Cellular fatty acid uptake is acutely regulated by membrane-associated fatty acid-binding proteins

Cellular fatty acid uptake is acutely regulated by membrane-associated fatty acid-binding proteins

Prostaglandins, Leukotrienes and Essential FattyAcids (2002) 67(2^3), 73^78 & 2002 Elsevier Science Ltd. All rights reserved. doi:10.1054/plef.401, av...

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Prostaglandins, Leukotrienes and Essential FattyAcids (2002) 67(2^3), 73^78 & 2002 Elsevier Science Ltd. All rights reserved. doi:10.1054/plef.401, available online at http://www.idealibrary.com on

Cellular fatty acid uptake is acutely regulated by membrane-associated fatty acid-binding proteins J. J. F. P. Luiken,1 A. Bonen,2 J. F. C. Glatz1 1

Department of Physiology, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht,The Netherlands and 2Department of Kinesiology, University of Waterloo,Waterloo, Ontario, Canada N2L 3G1

Summary Cellular long-chain fatty acid uptake is believed to occur largely by protein-mediated transmembrane transport of fattyacids, and also by passive diffusionaluptake.It ispostulated that the membrane proteins functionin trapping of fattyacidsfrom extracellular sources, whereafter their transmembrane translocation occurs by passive diffusion through the lipid bilayer.The key membrane-associated proteins involved are plasma membrane fatty acid-binding protein (FABPpm) and fatty acid translocase (FAT/CD36).Their plasma membrane contents are positively correlated with rates of fatty acid uptake. In studies with heart and skeletal muscle we observed that FAT/CD36 is regulated acutely, in that both contraction and insulin can translocate FAT/CD36 from an intracellular depot to the sarcolemma, thereby increasing the rate of fatty acid uptake.In addition, from studies with obese Zucker rats, an established rodent model of obesity and insulin resistance, evidence has been obtained that in heart, muscle and adipose tissue FAT/CD36 is permanently relocated from an intracellular pool to the plasma membrane, resulting in increased fatty acid uptake rates in this condition.These combined observations indicate that protein-mediated fatty acid uptake is a key step in cellular fatty acid utilization, and suggest that malfunctioning of the uptake process could be a critical factor in the pathogenesis of insulin resistance. & 2002 Elsevier Science Ltd. All rights reserved.

INTRODUCTION In recent years much progress has been made with respect to our understanding of the molecular mechanism by which long-chain fatty acids (LCFA) are taken up by cells. Compelling evidence is now available that LCFA can enter cells either by passive diffusion through the lipid bilayer or by protein-mediated transmembrane transport.1–3 The potential physiological importance of the latter is that it would represent a site of control, allowing changes in the presence and/or activity of these membrane proteins to regulate cellular LCFA uptake.

Received 12 November 2001 Accepted 3 May 2002 Correspondence to: Jan F. C. Glatz, PhD., Department of Physiology, CARIM, Maastricht University, P.O. Box 616, NL-6200 MD Maastricht,The Netherlands. Tel.: +31-43-388-1200; Fax: +31-43-388-4166; E-mail: [email protected]s.unimaas.nl Work presented at the 5th International Conference on Fatty Acids in Cell Signaling, Gargnano (Italy), September 2^4, 2001.

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In this report we discuss novel data indicating that one of the membrane-associated fatty acid-binding proteins (FABPs) identified, namely fatty acid translocase (FAT/ CD36), not only facilitates transmembrane transport of LCFA but in various cell types is also involved in the acute regulation of LCFA uptake. Moreover, alterations in cellular LCFA utilization, as occur in a number of pathological states, appear to be related to abnormalities in the presence or functioning of this protein.

MOLECULAR MECHANISM OF FATTY ACID UPTAKE Transmembrane transport of LCFA by a diffusional mechanism has been clearly demonstrated both in studies with model membrane systems and in studies with isolated cells (reviewed in refs. 1, 4–6). LCFA cross the phospholipid bilayer or cell membrane in the unionized form, and this apparent flip–flop mechanism is thought by some to occur with sufficient rapidity to explain observed rates of cellular LCFA uptake or release.1

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On the other hand, for at least three membraneassociated FABPs evidence is available from transfection studies that in their presence the rate of LCFA uptake is markedly increased. These proteins are 43-kDa plasma membrane FABP (FABPpm), a family of some five 60-kD fatty acid-transport proteins (FATP1-5), and 88-kD FAT/ CD36 (reviewed in refs. 7,8). However, the exact nature by which each of these three proteins facilitate transmembrane translocation of LCFA is not yet known, partly because their membrane topology is still unclear. Thus, FABPpm, which is a peripheral membrane protein,9 may exert its action solely by trapping of LCFA, whereafter LCFA cross the membrane by passive diffusion. FATP1-5 also possess acyl-coenzyme A synthetase activity, particularly for very-long-chain LCFAs,10,11 so that metabolic trapping might explain their facilitation of LCFA uptake. FAT/CD36 is a highly glycosylated, integral membrane protein that is believed to have only two transmembrane spanning regions, making it unlikely that it facilitates LCFA transport by channelling LCFAs through a pore in the membrane. In view of this notion it has been suggested that FAT/CD36 also operates mainly through trapping of LCFA to the plasma membrane.3,7 Taken together, these findings may suggest that passive diffusion and protein-mediated LCFA translocation coexist in such a manner that the membrane-associated proteins function in the trapping of LCFA from extracellular donors and their release to intracellular targets, or vice versa, whereby the actual transmembrane translocation step occurs by passive diffusion of LCFA through the lipid bilayer. This unified concept would be in agreement with virtually all experimental findings made so far on this controversial topic.3 Inside cells, LCFA are bound to cytoplasmic FABP (FABPc), which acts as an acceptor protein and is viewed as an intracellular counterpart of plasma albumin.3,12,13 The role of FABPc as intracellular LCFA acceptor and cytoplasmic carrier protein has now clearly been established. For instance, studies in vivo14 and with cardiac myocytes isolated from mice lacking the heart-type FABPc gene15 show that the rates of LCFA uptake and oxidation each are markedly (approximately 50%) reduced, while there is a compensatory upregulation of glucose uptake and oxidation. These data demonstrate the significance of FABPc for proper cellular LCFA utilization, but also reveal that FABPc is not indispensable. REGULATION OF CELLULAR FATTY ACID UPTAKE The direction of LCFA movement across the plasma membrane is determined by the transmembrane gradient of LCFA12,16 and, therefore, depends on the plasma supply of LCFA and the metabolic state of the cell. However, given the fact that both membrane-associated and

cytoplasmic FABPs increase the rate of LCFA translocation across the membrane, alterations in their presence and/or activity would have an impact on the actual rate of transport. Thus, chronic changes in tissue LCFA utilization, such as induced by exercise training, nutrition, and pharmacological manipulations, are paralleled by concomitant changes in the tissue content of membrane as well as cytoplasmic FABPs (reviewed in refs. 3, 12, 13). Conversely, changes in cellular content or functioning of FABPs, such as found in inherited FABP polymorphisms17,18 or experimentally induced by genetic manipulations (transgenic animals),14,19 lead to parallel limitations of the rate of LCFA uptake and utilization. It is well established that tissue LCFA utilization rates can change acutely, i.e., within minutes, for instance during the transition from a resting to contracting skeletal muscle. It is also known that these LCFAs originate from extracellular sources, while mobilization of intracellularly stored LCFA (triacylglycerols) also contribute to the increased rate of utilization.20 If such acute changes in LCFA uptake would also be mediated, at least in part, by FABPs, a mechanism must exist to acutely regulate the subcellular localization or activity of these proteins. It is conceivable that such a mechanism could resemble the manner by which glucose transport is acutely regulated. When the cellular need for glucose increases, for instance as induced by insulin (muscle, heart, and adipose tissue), the membrane glucose transporter GLUT4 is translocated from intracellular sites to the plasma membrane.21 To examine a possible analogy between the acute regulation of muscle glucose and LCFA uptake, we set out to study changes in LCFA uptake into skeletal and cardiac muscle following a contractioninduced increase in energetic need. GIANT MEMBRANE VESICLES AS MODEL SYSTEM In order to properly investigate cellular LCFA uptake it is important to dissect LCFA uptake from their subsequent metabolism as these are closely linked.22 To overcome this issue we have employed the so-called giant sarcolemmal vesicles in our studies on heart and muscle LCFA uptake. These vesicles are prepared by incubation of thinly sliced muscle strips in a buffer containing collagenase and a high concentration of KCl.23–26 The vesicles are formed by budding, are oriented right-side out, and are spherical with a diameter of 5–25 mm. Giant vesicles thus obtained do not contain cellular organelles like mitochondria, but do contain cytoplasmic constituents such as FABPc which can act as an intravesicular acceptor for LCFA. Thus, palmitate taken up by such vesicles could be completely recovered as unesterified LCFA.25,26 In previous studies giant sarcolemmal vesicles have already

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Cellular fatty acid uptake 75

been exploited successfully to reveal that LCFA uptake by skeletal and cardiac muscle mainly occurs by a protein-mediated uptake process, assisted by passive diffusion.25,26

CHANGES IN MUSCLE FATTY ACID UPTAKE FOLLOWING ELECTRICAL STIMULATION We studied LCFA uptake by giant vesicles prepared from rat lower leg muscles after short-term (30 min) electrical stimulation via the sciatic nerve. Vesicles prepared from the resting contralateral leg muscles served as control. Uptake of palmitate was 1.4-fold higher in giant vesicles from contracting muscles than in those from controls (Fig. 1).27 In parallel, the plasma membrane FAT/CD36 content was also 1.4-fold higher in contracting muscles. Importantly, these contraction-induced effects could be completely blunted in the presence of sulfo-N-succinimidyl-oleate (SSO), a specific inhibitor of FAT/CD36.26 In addition, when after cessation of contraction, the muscles were allowed to recover for about 20 min both palmitate uptake and vesicular FAT/CD36 content had decreased to similar values as found in the control muscles (Fig. 1). These short-term concomitant changes in vesicular LCFA uptake rate and FAT/CD36 content suggest that in response to increased muscle contraction, FAT/CD36 is recruited from (an) intracellular store(s) to be associated with the sarcolemma, thereby permitting a higher LCFA uptake rate, and that upon recovery of the muscle the protein is internalized and the rate of LCFA uptake similarly decreased. Further proof for this mechanism was obtained by subfractionation of the control and contracting muscles and assessment by immunoblot of


SIGNALLING PATHWAY FOR FAT/CD36 REDISTRIBUTION Besides by contraction, muscle metabolism is also affected by hormones, in particular insulin. Exposure of skeletal muscle29,30 and heart to insulin markedly increases the rate of LCFA uptake and esterification. In studies in which rat hindlimbs were perfused in the absence or presence of insulin, we recently observed that the resulting increase in LCFA uptake rate was accompanied by a translocation of FAT/CD36 from an intracellular depot to the sarcolemma.31 More recently, similar observations were made in isolated rat cardiac myocytes ( J.J.F.P. Luiken, unpublished observations). In further studies we have obtained evidence that the effects of muscle contraction and of insulin on both LCFA

FAT/CD36 distribution 100

* PM






(pmol/mg protein / 15 s)

Palmitate uptake rate

the presence of FAT/CD36 in both the sarcolemmal and an intracellular (endosomal) fraction, and the redistribution of this protein in response to contraction.27 The contraction-induced redistribution of FAT/CD36 then was also confirmed to occur in the heart. For these studies we employed isolated rat cardiac myocytes which were electrically stimulated in vitro using a newly developed device.28 With contracting myocytes initial palmitate uptake was 1.5-fold higher than with quiescent myocytes. This contraction-induced increase, just as in skeletal muscle, could be blocked by sulfo-N-succinimidyl-palmitate (SSP), thus suggesting the contractioninduced increase in LCFA uptake to be mediated by FAT/CD36.28 Whether FABPpm is also subject to contraction-induced recycling between an intracellular pool and the plasma membrane is not yet clear.




Rest Post-contracting Contracting



0 Postcontracting

Fig. 1 Evidence for acute regulation of long-chain fatty acid uptake by skeletal muscle. Rat hindlimb muscles were subjected to electrical stimulation through the sciatic nerve for 30 min, and studied immediately thereafter or following 20 min of recovery.The contralateral muscle served as control. Left panel.Palmitate uptake rate bygiant sarcolemmalvesiclesprepared fromresting muscle, from contracting skeletalmuscle, and from muscle obtained 20 min post-contraction. Right panel. Distribution of FAT/CD36 in resting muscle, contracting muscle, and 20 min postcontraction. Resting and contracting muscles were fractionated into plasma membrane (PM) and low-density microsomal (LDM) fractions and FAT/CD36 determined by Western blotting.Recovery data for post-contracting muscle were obtained by inference. Data represent means7SEM for 5^15 experiments, and were obtained from ref. 27.*Po0.05, contraction versus rest; **Po0.05, recovery versus contraction.

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uptake and FAT/CD36 translocation to the sarcolemma are additive (unpublished observations). This finding suggests that there are (at least) two separate intracellular pools from which FAT/CD36 can be recruited, one being sensitive to contraction and the other to insulin, or, alternatively, that there is a single depot from which FAT/ CD36 can be mobilized following two independent signal transduction cascades (Fig. 2). This mechanism, again, resembles the well-documented manner by which muscle contraction and insulin can independently translocate the glucose transporter GLUT4 from intracellular stores to the sarcolemma.21 The effects of insulin on FAT/CD36 translocation are mediated via the signalling protein PI3 kinase, in both muscle 31 and cardiac myocytes ( J.J.F.P. Luiken, unpublished observations). This became evident from the inhibition of the insulin-inducible LCFA uptake into cardiac myocytes and skeletal muscles, when the activity of PI3 kinase was inhibited by wortmannin or LY 294002.31 The protein kinase involved in the contractionmediated signalling pathway has not yet been unraveled. ALTERATIONS IN FATTY ACID UPTAKE AND FAT/ CD36 DISTRIBUTION IN DISEASE In view of the recent observations that insulin induces FAT/CD36 translocation in muscle, and of the quantita-

tive importance of skeletal muscle as target organ for this hormone, we set out to investigate whether in metabolic diseases in which lipid metabolism is altered, such as obesity and insulin resistance, the cellular distribution of FAT/CD36 is also affected. For this, we studied the obese Zucker rat, a well-established model for obesity and insulin resistance.32 LCFA uptake into giant vesicles prepared from heart and skeletal muscle were about 1.8-fold higher in obese animals when compared to lean littermates.33 Interestingly, these differences could not be associated with changes in FAT/CD36 mRNA or tissue protein contents, but in both tissues there was an increased abundance of FAT/ CD36 at the sarcolemma (1.6-fold in heart, 1.8-fold in muscle).33 Thus, it appears that in heart and muscle of obese Zucker rats the total cellular pool of FAT/CD36 is similar to that found in lean animals, but a larger proportion of the protein is permanently relocated to the cell surface at the expense of the intracellular storage compartment, resulting in higher LCFA uptake rates. This altered relocation could be the result of either an increased mobilization of FAT/CD36 or an impairment in the rate of endocytosis. In any case, the machinery regulating the subcellular distribution of FAT/CD36 might play a pivotal role in the etiology of obesity and insulin resistance.

Fig. 2 Schematic presentation of the cellular uptake and utilization of long-chain fatty acids (LCFA) illustrating the presumable roles of various lipid-binding proteins in this process. Following their dissociation from plasma albumin, the transmembrane translocation of LCFA most likely takes place either by passive diffusion through the lipid bilayer, or facilitated by membrane-associated proteins, or by a combination of both.This includes FABPpm acting as scavenger and FAT/CD36 acting as scavengerand/or transporter of LCFA.Not depicted here is FATP, which most likely is involved in fatty acyl-CoA synthesis.Intracellularly,LCFAwill be bound by cytoplasmic FABP and, after activation to fatty acyl-CoA, by acyl-CoA binding protein (ACBP; not shown). LCFA uptake can be modulated by recycling of FAT/CD36 between the plasma membrane and, at least in muscle, two distinct endosomal compartments. Alterations in redistribution of FAT/CD36 can be mediated by insulin, following the binding of this hormone to its receptor and involving PI3 kinase, or by muscle contraction, which may activate a yet unidentified protein kinase (PKX). FABPpm, plasma membrane fatty acid-binding protein; FAT, fatty acid translocase (CD36); FABPc, cytoplasmic fatty acid-binding protein; IR, insulin receptor; PKX, protein kinase X. Prostaglandins, Leukotrienes and Essential FattyAcids (2002) 67(2^3), 73^78

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Cellular fatty acid uptake 77

CONCLUDING REMARKS Both membrane-associated and cytoplasmic FABPs play central roles in the uptake and intracellular transport of LCFA. Their physiological significance most likely is that they may enhance the LCFA transport capacity and allow a careful control of the cellular handling of LCFA, thus performing a dual function of permissive transport to certain sites and sequestration from others. Our recent observation that LCFA uptake by heart and muscle is subject to short-term regulation involving the translocation of the membrane protein FAT/CD36 from an intracellular depot to the plasma membrane, schematically depicted in Fig. 2, indicates that the cellular FABPs not only facilitate but also regulate cellular LCFA metabolism. Moreover, the notion that the altered rates of LCFA metabolism observed in obese Zucker rats could be associated with changes in cellular FAT/CD36 distribution elicit the hypothesis that malfunctioning of the protein-mediated LCFA uptake process may be a critical factor in the pathogenesis of insulin resistance, and perhaps of other metabolic diseases in which lipid metabolism is affected. Future studies should be directed towards further unraveling the mechanism and regulation of cellular LCFA uptake, especially the signalling cascade(s) involved in the cellular redistribution of membrane transporters.

ACKNOWLEDGEMENTS Work in the authors’ laboratories was supported by the Netherlands Heart Foundation (Grant D98.012), the Canadian Institutes of Health Research, and the Ontario Heart and Stroke Foundation. Joost J.F.P. Luiken is a Dekker post-doctoral fellow of the Netherlands Heart Foundation.

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