractions in long chain fatty acid (LCFA) oxidation is unclear due to conflicting results obtained in Cd36 knockout mice and experiments using sulfo-N-succinimidyl oleate (SSO) for inhibition of CD36 mediated LCFA transport. We investigated effect of SSO on mitochondrial respiration and found that SSO substantially inhibits not only LCFA oxidation, but also oxidation of flavoprotein- and NADH-dependent substrates and generation of mitochondrial membrane potential. Experiments in rat liver, heart and kidney mitochondria demonstrated a direct effect on mitochondrial respiratory chain with the most pronounced inhibition of the complex III (IC₅₀ 4 μM SSO). The results presented here show that SSO is a potent and irreversible inhibitor of mitochondrial respiratory chain.     

Characterization of a heart-specific fatty acid transport protein

Fatty acids are a major source of energy for cardiac myocytes. Changes in fatty acid metabolism have been implicated as causal in diabetes and cardiac disease. The mechanism by which long chain fatty acids (LCFAs) enter cardiac myocytes is not well understood but appears to occur predominantly by protein-mediated transport. Here we report the cloning, expression pattern, and subcellular localization of a novel member of the fatty acid transport protein (FATP) family termed FATP6. FATP6 is principally expressed in the heart where it is the predominant FATP family member. Similar to other FATPs, transient and stable transfection of FATP6 into 293 cells enhanced uptake of LCFAs. FATP6 mRNA was localized to cardiac myocytes by in situ hybridization. Immunofluorescence microscopy of FATP6 in monkey and murine hearts revealed that the protein is exclusively located on the sarcolemma. FATP6 was restricted in its distribution to areas of the plasma membrane juxtaposed with small blood vessels. In these membrane domains FATP6 also colocalizes with another molecule involved in LCFA uptake, CD36. These findings suggest that FATP6 is involved in heart LCFA uptake, in which it may play a role in the pathogenesis of lipid-related cardiac disorders.     

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.     

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.     

FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires plasma membrane rafts

We previously reported that lipid rafts are involved in long-chain fatty acid (LCFA) uptake in 3T3-L1 adipocytes. The present data show that LCFA uptake does not depend on caveolae endocytosis because expression of a dominant negative mutant of dynamin had no effect on uptake of [3H]oleic acid, whereas it effectively prevented endocytosis of cholera toxin. Isolation of detergent-resistant membranes (DRMs) from 3T3-L1 cell homogenates revealed that FAT/CD36 was expressed in both DRMs and detergent-soluble membranes (DSMs), whereas FATP1 and FATP4 were present only in DSMs but not DRMs. Disruption of lipid rafts by cyclodextrin and specific inhibition of FAT/CD36 by sulfo-N-succinimidyl oleate (SSO) significantly decreased uptake of [3H]oleic acid, but simultaneous treatment had no additional or synergistic effects, suggesting that both treatments target the same mechanism. Indeed, subcellular fractionation demonstrated that plasma membrane fatty acid translocase (FAT/CD36) is exclusively located in lipid rafts, whereas intracellular FAT/CD36 cofractionated with DSMs. Binding assays confirmed that [3H]SSO predominantly binds to FAT/CD36 within plasma membrane DRMs. In conclusion, our data strongly suggest that FAT/CD36 mediates raft-dependent LCFA uptake. Plasma membrane lipid rafts might control LCFA uptake by regulating surface availability of FAT/CD36.     

Identification of fatty acid translocase on human skeletal muscle mitochondrial membranes: essential role in fatty acid oxidation

Fatty acid translocase (FAT/CD36) is a transport protein with a high affinity for long-chain fatty acids (LCFA). It was recently identified on rat skeletal muscle mitochondrial membranes and found to be required for palmitate uptake and oxidation. Our aim was to identify the presence and elucidate the role of FAT/CD36 on human skeletal muscle mitochondrial membranes. We demonstrate that FAT/CD36 is present in highly purified human skeletal mitochondria. Blocking of human muscle mitochondrial FAT/CD36 with the specific inhibitor sulfo-N-succimidyl-oleate (SSO) decreased palmitate oxidation in a dose-dependent manner. At maximal SSO concentrations (200 muM) palmitate oxidation was decreased by 95% (P<0.01), suggesting an important role for FAT/CD36 in LCFA transport across the mitochondrial membranes. SSO treatment of mitochondria did not affect mitochondrial octanoate oxidation and had no effect on maximal and submaximal carnitine palmitoyltransferase I (CPT I) activity. However, SSO treatment did inhibit palmitoylcarnitine oxidation by 92% (P<0.001), suggesting that FAT/CD36 may be playing a role downstream of CPT I activity, possibly in the transfer of palmitoylcarnitine from CPT I to carnitine-acylcarnitine translocase. These data provide new insight regarding human skeletal muscle mitochondrial fatty acid (FA) transport, and suggest that FAT/CD36 could be involved in the cellular and mitochondrial adaptations resulting in improved and/or impaired states of FA oxidation.     

Real-time quantification of fatty acid uptake using a novel fluorescence assay

Uptake of nonesterified long-chain fatty acids (LCFAs) into many cell types and organs such as liver, heart, intestine, and skeletal muscle occurs primarily through a saturable, protein-mediated mechanism. Membrane proteins that increase the uptake of LCFAs, such as FAT/CD36 and fatty acid transport proteins, represent significant therapeutic targets for the treatment of metabolic disorders, including type 2 diabetes. However, currently available methods for the quantification of LCFA uptake neither allow for real-time measurements of uptake kinetics nor are ideally suited for the development of LCFA uptake inhibitors in high-throughput screens. To address both problems, we developed a LCFA uptake assay using a fluorescently labeled fatty acid and a nontoxic cell-impermeable quenching agent that allows fatty acid transport to be measured in real time using fluorescence plate readers or standard fluorescence microscopy. With this assay, we faithfully reproduced known differentiation- and hormone-induced changes in LCFA uptake by 3T3-L1 cells and determined LCFA uptake kinetics with previously unobtainable temporal resolution. Applications of this novel assay should facilitate new insights into the biology of fatty acid uptake and provide new means for obesity-related drug discovery.     

Requirement for the heart-type fatty acid binding protein in cardiac fatty acid utilization.

Nonenzymatic cytosolic fatty acid binding proteins (FABPs) are abundantly expressed in many animal tissues with high rates of fatty acid metabolism. No physiological role has been demonstrated for any FABP, although these proteins have been implicated in transport of free long-chain fatty acids (LCFAs) and protection against LCFA toxicity. We report here that mice lacking heart-type FABP (H-FABP) exhibit a severe defect of peripheral (nonhepatic, non-fat) LCFA utilization. In these mice, the heart is unable to efficiently take up plasma LCFAs, which are normally its main fuel, and switches to glucose usage. Altered plasma levels of LCFAs, glucose, lactate and beta-hydroxybutyrate are consistent with depressed peripheral LCFA utilization, intensified carbohydrate usage, and increased hepatic LCFA oxidation; these changes are most pronounced under conditions favoring LCFA oxidation. H-FABP deficiency is only incompletely compensated, however, causing acute exercise intolerance and, at old age, a localized cardiac hypertrophy. These data establish a requirement for H-FABP in cardiac intracellular lipid transport and fuel selection and a major role in metabolic homeostasis. This new animal model should be particularly useful for investigating the significance of peripheral LCFA utilization for heart function, insulin sensitivity, and blood pressure.     

Binding of sulfosuccinimidyl fatty acids to adipocyte membrane proteins: Isolation and ammo-terminal sequence of an 88-kD protein implicated in transport of long-chain fatty acids

Summary We recently reported (Harmon et al., J. Membrane Biol. 124:261–268, 1991) that sulfo-N-succinimidyl derivatives of long-chain fatty acids (SS-FA) specifically inhibited transport of oleate by rat adipocytes. These compounds bound to an 85–90 kD membrane protein which was also labeled by another inhibitor of FA transport [³H]DIDS (4,4-diisothiocyanostilbene-2-2-sulfonate). These results indicated that the protein was a strong candidate as the transporter for long-chain fatty acids. In this report we determined that the apparent size of the protein is 88 kD and its isoelectric point is 6.9. We used [³H]SS-oleate (SSO), which specifically labels the 88-kD protein, to isolate it from rat adipocyte plasma membranes. Identification of 15 amino acids at the N-terminus region revealed strong sequence homology with two previously described membrane glycoproteins: CD36, a ubiquitous protein originally identified in platelets and PAS IV, a protein that is enriched in the apical membranes of lipidsecreting mammary cells during lactation. Antibody against PAS IV cross-reacted with the adipocyte protein. This, together with the N-terminal sequence homology, suggested that the adipocyte protein belongs to a family of related intrinsic membrane proteins which include CD36 and PAS IV.     

Labeling of adipocyte membranes by sulfo-N-succinimidyl derivatives of long-chain fatty acids: Inhibition of fatty acid transport

Summary Sulfo-N-succinimidyl derivatives of the long-chain fatty acids, oleic and myristic, were synthesized and covalently reacted with isolated rat adipocytes. The plasma membrane proteins labeled by these compounds and the effect of labeling on the transport of long-chain fatty acids were investigated. Sulfo-N-succinimidyl oleate (SSO) and myristate (SSM) inhibited the transport of fatty acids (by about 70%). Inhibition of fatty acid transport was not a result of alterations in cell integrity, as intracellular water volume was not changed. It did not reflect effects on fatty acid metabolism, since it was observed under conditions where greater than 90% of the fatty acid taken up was recovered in the free form. The inhibitory effect was specific to the fatty acid transport system, as the transport of glucose and the permeation of retinoic acid, a substance with structural similarities to long-chain fatty acids, were unaffected. Sulfosuccinimidyl oleate reacted exclusively with a plasma membrane protein with an apparent size of 85 kDa while sulfosuccinimidyl myristate also labeled a 75-kDa while sulfosuccinimidyl myristate also labeled a 75-kDa protein. These proteins were among the ones labeled by diisothiocyanodisulfonic acid (DIDS) which also inhibits fatty acid transport irreversibly. The data suggest that the 85-kDa protein, which is the only one labeled by all three inhibitors is involved in facilitating membrane permeation of long-chain fatty acids.     

Localization of adipocyte long-chain fatty acyl-CoA synthetase at the plasma membrane

Long-chain fatty acyl-CoA synthetase (FACS) catalyzes esterification of long-chain fatty acids (LCFAs) with coenzyme A (CoA), the first step in fatty acid metabolism. FACS has been shown to play a role in LCFA import into bacteria and implicated to function in mammalian cell LCFA import. In the present study, we demonstrate that FACS overexpression in fibroblasts increases LCFA uptake, and overexpression of both FACS and the fatty acid transport protein (FATP) have synergistic effects on LCFA uptake. To explore how FACS contributes to LCFA import, we examined the subcellular location of this enzyme in 3T3-L1 adipocytes which natively express this protein and which efficiently take up LCFAs. We demonstrate for the first time that FACS is an integral membrane protein. Subcellular fractionation of adipocytes by differential density centrifugation reveals immunoreactive and enzymatically active FACS in several membrane fractions, including the plasma membrane. Immunofluorescence studies on adipocyte plasma membrane lawns confirm that FACS resides at the plasma membrane of adipocytes, where it co-distributes with FATP. Taken together, our data support a model in which imported LCFAs are immediately esterified at the plasma membrane upon uptake, and in which FATP and FACS function coordinately to facilitate LCFA movement across the plasma membrane of mammalian cells.     

Gut expression and regulation of FAT/CD36: possible role in fatty acid transport in rat enterocytes

Fatty acid translocase (FAT)/CD36 is one of several putative plasma membrane long-chain fatty acid (LCFA) transport proteins; however, its role in intestinal absorption of LCFA is unknown. We hypothesized that FAT/CD36 would be differentially expressed along the longitudinal axis of the gut and during intestinal development, suggesting specificity of function. We found that intestinal mucosal FAT/CD36 mRNA levels varied by anatomic location along the longitudinal gut axis: stomach 45 +/- 7, duodenum 173 +/- 29, jejunum 238 +/- 17, ileum 117 +/- 14, and colon 9 +/- 1% (means +/- SE with 18S mRNA as control). FAT/CD36 protein levels were also higher in proximal compared with distal intestinal mucosa. Mucosal FAT/CD36 mRNA was also regulated during intestinal maturation, with a fourfold increase from neonatal to adult animals. In addition, FAT/CD36 mRNA levels and enterocyte LCFA uptake were rapidly downregulated by intraduodenal oleate infusion. These findings suggest that FAT/CD36 plays a role in the uptake of LCFA by small intestinal enterocytes. This may have important implications in understanding fatty acid absorption in human physiological and pathophysiological conditions.     

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.     

Long-chain fatty acid uptake into adipocytes depends on lipid raft function

This study investigates the role of lipid rafts and caveolae, a subclass of lipid raft microdomains, in the binding and uptake of long-chain fatty acids (LCFA) by 3T3-L1 cells during differentiation. Disruption of lipid rafts by beta-cyclodextrin (betaCD) or selective inhibition of caveolae by overexpression of a dominant-negative mutant of caveolin-3 (Cav(DGV)) resulted in disassembly of caveolae structures at the cell surface, as assessed by electron microscopy. While in 3T3-L1 fibroblasts, which express few caveolae, Cav(DGV) or betaCD had no effect on LCFA uptake, in 3T3-L1 adipocytes the same treatments decreased the level of [(3)H]oleic acid uptake by up to 55 +/- 8 and 49 +/- 7%, respectively. In contrast, cholesterol loading of 3T3-L1 adipocytes resulted in a 4-fold increase in the extent of caveolin-1 expression and a 1.7-fold increase in the level of LCFA uptake. Both the inhibitory and enhancing effects of these treatments were constantly increasing with the [(3)H]oleic acid incubation time up to 5 min. Incubation of 3T3-L1 adipocytes with [(3)H]stearate followed by isolation of a caveolin-1 positive detergent-resistant membrane (DRM) fraction revealed that [(3)H]stearate binds to caveolae. Fatty acid translocase (FAT/CD36) was found to be present in this DRM fraction as well. Our data thus strongly indicate a critical involvement of lipid rafts in the binding and uptake of LCFA into 3T3-L1 adipocytes. Furthermore, our findings suggest that caveolae play a pivotal role in lipid raft-dependent LCFA uptake. This transport mechanism is induced in conjunction with cell differentiation and might be mediated by FAT/CD36.     

Cardiac substrate uptake and metabolism in obesity and type-2 diabetes: Role of sarcolemmal substrate transporters

Cardiovascular disease is the primary cause of death in obesity and type-2 diabetes mellitus (T2DM). Alterations in substrate metabolism are believed to be involved in the development of both cardiac dysfunction and insulin resistance in these conditions. Under physiological circumstances the heart utilizes predominantly long-chain fatty acids (LCFAs) (60–70%), with the remainder covered by carbohydrates, i.e., glucose (20%) and lactate (10%). The cellular uptake of both LCFA and glucose is regulated by the sarcolemmal amount of specific transport proteins, i.e., fatty acid translocase (FAT)/CD36 and GLUT4, respectively. These transport proteins are not only present at the sarcolemma, but also in intracellular storage compartments. Both an increased workload and the hormone insulin induce translocation of FAT/CD36 and GLUT4 to the sarcolemma. In this review, recent findings on the insulin and contraction signalling pathways involved in substrate uptake and utilization by cardiac myocytes under physiological conditions are discussed. New insights in alterations in substrate uptake and utilization during insulin resistance and its progression towards T2DM suggest a pivotal role for substrate transporters. During the development of obesity towards T2DM alterations in cardiac lipid homeostasis were found to precede alterations in glucose homeostasis. In the early stages of T2DM, relocation of FAT/CD36 to the sarcolemma is associated with the myocardial accumulation of triacylglycerols (TAGs) eventually leading to an impaired insulin-stimulated GLUT4-translocation. These novel insights may result in new strategies for the prevention of development of cardiac dysfunction and insulin resistance in obesity and T2DM.     

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.