Plasma Membrane Fatty Acid-binding Protein Research Articles

Relationships Between Placental Lipid Activated/Transport-Related Factors and Macrosomia in Healthy Pregnancy.

To assess associations between infants with macrosomia and placental expression levels of lipid activated/transport-related factors and umbilical cord blood lipid concentrations in healthy pregnancy. We conducted a case-control study of 38 macrosomic neonates (MS group) and 39 normal-birth-weight newborns (NC group) in a healthy pregnancy. Cord blood lipid levels were measured by automatic biochemical analyzer, mRNA and protein expression levels of placental lipid activated/transport-related factors were determined by real-time polymerase chain reaction and western blot, respectively. Compared with NC group, cord blood total cholesterol (TC), low-density lipoprotein cholesterol (LDLC), and non-esterified fatty acid (NEFA) concentrations were decreased in the MS group. The mRNA and protein expression levels of placental peroxisome proliferator-activated receptors (PPARα, PPARγ), plasma membrane fatty acid-binding protein (FABPpm), and fatty acid translocase (FAT/CD36) were significantly higher in the MS group than the NC group. And there was a weak positive correlation between the expression of PPARγ, FABP4, and FABP3 mRNA in the placenta and the HDLC (rs = 0.439; P = 0.005), NEFA (rs = 0.342; P = 0.041), and TG (rs = 0.349; P = 0.034) levels in the cord blood in the MS group, respectively. After multivariate adjustment, the logistic regression analysis showed that high placental PPARα (adjusted odds ratio [AOR] = 3.022; 95% confidence interval [CI] 1.032-8.853) and FAT/CD36 (AOR=2.989; 95%CI 1.029-8.679) and low LDLC concentration in the cord blood (AOR=0.246; 95%CI 0.080-0.759) increased the risk of macrosomia. The increased PPARα and FAT/CD36 expression levels may influence the occurrence of fetal macrosomia through regulating placental lipid transport.

Maternal diet high in Omega-3 fatty acids upregulate genes involved in neurotrophin signalling in fetal brain during pregnancy in C57BL/6 mice

Neurotrophins play a critical role in the development, maintenance, and proper function of the brain. We investigated the effects of maternal diet high in omega (n)-3 polyunsaturated fatty acids (PUFA) on fatty acids composition and the gene expression of neurotrophins in fetal brain at different gestation stages. Female C57BL/6 mice (7-weeks old, n = 8/group) were fed a diet containing high, low or very low n-3 PUFA (9, 3 or 1% w/w, respectively), with an n-6:n-3 PUFA of 5:1, 20:1 and 40:1, respectively, for two weeks before mating and throughout pregnancy. Animals were sacrificed during pregnancy at gestation day 12.5 and 18.5 to determine placental and fetal-brain fatty acids composition. The gene expressions of endothelial lipase (EL) and plasma membrane fatty acid-binding protein (FABPpm) were measured in the placenta, while major facilitator superfamily domain-containing 2a (Mfsd2a), brain-derived neurotrophic factor (BDNF), tropomyosin-receptor kinase (TrK)-B, and cAMP response element-binding protein (CREB) were measured in fetal-brain, using qPCR. The protein expression of phosphorylated CREB (pCREB) was determined using ELISA. The high n-3 PUFA diet increased the mRNA expression of EL, FABPpm, and Mfsd2a at both gestation days, compared to other groups. Docosahexaenoic acid (DHA) and total n-3 PUFA were significantly higher in the high n-3 PUFA group, compared to the other groups at both gestation days. The high n-3 PUFA diet also increased the mRNA expressions of BDNF, TrKB and CREB, as well as the protein concentration of pCREB as gestation progressed, compared to the other groups. Our findings show for the first time that maternal diet high in n-3 PUFA increased the mRNA expression of Mfsd2a, which correlated with an increase in DHA accretion in the fetal-brain. A diet high in n-3 PUFA increased neurotrophin signalling in fetal-brain as gestation progressed, demonstrating the importance of n-3 PUFA during brain development.

Effects of nutritional status and diet composition on fatty acid transporters expression in zebrafish (Danio rerio)

This study investigated the expression of zebrafish (Danio rerio) fatty acid transporters, such as scavenger receptor CD36, plasma membrane fatty acid-binding protein (FABPpm), and fatty acid transport protein family (FATPs), in responses to nutritional status and diet composition. For diet composition study, three isonitrogenous (43% crude protein) purified diets were formulated to contain 6%, 12% and 18% crude lipid levels. Meanwhile five isonitrogenous and isoenergetic purified diets were formulated to contain different lipid sources including fish oil (FO; rich in n-3 HUFA), palmitic acid (PA; rich in C16:0), olive oil (OO; rich in C18:1n-9), sunflower oil (SO; rich in C18:2n-6) or perilla oil (PO; rich in C18:3n-3) as the sole lipid source. Results showed that liver CD36, FABPpm-a, FABPpm-b, FATP1-a and FATP6, intestine CD36, FABPpm-a and FABPpm-b, and muscle CD36 and FATP1-b were significantly increased during postprandial period. During starvation, liver CD36, FABPpm-a, FABPpm-b and FATP1-a, intestine FABPpm-a, FABPpm-b, FATP4 and FATP6, and muscle FATP1-b, FATP4 and FATP6 were significantly increased. The expression of some fatty acid transporters (including liver CD36 and FABPpm-b, intestine FABPpm-a, and muscle CD36, FABPpm-a, FATP1-a, FATP2 and FATP4) was up-regulated by refeeding, while the expression of other fatty acid transporters (liver FABPpm-a and intestine FATP1-a, FATP4 and FATP6) was down-regulated by refeeding. The expression of several fatty acid transporters (liver FATP2 and FATP4, and intestine CD36, FATP2 and FATP4) was significantly increased by high-lipid diet, whereas CD36, FATP1-b, FATP2 and FATP4 expression in the muscle were reduced by increasing dietary lipid level. Compared with FO group, the muscle of fish fed the diet with OO had a higher CD36 mRNA abundance, while the muscle of fish fed the diet with OO and PO had a higher FATP1-b expression. Compared with the FO group, the hepatic FATP2 mRNA was significantly increased in the PA group, while the intestine FATP2 mRNA was significantly increased in the SO and PO groups. In addition, intestine FATP4 expression in the PA and OO groups was lower compared with the FO group. The results indicate that zebrafish fatty acid transporters play a central role in coordinating the mobilization of fuel substrates in responses to nutritional status and diet composition.

Dynamic role of the transmembrane glycoprotein CD36 (SR-B2) in cellular fatty acid uptake and utilization

The widely expressed transmembrane glycoprotein, cluster of differentiation 36 (CD36), a scavenger receptor class B protein (SR-B2), serves many functions in lipid metabolism and signaling. Here, we review CD36's role in facilitating cellular long-chain fatty acid uptake across the plasma membrane, particularly in heart and skeletal muscles. CD36 acts in concert with other membrane proteins, such as peripheral plasma membrane fatty acid-binding protein, and is an intracellular docking site for cytoplasmic fatty acid-binding protein. The cellular fatty-acid uptake rate is governed primarily by the presence of CD36 at the cell surface, which is regulated by the subcellular vesicular recycling of CD36 from endosomes to the plasma membrane. CD36 has been implicated in dysregulated fatty acid and lipid metabolism in pathophysiological conditions, particularly in high-fat diet-induced insulin resistance and diabetic cardiomyopathy. Current research is exploring signaling pathways and vesicular trafficking routes involving CD36 to identify metabolic targets to manipulate the cellular utilization of fatty acids. Because of its rate-controlling function in the use of fatty acids in the heart and muscle, CD36 would be a preferable target to protect myocytes against lipotoxicity. Despite a poor understanding of its mechanism of action, CD36 has emerged as a pivotal membrane protein involved in whole-body lipid homeostasis.

Short-term starvation in silver pomfret (Pampus argenteus): molecular effects on lipid mobilization and utilization

To investigate the effects of short-term starvation on lipid metabolism, juvenile silver pomfret (Pampus argenteus) with an initial weight of 18.1 ± 1.53 g were starved for 6 days. Fish were sampled on days 0, 2, 4 and 6 of starvation (S0, S2, S4 and S6, respectively) and then analysed. The results showed that short-term starvation induced a significant decrease in visceral index, hepatosomatic index. The crude lipid content was also significantly decreased by 42.22% and 82.96% on S2 and S6 respectively. In addition, short-term starvation significantly decreased the plasma triacylglycerol, non-esterified fatty acid and low-density lipoprotein cholesterol. Moreover, short-term starvation significantly increased the expressions of hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) at the mRNA and protein levels in the muscle and the ATGL expression at the protein level in the liver. The expressions of fatty acid translocase (CD36) and plasma membrane fatty acid-binding protein (FABPpm) at the protein level in the muscle were significantly increased by short-term starvation, while the expressions of fatty acid transport protein 1 (FATP1), FABPpm and lipoprotein lipase (LPL) at the protein level in the liver were significantly increased by short-term starvation. Furthermore, the expressions of peroxisome proliferator activated receptor (PPAR) α and PPARγ at the mRNA and protein levels in the muscle were significantly elevated by short-term starvation. These findings suggested that short-term starvation increased lipid mobilization and utilization possibly through activation of lipolysis-related genes (HSL and ATGL), lipid uptake-related genes (LPL, CD36, FATP1 and FABPpm) and PPARs.

C2C12 골격근 세포에서 FAT/CD36 발현 조절에 있어 Insulin-like growth factor-I이 미치는 영향

본 연구에서는 C2C12 근육 세포의 분화 과정에 있어 IGF-I이 지방산의 수송을 담당하는 지방산 수송체인 FAT/CD36의 mRNA 및 단백질 발현에 미치는 영향에 대해 알아보았다. 그 결과 근육세포의 분화 과정에 있어 FAT/CD36의 단백질과 mRNA 발현이 분화 시간 의존적으로 유의하게 증가하였으며, IGF-I의 처리에 의해서도 유의하게 조절되었음을 알 수 있었다. 이는 IGF-I이 골격근 세포의 성장 및 분화를 촉진하여 근육 관련 유전자들의 발현을 조절하는 기능뿐만 아니라, 골격근의 주요 에너지원으로 사용되는 지방산의 수송을 담당하는 FAT/CD36의 발현에도 영향을 미친다는 것으로 해석할 수 있겠다. 향후 IGF-I이 골격근 세포에서 FAT/CD36의 발현에 영향을 미침으로써 골격근의 지방산 흡수와 산화율을 조절하는지, 그에 따라 지방대사에 어떠한 영향을 미치는지에 대한 연구가 필요할 것이며, 이와 관련된 신호전달 체계 및 기전에 대한 연구도 진행 되어야 할 것이다. Fatty acid transporters are key mediators of skeletal muscle lipid metabolism. Several protein groups have been implicated in cellular long-chain fatty acid uptake or oxidation, including fatty acid transporter proteins (FATPs), the plasma membrane fatty acid-binding protein (FABPpm), and the fatty acid translocase (FAT/CD36). FAT/CD36 is highly expressed in skeletal muscle and known to be regulated by various factors such as exercise and hormones. Insulin-like growth factor-I (IGF-I) is a well-known regulator of skeletal muscle cells. However, it has not been studied whether there is any interaction between IGF-I and FAT/CD36 in skeletal muscle cells. In this study, the effects of IGF-I treatment on FAT/CD36 induction were examined. Differentiated C2C12 cells were treated with 20 ng/ml of IGF-I at different time points. Treatment of C2C12 cells with IGF-I resulted in increased FAT/CD36 mRNA and protein expression. After 24 and 48 hr of IGF-I treatment, FAT/CD36 mRNA increased 89% and 24% respectively. The increase of both proteins returned to the control level after 72 hr of IGF-I treatment, suggesting that the FAT/CD36 gene is regulated pretranslationally by IGF-I in skeletal muscle cells. These results suggest that IGF-I can regulate the expression of FAT/CD36 in skeletal muscle cells. In conclusion, IGF-I induces a rapid transcriptional modification of the FAT/CD36 gene in C2C12 skeletal muscle cells and has modulating effects on fatty acid uptake proteins as well as oxidative proteins.

Acute cold and exercise training up-regulate similar aspects of fatty acid transport and catabolism in house sparrows (Passer domesticus).

Summit maximum thermoregulatory metabolic rate (Msum) and maximum exercise metabolic rate (MMR) both increase in response to acute cold or exercise training in birds. Because lipids are the main fuel supporting both thermogenesis and exercise in birds, adjustments to lipid transport and catabolic capacities may support elevated energy demands from cold and exercise training. To examine a potential mechanistic role for lipid transport and catabolism in organismal cross-training effects (exercise effects on both exercise and thermogenesis, and vice versa), we measured enzyme activities and mRNA and protein expression in pectoralis muscle for several key steps of lipid transport and catabolism pathways in house sparrows (Passer domesticus) during acute exercise and cold training. Both training protocols elevated pectoralis protein levels of fatty acid translocase (FAT/CD36), cytosolic fatty acid-binding protein, and citrate synthase (CS) activity. However, mRNA expression of FAT/CD36 and both mRNA and protein expression of plasma membrane fatty acid-binding protein did not change for either training group. CS activities in supracoracoideus, leg and heart, and carnitine palmitoyl transferase (CPT) and β-hydroxyacyl CoA-dehydrogenase activities in all muscles did not vary significantly with either training protocol. Both Msum and MMR were significantly positively correlated with CPT and CS activities. These data suggest that up-regulation of trans-sarcolemmal and intramyocyte lipid transport capacities and cellular metabolic intensities, along with previously documented increases in body and pectoralis muscle masses and pectoralis myostatin (a muscle growth inhibitor) levels, are common mechanisms underlying the training effects of both exercise and shivering in birds.

Plasma membrane phospholipase A2controls hepatocellular fatty acid uptake and is responsive to pharmacological modulation: implications for nonalcoholic steatohepatitis

Excess hepatic fat accumulation leads to nonalcoholic steatohepatitis (NASH), a serious threat to health for which no effective treatment is available. However, the mechanism responsible for fatty acid uptake by hepatocytes remains unclear. Using the human hepatocyte-derived tumor cell line HepG2, we found that fatty acid influx is mediated by a heterotetrameric plasma membrane protein complex consisting of plasma membrane fatty acid-binding protein, caveolin-1, CD36, and calcium-independent membrane phospholipase A2 (iPLA2β). Blocking iPLA2β with the bile acid-phospholipid conjugate ursodeoxycholate-lysophosphatidylethanolamide (UDCA-LPE) caused the dissociation of the complex, thereby inhibiting fatty acid influx (IC50 47 μM), and suppressed the synthesis of all subunits through a reduction in lysophosphatidylcholine from 8.0 to 3.5 μmol/mg of protein and corresponding depletion of phosphorylated c-Jun N-terminal kinase. These findings were substantiated by an observed 56.5% decrease in fatty acid influx in isolated hepatocytes derived from iPLA2β-knockout mice. Moreover, steatosis and inflammation were abrogated by UDCA-LPE treatment in a cellular model of NASH. Thus, iPLA2β acts as an upstream checkpoint for mechanisms that regulate fatty acid uptake, and its inhibition by UDCA-LPE qualifies this nontoxic compound as a therapeutic candidate for the treatment of NASH.-Stremmel, W., Staffer, S., Wannhoff, A., Pathil, A., Chamulitrat, W. Plasma membrane phospholipase A2 controls hepatocellular fatty acid uptake and is responsive to pharmacological modulation: implications for nonalcoholic steatohepatitis.

Seasonal upregulation of fatty acid transporters in flight muscles of migratory white-throated sparrows (Zonotrichia albicollis)

Endurance flights of birds, some known to last several days, can only be sustained by high rates of fatty acid uptake by flight muscles. Previous research in migratory shorebirds indicates that this is made possible in part by very high concentrations of cytosolic heart-type fatty acid binding protein (H-FABP), which is substantially upregulated during migratory seasons. We investigated if H-FABP and other components of muscle fatty acid transport also increase during these seasons in a passerine species, the white-throated sparrow (Zonotrichia albicollis). Fatty acid translocase (FAT/CD36) and plasma-membrane fatty acid binding protein (FABPpm) are well characterized mammalian proteins that facilitate transport of fatty acid through the muscle membrane, and in this study they were identified for the first time in birds. We used quantitative PCR to measure mRNA of FAT/CD36, FABPpm and H-FABP and immunoblotting to measure protein expression of FABPpm and H-FABP in the pectoralis muscles of sparrows captured in migratory (spring, fall) and non-migratory (winter) seasons. During migratory seasons, mRNA expression of these genes increased 70-1000% above wintering levels, while protein expression of H-FABP and FABPpm increased 43% and 110% above wintering levels. Activities of key metabolic enzymes, 3-hydroxyacyl-CoA-dehydrogenase (HOAD), carnitine palmitoyl transferase II (CPT II), and citrate synthase (CS) also increased (90-110%) in pectoralis muscles of migrant birds. These results support the hypothesis that enhanced protein-mediated transport of fatty acids from the circulation into muscle is a key component of the changes in muscle biochemistry required for migration in birds.

Functional Analysis of Long-chain Acyl-CoA Synthetase 1 in 3T3-L1 Adipocytes

ACSL1 (acyl-CoA synthetase 1), the major acyl-CoA synthetase of adipocytes, has been proposed to function in adipocytes as mediating free fatty acid influx, esterification, and storage as triglyceride. To test this hypothesis, ACSL1 was stably silenced (knockdown (kd)) in 3T3-L1 cells, differentiated into adipocytes, and evaluated for changes in lipid metabolism. Surprisingly, ACSL1-silenced adipocytes exhibited no significant changes in basal or insulin-stimulated long-chain fatty acid uptake, lipid droplet size, or tri-, di-, or monoacylglycerol levels when compared with a control adipocyte line. However, ACSL1 kd adipocytes displayed a 7-fold increase in basal and a approximately 15% increase in forskolin-stimulated fatty acid efflux without any change in glycerol release, indicating a role for the protein in fatty acid reesterification following lipolysis. Consistent with this proposition, ACSL1 kd cells exhibited a decrease in activation and phosphorylation of AMP-activated protein kinase and its primary substrate acetyl-CoA carboxylase. Moreover, ACSL1 kd adipocytes displayed an increase in phosphorylated protein kinase C and phosphorylated JNK, attenuated insulin signaling, and a decrease in insulin-stimulated glucose uptake. These findings identify a primary role of ACSL1 in adipocytes not in control of lipid influx, as previously considered, but in lipid efflux and fatty acid-induced insulin resistance.

Etomoxir-induced partial carnitine palmitoyltransferase-I (CPT-I) inhibition in vivo does not alter cardiac long-chain fatty acid uptake and oxidation rates

Although CPT-I (carnitine palmitoyltransferase-I) is generally regarded to present a major rate-controlling site in mitochondrial beta-oxidation, it is incompletely understood whether CPT-I is rate-limiting in the overall LCFA (long-chain fatty acid) flux in the heart. Another important site of regulation of the LCFA flux in the heart is trans-sarcolemmal LCFA transport facilitated by CD36 and FABPpm (plasma membrane fatty acid-binding protein). Therefore, we explored to what extent a chronic pharmacological blockade of the LCFA flux at the level of mitochondrial entry of LCFA-CoA would affect sarcolemmal LCFA uptake. Rats were injected daily with saline or etomoxir, a specific CPT-I inhibitor, for 8 days at 20 mg/kg of body mass. Etomoxir-treated rats displayed a 44% reduced cardiac CPT-I activity. Sarcolemmal contents of CD36 and FABPpm, as well as the LCFA transport capacity, were not altered in the hearts of etomoxir-treated versus control rats. Furthermore, rates of LCFA uptake and oxidation, and glucose uptake by cardiac myocytes from etomoxir-treated rats were not different from control rats, neither under basal nor under acutely induced maximal metabolic demands. Finally, hearts from etomoxir-treated rats did not display triacylglycerol accumulation. Therefore CPT-I appears not to present a major rate-controlling site in total cardiac LCFA flux. It is likely that sarcolemmal LCFA entry rather than mitochondrial LCFA-CoA entry is a promising target for normalizing LCFA flux in cardiac metabolic diseases.

11β-HSD1 inhibition improves triglyceridemia through reduced liver VLDL secretion and partitions lipids toward oxidative tissues

Tissue-specific alterations in 11beta-hydroxysteroid dehydrogenase (HSD) type 1 activity, which amplifies glucocorticoid action, are thought to contribute to some of the metabolic complications of obesity. The present study tested whether hypertriglyceridemia is one such complication by investigating the effects of an 11beta-HSD1 inhibitor (compound A, 3 mgxkg(-1)xday(-1), 21 days) on triglyceride (TG) metabolism in a rat model of diet-induced obesity. The dose of compound A used did not affect food intake or final body weight. Compound A improved fasting triglyceridemia (-42%) through a robust reduction (-41%) in hepatic TG secretion rate, without change in plasma TG clearance rate. Uptake of TG-derived fatty acids was, however, increased in oxidative tissues, including red gastrocnemius (+47%), heart (+39%), and brown adipose tissue (BAT, +46%) at the expense of the liver, with a concomitant increase in plasma membrane fatty acid-binding protein. Lipid oxidation products were increased in red gastrocnemius (+35%) and heart (+33%), as were levels of uncoupling protein 1 mRNA in BAT (+48%), and carnitine palmitoyltransferase 1 activity tended to be increased in some oxidative tissues. These findings demonstrate that pharmacological inhibition of 11beta-HSD1 at a dose that does not affect food intake improves triglyceridemia by reducing hepatic very low density lipoprotein-TG secretion, with a shift in the pattern of TG-derived fatty acid uptake toward oxidative tissues, in which lipid accumulation is prevented by increased lipid oxidation.

Two weeks of high-intensity aerobic interval training increases the capacity for fat oxidation during exercise in women

Our aim was to examine the effects of seven high-intensity aerobic interval training (HIIT) sessions over 2 wk on skeletal muscle fuel content, mitochondrial enzyme activities, fatty acid transport proteins, peak O(2) consumption (Vo(2 peak)), and whole body metabolic, hormonal, and cardiovascular responses to exercise. Eight women (22.1 +/- 0.2 yr old, 65.0 +/- 2.2 kg body wt, 2.36 +/- 0.24 l/min Vo(2 peak)) performed a Vo(2 peak) test and a 60-min cycling trial at approximately 60% Vo(2 peak) before and after training. Each session consisted of ten 4-min bouts at approximately 90% Vo(2 peak) with 2 min of rest between intervals. Training increased Vo(2 peak) by 13%. After HIIT, plasma epinephrine and heart rate were lower during the final 30 min of the 60-min cycling trial at approximately 60% pretraining Vo(2 peak). Exercise whole body fat oxidation increased by 36% (from 15.0 +/- 2.4 to 20.4 +/- 2.5 g) after HIIT. Resting muscle glycogen and triacylglycerol contents were unaffected by HIIT, but net glycogen use was reduced during the posttraining 60-min cycling trial. HIIT significantly increased muscle mitochondrial beta-hydroxyacyl-CoA dehydrogenase (15.44 +/- 1.57 and 20.35 +/- 1.40 mmol.min(-1).kg wet mass(-1) before and after training, respectively) and citrate synthase (24.45 +/- 1.89 and 29.31 +/- 1.64 mmol.min(-1).kg wet mass(-1) before and after training, respectively) maximal activities by 32% and 20%, while cytoplasmic hormone-sensitive lipase protein content was not significantly increased. Total muscle plasma membrane fatty acid-binding protein content increased significantly (25%), whereas fatty acid translocase/CD36 content was unaffected after HIIT. In summary, seven sessions of HIIT over 2 wk induced marked increases in whole body and skeletal muscle capacity for fatty acid oxidation during exercise in moderately active women.

Regulation of Sarcolemmal Transport of Substrates in the Healthy and Diseased Heart

Long-chain fatty acids and glucose are the predominant substrates for cardiac metabolic energy production. While in the healthy heart there is a distinctive and very finely tuned balance between the utilization of these metabolic substrates, in chronic cardiac disease this balance is upset to the use of primarily glucose (e.g., cardiac hypertrophy and failure) or primarily fatty acids (e.g., diabetic cardiomyopathy). Cardiac substrate preference is regulated not only at the level of mitochondrial oxidation (Randle cycle) but also at the level of sarcolemmal uptake of substrates. The latter occurs by translocation of specific substrate transporters, namely fatty acid translocase/CD36 and plasma membrane fatty acid-binding protein (FABPpm) to regulate fatty acid transport, and GLUT4 to regulate glucose transport, from intracellular storage pools to the sarcolemma. Both insulin and cardiac muscle contractions increase the cellular uptake of fatty acids and glucose simultaneously by these mechanisms. Although the signal transduction pathways involved in eliciting substrate transporter trafficking have only partly been disclosed, recent studies indicate the feasibility of selective recruitment of either CD36 or GLUT4 to the sarcolemma, thereby increasing the uptake of a single class of substrates and thus altering the substrate preference of cardiac muscle cells. As a result, selective modulation of the sarcolemmal localization of fatty acid- and/or glucose transporters holds promise as a therapeutic tool to rectify a disruption of the cardiac substrate balance occurring in chronic cardiac disease.

Studies of plasma membrane fatty acid-binding protein and other lipid-binding proteins in human skeletal muscle.

The first putative fatty acid transporter identified was plasma membrane fatty acid-binding protein (FABPpm). Later it was demonstrated that this protein is identical to the mitochondrial isoform of the enzyme aspartate aminotransferase. In recent years data from several cell types have emerged, indicating that FABPpm plays a role in the transport of long-chain saturated and unsaturated fatty acids. In the limited number of studies in human skeletal muscle it has been demonstrated that dietary composition and exercise training can influence the content of FABPpm. Ingestion of a fat-rich diet induces an increase in FABPpm protein content in human skeletal muscle in contrast to the decrease seen during consumption of a carbohydrate-rich diet. A similar effect of a fat-rich diet is also observed for cytosolic fatty acid-binding protein and fatty acid translocase/CD36 protein expression. Exercise training up regulates FABPpm protein content in skeletal muscle, but only in male subjects; no significant differences were observed in muscle FABPpm content in a cross-sectional study of female volunteers of varying training status, even though muscle FABPpm content did not depend on gender in the untrained state. A higher utilization of plasma long-chain fatty acids during exercise in males compared with females could explain the gender-dependent influence of exercise training on FABPpm. The mechanisms involved in the regulation of the function and expression of FABPpm protein remain to be clarified.

Regulation of cardiac long-chain fatty acid and glucose uptake by translocation of substrate transporters

Cardiac uptake of long-chain fatty acids (FA) is mediated predominantly by two membrane-associated proteins, the 43-kDa plasma membrane fatty acid-binding protein (FABPpm) and the 88-kDa fatty acid translocase/CD36 (FAT/CD36). While FABPpm is present constitutively in the sarcolemma, FAT/CD36 is recycled between an intracellular membrane compartment and the sarcolemma. Since the amount of sarcolemmal FAT/CD36 is a major determinant of cellular FA uptake, understanding of the regulation of its recycling is likely to provide new insights into altering substrate preference of the heart. FAT/CD36 recycling displays a remarkable similarity with that of the two glucose transporters (GLUT) in the heart, GLUT1 and GLUT4. Translocation of all three transporters is induced by insulin and by contraction, which stimuli activate distinct signalling cascades. The insulin pathway involves phosphatidylinositol-3 kinase (PI3K) whilst the contraction pathway is dependent on AMP-activated protein kinase (AMPK). For the identification of additional protein components involved in the regulation of FAT/CD36 recycling, valuable lessons can be learned from GLUT1 and GLUT4 recycling. Especially GLUT4 recycling is an intensively studied process in which a number of signalling proteins, both upstream and downstream from PI3 K and AMPK, have been identified, as well as proteins that are part of the translocation machinery involving Rab GTPases and soluble N-ethylmaleimide attachment protein receptors (SNAREs). Comparison of the magnitude of the effects of insulin and contraction on substrate uptake and on transporter appearance in the sarcolemma have revealed that FAT/CD36 recycling resembles GLUT1 recycling more closely than that of GLUT4. This pinpoints the recycling compartment and excludes a pre-endosomal storage compartment as the intracellular storage site for FAT/CD36. Further research will probably establish whether FAT/CD36 translocation is (partly) coupled to that of one or both GLUTs or, alternatively, whether it is a distinct process that also can be induced independently of GLUT1 or GLUT4 movement. In the latter case, a unique set of proteins would be dedicated to FAT/CD36 recycling, which would then provide an attractive target for manipulating cardiac substrate preference.

Palmitate movement across red and white muscle membranes of rainbow trout.

We examined the movement of [3H]palmitate across giant sarcolemmal vesicles prepared from red and white muscle of rainbow trout (Oncorhynchus mykiss). Red and white muscle fatty acid carriers have similar affinities for palmitate (apparent Km = 26 +/- 6 and 33 +/- 8 nM, respectively); however, red muscle has a higher maximal uptake compared with white muscle (Vmax = 476 +/- 41 vs. 229 +/- 23 pmol.mg protein-1.s-1, respectively). Phloretin (250 microM) inhibited palmitate influx in red and white muscle vesicles by approximately 40%, HgCl2 (2.5 mM) inhibited palmitate uptake by 20-30%, and the anion-exchange inhibitor DIDS (250 microM) inhibited palmitate influx in red and white muscle vesicles by approximately 15 and 30%, respectively. Western blot analysis of red and white muscle vesicles did not detect a mammalian-type fatty acid transporter (FAT); however, preincubation of vesicles with sulfo-N-succinimidyloleate, a specific inhibitor of FAT in rats, reduced palmitate uptake in red and white muscle vesicles by approximately 15 and 25%, respectively. A mammalian-type plasma membrane fatty acid-binding protein was identified in trout muscle using Western blotting, but the protein differed in size between red and white muscle. At low concentrations of free palmitate (2.5 nM), addition of high concentrations (111 microM total) of oleate (18:0) caused approximately 50% reduction in palmitate uptake by red and white muscle vesicles, but high concentrations (100 microM) of octanoate (8:0) caused no inhibition of uptake. Five days of aerobic swimming at approximately 2 body lengths/s and 9 days of chronic cortisol elevation in vivo, both of which stimulate lipid metabolism, had no effect on the rate of palmitate movement in red or white muscle vesicles.

Aging is associated with elevated muscle triglyceride content and increased insulin-stimulated fatty acid uptake.

The purpose of the present study was to examine the utilization of fatty acids (FA) and muscle substrates by skeletal muscle in young, middle-aged, and old adult rats under hyperglycemic and hyperinsulinemic conditions. Male Fischer 344 x Brown Norway rats aged 5, 15, or 24 mo underwent hindlimb perfusion with a medium of 20 mM glucose, 1 mM palmitate, 1,000 microU/ml insulin, [1-14C]palmitate, and [3-3H]glucose. Glucose uptake and palmitate delivery were similar among age groups. Palmitate uptake and oxidation as well as muscle protein concentration of fatty acid translocase (FAT/CD36) and plasma membrane fatty acid-binding protein (FABPPM) were significantly increased (P < or = 0.05) in 24- vs. 5- and 15-mo-old animals. Compared with 5- and 15-mo-old animals, pre- and postperfusion muscle triglyceride (TG) levels were significantly (P < 0.05) elevated 72-145% in red and 112-129% in white muscles of 24-mo-old animals. Palmitate uptake was associated with total preperfusion TG concentration (r2 = 0.27, P < 0.05) and total TG synthesis rate (r2 = 0.68, P < 0.05). These results indicate that, under insulin-stimulated conditions, FA uptake is significantly increased in old animals, which is associated with increased rates of TG synthesis and may contribute to the accumulation of TG in muscle of old animals.

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.

Exercise and insulin increase muscle fatty acid uptake by recruiting putative fatty acid transporters to the sarcolemma

Skeletal muscle metabolic energy, needed to maintain contractile activity, is mainly obtained from glucose and long-chain fatty acids. Recent studies have revealed a remarkable parallel between the regulation of uptake of glucose and fatty acids by muscle, in that each is mediated by sarcolemmal transporters that are recruited from an intracellular storage site. The focus of this review is to describe newly obtained insights on the recruitment of fatty acid transporters and their malfunctioning in diabetes. The major fatty acid transporter involved is fatty acid translocase (CD36). Translocation of this protein to the membrane is triggered by muscle contraction and by insulin, and presumably occurs from distinct intracellular pools. This resembles the well documented exercise and insulin-induced recruitment of glucose transporter-4. Whether another transporter, plasma membrane fatty acid-binding protein, is also subject to such recycling is not yet clear. In a rodent model of insulin-dependent (type 1) diabetes, the increased rate of muscle fatty acid uptake could be associated with an increased total amount of fatty acid translocase (CD36). In a model of non-insulin dependent (type 2) diabetes, this increased rate could be associated with a permanent relocalization of fatty acid translocase to the sarcolemma. These findings indicate a pivotal role for the membrane transporter fatty acid translocase in the exercise and insulin-induced increases of muscle fatty acid uptake and utilization, and suggest that malfunctioning of the cellular recycling of fatty acid translocase is involved in the etiology of insulin resistance and type 2 diabetes.