Fatty acid transport in adipocytes monitored by imaging intracellular free fatty acid levels

Transport of free fatty acids (FFA) across the adipocyte plasma membrane is critical for maintaining homeostasis. To determine the membrane's role in regulating transport we describe here the first measurements of the intracellular (unbound) FFA concentration ([FFA(i)]) and their use in monitoring transport of FFA across 3T3F442A adipocytes. [FFA(i)] was measured by microinjecting cells with ADIFAB, a fluorescently labeled fatty acid-binding protein that is used to measure unbound FFA levels. We used ratio fluorescence microscopy of intracellular ADIFAB to image unbound FFA levels and determined the time course of [FFA(i)] in response to changing the extracellular unbound FFA concentration ([FFA(o)]). [FFA(o)] was clamped at defined levels using complexes of FFA and bovine serum albumin. We show that FFA influx is slow, requiring about 300 s to reach steady state (rate constant approximately 0.02 s(-1)) and saturable (K(o) approximately 200 nm). Efflux is twice as fast as influx, for zero [FFA(o)], but decreases with increasing [FFA(o)]. Surprisingly, at steady state [FFA(i)] is 2-5-fold (average 2-fold) greater than [FFA(o)] and this [FFA(i)]/[FFA(o)] gradient is abolished by depleting cellular ATP. Our results indicate that FFA transport across adipocyte membranes is highly regulated, involving an ATP-driven pump and a mechanism for gating efflux that is sensitive to [FFA(o)]. These characteristics are well described by a membrane carrier model but are not consistent with FFA transport across the membrane's lipid phase. We suggest that these characteristics are important in regulating circulating FFA levels by the adipocyte.     

Free fatty acid transport across adipocytes is mediated by an unknown membrane protein pump

The role of cell membranes in regulating the flux of long chain free fatty acids (FFA) into and out of adipocytes is intensely debated. Four different membrane proteins including, FABPpm, CD36/FAT, caveolin-1, and FATP have been identified as facilitating FFA transport. Moreover, CD36 and caveolin-1 are also reported to mediate transport in conjunction with lipid rafts. The principal evidence for these findings is a correlation of the level of FFA uptake with the expression level of these proteins and with the integrity of lipid rafts. The 3T3-L1 and 3T3-F442A cell lines in their preadipocyte states reveal little or no expression of these proteins and correspondingly low levels of uptake. Here we have microinjected the adipocyte and preadipocyte cell lines with ADIFAB, the fluorescent indicator of FFA. The ADIFAB fluorescence allowed us to monitor the intracellular unbound FFA concentration during FFA influx and efflux. We show that these measurements of transport, in contrast to FFA uptake measurements, correlate neither with expression of these proteins nor with lipid raft integrity in preadipocytes and adipocytes. Transport characteristics, including the generation of an ATP-dependent FFA concentration gradient, are virtually identical in adipocytes and preadipocytes. We suggest that the origin of the discrepancy between uptake and our measurements is that most of the FFA transported into the cells is lost during the uptake but not in the transport protocols. We conclude that long chain fatty acid transport in adipocytes is very likely mediated by an as-yet-unidentified membrane protein pump.     

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.     

Fatty acid interactions with native and mutant fatty acid binding proteins

The interactions of long chain fatty acids (FA) with wild type (WT) fatty acid binding proteins (FABP) and engineered FABP mutants have been monitored to determine the equilibrium binding constants as well as the rate constants for binding and dissociation. These measurements have been done using the fluorescent probes, ADIFAB and ADIFAB2, that allow the determination of the free fatty acid (FFA) concentration in the reaction of FA with proteins and membranes. The results of these studies indicate that for WT proteins from adipocyte, heart, intestine, and liver, Kd values are in the nM range and affinities decrease with increasing aqueous solubility of the FA. Binding affinities for heart and liver are generally greater than those for adipocyte and intestine. Moreover, measurements of the rate constants indicate that binding equilibrium at 37øC is achieved within seconds for all FA and FABPs. These results, together with the level of serum (unbound) FFA, suggests a buffering action of FABPs that helps to maintain the intracellular concentration of FFA so that the flux of FFA between serum and cells occurs down a concentration gradient. Measurements of the temperature dependence of binding reveal that the free energy is predominately enthalpic and that the enthalpy of the reaction results from FA-FABP interactions within the binding cavity. The nature of these interactions were investigated by determining the thermodynamics of binding to engineered point mutants of the intestinal FABP. These measurements showed that binding affinities did not report accurately the changes in protein-FA interactions because changes in the binding entropy and enthalpy tend to compensate. For example, an alanine substitution for arginine 106 yields a 30 fold increase in binding affinity, because the loss in enthalpy due to the elimination of the favorable interaction between the FA carboxylate and Arg106, is more than compensated for by an increase in entropy. Thus understanding the effects of amino acid replacements on FA-FABP interactions requires measurements of enthalpy and entropy, in addition to affinity.     

Fatty acid-albumin complexes and the determination of the transport of long chain free fatty acids across membranes

Understanding the mechanism that governs the transport of long chain free fatty acids (FFA) across lipid bilayers is critical for understanding transport across cell membranes. Conflicting results have been reported for lipid vesicles; most investigators report that flip-flop occurs within the resolution time of the method (<5 ms) and that dissociation from the membrane is rate limiting, while other studies find that flip-flop is rate limiting and on the order of seconds. We have reinvestigated this problem and find that the methods used in studies reporting rapid flip-flop have not been interpreted correctly. We find that accurate information about transport of FFA across lipid vesicles requires that FFA be delivered to the vesicles as complexes with albumin (BSA). For example, we find that stopped-flow mixing of uncomplexed FFA with small unilamellar vesicles (SUV) containing pyranine yields the very fast influx rates reported previously (>100 s(-1)). However, these influx rates increase linearly with lipid vesicle concentration and can therefore not, as previously interpreted, represent flip-flop. In contrast, measurements of influx rates in SUV and giant unilamellar vesicles performed with oleate-BSA complexes reveal no dependence on vesicle concentration and yield influx rate constants of approximately 4 and approximately 0.5 s(-1), respectively. Rate constants for efflux and dissociation were determined from the transfer of oleate from vesicles to BSA and reveal similar influx and efflux but dissociation rate constants that are approximately 5-10-fold greater. We conclude that flip-flop is rate limiting for transport of FFA across lipid vesicles and slows with an increasing radius of curvature. These results, in contrast to those reporting that flip-flop is extremely fast, indicate that the lipid bilayer portion of biological membranes may present a significant barrier to transport of FFA across cell membranes.     

FAT/CD36 is located on the outer mitochondrial membrane, upstream of long-chain acyl-CoA synthetase, and regulates palmitate oxidation

FAT/CD36 (fatty acid translocase/Cluster of Differentiation 36), a plasma membrane fatty-acid transport protein, has been found on mitochondrial membranes; however, it remains unclear where FAT/CD36 resides on this organelle or its functional role within mitochondria. In the present study, we demonstrate, using several different approaches, that in skeletal muscle FAT/CD36 resides on the OMM (outer mitochondrial membrane). To determine the functional role of mitochondrial FAT/CD36 in this tissue, we determined oxygen consumption rates in permeabilized muscle fibres in WT (wild-type) and FAT/CD36-KO (knockout) mice using a variety of substrates. Despite comparable muscle mitochondrial content, as assessed by unaltered mtDNA (mitochondrial DNA), citrate synthase, β-hydroxyacyl-CoA dehydrogenase, cytochrome c oxidase complex IV and respiratory capacities [maximal OXPHOS (oxidative phosphorylation) respiration] in WT and KO mice, palmitate-supported respiration was 34% lower in KO animals. In contrast, palmitoyl-CoA-supported respiration was unchanged. These results indicate that FAT/CD36 is key for palmitate-supported respiration. Therefore we propose a working model of mitochondrial fatty-acid transport, in which FAT/CD36 is positioned on the OMM, upstream of long-chain acyl-CoA synthetase, thereby contributing to the regulation of mitochondrial fatty-acid transport. We further support this model by providing evidence that FAT/CD36 is not located in mitochondrial contact sites, and therefore does not directly interact with carnitine palmitoyltransferase-I as original proposed.     

Purification, immunochemical quantification and localization in rat heart of putative fatty acid translocase (FAT/CD36)

Evidence is accumulating that the heavily glycosylated integral membrane protein fatty acid translocase (FAT/CD36) is involved in the transport of long-chain fatty acids across the sarcolemma of heart muscle cells. The aim of this study was to analyse the distribution between FAT/CD36 present in cardiac myocytes and endothelial cells. We therefore developed a method to purify FAT/CD36 from total rat heart and isolated cardiomyocytes, and used the proteins as standards in an immunochemical assay. Two steps, chromatography on wheat germ agglutinin-agarose and anion-exchange chromatography on Q-Sepharose fast flow, were sufficient for obtaining the protein in a > 95% pure form. When used to isolate FAT/CD36 from total heart tissue, the FAT/CD36 yield of the method was 9% and the purification factor was 64. Purifying FAT/CD36 from isolated cardiomyocytes yielded the same 88 kDa protein band on SDS-PAGE gels and reactivity of this band on western blots was comparable to that of the FAT/CD36 isolated from total hearts. Quantifying FAT/CD36 contents by western blotting showed that the amounts of FAT/CD36 that are present in isolated cardiomyocytes (10 ± 3 μg/mg protein) and total hearts (14 ± 4 μg/mg protein) are of comparable magnitude. Immunofluorescence labelling showed that at least a part of the FAT/CD36 present in the cardiomyocyte is associated with the sarcolemma. This study established that FAT/CD36 is a relatively abundant protein in the cardiomyocyte. In addition, the further developed purification procedure is the first method for isolating FAT/CD36 from rat heart and cardiomyocyte FAT/CD36.     

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.     

Caveolin-1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts

Several lines of evidence suggest that lipid rafts are involved in cellular fatty acid uptake and influence fatty acid translocase (FAT/CD36) function. However, it remains unknown whether caveolae, a specialized raft type, are required for this mechanism. Here, we show that wild-type (WT) mouse embryonic fibroblasts (MEFs) and caveolin-1 knockout (KO) MEFs, which are devoid of caveolae, have comparable overall expression of FAT/CD36 protein but altered subcellular FAT/CD36 localization and function. In WT MEFs, FAT/CD36 was isolated with both lipid raft enriched detergent-resistant membranes (DRMs) and detergent-soluble membranes (DSMs), whereas in cav-1 KO cells it was exclusively associated with DSMs. Subcellular fractionation demonstrated that FAT/CD36 in WT MEFs was localized intracellularly and at the plasma membrane level while in cav-1 KO MEFs it was absent from the plasma membrane. This mistargeting of FAT/CD36 in cav-1 KO cells resulted in reduced fatty acid uptake compared to WT controls. Adenoviral expression of caveolin-1 in KO MEFs induced caveolae formation, redirection of FAT/CD36 to the plasma membrane and rescue of fatty acid uptake. In conclusion, our data provide evidence that caveolin-1 is necessary to target FAT/CD36 to the plasma membrane. Caveolin-1 may influence fatty acid uptake by regulating surface availability of FAT/CD36.     

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

Cellular long-chain fatty acid uptake is believed to occur largely by protein-mediated transmembrane transport of fatty acids, and also by passive diffusional uptake. It is postulated that the membrane proteins function in trapping of fatty acids from 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 (FABP(pm)) 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.     

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.     

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.     

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.     

A null mutation in skeletal muscle FAT/CD36 reveals its essential role in insulin- and AICAR-stimulated fatty acid metabolism

Fatty acid translocase (FAT)/CD36 is involved in regulating the uptake of long-chain fatty acids into muscle cells. However, the contribution of FAT/CD36 to fatty acid metabolism remains unknown. We examined the role of FAT/CD36 on fatty acid metabolism in perfused muscles (soleus and red and white gastrocnemius) of wild-type (WT) and FAT/CD36 null (KO) mice. In general, in muscles of KO mice, 1) insulin sensitivity and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) sensitivity were normal, 2) key enzymes involved in fatty acid oxidation were altered minimally or not at all, and 3) except for an increase in soleus muscle FATP1 and FATP4, these fatty acid transporters were not altered in red and white gastrocnemius muscles, whereas plasma membrane-bound fatty acid binding protein was not altered in any muscle. In KO muscles perfused under basal conditions (i.e., no insulin, no AICAR), rates of hindquarter fatty acid oxidation were reduced by 26%. Similarly, in oxidative but not glycolytic muscles, the basal rates of triacylglycerol esterification were reduced by 40%. When muscles were perfused with insulin, the net increase in fatty acid esterification was threefold greater in the oxidative muscles of WT mice compared with the oxidative muscles in KO mice. With AICAR-stimulation, the net increase in fatty acid oxidation by hindquarter muscles was 3.7-fold greater in WT compared with KO mice. In conclusion, the present studies demonstrate that FAT/CD36 has a critical role in regulating fatty acid esterification and oxidation, particularly during stimulation with insulin or AICAR.     

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.     

Cellular cholesterol stimulates acute uptake of palmitate by redistribution of fatty acid translocase in type II pneumocytes

Cholesterol is an abundant lipid of lung surfactant, where its concentration changes relative to phospholipids in response to certain physiological conditions. We investigated the effect of the cellular cholesterol content on uptake and esterification of palmitic acid, and on cellular distribution of fatty acid translocase (FAT/CD36) in alveolar type II cells. Incubation of type II cells with methyl-beta-cyclodextrin-cholesterol complexes increased the cholesterol content of lamellar bodies. The palmitate uptake of type II cells increased in parallel with the cellular cholesterol content. The content of FAT/CD36 increased in membranes and decreased in cytosol in type II cells. The detergent-insoluble fraction (DIGs), isolated from type II cells, was enriched in FAT/CD36 and caveolin-1 after increasing the cellular cholesterol. The total incorporation of labeled palmitic acid into glycerolipids and cholesterol ester (CE) increased by a factor of about 10 when the amount of unbound (14)C-palmitic acid added to type II cells was increased by a factor of about 1000. Under these conditions, a small but significant increase of the palmitate incorporation into PL occurred. Independent from the amount of added palmitate, palmitate incorporation into triacylglycerol decreased and palmitate incorporation into cholesterol ester increased about 40-65-fold. The beta-oxidation of palmitate significantly decreased. We conclude that alveolar type II cells respond to an increase of the cholesterol level with (i) cellular redistribution of FAT/CD36 into DIGs causing enhanced palmitate uptake and increased cholesterol ester-formation, (ii) storage of cholesterol in lamellar bodies, and (iii) induction of the formation of caveolae-like microdomains in the surface membrane, a structure possibly involved in a lamellar body-independent efflux of free cholesterol via the high-density lipoprotein-specific pathway.     

Contraction-induced skeletal muscle FAT/CD36 trafficking and FA uptake is AMPK independent

The aim of this study was to investigate the molecular mechanisms regulating FA translocase CD36 (FAT/CD36) translocation and FA uptake in skeletal muscle during contractions. In one model, wild-type (WT) and AMP-dependent protein kinase kinase dead (AMPK KD) mice were exercised or extensor digitorum longus (EDL) and soleus (SOL) muscles were contracted, ex vivo. In separate studies, FAT/CD36 translocation and FA uptake in response to muscle contractions were investigated in the perfused rat hindlimb. Exercise induced a similar increase in skeletal muscle cell surface membrane FAT/CD36 content in WT (+34%) and AMPK KD (+37%) mice. In contrast, 5-aminoimidazole-4-carboxamide ribonucleoside only induced an increase in cell surface FAT/CD36 content in WT (+29%) mice. Furthermore, in the perfused rat hindlimb, muscle contraction induced a rapid (1 min, +15%) and sustained (10 min, +24%) FAT/CD36 relocation to cell surface membranes. The increase in cell surface FAT/CD36 protein content with muscle contractions was associated with increased FA uptake, both in EDL and SOL muscle from WT and AMPK KD mice and in the perfused rat hindlimb. This suggests that AMPK is not essential in regulation of FAT/CD36 translocation and FA uptake in skeletal muscle during contractions. However, AMPK could be important in regulation of FAT/CD36 distribution in other physiological situations.     

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.     

Activation of AMPKα2 Is Not Required for Mitochondrial FAT/CD36 Accumulation during Exercise

Exercise has been shown to induce the translocation of fatty acid translocase (FAT/CD36), a fatty acid transport protein, to both plasma and mitochondrial membranes. While previous studies have examined signals involved in the induction of FAT/CD36 translocation to sarcolemmal membranes, to date the signaling events responsible for FAT/CD36 accumulation on mitochondrial membranes have not been investigated. In the current study muscle contraction rapidly increased FAT/CD36 on plasma membranes (7.5 minutes), while in contrast, FAT/CD36 only increased on mitochondrial membranes after 22.5 minutes of muscle contraction, a response that was exercise-intensity dependent. Considering that previous research has shown that AMP activated protein kinase (AMPK) α2 is not required for FAT/CD36 translocation to the plasma membrane, we investigated whether AMPK α2 signaling is necessary for mitochondrial FAT/CD36 accumulation. Administration of 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) induced AMPK phosphorylation, and resulted in FAT/CD36 accumulation on SS mitochondria, suggesting AMPK signaling may mediate this response. However, SS mitochondrial FAT/CD36 increased following acute treadmill running in both wild-type (WT) and AMPKα 2 kinase dead (KD) mice. These data suggest that AMPK signaling is not required for SS mitochondrial FAT/CD36 accumulation. The current data also implicates alternative signaling pathways that are exercise-intensity dependent, as IMF mitochondrial FAT/CD36 content only occurred at a higher power output. Taken altogether the current data suggests that activation of AMPK signaling is sufficient but not required for exercise-induced accumulation in mitochondrial FAT/CD36.