Myocardial Long-chain Fatty Acid Research Articles

Fatty acid metabolism in adipocytes: functional analysis of fatty acid transport proteins 1 and 4

The role of fatty acid transport protein 1 (FATP1) and FATP4 in facilitating adipocyte fatty acid metabolism was investigated using stable FATP1 or FATP4 knockdown (kd) 3T3-L1 cell lines derived from retrovirus-delivered short hairpin RNA (shRNA). Decreased expression of FATP1 or FATP4 did not affect preadipocyte differentiation or the expression of FATP1 (in FATP4 kd), FATP4 (in FATP1 kd), fatty acid translocase, acyl-coenzyme A synthetase 1, and adipocyte fatty acid binding protein but did lead to increased levels of peroxisome proliferator-activated receptor gamma and CCAAT/enhancer binding protein alpha. Both FATP1 and FATP4 kd adipocytes exhibited reduced triacylglycerol deposition and corresponding reductions in diacylglycerol and monoacylglycerol levels compared with control cells. FATP1 kd adipocytes displayed an approximately 25% reduction in basal (3)H-labeled fatty acid uptake and a complete loss of insulin-stimulated (3)H-labeled fatty acid uptake compared with control adipocytes. In contrast, FATP4 kd adipocytes as well as HEK-293 cells overexpressing FATP4 did not display any changes in fatty acid influx. FATP4 kd cells exhibited increased basal lipolysis, whereas FATP1 kd cells exhibited no change in lipolytic capacity. Consistent with reduced triacylglycerol accumulation, FATP1 and FATP4 kd adipocytes exhibited enhanced 2-deoxyglucose uptake compared with control adipocytes. These findings define unique and distinct roles for FATP1 and FATP4 in adipose fatty acid metabolism.

AMPK-mediated increase in myocardial long-chain fatty acid uptake critically depends on sarcolemmal CD36

CD36, also named fatty acid translocase, has been identified as a putative membrane transporter for long-chain fatty acids (LCFA). In the heart, contraction-induced 5′ AMP-activated protein kinase (AMPK) signaling regulates cellular LCFA uptake through translocation of CD36 and possibly of other LCFA transporters from intracellular storage compartments to the sarcolemma. In this study, isolated cardiomyocytes from CD36 +/+- and CD36 −/− mice were used to investigate to what extent basal and AMPK-mediated LCFA uptake are CD36-dependent. Basal LCFA uptake was not altered in CD36 −/− cardiomyocytes, most likely resulting from a (1.8-fold) compensatory upregulation of fatty acid-transport protein-1. The stimulatory effect of contraction-mimetic stimuli, oligomycin (2.5-fold) and dipyridamole (1.6-fold), on LCFA uptake into CD36 +/+ cardiomyocytes was almost completely lost in CD36 −/− cardiomyocytes, despite that AMPK signaling was fully intact. CD36 is almost entirely responsible for AMPK-mediated stimulation of LCFA uptake in cardiomyocytes, indicating a pivotal role for CD36 in mediating changes in cardiac LCFA fluxes.

Enhanced sarcolemmal FAT/CD36 content and triacylglycerol storage in cardiac myocytes from obese zucker rats.

In obesity, the development of cardiomyopathy is associated with the accumulation of myocardial triacylglycerols (TAGs), possibly stemming from elevation of myocardial long-chain fatty acid (LCFA) uptake. Because LCFA uptake is regulated by insulin and contractions, we examined in cardiac myocytes from lean and obese Zucker rats the effects of insulin and the contraction-mimetic agent oligomycin on the initial rate of LCFA uptake, subcellular distribution of FAT/CD36, and LCFA metabolism. In cardiac myocytes from obese Zucker rats, under basal conditions, FAT/CD36 was relocated to the sarcolemma at the expense of intracellular stores. In addition, the LCFA uptake rate, LCFA esterification rate into TAGs, and the intracellular unesterified LCFA concentration each were significantly increased. All these metabolic processes were normalized by the FAT/CD36 inhibitor sulfo-N-succinimidyloleate, indicating its antidiabetic potential. In cardiac myocytes isolated from lean rats, in vitro administration of insulin induced the translocation of FAT/CD36 to the sarcolemma and stimulated initial rates of LCFA uptake and TAG esterification. In contrast, in myocytes from obese rats, insulin failed to alter the subcellular localization of FAT/CD36 and the rates of LCFA uptake and TAG esterification. In cardiac myocytes from lean and obese animals, oligomycin stimulated the initial rates of LCFA uptake and oxidation, although oligomycin only induced the translocation of FAT/CD36 to the sarcolemma in lean rats. The present results indicate that in cardiac myocytes from obese Zucker rats, a permanent relocation of FAT/CD36 to the sarcolemma is responsible for myocardial TAG accumulation. Furthermore, in vitro these cardiac myocytes, although sensitive to contraction-like stimulation, were completely insensitive to insulin, as the basal conditions in hyperinsulinemic, obese animals resemble the insulin-stimulated condition in lean littermates.

Myocardial carnitine palmitoyltransferase I expression and long-chain fatty acid oxidation in fetal and newborn lambs.

Carnitine palmitoyltransferase I (CPT I) catalyzes the conversion of acyl-CoA to acylcarnitine at the outer mitochondrial membrane and is a key enzyme in the control of long-chain fatty acid (LC-FA) oxidation. Because myocardial LC-FA oxidation increases dramatically after birth, we determined the extent to which CPT I expression contributes to these changes in the perinatal lamb. We measured the steady-state level of transcripts of the CPT1A and CPT1B genes, which encode the liver (L-CPT I) and muscle CPT I (M-CPT I) isoforms, respectively, as well as the amount of these proteins, their total activity, and the amount of carnitine in left ventricular tissue from fetal and newborn lambs. We compared these data with previously obtained myocardial FA oxidation rates in vivo in the same model. The results showed that CPT1B was already expressed before birth and that total CPT I expression transiently increased after birth. The protein level of M-CPT I was high throughout development, whereas that of L-CPT I was only transiently upregulated in the first week after birth. The total CPT I activity in vitro also increased after birth. However, the increase in myocardial FA oxidation measured in vivo (112-fold) by far exceeded the increase in gene expression (2.2-fold), protein amount (1.1-fold), and enzyme activity (1.2-fold) in vitro. In conclusion, these results stress the importance of substrate supply per se in the postnatal increase in myocardial FA oxidation. M-CPT I is expressed throughout perinatal development, making it a primary target for metabolic modulation of myocardial FA oxidation.

Influence of CD36 Deficiency on Heart Disease in Children

The physiological role of the CD36 molecule in pediatric heart disease has not been fully investigated. The CD36 antigen in platelets and monocytes was measured by flow cytometry in 189 patients with various heart diseases; 15 (7.9%) had a diagnosis of CD36 deficiency (type I: 2[1 boy, 1 girl], type II: 13 [6 boys, 7 girls]). The prevalence in each heart disease was as follows: group A (congenital heart disease) 7.6% (9/118, type II: 9 [6 boys, 3 girls]); group B (myocardial disease) 20.0% (3/15, I: 1 girl, II: 2[1 boy, 1 girl]), group C (Kawasaki disease) 4.9% (2/41, II: 2 [1 boy, 1 girl]), group D (arrhythmia): 6.7% (1/15, I: 1 boy). Three patients in group B had transient myocardial damage, which was thought to be related to abnormal myocardial long-chain fatty acid metabolism. The frequency of CD36 deficiency in childhood heart disease was almost identical to that of healthy individuals. Some patients with CD36 deficiency may be susceptible to myocardial damage in the presence of disadvantageous conditions, such as serious infections or massive steroid therapy.

Characterization of altered myocardial fatty acid metabolism in patients with inherited cardiomyopathy.

Inherited defects in myocardial long-chain fatty acid metabolism are increasingly recognized as a cause of cardiomyopathy and sudden death in children. To evaluate whether the phenotypic expression of these genetic diseases could be delineated using positron emission tomography (PET), 11 patients with inherited defects in fatty acid metabolism were evaluated and results were compared with those of 6 nonaffected siblings. Myocardial perfusion, myocardial oxygen consumption (MVO2), and long-chain fatty acid metabolism were determined noninvasively with PET using quantitative mathematical models. There were no differences in haemodynamics, perfusion, MVO2 or plasma substrate levels between groups. Patients with defects in enzymes of fatty acid beta-oxidation (acyl-CoA dehydrogenase and 3-hydroxyacyl-CoA dehydrogenase deficiencies) (n = 5) had diminished myocardial palmitate oxidation compared with healthy siblings (3.2 +/- 3.0 vs. 13.0 +/- 5.6 nmol/g per min, p < 0.03) and a decrease in the percentage of MVO2 accounted for by palmitate (2% +/- 3% vs. 9% +/- 5%, p < 0.04). In these patients, extracted palmitate was shunted into a slow-turnover compartment (predominantly reflecting esterification to triglycerides) with expansion of palmitate in that pool (185 +/- 246 compared with 27 +/- 67 nmol/g in healthy siblings,p < 0.02). In contrast, myocardium of patients with carnitine deficiency (n = 6) (all on oral carnitine therapy) had normal palmitate extraction but expansion of the interstitial/cytosolic fatty acid pool (617 +/- 399 vs. 261 +/- 73 nmol/g in healthy siblings, p < 0.04), suggesting different mechanisms for handling upstream fatty acyl intermediates. Thus, PET can be used to noninvasively assess abnormal myocardial handling of fatty acids in patients with inherited defects of metabolism. This approach should be useful in the assessment of altered myocardial fatty acid metabolism associated with cardiomyopathy as well as for evaluating the efficacy of therapeutic interventions in affected patients.

Defect in human myocardial long-chain fatty acid uptake is caused by FAT/CD36 mutations

Because of the importance of long-chain fatty acids (LCFAs) as a myocardial energy substrate, myocardial LCFA metabolism has been of particular interest for the understanding of cardiac pathophysiology. Recently, by using radiolabeled LCFA analogues, myocardial LCFA metabolism has been clinically evaluated, which revealed a total defect of myocardial LCFA accumulation in a small number of subjects. The mechanism for the cellular LCFA uptake process is still disputable, but recent results suggest that fatty acid translocase (FAT)/CD36 is a transporter in the heart. In the present study, we analyzed mutations and protein expression of the FAT/CD36 gene in 47 patients who showed total lack of the accumulation of a radiolabeled LCFA analogue in the heart. All the patients carried two mutations in the FAT/CD36 gene, and expression of the FAT/CD36 protein was not detected on either platelet or monocyte membranes. Our results showed the link between mutations of the FAT/CD36 gene and a defect in the accumulation of LCFAs in the human heart.—Tanaka, T., T. Nakata, T. Oka, T. Ogawa, F. Okamoto, Y. Kusaka, K. Sohmiya, K. Shimamoto, and K. Itakura. Defect in human myocardial long-chain fatty acid uptake is caused by FAT/CD36 mutations. J. Lipid Res. 2001. 42: 751–759.

CD36 Deficiency has Little Influence on the Pathophysiology of Hypertrophic Cardiomyopathy

CD36 is homologous with myocardial long-chain fatty acid (LCFA) binding protein and has been suggested to relate to myocardial fatty acid metabolism. Myocardial scintigraphy with iodine-123 15- (p -iodophenyl)-3-(R, S)-methylpentadecanoic acid (BMIPP) revealed an impairment in LCFA metabolism chiefly in the hypertrophic myocardium in hypertrophic cardiomyopathy (HCM). Recently, the incidence of CD36 deficiency has been reported to be high in HCM patients, and CD36 deficiency was proposed as an etiology of hereditary HCM. However, the pathophysiological effect of CD36 deficiency on HCM has not been fully investigated. We analysed the expression of CD36 antigens on both platelets and monocytes obtained from 82 patients with HCM using two-color flow cytometry. Among the study patients, seven patients (8.5%) demonstrated type II CD36 deficiency, whereas type I CD36 deficiency was not detected. Two of 23 patients (8.7%) with a family history of HCM and five of 59 patients (8.5%) without a family history of HCM showed type II CD36 deficiency respectively. Contrary to the previous report, three of 53 patients with asymmetric septal hypertrophy (ASH) (5.7%) and four of 29 patients without ASH (13.8%) showed CD36 deficiency. Moreover, clinical characteristics, scintigraphic findings, echocardiographic data, and hemodynamic findings disclosed no significant differences between the HCM patients showing normal CD36 expression and those with CD36 deficiency. The incidence of CD36 deficiency in HCM patients is not higher than in the general population. Therefore, CD36 deficiency is not a characteristic factor of HCM and has little influence on the pathyphysiology of HCM.

Beta-Methyl-p-(123I)-iodophenyl pentadecanoic acid single-photon emission computed tomography in cardiomyopathy.

beta-Methyl-p-(123I)-iodophenyl pentadecanoic acid (BMIPP) is one of the branched-chain free fatty acids, which has suitable characteristics for myocardial SPECT because of higher uptake and longer retention in the myocardium. Recent advances of BMIPP myocardial SPECT for evaluating cardiomyopathy were reviewed. BMIPP defects were observed in 80% patients with hypertrophic cardiomyopathy (HCM). Moreover, BMIPP uptake was reduced at sites that corresponded with hypertrophic areas, where thallium uptake was increased. The correlations between severity score and septal wall thickness and LV function were better with BMIPP SPECT, suggesting that BMIPP is more suitable for the assessment of myocardial integrity in HCM. The dissociation between BMIPP and thallium defects was not observed frequently in dilated cardiomyopathy (DCM). We carried out BMIPP myocardial SPECT to evaluate the therapeutic effects of co-enzyme Q10 on DCM patients. Hearts to the mediastinum ratio and BMIPP defect scores were significantly decreased after co-enzyme Q10 treatment. BMIPP myocardial SPECT was confirmed to be sensitive in evaluating the therapeutic effect for the perspective of metabolic SPECT imaging. Recently, a lack of myocardial uptake of BMIPP has been found in a small subset of patients (0.3%-1.2%). Cardiac radionuclide imaging using BMIPP and 18F-FDG were performed on patients with type I CD36 deficiency. The percent dose uptake of 18F-FDG was significantly higher than in normal controls. CD functions as a major myocardial long-chain fatty acid transporter and its absence may lead to a compensatory upregulation of myocardial glucose uptake. An increased frequency of CD36 deficiency was demonstrated in cardiomyopathy. Therefore, fatty acid transport proteins and their related gene defects in relation to BMIPP uptake may become an important issue in the future.

CD36 abnormality and impaired myocardial long-chain fatty acid uptake in patients with hypertrophic cardiomyopathy.

Some patients with hypertrophic cardiomyopathy (HCM) demonstrate abnormal myocardial long-chain fatty acid (LCFA) metabolism. However, the exact mechanism involved is unknown. Recently, it was proposed that myocardial cells take up LCFAs via a specific mechanism, in which the CD36 molecule has been implicated as a possible candidate molecule. In addition, a high prevalence of CD36 deficiency was also found in a small number of HCM patients. Accordingly, the investigation of abnormality of the CD36 molecule in a large number of HCM patients may be useful in finding the possible cause of HCM. Moreover, the analysis of myocardial LCFA uptake in patients with molecular abnormalities may be helpful in understanding the possible function of this molecule. In this study, in order to discover the relationship between HCM and the CD36 molecular abnormality, the expression level of platelet CD36 and CD36 cDNA in 55 HCM patients was analyzed. Twelve patients showed negligible (<5%) CD36 expression on their platelets. Among them, one was found to be homozygous for the C-478-->T substitution and 6 were heterozygous for the C-478-->T substitution. In 9 patients, CD36 was expressed by less than 50% of the platelets. One of them was found to be heterozygous for the C-478-->T substitution. Two other patients were also found to be heterozygous for this point mutation, although their platelets expressed CD36. Thus, 23 out of 55 (41.8%) HCM patients had negligible (<5%) or reduced (<50%) levels of CD36 expression on platelets, or had a point mutation of CD36 cDNA. These 55 HCM patients were also evaluated with myocardial scintigraphy both for LCFA uptake and perfusion, which showed a moderate to severe discrepancy between myocardial LCFA accumulation and myocardial perfusion in 95.5% of the patients (21/23). On the other hand, 70% of the patients with normal (>90%) CD36 expression (14/20) did not show any severe discrepancies between myocardial LCFA accumulation and myocardial perfusion. These data could suggest that abnormal myocardial LCFA metabolism seen in HCM patients may be related to abnormality of the CD36 molecule, and that abnormalities of this molecule may be linked to the cause of some types of HCM.

Is CD36 Deficiency an Etiology of Hereditary Hypertrophic Cardiomyopathy?

Hereditary hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease, but the genetic defects are still unclear in many cases. Reduced myocardial long-chain fatty acid (LCFA) uptake has been demonstrated in patients with some types of HCM. In addition, a possible relationship between a shift of myocardial substrate utilization and cardiac hypertrophy has been suggested by experimental studies. Myocardial uptake of LCFAs occurs via a specific transporter, which is homologous with human CD36. CD36 deficiency has also been reported in some individuals, and is transmitted as an autosomal dominant trait like HCM. In this study, we analyzed CD36 in 47 patients with HCM [29 with asymmetric septal hypertrophy (ASH) and 18 without ASH], 11 patients with dilated cardiomyopathy (DCM), and 26 patients with pressure-overload cardiac hypertrophy. Eleven patients (37.9%) who had HCM with ASH, one (9.1%) with DCM, and two (7.7%) with pressure-overload hypertrophy showed CD36 deficiency, while none of the HCM patients without ASH had CD36 deficiency. One patient who had HCM with ASH and CD36 deficiency showed no myocardial LCFA uptake, although myocardial perfusion was normal. Reduced myocardial LCFA uptake despite normal myocardial perfusion was demonstrated in the other HCM patients with ASH and CD36 deficiency. Based on the high prevalence of CD36 deficiency in HCM patients with ASH, we hypothesize that this deficiency might be one etiology of hereditary HCM.

Isolation of myocardial membrane long-chain fatty acid-binding protein: Homology with a rat membrane protein implicated in the binding or transport of long-chain fatty acids

Abnormal myocardial long-chain fatty acid uptake is suspected of being involved in certain types of heart disease, but the mechanism by which the heart takes up long-chain fatty acids remains unclear. The sulfo-N-succinimidyl derivatives of long-chain fatty acids have been reported to undergo covalent binding to a membrane protein and to irreversibly inhibit the transport of long-chain fatty acids by rat adipocytes (Harmon et al., 1991). It has been suggested that the membrane protein bound by these derivatives is a candidate transporter for long-chain fatty acids in adipocytes. However, myocardial membrane long-chain fatty acid-binding proteins have not yet been fully investigated. Rat hearts were isolated and perfused with a sulfo-N-succinimidyl derivative of tritium-labeled palmitate ([3H]SSP). Then the [3H]SSP-binding protein was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography and histological autoradiography. Myocardial palmitic acid uptake was examined after pretreatment of isolated perfused rat hearts with SSP. The SSP-binding protein was isolated from bovine hearts by successive chromatography, and the amino acid sequences of lysylendopeptidase-digested peptide fragments were determined. SDS-PAGE autoradiography revealed that [3H]SSP bound to an 85-90 kDa protein derived from the myocardial microsomal fraction, and histological autoradiography demonstrated that [3H]SSP radioactivity was localized to the myocardial cell membrane. Pre-incubation with SSP inhibited palmitic acid uptake by isolated perfused rat hearts. A [3H]SSP-binding protein was also found in canine and bovine hearts, and was isolated from the bovine cardiac membrane fraction. Amino acid sequencing revealed that four peptide fragments showed strong sequence homology with rat adipocyte membrane protein, which is implicated in the binding or transport of long-chain fatty acids (Abumrad et al., 1993). We conclude that the SSP-binding protein is localized to the myocardial cell membrane and might be involved in the uptake or transport of long-chain fatty acids.

Effect of sulfo-N-succinimidyl palmitate on the rat heart: Myocardial long-chain fatty acid uptake and cardiac hypertrophy

Abnormal long-chain fatty acid metabolism has been suggested as having a role in the genesis of certain cardiac diseases, and depressed myocardial long-chain fatty acid uptake has been clinically demonstrated in some patients with hypertrophic cardiomyopathy. However, the site where long-chain fatty acid metabolism is affected in cardiomyopathy remains unclear. Although cardiac hypertrophy is reported to be induced in rats by a fat-free diet, little is known of the consequences of depressed myocardial long-chain fatty acid uptake. Sulfo-N-succinimidyl derivatives of long-chain fatty acids have been shown to irreversibly inhibit long-chain fatty acid transport. To investigate the possible linkage of abnormal long-chain fatty acid uptake with cardiac hypertrophy, myocardial long-chain fatty acid uptake was blocked in rats using a sulfo-N-succinimidyl derivative of palmitate (SSP). SSP was intraperitoneally administered to rats for 12 weeks, and its effects on physiological parameters, and cardiac morphology were studied, SSP treatment (20 mg/kg) caused a 12% increase in heart weight (663.7 +/- 33.6 mg in controls v 741.2 +/- 26.5 mg after SSP treatment) and an 11% increase in the heart weight to body weight ratio (2.46 +/- 0.10 in controls v 2.72 +/- 0.17 after SSP) without any significant change of body weight. No significant differences were observed in blood pressure, heart rate, and serum hormones (insulin and triiodothyronine) between the control and SSP-treated groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Competition between palmitate and ketone bodies as fuels for the heart: study with positron emission tomography

To test the ability of ketone bodies to inhibit myocardial fatty acid oxidation in vivo, the myocardial clearance kinetics of [1-11C]palmitate was assessed with positron emission tomography in six fasted volunteers and six instrumented dogs, studied repeatedly before and during infusion of 3-hydroxybutyrate (17 mumol.kg-1 x min-1). With the use of multiexponential fitting of tissue time-activity curves, the size, half time (T1/2), and index of the early rapid phase of 11C myocardial clearance, reflecting palmitate oxidation, were calculated. In humans, the relative size (-28%, P < 0.001) and index (-37%, P < 0.01) of the early rapid phase decreased significantly during infusion of 3-hydroxybutyrate, consistent with decreased fatty acid oxidation. Paradoxically, T1/2 decreased from 10.1 +/- 1.6 to 7.4 +/- 1.1 min (P < 0.01). To elucidate possible mechanisms, multiple coronary arteriovenous samples were obtained from the dogs to assess the efflux of oxidized and nonmetabolized tracer. Infusion of 3-hydroxybutyrate resulted in decreased myocardial [11C]CO2 production (-40%, P < 0.05) and reduced palmitate retention (-38%, P < 0.05). In three dogs, the arteriovenous difference in radiolabeled palmitate became negative 10 min after injection, indicating backdiffusion of nonmetabolized tracer from the myocardium. Thus a steady-state infusion of 3-hydroxybutyrate, resulting in physiological plasma levels, alters [1-11C]palmitate kinetics in vivo by decreasing myocardial long-chain fatty acid oxidation and by increasing backdiffusion of nonmetabolized tracer.