Joost J. F. P. Luiken

Membrane Fatty Acid Transporters as Regulators of Lipid Metabolism: Implications for Metabolic Disease

Long-chain fatty acids and lipids serve a wide variety of functions in mammalian homeostasis, particularly in the formation and dynamic properties of biological membranes and as fuels for energy production in tissues such as heart and skeletal muscle. On the other hand, long-chain fatty acid metabolites may exert toxic effects on cellular functions and cause cell injury. Therefore, fatty acid uptake into the cell and intracellular handling need to be carefully controlled. In the last few years, our knowledge of the regulation of cellular fatty acid uptake has dramatically increased. Notably, fatty acid uptake was found to occur by a mechanism that resembles that of cellular glucose uptake. Thus, following an acute stimulus, particularly insulin or muscle contraction, specific fatty acid transporters translocate from intracellular stores to the plasma membrane to facilitate fatty acid uptake, just as these same stimuli recruit glucose transporters to increase glucose uptake. This regulatory mechanism is important to clear lipids from the circulation postprandially and to rapidly facilitate substrate provision when the metabolic demands of heart and muscle are increased by contractile activity. Studies in both humans and animal models have implicated fatty acid transporters in the pathogenesis of diseases such as the progression of obesity to insulin resistance and type 2 diabetes. As a result, membrane fatty acid transporters are now being regarded as a promising therapeutic target to redirect lipid fluxes in the body in an organ-specific fashion.

Triacylglycerol accumulation in human obesity and type 2 diabetes is associated with increased rates of skeletal muscle fatty acid transport and increased sarcolemmal FAT/CD36

We examined whether, in human obesity and type 2 diabetes, long chain fatty acid (LCFA) transport into skeletal muscle is upregulated and contributes to an excess intramuscular triacylglycerol accumulation. In giant sarcolemmal vesicles prepared from human skeletal muscle, LCFA transport rates were upregulated ~4‐fold and were associated with an increased intramuscular triacylglycerol content in obese individuals and in type 2 diabetics. In these individuals, the increased sarcolemmal LCFA transport rate was not associated with an altered expression of FAT/CD36 or FABPpm. Instead, the increase in the LCFA transport rate was associated with an increase in sarcolemmal FAT/CD36 but not sarcolemmal FABPpm. Rates of fatty acid esterification were increased threefold in isolated human muscle strips obtained from obese subjects, while concomitantly rates of fatty acid oxidation were not altered. Thus, the increased rate of fatty acid transport may contribute to the increased rates of triacylglycerol accumulation in human skeletal muscle. The altered FAT/CD36 trafficking in muscle from obese subjects and type 2 diabetics juxtaposes the known alterations in GLUT4 trafficking, i.e., GLUT4 is known to be retained in its intracellular depots while FAT/CD36 is retained at the sarcolemma. This redistribution of FAT/CD36 to the sarcolemma may contribute to the etiology of insulin resistance in human muscle, and hence, FAT/CD36 provides another potential therapeutic target for the prevention and/or treatment of insulin resistance.

Modest PGC-1α Overexpression in Muscle in Vivo Is Sufficient to Increase Insulin Sensitivity and Palmitate Oxidation in Subsarcolemmal, Not Intermyofibrillar, Mitochondria

PGC-1α overexpression in skeletal muscle, in vivo, has yielded disappointing and unexpected effects, including disrupted cellular integrity and insulin resistance. These unanticipated results may stem from an excessive PGC-1α overexpression in transgenic animals. Therefore, we examined the effects of a modest PGC-1α overexpression in a single rat muscle, in vivo, on fuel-handling proteins and insulin sensitivity. We also examined whether modest PGC-1α overexpression selectively targeted subsarcolemmal (SS) mitochondrial proteins and fatty acid oxidation, because SS mitochondria are metabolically more plastic than intermyofibrillar (IMF) mitochondria. Among metabolically heterogeneous rat hindlimb muscles, PGC-1α was highly correlated with their oxidative fiber content and with substrate transport proteins (GLUT4, FABPpm, and FAT/CD36) and mitochondrial proteins (COXIV and mTFA) but not with insulin-signaling proteins (phosphatidylinositol 3-kinase, IRS-1, and Akt2), nor with 5′-AMP-activated protein kinase, α2 subunit, and HSL. Transfection of PGC-1α into the red (RTA) and white tibialis anterior (WTA) compartments of the tibialis anterior muscle increased PGC-1α protein by 23-25%. This also induced the up-regulation of transport proteins (FAT/CD36, 35-195%; GLUT4, 20-32%) and 5′-AMP-activated protein kinase, α2 subunit (37-48%), but not other proteins (FABPpm, IRS-1, phosphatidylinositol 3-kinase, Akt2, and HSL). SS and IMF mitochondrial proteins were also up-regulated, including COXIV (15-75%), FAT/CD36 (17-30%), and mTFA (15-85%). PGC-1α overexpression also increased palmitate oxidation in SS (RTA, +116%; WTA, +40%) but not in IMF mitochondria, and increased insulin-stimulated phosphorylation of AKT2 (28-43%) and rates of glucose transport (RTA, +20%; WTA, +38%). Thus, in skeletal muscle in vivo, a modest PGC-1α overexpression up-regulated selected plasmalemmal and mitochondrial fuel-handling proteins, increased SS (not IMF) mitochondrial fatty acid oxidation, and improved insulin sensitivity.

Mitochondrial long chain fatty acid oxidation, fatty acid translocase/CD36 content and carnitine palmitoyltransferase I activity in human skeletal muscle during aerobic exercise

Mitochondrial fatty acid transport is a rate‐limiting step in long chain fatty acid (LCFA) oxidation. In rat skeletal muscle, the transport of LCFA at the level of mitochondria is regulated by carnitine palmitoyltransferase I (CPTI) activity and the content of malonyl‐CoA (M‐CoA); however, this relationship is not consistently observed in humans. Recently, fatty acid translocase (FAT)/CD36 was identified on mitochondria isolated from rat and human skeletal muscle and found to be involved in LCFA oxidation. The present study investigated the effects of exercise (120 min of cycling at ∼60%) on CPTI palmitoyl‐CoA and M‐CoA kinetics, and on the presence and functional significance of FAT/CD36 on skeletal muscle mitochondria. Whole body fat oxidation rates progressively increased during exercise (P < 0.05), and concomitantly M‐CoA inhibition of CPTI was progressively attenuated. Compared to rest, 120 min of cycling reduced (P < 0.05) the inhibition of 0.7, 2, 5 and 10 μm M‐CoA by 16%, 21%, 30% and 34%, respectively. Whole body fat oxidation and palmitate oxidation rates in isolated mitochondria progressively increased (P < 0.05) during exercise, and were positively correlated (r= 0.78). Mitochondrial FAT/CD36 protein increased by 63% (P < 0.05) during exercise and was significantly (P < 0.05) correlated with mitochondrial palmitate oxidation rates at all time points (r= 0.41). However, the strongest (P < 0.05) correlation was observed following 120 min of cycling (r= 0.63). Importantly, the addition of sulfo‐N‐succimidyloleate, a specific inhibitor of FAT/CD36, reduced mitochondrial palmitate oxidation to ∼20%, indicating FAT/CD36 is functionally significant with respect to LCFA oxidation. We hypothesize that exercise‐induced increases in fatty acid oxidation occur as a result of an increased ability to transport LCFA into mitochondria. We further suggest that decreased CPTI M‐CoA sensitivity and increased mitochondrial FAT/CD36 protein are both important for increasing whole body fatty acid oxidation during prolonged exercise.

Regulation of fatty acid transport by fatty acid translocase/CD36

Fatty acid (FA) translocase (FAT)/CD36 is a key protein involved in regulating the uptake of FA across the plasma membrane in heart and skeletal muscle. A null mutation of FAT/CD36 reduces FA uptake rates and metabolism, while its overexpression increases FA uptake rates and metabolism. FA uptake into the myocyte may be regulated (a) by altering the expression of FAT/CD36, thereby increasing the plasmalemmal content of this protein (i.e. streptozotocin-induced diabetes, chronic muscle stimulation), or (b) by relocating this protein to the plasma membrane, without altering its expression (i.e. obese Zucker rats). By repressing FAT/CD36 expression, and thereby lowering the plasmalemmal FAT/CD36 (i.e. leptin-treated animals), the rate of FA transport is reduced. Within minutes of beginning muscle contraction or being exposed to insulin FA transport is increased. This increase is a result of the contraction- and insulin-induced translocation of FAT/CD36 from an intracellular depot to the cell surface. Neither PPAR alpha nor PPAR gamma activation alter FAT/CD36 expression in muscle, despite the fact that PPAR alpha activation increases FAT/CD36 by 80% in liver. A novel observation is that FAT/CD36 also appears to be involved in mitochondrial FA oxidation, as this protein is located on the mitochondrial membrane and seems to be required to participate in moving FA across the mitochondrial membrane. Clearly, FAT/CD36 has an important role in FA homeostasis in skeletal muscle and the heart.

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.

Uncoupling protein 3 as a mitochondrial fatty acid anion exporter

In contrast to UCP1, the primary function of UCP3 is not the dissipation of energy. Rather, several lines of evidence suggest that UCP3 is related to cellular long‐chain fatty acid homeostasis. If long‐chain fatty acids enter the mitochondrial matrix in their non‐esterified form, they cannot be metabolized and may exert deleterious effects. To test the feasibility that UCP3 exports fatty acid anions, we systematically interfered at distinct steps in the fatty acid metabolism pathway, thereby creating conditions in which the entry of (non‐esterified) fatty acids into the mitochondrial matrix is enhanced. First, reducing the cellular fatty acid binding capacity, known to increase cytosolic concentrations of non‐esterified fatty acids, up‐regulated UCP3 5.3‐fold. Second, inhibition of mitochondrial entry of esterified long‐chain fatty acids up‐ regulated UCP3 by 1.9‐fold. Third, high‐fat diets, to increase mitochondrial supply of non‐ esterified long‐chain fatty acids exceeding oxidative capacity, up‐regulated UCP3 twofold. However, feeding a similar amount of medium‐chain fatty acids, which can be oxidized inside the mitochondrial matrix and therefore do not need to be exported from the matrix, did not affect UCP3 protein levels. These data are compatible with a physiological function of UCP3 in facilitating outward transport of long‐chain fatty acid anions, which cannot be oxidized, from the mitochondrial matrix.

Regulation of sarcolemmal glucose and fatty acid transporters in cardiac disease

Circulating long-chain fatty acids (LCFA) and glucose are the main sources for energy production in the heart. In the healthy heart the ratio of glucose and LCFA oxidation is sensitively balanced and chronic alterations in this substrate mix are closely associated with cardiac dysfunction. While it has been accepted for several years that cardiac glucose uptake is mediated by facilitated transport, i.e. by means of the glucose transport proteins GLUT1 and GLUT4, only in the last few years it has become clear that proteins with high-affinity binding sites to LCFA, referred to as LCFA transporters, are responsible for bulk LCFA uptake. Similar to the GLUTs, the LCFA transporters CD36 and FABP(pm) can be recruited from an intracellular storage compartment to the sarcolemma to increase the rate of substrate uptake. Permanent relocation of LCFA transporters, mainly CD36, from intracellular stores to the sarcolemma is accompanied by accumulation of lipids and lipid metabolites in the heart. As a consequence, insulin signalling and glucose utilization are impaired, leading to decreased contractile activity of the heart. These observations underline the particular role and interplay of substrate carriers for glucose and LCFA in modulating cardiac metabolism, and the development of heart failure. The signalling and trafficking pathways and subcellular machinery regulating translocation of glucose and LCFA transporters are beginning to be unravelled. More knowledge on substrate transporter recycling, especially the similarities and differences between glucose and LCFA transporters, is expected to enable novel therapies aimed at changing the subcellular distribution of glucose and LCFA transporters, thereby manipulating the substrate preference of the diseased heart to help restore cardiac function.

Sulfo-N-succinimidyl esters of long chain fatty acids specifically inhibit fatty acid translocase (FAT/CD36)-mediated cellular fatty acid uptake.

Sulfo-N-succinimidyl esters of LCFAs are a powerful tool to investigate the functional significance of plasmalemmal proteins in the LCFA uptake process. This notion is based on the following observations. First, sulfo-N-succinimidyl oleate (SSO) was found to inhibit the bulk of LCFA uptake into various cell types, i.e. rat adipocytes, type II pneumocytes and cardiac myocytes. Second, using cardiac giant membrane vesicles, in which LCFA uptake can be investigated in the absence of mitochondrial β-oxidation, SSO retained the ability to largely inhibit LCFA uptake, indicating that inhibition of LCFA transsarcolemmal transport is its primary action. Third, SSO has no inhibitory effect on glucose and octanoate uptake into giant membrane vesicles derived from heart and skeletal muscle, indicating that its action is specific for LCFA uptake. Finally, SSO specifically binds to the 88 kDa plasmalemmal fatty acid transporter FAT, a rat homologue of human CD36, resulting in an arrest of the transport function of this protein.In addition to its inhibitory action at the plasma membrane level, evidence is presented for the lack of a direct inhibitory effect on subsequent LCFA metabolism. First, the relative contribution of oxidation and esterification to LCFA uptake is not altered in the presence of SSO. Second, isoproterenol-mediated channeling of LCFAs into oxidative pathways is not affected by sulfo-N-succinimidyl palmitate (SSP). As an example of its application we used SSP to study the role of FAT/CD36 in contraction- and insulin-stimulated LCFA uptake by cardiac myocytes , showing that this transporter is a primary site of regulation of cellular LCFA utilization.

Apical CD36 immunolocalization in human and porcine taste buds from circumvallate and foliate papillae.

CD36 is the receptor for long chain fatty acids (LCFA), and is expressed in lingual taste cells from rodents. In these animals, CD36 has been proposed to play an important role in oral detection of LCFA, and subsequently, determines their dietary fat preference. Humans also seem to detect LCFA in the oral cavity, however, information on the molecular mechanism of this human orosensory LCFA recognition is currently lacking. The aim of our study was to investigate whether CD36 is also expressed in lingual human and porcine taste buds cells. Using fluorescence immunohistochemistry, apical CD36 expression was revealed in human and porcine taste bud cells from circumvallate and foliate papillae. These data suggest CD36 as the putative orosensory receptor for dietary LCFA in human, and, therefore, may be involved in our preference for fatty foods.