Phosphatidylinositol 4-kinase IIIß mediates contraction-induced GLUT4 translocation and shows its anti-diabetic action in cardiomyocytes.

In the diabetic heart, long-chain fatty acid (LCFA) uptake is increased at the expense of glucose uptake. This metabolic shift ultimately leads to insulin resistance and a reduced cardiac function. Therefore, signaling kinases that mediate glucose uptake without simultaneously stimulating LCFA uptake could be considered attractive anti-diabetic targets. Phosphatidylinositol-4-kinase-IIIß (PI4KIIIß) is a lipid kinase downstream of protein kinase D1 (PKD1) that mediates Golgi-to-plasma membrane vesicular trafficking in HeLa-cells. In this study, we evaluated whether PI4KIIIß is involved in myocellular GLUT4 translocation induced by contraction or oligomycin (an F1F0-ATP synthase inhibitor that activates contraction-like signaling). Pharmacological targeting, with compound MI14, or genetic silencing of PI4KIIIß inhibited contraction/oligomycin-stimulated GLUT4 translocation and glucose uptake in cardiomyocytes but did not affect CD36 translocation nor LCFA uptake. Addition of the PI4KIIIß enzymatic reaction product phosphatidylinositol-4-phosphate restored oligomycin-stimulated glucose uptake in the presence of MI14. PI4KIIIß activation by PKD1 involves Ser294 phosphorylation and altered its localization with unchanged enzymatic activity. Adenoviral PI4KIIIß overexpression stimulated glucose uptake, but did not activate hypertrophic signaling, indicating that unlike PKD1, PI4KIIIß is selectively involved in GLUT4 translocation. Finally, PI4KIIIß overexpression prevented insulin resistance and contractile dysfunction in lipid-overexposed cardiomyocytes. Together, our studies identify PI4KIIIß as positive and selective regulator of GLUT4 translocation in response to contraction-like signaling, suggesting PI4KIIIß as a promising target to rescue defective glucose uptake in diabetics.

Increased intramyocellular lipids but unaltered in vivo mitochondrial oxidative phosphorylation in skeletal muscle of adipose triglyceride lipase-deficient mice.

Adipose triglyceride lipase (ATGL) is a lipolytic enzyme that is highly specific for triglyceride hydrolysis. The ATGL-knockout mouse (ATGL(-/-)) accumulates lipid droplets in various tissues, including skeletal muscle, and has poor maximal running velocity and endurance capacity. In this study, we tested whether abnormal lipid accumulation in skeletal muscle impairs mitochondrial oxidative phosphorylation, and hence, explains the poor muscle performance of ATGL(-/-) mice. In vivo ¹H magnetic resonance spectroscopy of the tibialis anterior of ATGL(-/-) mice revealed that its intramyocellular lipid pool is approximately sixfold higher than in WT controls (P = 0.0007). In skeletal muscle of ATGL(-/-) mice, glycogen content was decreased by 30% (P < 0.05). In vivo ³¹P magnetic resonance spectra of resting muscles showed that WT and ATGL(-/-) mice have a similar energy status: [PCr], [P(i)], PCr/ATP ratio, PCr/P(i) ratio, and intracellular pH. Electrostimulated muscles from WT and ATGL(-/-) mice showed the same PCr depletion and pH reduction. Moreover, the monoexponential fitting of the PCr recovery curve yielded similar PCr recovery times (τPCr; 54.1 ± 6.1 s for the ATGL(-/-) and 58.1 ± 5.8 s for the WT), which means that overall muscular mitochondrial oxidative capacity was comparable between the genotypes. Despite similar in vivo mitochondrial oxidative capacities, the electrostimulated muscles from ATGL(-/-) mice displayed significantly lower force production and increased muscle relaxation time than the WT. These findings suggest that mechanisms other than mitochondrial dysfunction cause the impaired muscle performance of ATGL(-/-) mice.

Exercise-induced modulation of cardiac lipid content in healthy lean young men.

Cardiac lipid accumulation is associated with decreased cardiac function and energy status (PCr/ATP). It has been suggested that elevated plasma fatty acid (FA) concentrations are responsible for the cardiac lipid accumulation. Therefore, the aim of the present study was to investigate if elevating plasma FA concentrations by exercise results in an increased cardiac lipid content, and if this influences cardiac function and energy status. Eleven male subjects (age 25.4 ± 1.1 years, BMI 23.6 ± 0.8 kg/m²) performed a 2-h cycling protocol, once while staying fasted and once while ingesting glucose, to create a state of high versus low plasma FA concentrations, respectively. Cardiac lipid content was measured by proton magnetic resonance spectroscopy (¹H-MRS) at baseline, directly after exercise and again 4 h post-exercise, together with systolic function (by multi-slice cine-MRI) and cardiac energy status (by ³¹P-MRS). Plasma FA concentrations were increased threefold during exercise and ninefold during recovery in the fasted state compared with the glucose-fed state (p < 0.01). Cardiac lipid content was elevated at the end of the fasted test day (from 0.26 ± 0.04 to 0.44 ± 0.04%, p = 0.003), while it did not change with glucose supplementation (from 0.32 ± 0.03 to 0.26 ± 0.05%, p = 0.272). Furthermore, PCr/ATP was decreased by 32% in the high plasma FA state compared with the low FA state (n = 6, p = 0.014). However, in the high FA state, the ejection fraction 4 h post-exercise was higher compared with the low FA state (63 ± 2 vs. 59 ± 2%, p = 0.018). Elevated plasma FA concentrations, induced by exercise in the fasted state, lead to increased cardiac lipid content, but do not acutely hamper systolic function. Although the lower cardiac energy status is in line with a lipotoxic action of cardiac lipid content, a causal relationship cannot be proven.

Brain-specific fatty acid-binding protein is elevated in serum of patients with dementia-related diseases.

BACKGROUND: There is a need for biomarkers in accessible matrices, such as blood, for the diagnosis of neurodegenerative diseases. The aim of this study was to measure the serum levels of brain-type fatty acid-binding protein (FABP) and heart-type FABP in patients with dementia-involving diseases. METHODS: Brain- and heart-type FABP were measured in serum samples from patients with either Alzheimer's disease (AD) (n = 31), Parkinson's disease (PD, n = 43), or other cognitive disorders (OCD, n = 42) and in 52 healthy controls. The localization of brain- and heart-type FABP was determined in brain sections by immunohistochemistry. RESULTS: Brain-type FABP levels were elevated in serum of 29%, 35%, and 24% of the patients with AD, PD, and OCD, respectively, and in 2% of the healthy donors. Heart-type FABP serum levels were not different amongst the patient groups. Brain-type and heart-type FABP expression was observed in reactive astrocytes in brain sections of patients with AD. CONCLUSIONS: In contrast to heart-type FABP, serum levels of brain-type FABP are elevated in a significant proportion of patients with various neurodegenerative diseases and can therefore have importance for defining subgroups of these patients.

Diagnosing acute coronary syndrome in primary care: comparison of the physicians' risk estimation and a clinical decision rule.

BACKGROUND: Diagnosing acute coronary syndrome (ACS) in a primary care setting poses a diagnostic dilemma for physicians. OBJECTIVE: We directly compared the diagnostic accuracy of a clinical decision rule (CDR) based on history taking and physical examination in suspected ACS with the risk estimates of the attending GP. METHODS: In a prospective multicenter study, patients suspected of ACS were included by the GP. GPs were asked to estimate the probability (0%-100%) of the presence of ACS. GPs collected patient data, but they were not aware of the CDR and did not score the patient accordingly. RESULTS: Two hundred and ninety-eight patients were included (52% female, mean age 66 years, 22% ACS). The area under the receiver operating characteristic (ROC) curve (AUC) was 0.75 [95% confidence interval (CI) 0.68-0.82] for the GP risk estimate and 0.66 (95% CI 0.58-0.73) for the CDR. There was an agreement between the risk estimation of the GP and a CDR in 51% and the prevalence of ACS in predefined low-, intermediate- and high-risk groups was similar for the GP and CDR estimates. In the low-risk group, according to the GP, four patients (8.2%) suffered an ACS. These four patients were all identified by the decision rule as high risk. CONCLUSIONS: The GP classified patients as ACS or no ACS more adequately than the CDR, judged by the AUC. However, the use of a CDR in patients that are considered at low risk for ACS by the GP could reduce the amount of missed myocardial infarctions.

Requirement for distinct vesicle-associated membrane proteins in insulin- and AMP-activated protein kinase (AMPK)-induced translocation of GLUT4 and CD36 in cultured cardiomyocytes.

AIMS/HYPOTHESIS: Upon stimulation of insulin signalling or contraction-induced AMP-activated protein kinase (AMPK) activation, the glucose transporter GLUT4 and the long-chain fatty acid (LCFA) transporter CD36 similarly translocate from intracellular compartments to the plasma membrane of cardiomyocytes to increase uptake of glucose and LCFA, respectively. This similarity in regulation of GLUT4 traffic and CD36 traffic suggests that the same families of trafficking proteins, including vesicle-associated membrane proteins (VAMPs), are involved in both processes. While several VAMPs have been implicated in GLUT4 traffic, nothing is known about the putative function of VAMPs in CD36 traffic. Therefore, we compared the involvement of the myocardially produced VAMP isoforms in insulin- or contraction-induced GLUT4 and CD36 translocation. METHODS: Five VAMP isoforms were silenced in HL-1 cardiomyocytes. The cells were treated with insulin or the contraction-like AMPK activator oligomycin or were electrically stimulated to contract. Subsequently, GLUT4 and CD36 translocation as well as substrate uptake were measured. RESULTS: Three VAMPs were demonstrated to be necessary for both GLUT4 and CD36 translocation, either specifically in insulin-treated cells (VAMP2, VAMP5) or in oligomycin/contraction-treated cells (VAMP3). In addition, there are VAMPs specifically involved in either GLUT4 traffic (VAMP7 mediates basal GLUT4 retention) or CD36 traffic (VAMP4 mediates insulin- and oligomycin/contraction-induced CD36 translocation). CONCLUSIONS/INTERPRETATION: The involvement of distinct VAMP isoforms in both GLUT4 and CD36 translocation indicates that CD36 translocation, just like GLUT4 translocation, is a vesicle-mediated process dependent on soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. The ability of other VAMPs to discriminate between GLUT4 and CD36 translocation allows the notion that myocardial substrate preference can be modulated by these VAMPs.

Increased levels of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1alpha) improve lipid utilisation, insulin signalling and glucose transport in skeletal muscle of lean and insulin-resistant obese Zucker rats.

AIMS/HYPOTHESIS: Reductions in peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1alpha) levels have been associated with the skeletal muscle insulin resistance. However, in vivo, the therapeutic potential of PGC-1alpha has met with failure, as supra-physiological overexpression of PGC-1alpha induced insulin resistance, due to fatty acid translocase (FAT)-mediated lipid accumulation. Based on physiological and metabolic considerations, we hypothesised that a modest increase in PGC-1alpha levels would limit FAT upregulation and improve lipid metabolism and insulin sensitivity, although these effects may differ in lean and insulin-resistant muscle. METHODS: Pgc-1alpha was transfected into lean and obese Zucker rat muscles. Two weeks later we examined mitochondrial biogenesis, intramuscular lipids (triacylglycerol, diacylglycerol, ceramide), GLUT4 and FAT levels, insulin-stimulated glucose transport and signalling protein phosphorylation (thymoma viral proto-oncogene 2 [Akt2], Akt substrate of 160 kDa [AS160]), and fatty acid oxidation in subsarcolemmal and intermyofibrillar mitochondria. RESULTS: Electrotransfection yielded physiologically relevant increases in Pgc-1alpha (also known as Ppargc1a) mRNA and protein ( approximately 25%) in lean and obese muscle. This induced mitochondrial biogenesis, and increased FAT and GLUT4 levels, insulin-stimulated glucose transport, and Akt2 and AS160 phosphorylation in lean and obese animals, while bioactive intramuscular lipids were only reduced in obese muscle. Concurrently, PGC-1alpha increased palmitate oxidation in subsarcolemmal, but not in intermyofibrillar mitochondria, in both groups. In obese compared with lean animals, the PGC-1alpha-induced improvement in insulin-stimulated glucose transport was smaller, but intramuscular lipid reduction was greater. CONCLUSIONS/INTERPRETATIONS: Increases in PGC-1alpha levels, similar to those that can be induced by physiological stimuli, altered intramuscular lipids and improved fatty acid oxidation, insulin signalling and insulin-stimulated glucose transport, albeit to different extents in lean and insulin-resistant muscle. These positive effects are probably attributable to limiting the PGC-1alpha-induced increase in FAT, thereby preventing bioactive lipid accumulation as has occurred in transgenic PGC-1alpha animals.

Pyruvate dehydrogenase kinase 4 expression is synergistically induced by AMP-activated protein kinase and fatty acids.

Organs are flexible as to which substrates they will use to maintain energy homeostasis. Under well-fed conditions, glucose is a preferred substrate for oxidation. During fasting, fatty acid oxidation will become a more important energy source. Glucose oxidation is decreased by fatty acids, a process in which the pyruvate dehydrogenase complex (PDH) and its regulator pyruvate dehydrogenase kinase 4 (PDK4) play important roles. It is currently unknown how energy status influences PDH activity. We show that AMP-activated protein kinase (AMPK) activation by hypoxia and AICAR treatment combined with fatty acid administration synergistically induce PDK4 expression. We provide evidence that AMPK activation modulates ligand-dependent activation of peroxisome proliferator-activated receptor. Finally, we show that this synergistic induction of PDK4 decreases cellular glucose oxidation. In conclusion, AMPK and fatty acids play a direct role in fuel selection in response to cellular energy status in order to spare glucose.

Assessing fatty acid oxidation flux in rodent cardiomyocyte models.

The healthy adult heart primarily relies on fatty acid oxidation (FAO) for energy production but instantaneously adapts its substrate preference in response to physiological or pathological challenges. Accurate FAO measurements are crucial to investigate early metabolic (mal)adaptations. While measurements in intact cardiomyocytes offer greater physiological relevance, current FAO protocols mainly employ cell-free systems and/or require expensive equipment. Here, we present an easy-to-use, inexpensive, and sensitive method to measure, compare and modulate FAO in various cardiomyocyte models. Basal FAO was 2-fold higher in fresh versus cultured adult rat cardiomyocytes (aRCM), while OXPHOS protein levels were maintained. Basal FAO was higher in cultured (3-fold) and fresh (8-fold) aRCM, versus widely used neonatal rat cardiomyocytes (nRCM) and mouse HL1 cardiomyocytes. Moreover, we utilized chemical and pharmacological treatments in order to modulate the FAO flux at different cellular signalling levels. Our data indicate that caution should be taken when studying metabolism in nRCM and HL1 cell models, as these display significantly lower FAO than aRCM. Accurate FAO measurement in cultured aRCM opens new avenues for studying the complex cardiomyocyte metabolic responses to mechanical, nutritional, pharmacological, and genetic manipulations.

A continuous displacement immunoassay for human heart-type fatty acid-binding protein in plasma.

Human heart-type fatty acid-binding protein (FABP) is suggested as an early plasma marker of acute myocardial infarction (AMI), and several studies have proved that, for early diagnosis of AMI, FABP performs better than myoglobin, which is a more often used early marker protein. Because serial measurement of biochemical markers in plasma is now universally accepted as an important determinant in AMI diagnosis, a rapid and continuous measuring method for FABP would be desirable. The aim of the present study was to develop an immunoassay based on the principle of displacement and using a column for rapid and continuous measurement of FABP in plasma. Glass columns filled with Sepharose-bound FABP were loaded with a horseradish peroxidase (HRP)-labeled antibody (Ab) and equilibrated with human plasma. After reaching a stable baseline, human plasma spiked with FABP or plasma from AMI patients was added. The Ab-HRP complex dissociated due to the presence of FABP in the plasma and was subsequently quantified. For plasma from AMI patients (n=5), the Ab-HRP level thus measured correlated with the corresponding plasma FABP concentration (R=0.96). The results of this study show the feasibility of a sensor for continuous monitoring of FABP in plasma.

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.

Development of a displacement immunoassay for human heart-type fatty acid-binding protein in plasma: the basic conditions.

To risk-stratify patients with chest pain who are admitted to emergency rooms and for whom initial evaluation is not conclusive, the use of cardiac markers has become a standard procedure. A recently introduced early plasma marker for acute myocardial infarction (AMI) is the 14.5-kDa cytoplasmic heart-type fatty acid-binding protein (FABP). To fully exploit its early release from injured myocardium, a rapid method for repeated measurements or continuous monitoring of FABP in plasma is desirable. Such an on-line method could be an immunosensor based on displacement. The aim of the present study was to further investigate the principles underlying the displacement assay of FABP, both in buffer and in plasma. Batches of sepharose-bound FABP were loaded with an antibody-horseradish peroxidase (HRP) conjugate (anti-FABP). Continuous measurement of FABP was mimicked by repeated addition of FABP containing solutions followed by several washing steps. In the presence of free FABP the antibody-HRP complex dissociated and was subsequently quantified. Significant displacement in the presence of free FABP was observed in both buffer and human plasma. Anti-FABP could be intermittently displaced in the same batch, for at least 9 h, and the displacement was concentration-dependent. These results show the feasibility of a sensor based on the displacement principle to be used for the diagnosis of AMI in emergency medicine.

Long-chain fatty acid uptake by skeletal muscle is impaired in homozygous, but not heterozygous, heart-type-FABP null mice.

Previous studies with cardiac myocytes from homozygous heart-type fatty acid (FA)-binding protein (H-FABP) -/- mice have indicated that this intracellular receptor protein for long-chain FA is involved in the cellular uptake of these substrates. Based on the knowledge that muscle FA uptake is a process highly sensitive to regulation by hormonal and mechanical stimuli, we studied whether H-FABP would play a role in this regulation. A suitable model system to answer this question is provided by H-FABP +/- mice, because in hindlimb muscles the content of H-FABP was measured to be 34% compared to wild-type mice. In these H-FABP +/- skeletal muscles, just as in H-FABP -/- muscles, contents of FA transporters, i.e., 43-kDa FABPpm and 88-kDa FAT/CD36, were similar compared to wild-type muscles, excluding possible compensatory mechanisms at the sarcolemmal level. Palmitate uptake rates were measured in giant vesicles prepared from hindlimb muscles of H-FABP -/-, H-FABP +/-, and H-FABP +/+ mice. For comparison, giant vesicles were isolated from liver, the tissue of which expresses a distinct type of FABP (i.e., L-FABP). Whereas in H-FABP -/- skeletal muscle FA uptake was reduced by 42-45%, FA uptake by H-FABP +/- skeletal muscle was not different from that in wild-type mice. In contrast, in liver from H-FABP -/- and from H-FABP +/- mice, FA uptake was not altered compared to wild-type animals, indicating that changes in FA uptake are restricted to H-FABP expressing tissues. It is concluded that H-FABP plays an important, yet merely permissive, role in FA uptake into muscle tissues.

Diagnostic value of a heart-type fatty acid-binding protein (H-FABP) bedside test in suspected acute coronary syndrome in primary care.

BACKGROUND: To determine the diagnostic accuracy of a rapid heart-type fatty acid-binding protein (H-FABP) test in patients suspected of acute coronary syndrome (ACS) in primary care. METHODS: General practitioners included 298 patients suspected of ACS. In all patients, whether referred to hospital or not, ECG and cardiac biomarker testing was performed. ACS was determined in accordance with international guidelines. Multivariate analysis was used to determine the value of H-FABP in addition to clinical findings. RESULTS: Mean patient age was 66 years (SD 14), 52% was female and 66 patients (22%) were diagnosed with ACS. The H-FABP bedside test was performed within 24h (median 3.1, IQR 1.5 to 7.1) after symptom onset. The positive predictive value (PPV) of H-FABP was 65% (95% confidence interval (CI) 50-78). The negative predictive value (NPV) was 85% (95% CI 80-88). Sensitivity was 39% (29-51%) and specificity 94% (90-96%). Within 6h after symptom onset, the PPV was 72% (55-84) and the NPV was 83% (77-88), sensitivity 43% (31-57%) and specificity 94% (89-97%). Adding the H-FABP test to a diagnostic model for ACS led to an increase in the area under the receiver operating curve from 0.66 (95% CI 0.58-0.73) to 0.75 (95% CI 0.68-0.82). CONCLUSION: The H-FABP rapid test provides modest additional diagnostic certainty in primary care. It cannot be used to safely exclude rule out ACS. The test can only be used safely in patients otherwise NOT referred to hospital by the GP, as an extra precaution not to miss ACS ('rule in').

Caffeine-stimulated fatty acid oxidation is blunted in CD36 null mice.

AIM: The increase in skeletal muscle fatty acid metabolism during exercise has been associated with the release of calcium. We examined whether this increase in fatty acid oxidation was attributable to a calcium-induced translocation of the fatty acid transporter CD36 to the sarcolemma, thereby providing an enhanced influx of fatty acids to increase their oxidation. METHODS: Calcium release was triggered by caffeine (3 mm) to examine fatty acid oxidation in intact soleus muscles of WT and CD36-KO mice, while fatty acid transport and mitochondrial fatty acid oxidation were examined in giant vesicles and isolated mitochondria, respectively, from caffeine-perfused hindlimb muscles of WT and CD36-KO mice. Western blotting was used to examine calcium-induced signalling. RESULTS: In WT, caffeine stimulated muscle palmitate oxidation (+136%), but this was blunted in CD36-KO mice (-70%). Dantrolene inhibited (WT) or abolished (CD36-KO) caffeine-induced palmitate oxidation. In muscle, caffeine-stimulated palmitate oxidation was not attributable to altered mitochondrial palmitate oxidation. Instead, in WT, caffeine increased palmitate transport (+55%) and the translocation of fatty acid transporters CD36, FABPpm, FATP1 and FATP4 (26-70%) to the sarcolemma. In CD36-KO mice, caffeine-stimulated FABPpm, and FATP1 and 4 translocations were normal, but palmitate transport was blunted (-70%), comparable to the reductions in muscle palmitate oxidation. Caffeine did not alter the calcium-/calmodulin-dependent protein kinase II phosphorylation but did increase the phosphorylation of AMPK and acetyl-CoA carboxylase comparably in WT and CD36-KO. CONCLUSION: These studies indicate that sarcolemmal CD36-mediated fatty acid transport is a primary mediator of the calcium-induced increase in muscle fatty acid oxidation.

Signaling role of CD36 in platelet activation and thrombus formation on immobilized thrombospondin or oxidized low-density lipoprotein.

BACKGROUND AND OBJECTIVE: Platelets abundantly express glycoprotein CD36 with thrombospondin-1 (TSP1) and oxidized low-density lipoprotein (oxLDL) as proposed ligands. How these agents promote platelet activation is still poorly understood. METHODS AND RESULTS: Both TSP1 and oxLDL caused limited activation of platelets in suspension. However, immobilized TSP1 and oxLDL, but not LDL, strongly supported platelet adhesion and spreading with a major role of CD36. Platelet spreading was accompanied by potent Ca(2+) rises, and resulted in exposure of P-selectin and integrin activation, all in a CD36-dependent manner with additional contributions of α(IIb) ß(3) and ADP receptor stimulation. Signaling responses via CD36 involved activation of the protein tyrosine kinase Syk. In whole blood perfusion, co-coating of TSP1 or oxLDL with collagen enhanced thrombus formation at high-shear flow conditions, with increased expression on platelets of activated α(IIb) ß(3), P-selectin and phosphatidylserine, again in a CD36-dependent way. CONCLUSIONS: Immobilized TSP1 and oxLDL activate platelets partly via CD36 through a Syk kinase-dependent Ca(2+) signaling mechanism, which enhances collagen-dependent thrombus formation under flow. These findings provide novel insight into the role of CD36 in hemostasis.

Contribution of FAT/CD36 to the regulation of skeletal muscle fatty acid oxidation: an overview.

Long chain fatty acids (LCFAs) are an important substrate for ATP production within the skeletal muscle. The process of LCFA delivery from adipose tissue to muscle mitochondria involves many regulatory steps. Recently, it has been recognized that LCFA oxidation is not only dependent on LCFA delivery to the muscle, but also on regulatory steps within the muscle. Increasing selected fatty acid binding proteins/transporters on the plasma membrane facilitates a very rapid LCFA increase into the muscle, independent of any changes in LCFA delivery to the muscle. Such a mechanism of LCFA transporter translocation is activated by muscle contraction. Intramuscular triacylglycerols may also be hydrolysed to provide fatty acids for mitochondrial oxidation, particularly during exercise, when hormone-sensitive lipase and other enzymes are activated. Mitochondrial LCFA entry is also highly regulated. This however does not involve only the malonyl CoA carnitine palmitoyltransferase-I (CPTI) axis. Exercise-induced fatty acid entry into mitochondria is also regulated by at least one of the proteins (FAT/CD36) that also regulates plasma membrane fatty acid transport. Among individuals, differences in mitochondrial fatty acid oxidation appear to be correlated with the content of mitochondrial CPTI and FAT/CD36. This paper provides a brief overview of mechanisms that regulate LCFA uptake and oxidation in skeletal muscle during exercise and in obesity. We focus largely on our own work on FAT/CD36, which contributes to regulating, in a coordinated fashion, LCFA uptake across the plasma membrane and the mitochondrial membrane. Very little is known about the roles of FATP1-6 on fatty acid transport in skeletal muscle.

Insulin acutely upregulates protein expression of the fatty acid transporter CD36 in human skeletal muscle in vivo.

Enhanced fatty acid uptake may lead to the accumulation of lipid intermediates. This is related to insulin resistance and type 2 diabetes mellitus. Rodent studies suggest that fatty acid transporters are acutely regulated by insulin. We investigated differences in fatty acid transporter content before and at the end of a hyperinsulinemic euglycemic clamp in skeletal muscle (m. vastus lateralis) of obese, glucose-intolerant men (IGT) and obese normal glucose tolerant controls (NGT). The fatty acid transporter FAT/CD36 protein content increased 1.5-fold (P < 0.05) after 3-hrs of insulin stimulation with no difference between IGT and control subjects. No change was seen in cytosolic fatty acid binding protein (FABPc) protein content. The increase in FAT/CD36 protein content was positively related to insulin resistance as measured during the clamp (r = 0.56, P < 0.05). An increase in FAT/CD36 protein content in skeletal muscle may result in a higher fractional extraction of fatty acids (larger relative uptake) after a meal, enhancing triglyceride accumulation in the muscle. We conclude that also in obese humans the FAT/CD36 protein content in skeletal muscle is dynamically regulated by insulin in vivo on the short term.

Skeletal muscle fatty acid transporter protein expression in type 2 diabetes patients compared with overweight, sedentary men and age-matched, endurance-trained cyclists.

AIM: Membrane fatty acid transporters can modulate the balance between fatty acid uptake and subsequent storage and/or oxidation in muscle tissue. As such, skeletal muscle fatty acid transporter protein expression could play an important role in the etiology of insulin resistance and/or type 2 diabetes. METHODS: In the present study, fatty acid translocase (FAT/CD36), plasma membrane-bound fatty acid-binding protein (FABPpm) and fatty acid transport protein 1 (FATP1) mRNA and protein expression were assessed in muscle tissue obtained from 10 sedentary, overweight type 2 diabetes patients (60 +/- 2 years), 10 sedentary, weight-matched normoglycemic controls (60 +/- 2 years) and 10 age-matched, endurance trained cyclists (57 +/- 1 years). RESULTS: Both FAT/CD36 and FATP1 mRNA and protein expression did not differ between groups. In contrast, FABPpm mRNA and protein expression were approx. 30-40% higher in the trained men compared with the diabetes patients (P < 0.01) and sedentary controls (P < 0.05). CONCLUSIONS: Skeletal muscle FAT/CD36, FABPpm and FATP1 mRNA and protein expression are not up- or downregulated in a sedentary and/or insulin resistant state. In contrast, FABPpm expression is upregulated in the endurance trained state and likely instrumental to allow greater fatty acid oxidation rates.