J. J. F. P. Luiken

Palmitate transport and fatty acid transporters in red and white muscles

We performed studies 1) to investigate the kinetics of palmitate transport into giant sarcolemmal vesicles, 2) to determine whether the transport capacity is greater in red muscles than in white muscles, and 3) to determine whether putative long-chain fatty acid (LCFA) transporters are more abundant in red than in white muscles. For these studies we used giant sarcolemmal vesicles, which contained cytoplasmic fatty acid binding protein (FABPc), an intravesicular fatty acid sink. Intravesicular FABPcconcentrations were sufficiently high so as not to limit the uptake of palmitate under conditions of maximal palmitate uptake (i.e., 4.5-fold excess in white and 31.3-fold excess in red muscle vesicles). All of the palmitate taken up was recovered as unesterified palmitate. Palmitate uptake was reduced by phloretin (-50%), sulfo- N-succinimidyl oleate (-43%), anti-plasma membrane-bound FABP (FABPpm, -30%), trypsin (-45%), and when incubation temperature was lowered to 0°C (-70%). Palmitate uptake was also reduced by excess oleate (-65%), but not by excess octanoate or by glucose. Kinetic studies showed that maximal transport was 1.8-fold greater in red vesicles than in white vesicles. The Michaelis-Menten constant in both types of vesicles was ∼6 nM. Fatty acid transport protein mRNA and fatty acid translocase (FAT) mRNA were about fivefold greater in red muscles than in white muscles. FAT/CD36 and FABPpm proteins in red vesicles or in homogenates were greater than in white vesicles or homogenates ( P < 0.05). These studies provide the first evidence of a protein-mediated LCFA transport system in skeletal muscle. In this tissue, palmitate transport rates are greater in red than in white muscles because more LCFA transporters are available.We performed studies 1) to investigate the kinetics of palmitate transport into giant sarcolemmal vesicles, 2) to determine whether the transport capacity is greater in red muscles than in white muscles, and 3) to determine whether putative long-chain fatty acid (LCFA) transporters are more abundant in red than in white muscles. For these studies we used giant sarcolemmal vesicles, which contained cytoplasmic fatty acid binding protein (FABPc), an intravesicular fatty acid sink. Intravesicular FABPc concentrations were sufficiently high so as not to limit the uptake of palmitate under conditions of maximal palmitate uptake (i.e., 4.5-fold excess in white and 31.3-fold excess in red muscle vesicles). All of the palmitate taken up was recovered as unesterified palmitate. Palmitate uptake was reduced by phloretin (-50%), sulfo-N-succinimidyl oleate (-43%), anti-plasma membrane-bound FABP (FABPpm, -30%), trypsin (-45%), and when incubation temperature was lowered to 0 degrees C (-70%). Palmitate uptake was also reduced by excess oleate (-65%), but not by excess octanoate or by glucose. Kinetic studies showed that maximal transport was 1.8-fold greater in red vesicles than in white vesicles. The Michaelis-Menten constant in both types of vesicles was approximately 6 nM. Fatty acid transport protein mRNA and fatty acid translocase (FAT) mRNA were about fivefold greater in red muscles than in white muscles. FAT/CD36 and FABPpm proteins in red vesicles or in homogenates were greater than in white vesicles or homogenates (P < 0.05). These studies provide the first evidence of a protein-mediated LCFA transport system in skeletal muscle. In this tissue, palmitate transport rates are greater in red than in white muscles because more LCFA transporters are available.

Cellular fatty acid transport in heart and skeletal muscle as facilitated by proteins.

Despite the importance of long-chain fatty acids (FA) as fuels for heart and skeletal muscles, the mechanism of their cellular uptake has not yet been clarified. There is dispute as to whether FA are taken up by the muscle cellsvia passive diffusion and/or carrier-mediated transport. Kinetic studies of FA uptake by cardiac myocytes and the use of membrane protein-modifying agents have suggested the bulk of FA uptake is due to a protein component. Three membrane-associated FA-binding proteins were proposed to play a role in FA uptake, a 40-kDa plasma membrane FA-binding protein (FABPpm), an 88-kDa FA translocase (FAT/CD36), and a 60-kDa FA transport protein (FATP). In cardiac and skeletal myocytes the intracellular carrier for FA is cytoplasmic heart-type FA-binding protein (H-FABP), which likely transports FA from the sarcolemma to their intracellular sites of metabolism. A scenario is discussed in which FABPpm, FAT/CD36, and H-FABP, probably assisted by an albumin-binding protein, cooperate in the translocation of FA across the sarcolemma.

Role of membrane-associated and cytoplasmic fatty acid-binding proteins in cellular fatty acid metabolism

A number of membrane-associated and cytoplasmic fatty acid-binding proteins (FABPs) are now being implicated in the cellular uptake and intracellular transport of long-chain fatty acids (FA). These proteins each have the capacity of non-covalent binding of FA, are present in tissues actively involved in FA metabolism, and are upregulated in conditions of increased cellular FA metabolism. To date, five distinct membrane FABPs have been described, ranging in mass from 22 to 88 kDa and each showing a characteristic tissue distribution. Evidence for involvement in cellular fatty acid uptake has been provided for several of them, because it was recently found that isolated cell lines transfected with 88-kDa putative fatty acid translocase (FAT; homologous to CD36) or with 63-kDa fatty acid-transport protein show an increased rate of FA uptake. The (at least nine) FABPs of cytoplasmic origin belong to a family of small (14-15 kDa) lipid binding proteins, all having a similar tertiairy structure but differing in binding properties and in tissue occurrence. The biological functions of the various FABPs, possibly exerted in a concerted action among them, comprise solubilization and compartmentalization of FA, facilitation of the cellular uptake and intracellular trafficking of FA, and modulation of mitosis, cell growth, and cell differentiation. In addition, the FABPs have been suggested to participate in and/or modulate FA-mediated signal transduction pathways and FA regulation of gene expression, and to prevent local high FA concentrations thereby contributing to the protection of cells against the toxic effects of FA. In conclusion, long-chain fatty acids are subject to continuous interaction with multiple proteins, which interplay influences their cellular metabolism.

Muscle palmitate uptake and binding are saturable and inhibited by antibodies to FABP(PM).

Studies show that uptake of long-chain fatty acids (LCFA) across the plasma membranes (PM) may occur partly via a carrier-mediated process and that the plasma membrane fatty acid-binding protein (FABPPM) may be a component of this system. To test the hypothesis that FABPPM is involved in transsarcolemmal transport of LCFA in muscle, we measured palmitate uptake in giant sarcolemmal vesicles and palmitate binding to PM proteins in rat muscles, (1) in the presence of increasing amounts of unbound palmitate and (2) in the absence or presence of antibody to FABPPM. Both palmitate uptake and binding were found to be saturable functions of the unbound palmitate concentration with calculated Vmax values of 10.5 ± 1.2 pmol/mg protein/15 sec and 45.6 ± 2.9 nmol/mg protein/15 min and Km values of 12.8 ± 3.8 and 18.4 ± 1.8 nmol/L, respectively. The Vmax values for both palmitate uptake and binding were significantly decreased by 75-79% in the presence of a polyclonal antibody to the rat hepatic FABPPM. Antibody inhibition was found to be dose-dependent and specific to LCFA. Glucose uptake was not affected by the presence of the antibody to FABPPM. Palmitate uptake and binding were also inhibited in the presence of trypsin and phloretin. These results support the hypothesis that transsarcolemmal LCFA transport occurs in part by a carrier-mediated process and that FABPPM is a component of this process in muscle.

Skeletal muscle fatty acid transport and transporters.

While it has long been assumed that long chain fatty acids (LCFA) can freely diffuse across the plasma membrane, recent work has shown that LCFA uptake also involves a protein-mediated mechanism. Three putative LCFA transporters have been identified (FABPpm, FATP, and FAT/CD36), and all are expressed in rodent and human muscles. In a new model system (giant vesicles), we have demonstrated that (a) LCFA transport rates are scaled with the oxidative capacity of heart and muscle, (b) only FABPpm and FAT/CD36, but not FATP1, correlate with vesicular LCFA transport, and (c) LCFA transport can be increased by increasing (1) the FAT/CD36 protein of muscle (chronic adaptation) or (2) via the translocation of FAT/CD36 from an intracellular pool to the plasma membrane during muscle contraction (acute adaptation).

Coordinately regulated expression of FAT/CD36 and FACS1 in rat skeletal muscle

Protein-mediated fatty acid uptake and intracellular fatty acid activation are key steps in fatty acid metabolism in muscle.We have examined (a) the abundance of fatty acid translocase (FAT/CD36) mRNA (a fatty acid transporter) and long-chain acyl CoA synthetase (FACS1) mRNA in metabolically heterogeneous muscles (soleus (SOL), red (RG) and white gastrocnemius (WG)), and (b) whether FAT/CD36 and FACS1 mRNAs were coordinately upregulated in red (RTA) and white tibialis muscles (WTA) that had been chronically stimulated for varying periods of time (0.25, 1, 6 and 24 h/day) for 7 days. FAT/CD36 mRNA and FACS1 mRNA abundance were scaled with (a) the oxidative capacity of muscle (SOL > RG > WG) (p < 0.05), (b) the rates of fatty acid oxidation in red and white muscles, and (c) fatty acid uptake by sarcolemmal vesicles, derived from red and white muscles. In chronically stimulated muscles (RTA and WTA), FAT/CD36 mRNA and FACS1 mRNA were up-regulated in relation to the quantity of muscle contractile activity (p < 0.05). FAT/CD36 mRNA and FACS1 mRNA up-regulation was highly correlated (r = 0.98). The coordinated expression of FAT/CD36 and FACS is likely a functional adaptive response to facilitate a greater rate of fatty acid activation in response to a greater rate of fatty acid transport, either among different types of muscles or in muscles in which capacity for fatty acid metabolism has been enhanced.