Gene targeting implicates Cdc42 GTPase in GPVI and non-GPVI mediated platelet filopodia formation, secretion and aggregation.

BACKGROUND: Cdc42 and Rac1, members of the Rho family of small GTPases, play critical roles in actin cytoskeleton regulation. We have shown previously that Rac1 is involved in regulation of platelet secretion and aggregation. However, the role of Cdc42 in platelet activation remains controversial. This study was undertaken to better understand the role of Cdc42 in platelet activation. METHODOLOGY/PRINCIPAL FINDINGS: We utilized the Mx-cre;Cdc42(lox/lox) inducible mice with transient Cdc42 deletion to investigate the involvement of Cdc42 in platelet function. The Cdc42-deficient mice exhibited a significantly reduced platelet count than the matching Cdc42(+/+) mice. Platelets isolated from Cdc42(-/-), as compared to Cdc42(+/+), mice exhibited (a) diminished phosphorylation of PAK1/2, an effector molecule of Cdc42, (b) inhibition of filopodia formation on immobilized CRP or fibrinogen, (c) inhibition of CRP- or thrombin-induced secretion of ATP and release of P-selectin, (d) inhibition of CRP, collagen or thrombin induced platelet aggregation, and (e) minimal phosphorylation of Akt upon stimulation with CRP or thrombin. The bleeding times were significantly prolonged in Cdc42(-/-) mice compared with Cdc42(+/+) mice. CONCLUSION/SIGNIFICANCE: Our data demonstrate that Cdc42 is required for platelet filopodia formation, secretion and aggregation and therefore plays a critical role in platelet mediated hemostasis and thrombosis.

Uptake of oxidized low density lipoprotein by CD36 occurs by an actin-dependent pathway distinct from macropinocytosis.

The class B scavenger receptor CD36 has numerous ligands that include modified forms of low density lipoprotein, fibrillar amyloid, apoptotic cells, and Plasmodium falciparum-infected red blood cells, linking this molecule to atherosclerosis, Alzheimer disease, malaria, and other diseases. We studied the signaling events that follow receptor engagement and lead to CD36 and ligand internalization. We show that oxidized low density lipoprotein or antibody-induced clustering of CD36 triggers macropinocytosis and internalization of the receptor-ligand complex. Remarkably, however, CD36 internalization is independent of macropinocytosis and occurs by a novel endocytic mechanism that depends on actin, but not dynamin. This actin-driven endocytosis requires the activation Src family kinases, JNK, and Rho family GTPases, but, unlike macropinocytosis, it is not affected by inhibitors of phosphatidylinositol 3-kinase or Na/H exchange. Manipulation of this unique mode of internalization may prove helpful in the prevention and management of the wide range of diseases in which CD36 is implicated.

Cardiac and skeletal muscle fatty acid transport and transporters and triacylglycerol and fatty acid oxidation in lean and Zucker diabetic fatty rats.

We examined fatty acid transporters, transport, and metabolism in hearts and red and white muscles of lean and insulin-resistant (week 6) and type 2 diabetic (week 24) Zucker diabetic fatty (ZDF) rats. Cardiac fatty acid transport was similar in lean and ZDF hearts at week 6 but was reduced at week 24 (-40%) in lean but not ZDF hearts. Red muscle of ZDF rats exhibited an early susceptibility to upregulation (+66%) of fatty acid transport at week 6 that was increased by 50% in lean and ZDF rats at week 24 but remained 44% greater in red muscle of ZDF rats. In white muscle, no differences were observed in fatty acid transport between groups or from week 6 to week 24. In all tissues (heart and red and white muscle), FAT/CD36 protein and plasmalemmal content paralleled the changes in fatty acid transport. Triacylglycerol content in red and white muscles, but not heart, in lean and ZDF rats correlated with fatty acid transport (r = 0.91) and sarcolemmal FAT/CD36 (r = 0.98). Red and white muscle fatty acid oxidation by isolated mitochondria was not impaired in ZDF rats but was reduced by 18-24% in red muscle of lean rats at week 24. Thus, in red, but not white, muscle of insulin-resistant and type 2 diabetic animals, a marked upregulation in fatty acid transport and intramuscular triacylglycerol was associated with increased levels of FAT/CD36 expression and plasmalemmal content. In heart, greater rates of fatty acid transport and FAT/CD36 in ZDF rats (week 24) were attributable to the inhibition of age-related reductions in these parameters. However, intramuscular triacylglycerol did not accumulate in hearts of ZDF rats. Thus insulin resistance and type 2 diabetes are accompanied by tissue-specific differences in FAT/CD36 and fatty acid transport and metabolism. Upregulation of fatty acid transport increased red muscle, but not cardiac, triacylglycerol accumulation. White muscle lipid metabolism dysregulation was not observed.

FAT/CD36 expression is not ablated in spontaneously hypertensive rats.

There is doubt whether spontaneously hypertensive rats (SHR; North American strain) are null for fatty acid translocase (FAT/CD36). Therefore, we examined whether FAT/CD36 is expressed in heart, muscle, liver and adipose tissue in SHR. Insulin resistance was present in SHR skeletal muscle. We confirmed that SHR expressed aberrant FAT mRNAs in key metabolic tissues; namely, the major 2.9 kb transcript was not expressed, but 3.8 and 5.4 kb transcripts were present. Despite this, FAT/CD36 protein was expressed in all tissues, although there were tissue-specific reductions in FAT/CD36 protein expression and plasmalemmal content, ranging from 26-85%. Fatty acid transport was reduced in adipose tissue (-50%) and was increased in liver (+47%). Normal rates of fatty acid transport occurred in heart and muscle, possibly due to compensatory upregulation of plasmalemmal fatty acid binding protein (FABPpm) in red (+123%) and white muscle (+110%). In conclusion, SHRs (North American strain) are not a natural FAT/CD36 null model, the North American strain of SHR express FAT/CD36, albeit at reduced levels.

Rosiglitazone increases fatty acid oxidation and fatty acid translocase (FAT/CD36) but not carnitine palmitoyltransferase I in rat muscle mitochondria.

Peroxisome proliferator-activated receptors (PPARs) alter the expression of genes involved in regulating lipid metabolism. Rosiglitazone, a PPARgamma agonist, induces tissue-specific effects on lipid metabolism; however, its mode of action in skeletal muscle remains unclear. Since fatty acid translocase (FAT/CD36) was recently identified as a possible regulator of skeletal muscle fatty acid transport and mitochondrial fatty acid oxidation, we examined in this tissue the effects of rosiglitazone infusion (7 days, 1 mg day(-1)) on FAT/CD36 mRNA and protein, its plasmalemmal content and fatty acid transport. In addition, in isolated subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria we examined rates of fatty acid oxidation, FAT/CD36 and carnitine palmitoyltransferase I (CPTI) protein, and CPTI and beta-hydroxyacyl CoA dehydrogenase (beta-HAD) activities. Rosiglitazone did not alter FAT/CD36 mRNA or protein expression, FAT/CD36 plasmalemmal content, or the rate of fatty acid transport into muscle (P > 0.05). In contrast, rosiglitazone increased the rates of fatty acid oxidation in both SS (+21%) and IMF mitochondria (+36%). This was accompanied by concomitant increases in FAT/CD36 in subsarcolemmal (SS) (+43%) and intermyofibrillar (IMF) mitochondria (+46%), while SS and IMF CPTI protein content, and CPTI submaximal and maximal activities (P > 0.05) were not altered. Similarly, citrate synthase (CS) and beta-HAD activities were also not altered by rosiglitazone in SS and IMF mitochondria (P > 0.05). These studies provide another example whereby changes in mitochondrial fatty oxidation are associated with concomitant changes in mitochondrial FAT/CD36 independent of any changes in CPTI. Moreover, these studies identify for the first time a mechanism by which rosiglitazone stimulates fatty acid oxidation in skeletal muscle, namely the chronic, subcellular relocation of FAT/CD36 to mitochondria.

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.

Skeletal muscle mitochondrial FAT/CD36 content and palmitate oxidation are not decreased in obese women.

A reduction in fatty acid oxidation has been associated with lipid accumulation and insulin resistance in the skeletal muscle of obese individuals. We examined whether this decrease in fatty acid oxidation was attributable to a reduction in muscle mitochondrial content and/or a dysfunction in fatty acid oxidation within mitochondria obtained from skeletal muscle of age-matched, lean [body mass index (BMI) = 23.3 +/- 0.7 kg/m2] and obese women (BMI = 37.6 +/- 2.2 kg/m2). The mitochondrial marker enzymes citrate synthase (-34%), beta-hydroxyacyl-CoA dehydrogenase (-17%), and cytochrome c oxidase (-32%) were reduced (P < 0.05) in obese participants, indicating that mitochondrial content was diminished. Obesity did not alter the ability of isolated mitochondria to oxidize palmitate; however, fatty acid oxidation was reduced at the whole muscle level by 28% (P < 0.05) in the obese. Mitochondrial fatty acid translocase (FAT/CD36) did not differ in lean and obese individuals, but mitochondrial FAT/CD36 was correlated with mitochondrial fatty acid oxidation (r = 0.67, P < 0.05). We conclude that the reduction in fatty acid oxidation in obese individuals is attributable to a decrease in mitochondrial content, not to an intrinsic defect in the mitochondria obtained from skeletal muscle of obese individuals. In addition, it appears that mitochondrial FAT/CD36 may be involved in regulating fatty acid oxidation in human skeletal muscle.

The Fab fragment of a novel anti-GPVI monoclonal antibody, OM4, reduces in vivo thrombosis without bleeding risk in rats.

BACKGROUND: Inhibition of GPVI has been proposed as a useful antithrombotic strategy; however, in vivo proof-of-concept animal studies targeting GPVI are lacking. We evaluated a novel anti-human GPVI monoclonal antibody OM4 Fab in rats. METHODS AND RESULTS: OM4 Fab specifically inhibited collagen-induced aggregation of rat platelets in vitro with an IC50 of 20 to 30 microg/mL but not ADP and AA-induced platelet aggregation. After intravenous administration of OM4 Fab, a rapid inhibition of ex vivo platelet aggregation was observed with a gradual recovery within 60 to 90 minutes which corresponded to the decline in OM4 Fab plasma concentration and time-dependent decrease in platelet-bound OM4 Fab. In contrast to previous reports in mice, intravenous OM4 Fab did not deplete platelet GPVI. Injection of OM4 IgG caused acute thrombocytopenia. In a modified Folts model of cyclic flow reduction in rat carotid artery, the number of complete occlusions was significantly reduced by intravenous administration of OM4 Fab (20 mg/kg) before or after mechanical injury to the vessel, without prolongation of bleeding time. CONCLUSION: Fab fragment of the monoclonal antibody OM4 effectively inhibits collagen induced platelet aggregation in vitro and ex vivo, and in vivo thrombosis in rats without prolonging bleeding time. Antibodies against GPVI may have therapeutic potential, inhibiting thrombosis without prolonging bleeding time.

Highly potent anti-human GPVI monoclonal antibodies derived from GPVI knockout mouse immunization.

Recent progress in the understanding of thrombus formation has suggested an important role for glycoprotein (GP) VI in this process. To clarify the exact role in detail, it is necessary to use specific, high affinity inhibitory antibodies. However, possibly due to the conserved structure of GPVI among species, it has been difficult to obtain potent antibodies. In this study, we developed highly potent anti-human GPVI monoclonal antibodies using GPVI knockout mice for immunization. Fab fragments of these antibodies, named OM1 and OM2, potently inhibit collagen-induced platelet aggregation. The IC(50) values for OM1 and OM2 are 0.6+/-0.05 and 1.7+/-0.5 microg/mL, respectively, showing potency greater than, or equal to that of abciximab (1.7+/-0.3 microg/mL), an anti-GPIIb/IIIa antibody. Fab fragments of OM1 and OM2 also potently inhibit collagen-induced ATP release, thromboxane A(2) formation, and platelet adhesion to immobilized collagen under static and flow conditions. Interestingly, platelet aggregation induced with collagen-related peptide was potently inhibited by OM2 but not OM1, indicating that OM1 recognizes an epitope that is different from collagen-related peptide-binding site on GPVI. These results suggest that OM1 and OM2 may be useful tools to understand the role of GPVI in thrombus formation. Furthermore, these antibodies have the potential to be developed as a new class of therapeutic tool.

Importance of the carboxyl terminus of FAT/CD36 for plasma membrane localization and function in long-chain fatty acid uptake.

This study investigates the role of the cytoplasmic C terminus of fatty acid translocase (FAT/CD36) in localization of the molecule to the plasma membrane, its insertion into lipid rafts, and its ability to enhance long-chain fatty acid uptake in transfected H4IIE rat hepatoma cells. In these cells, wild-type FAT/CD36 is localized to both lipid raft and nonraft domains of the plasma membrane. Interestingly, a FAT/CD36 truncation mutant lacking the final 10 amino acids of the cytoplasmic C terminus was retained within the cell in detergent-resistant membranes, and unlike wild-type FAT/CD36, it did not enhance oleate uptake. Furthermore, expression of FAT/CD36 in these cells increased the incorporation of oleate into diacylglycerol, a property that was not shared by truncated FAT/CD36. To examine whether the C terminus itself has an intrinsic ability to dictate the plasma membrane localization of FAT/CD36, this region was fused in-frame to enhanced green fluorescent protein (EGFP). This domain was sufficient to attach EGFP to cellular membranes, suggesting an involvement in the intracellular traffic of the molecule. We conclude that the C terminus of FAT/CD36 is required for localization of the receptor to the cell surface and its ability to enhance cellular oleate uptake.

Ex vivo evaluation of anti-GPVI antibody in cynomolgus monkeys: dissociation between anti-platelet aggregatory effect and bleeding time.

Recent progress in the understanding of thrombus formation has suggested an important role of glycoprotein (GP)VI. In contrast to its pivotal role in collagen-induced platelet activation, it has been suggested that its blockade does not induce massive bleeding tendency. To demonstrate the dissociation between inhibitory effect on platelet aggregation and bleeding by GPVI blockade, we examined the effects of Fab fragment of OM2, an anti-human GPVI monoclonal antibody on ex vivo collagen-induced platelet aggregation and skin bleeding time after intravenous injection in cynomolgus monkeys. In a dose-escalation study, OM2 potently (> 80%) inhibited collagen-induced platelet aggregation at the cumulative dose of 0.2 mg/kg with a slight prolongation of bleeding time (1.3 times baseline value). Furthermore, at 18.8 mg/kg, the highest dose tested, prolongation of bleeding time was still mild (1.9 times). In contrast, abciximab, Fab fragment of anti-GPIIb/IIIa antibody prolonged bleeding time by 5.0 times at 0.35 mg/kg, the lowest effective dose on platelet aggregation. In a pharmacodynamic study, a bolus injection of OM2 at 0.4 mg/kg produced potent inhibition of collagen-induced aggregation up to six hours after injection, showing longer half-life than that of abciximab. The injection of OM2 Fab did not induce thrombocytopenia and GPVI depletion in monkeys. These results suggest that blockade of GPVI by antibody can exert a potent inhibitory effect on collagen-induced platelet aggregation with a milder prolongation of bleeding time than blockade of GPIIb/IIIa. This study indicates that OM2 has the potential to be developed as a new class of therapeutic tool.

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.

Prolonged AMPK activation increases the expression of fatty acid transporters in cardiac myocytes and perfused hearts.

Recently, fatty acid transport across the plasma membrane has been shown to be a key process that contributes to the regulation of fatty acid metabolism in the heart. Since AMP kinase activation by 5-aminoimidazole-4-carboxamide-1-beta-D: -ribofuranoside (AICAR) stimulates fatty acid oxidation, as well as the expression of selected proteins involved with energy provision, we examined (a) whether AICAR induced the expression of the fatty acid transporters FABPpm and FAT/CD36 in cardiac myocytes and in perfused hearts and (b) the signaling pathway involved. Incubation of cardiac myocytes with AICAR increased the protein expression of the fatty acid transporter FABPpm after 90 min (+27%, P < 0.05) and this protein remained stably overexpressed until 180 min. Similarly, FAT/CD36 protein expression was increased after 60 min (+38%, P < 0.05) and remained overexpressed thereafter. Protein overexpression, which occurred via transcriptional mechanisms, was dependent on the AICAR concentration, with optimal induction occurring at AICAR concentrations 1-5 mM for FABPpm and at 2-8 mM for FAT/CD36. The AICAR (2 h, 2 mM AICAR) effects on FABPpm and FAT/CD36 protein expression were similar in perfused hearts and in cardiac myocytes. AICAR also induced the plasmalemmal content of FAT/CD36 (+49%) and FABPpm (+42%) (P < 0.05). This was accompanied by a marked increase in the rate of palmitate transport (2.5 fold) into giant sarcolemmal vesicles, as well as by increased rates of palmitate oxidation in cardiac myocytes. When the AICAR-induced AMPK phosphorylation was blocked, neither FAT/CD36 nor FABPpm were overexpressed, nor were palmitate uptake and oxidation increased. This study has revealed that AMPK activation stimulates the protein expression of both fatty acid transporters, FAT/CD36 and FABPpm in (a) time- and (b) dose-dependent manner via (c) the AMPK signaling pathway. AICAR also (d) increased the plasmalemmal content of FAT/CD36 and FABPm, thereby (e) increasing the rates of fatty acid transport. Thus, activation of AMPK is a key mechanism regulating the expression as well as the plasmalemmal localization of fatty acid transporters.

Tissue-specific and fatty acid transporter-specific changes in heart and soleus muscle over a 1-yr period.

Rates of fatty acid oxidation increase rapidly in both rat heart and skeletal muscle in the early postnatal period. Therefore, we examined in heart and soleus muscle, (a) whether there were rapid changes in fatty acid transporter (FAT/CD36, FABPpm) mRNA and protein expression early in life (days 10 -36) and thereafter (days 84, 160, 365), and (b) whether the rates of fatty acid transport and the plasmalemmal content of FAT/CD36 and FABPpm were altered. Protein expression was altered rapidly from day 10-36 in both heart (FAT/CD36 only, +21%, P < 0.05)) and soleus muscle (FAT/CD36 + 100%, P < 0.05; FABPpm -20%, P < 0.05), with no further changes thereafter (P < 0.05). Rates of fatty acid transport (day 10 vs day 160) were increased in heart (+33%, P < 0.05) and muscle (+85%, P < 0.05), and were associated with concomitant increases in plasmalemmal FABPpm (+44%, P < 0.05) and FAT/CD36 (+16%, P < 0.05) in the heart, and only plasmalemmal FAT/CD36 in muscle (+90%, P < 0.05). Therefore, known changes in the rates of fatty acid oxidation in heart and muscle early in life appear to be accompanied by a concurrent upregulation in the rates of fatty acid transport and the expression of FAT/CD36 in heart and muscle, as well as an increase in plasmalemmal FAT/CD36 and FABPpm in the heart, and only plasmalemmal FAT/CD36 in soleus muscle. We speculate that the rapid upregulation of fatty acid transport rates in heart and muscle are needed to support the increased rates of fatty oxidation that have been previously observed in these tissues.

Fatty acid transport and FAT/CD36 are increased in red but not in white skeletal muscle of ZDF rats.

An increased rate of fatty acid transport into skeletal muscle has been has been linked to the accumulation of intramuscular lipids and insulin resistance, and red muscles are more susceptible than white muscles in developing fatty acid-mediated insulin resistance. Therefore, we examined in Zucker diabetic fatty (ZDF) rats, relative to lean rats, 1) whether rates of fatty acid transport and transporters (FAT/CD36 and FABPpm) were upregulated in skeletal muscle during the transition from insulin resistance (week 6) to type 2 diabetes (weeks 12 and 24), 2) whether such changes occurred primarily in red skeletal muscle, and 3) whether changes in FAT/CD36 and GLUT4 were correlated. In red muscles of ZDF compared with lean rats, the rates of fatty acid transport were upregulated (+66%) early in life (week 6). Compared with the increase in fatty acid transport in lean red muscle from weeks 12-24 (+57%), the increase in fatty acid transport rate in ZDF red muscle was 50% greater during this same period. In contrast, no differences in fatty acid transport rates were observed in the white muscles of lean and ZDF rats at any time (weeks 6-24). In red muscle only, there was an inverse relationship between FAT/CD36 and GLUT4 protein expression as well as their plasmalemmal content. These studies have shown that, 1) before the onset of diabetes, as well as during diabetes, fatty acid transport and FAT/CD36 expression and plasmalemmal content are upregulated in ZDF rats, but importantly, 2) these changes occurred only in red, not white, muscles of ZDF rats.

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 microg/mg protein) and total hearts (14 +/- 4 microg/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.

Differential effects of contraction and PPAR agonists on the expression of fatty acid transporters in rat skeletal muscle.

We have examined over the course of a 1-week period the independent and combined effects of chronically increased muscle contraction and the peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma activators, Wy 14,643 and rosiglitazone, on the expression and plasmalemmal content of the fatty acid transporters, FAT/CD36 and FABPpm, as well as on the rate of fatty acid transport. In resting muscle, the activation of either PPARalpha or PPARgamma failed to induce the protein expression of FAT/CD36. PPARalpha activation also failed to induce the protein expression of FABPpm. In contrast, PPARgamma activation induced the expression of FABPpm protein (40%; P < 0.05). Chronic muscle contraction increased the protein expression of FAT/CD36 (approximately 50%; P < 0.05), whereas FABPpm was slightly increased (12%; P < 0.05). Neither PPARalpha nor PPARgamma activation altered the contraction-induced expression of FAT/CD36 or FABPpm. Changes in protein expression of FAT/CD36 or FABPpm, induced by either contractions or by administration of rosiglitazone, were largely attributable to increased transcription. The contraction-induced increments in FAT/CD36 were accompanied by parallel increments in plasmalemmal FAT/CD36 and in rates of fatty acid transport (P < 0.05). Up-regulation of FABPpm expression was, however, accompanied by a reduction in plasmalemmal FABPpm, which did not affect the rates of long chain fatty acid (LCFA) transport. These studies have shown that in skeletal muscle (i) neither PPARalpha nor PPARgamma activation alters FAT/CD36 expression, (ii) PPARgamma activation selectively up-regulates FABPpm expression and (iii) contraction-induced up-regulation of LCFA transport does not appear to occur via activation of either PPARalpha or PPARgamma.

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 approximately 60% V(O2peak)) 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 microM 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 approximately 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.

GPVI-deficient mice lack collagen responses and are protected against experimentally induced pulmonary thromboembolism.

Platelet glycoprotein VI (GPVI) is now considered to be a major player in platelet-collagen adhesive interactions leading to thrombus formation. GPVI blockade, or its depletion, has been shown in mice to result in complete protection against arterial thrombosis, without significant prolongation of bleeding time. GPVI may therefore represent a useful antithrombotic target. In order to reaffirm the role of GPVI in platelet-collagen interactions, we developed GPVI(null) mice by targeted disruption methodology. GPVI(null) mice platelets failed to respond to a high dose of fibrillar collagen, or convulxin, a GPVI agonist, but showed a normal response to other agonists such as ADP, PMA and arachidonic acid. We report, for the first time, that a proportion of GPVI(null) mice is protected against lethal thromboembolism, induced by the infusion of a mixture of collagen and epinephrine. Greater than 55% of GPVI(null) mice survived the challenge, whereas the maximal survival from the other genotypes was 17% (n=18 per genotype). Washed platelets obtained from GPVI(null) mice showed >90% reduction in adhesion to fibrillar collagen under static conditions. Platelet adhesion to collagen under dynamic conditions using a high shear rate (2600 s(-1)) was dramatically reduced using blood from GPVI(null) mice, while platelets from wild-type and heterozygous animals showed a similar amount of adhesion. Animals from each genotype had essentially similar tail bleeding time, suggesting that a complete deficiency of GPVI, at least in mice, does not result in an enhanced bleeding tendency. These observations clearly establish that blockade of GPVI may attenuate platelet-collagen interactions without adversely affecting the bleeding time.

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.