Endothelial cell CD36 regulates membrane ceramide formation, exosome fatty acid transfer and circulating fatty acid levels.

Endothelial cell (EC) CD36 controls tissue fatty acid (FA) uptake. Here we examine how ECs transfer FAs. FA interaction with apical membrane CD36 induces Src phosphorylation of caveolin-1 tyrosine-14 (Cav-1Y14) and ceramide generation in caveolae. Ensuing fission of caveolae yields vesicles containing FAs, CD36 and ceramide that are secreted basolaterally as small (80-100 nm) exosome-like extracellular vesicles (sEVs). We visualize in transwells EC transfer of FAs in sEVs to underlying myotubes. In mice with EC-expression of the exosome marker emeraldGFP-CD63, muscle fibers accumulate circulating FAs in emGFP-labeled puncta. The FA-sEV pathway is mapped through its suppression by CD36 depletion, blocking actin-remodeling, Src inhibition, Cav-1Y14 mutation, and neutral sphingomyelinase 2 inhibition. Suppression of sEV formation in mice reduces muscle FA uptake, raises circulating FAs, which remain in blood vessels, and lowers glucose, mimicking prominent Cd36-/- mice phenotypes. The findings show that FA uptake influences membrane ceramide, endocytosis, and EC communication with parenchymal cells.

CD36 gene deletion reduces fat preference and intake but not post-oral fat conditioning in mice.

Several findings suggest the existence of a "fatty" taste, and the CD36 fatty acid translocase is a candidate taste receptor. The present study compared fat preference and acceptance in CD36 knockout (KO) and wild-type (WT) mice using nutritive (triglyceride and fatty acid) and nonnutritive (Sefa Soyate oil) emulsions. In two-bottle tests (24 h/day) naive KO mice, unlike WT mice, displayed little or no preference for dilute soybean oil, linoleic acid, or Sefa Soyate emulsions. At high concentrations (2.5-20%), KO mice developed significant soybean oil preferences, although they consumed less oil than WT mice. The postoral actions of fat likely conditioned these preferences. KO mice, like WT mice, learned to prefer a flavored solution paired with intragastric soybean oil infusions. These findings support CD36 mediation of a gustatory component to fat preference but demonstrate that it is not essential for fat-conditioned flavor preferences. The finding that oil-naive KO mice failed to prefer a nonnutritive oil, assumed to provide texture rather than taste cues, requires explanation. Finally, CD36 deletion decreased fat consumption and enhanced the ability of the mice to compensate for the calories provided by their optional fat intake.

CD36-dependent fatty acid uptake regulates expression of peroxisome proliferator activated receptors.

CD36 is an important regulator of lipid metabolism in vivo due to its role in the facilitated uptake of long-chain FAs (fatty acids). CD36-deficient mice display reduced TAG (triacylglycerol) in muscle, but elevated hepatic TAG. Also, insulin sensitivity is enhanced peripherally, while it appears impaired in the liver. Tissues such as muscle, which normally express high levels of CD36, shift to high glucose utilization in CD36 deficiency, so we hypothesized that this shift must involve adaptive changes in the PPAR (peroxisome-proliferator-activated receptor) transcription factors which regulate FA metabolism. To test this, we examined mRNA levels for the three PPAR isoforms in tissues of WT (wild-type) and CD36-deficient mice following the administration of saline, glucose or olive oil by intragastric gavage. Compared with WT mice, CD36-null mice had 5-10-fold increased PPAR mRNA in adipose tissue in the basal state, and did not exhibit diet-induced changes. Correlations between adipose PPAR mRNA abundance and plasma lipids were observed in WT mice, but not in CD36-null mice. The opposite was true for hepatic PPAR mRNA levels, which correlated with plasma FA, TAG and/or glucose only in CD36-null mice. No significant differences were observed in PPAR mRNA levels in the intestine, where CD36 does not impact on FA uptake. The data suggest that CD36 and the PPARs are components of the FA-sensing machinery to respond to changes in FA flux in a tissue-specific manner.

The impact of overexpression and deficiency of fatty acid translocase (FAT)/CD36.

Fatty acid translocase (FAT)/CD36 has been associated with diverse normal and pathologic processes. These include scavenger receptor functions (uptake of apoptotic cells and modified lipid), lipid metabolism and fatty acid transport, adhesion, angiogenesis, modulation of inflammation, transforming growth factor-beta activation, atherosclerosis, diabetes and cardiomyopathy. Although CD36 was identified more than 25 years ago, it is only with the advent of recent genetic technology that in vivo evidence has emerged for its physiologic and pathologic relevance. As these in vivo studies are expanded, we will gain further insight into the mechanism(s) by which CD36 transmits a cellular signal, and this will allow the design of specific therapeutics that impact on a particular function of CD36.

The gene-nutrient-gene loop.

It is increasingly apparent that the genetic influence on the development and severity of a particular phenotype (e.g. diabetes, hyperlipidemia, hypertension, and coronary heart disease) can be strongly modulated by diet. In turn, the response of the phenotype to dietary intervention is determined by the individual genotype. The reviews in this issue provide striking examples of recent progress related to the molecular basis of nutrient-gene interactions. As our understanding of these interactions improves, we should be better equipped to identify individuals at risk of specific pathologies and make a better assessment of the risk involved. Nutritional support could then be tailored to the individual genotype to favour beneficial phenotypic expression or to suppress that leading to pathology and disease.

Role of CD36 in membrane transport and utilization of long-chain fatty acids by different tissues.

The transmembrane glycoprotein CD36 has been identified in isolated cell studies as a putative transporter of long-chain fatty acids. To examine the physiological role of CD36, we studied FA uptake and metabolism by tissues of CD36 null mice after injection with two fatty acid analogs. Compared to controls, uptake was substantially reduced (50-80%) in heart, skeletal muscle, and adipose tissues of null mice. The reduction in uptake was associated with a large decrease in fatty acid incorporation into triglycerides, which could be accounted for by an accumulation of diacylglycerides. Thus CD36 facilitates a major fraction of fatty acid uptake by myocardial, skeletal muscle, and adipose tissues, where it is highly expressed. Its role in other tissues where its expression is low and cell-specific could not be determined in these studies.

Defective fatty acid uptake in the spontaneously hypertensive rat is a primary determinant of altered glucose metabolism, hyperinsulinemia, and myocardial hypertrophy.

Genetic linkage studies implicated deficiency of CD36, a membrane fatty acid (FA) transporter, in the hypertriglyceridemia and hyperinsulinemia of the spontaneously hypertensive rat (SHR). In this study we determined whether loss of CD36 function in FA uptake is a primary determinant of the SHR phenotype. In vivo, tissue distribution of iodinated, poorly oxidized beta-methyliodophenyl pentadecanoic acid (BMIPP) was examined 2 h after its intravenous injection. Fatty acid transport was also measured in vitro over 20 to 120 s in isolated adipocytes and cardiomyocytes obtained from SHR and from a congenic line (SHRchr4) that incorporates a piece of chromosome 4 containing wild-type CD36. SHR heart and adipose tissue exhibited defects in FA uptake and in conversion of diglycerides to triglycerides that are similar to those observed in the CD36 null mouse. However, a key difference in SHR tissues is that fatty acid oxidation is much more severely impaired than fatty acid esterification, which may underlie the 4-5-fold accumulation of free BMIPP measured in SHR muscle. Studies with isolated adipocytes and cardiomyocytes directly confirmed both the defect in FA transport and the fact that it is underestimated by BMIPP. Heart, oxidative muscle, and adipose tissue in the SHR exhibited a large increase in glucose uptake measured in vivo using [(18)F]fluorodeoxyglucose. Supplementation of the diet with short-chain fatty acids, which do not require CD36-facilitated transport, eliminated the increase in glucose uptake, the hyperinsulinemia, and the heart hypertrophy in the SHR. This indicated that lack of metabolic energy consequent to deficient FA uptake is the primary defect responsible for these abnormalities. Hypertension was not alleviated by the supplemented diet suggesting it is unrelated to fuel supply and any contribution of CD36 deficiency to this trait may be more complex to determine. It may be worth exploring whether short-chain FA supplementation can reverse some of the deleterious effects of CD36 deficiency in humans, which may include hypertrophic cardiomyopathy.

Transgenic rescue of defective Cd36 ameliorates insulin resistance in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) display several features of the human insulin-resistance syndromes. Cd36 deficiency is genetically linked to insulin resistance in SHR. We show that transgenic expression of Cd36 in SHR ameliorates insulin resistance and lowers serum fatty acids. Our results provide direct evidence that Cd36 deficiency can promote defective insulin action and disordered fatty-acid metabolism in spontaneous hypertension.

Lipid transporters: membrane transport systems for cholesterol and fatty acids.

Lipophilic molecules can passively diffuse across cell membranes, a process that is driven by the concentration gradient, by availability of acceptors to facilitate desorption from the bilayer, and by cellular metabolism. However, evidence has accumulated that supports the existence of specialized, protein-facilitated membrane transport systems for many lipophilic molecules. This has generated considerable debate regarding why such systems need to exist. The present review summarizes recent developments related to the membrane transport systems for cholesterol and fatty acids, which have been shown to involve structurally related proteins. General similarities of the cholesterol and fatty acid systems to other lipid transport systems (briefly discussed in the Introduction section) are highlighted in the Conclusion section. The overall aim of the present review is to illustrate why lipid transporters are needed in vivo, and how they accomplish specific functions that can not be met by lipid diffusion alone.

Defective uptake and utilization of long chain fatty acids in muscle and adipose tissues of CD36 knockout mice.

The transmembrane protein CD36 has been identified in isolated cell studies as a putative transporter of long chain fatty acids. In humans, an association between CD36 deficiency and defective myocardial uptake of the fatty acid analog 15-(p-iodophenyl)-3-(R, S)-methyl pentadecanoic acid (BMIPP) has been reported. To determine whether this association represents a causal link and to assess the physiological role of CD36, we compared tissue uptake and metabolism of two iodinated fatty acid analogs BMIPP and 15-(p-iodophenyl) pentadecanoic acid (IPPA) in CD36 null and wild type mice. We also investigated the uptake and lipid incorporation of palmitate by adipocytes isolated from both groups. Compared with wild type, uptake of BMIPP and IPPA was reduced in heart (50-80%), skeletal muscle (40-75%), and adipose tissues (60-70%) of null mice. The reduction was associated with a 50-68% decrease in label incorporation into triglycerides and in 2-3-fold accumulation of label in diglycerides. Identical results were obtained from studies of [(3)H]palmitate uptake in isolated adipocytes. The block in diglyceride to triglyceride conversion could not be explained by changes in specific activities of the key enzymes long chain acyl-CoA synthetase and diacylglycerol acyltransferase, which were similar in tissues from wild type and null mice. It is concluded that CD36 facilitates a large fraction of fatty acid uptake by heart, skeletal muscle, and adipose tissues and that CD36 deficiency in humans is the cause of the reported defect in myocardial BMIPP uptake. In CD36-expressing tissues, uptake regulates fatty acid esterification at the level of diacylglycerol acyltransferase by determining fatty acyl-CoA supply. The membrane transport step may represent an important control site for fatty acid metabolism in vivo.

Muscle-specific overexpression of FAT/CD36 enhances fatty acid oxidation by contracting muscle, reduces plasma triglycerides and fatty acids, and increases plasma glucose and insulin.

Increasing evidence has implicated the membrane protein CD36 (FAT) in binding and transport of long chain fatty acids (FA). To determine the physiological role of CD36, we examined effects of its overexpression in muscle, a tissue that depends on FA for its energy needs and is responsible for clearing a major fraction of circulating FA. Mice with CD36 overexpression in muscle were generated using the promoter of the muscle creatine kinase gene (MCK). Transgenic (MCK-CD36) mice had a slightly lower body weight than control litter mates. This reflected a leaner body mass with less overall adipose tissue, as evidenced by magnetic resonance spectroscopy. Soleus muscles from transgenic animals exhibited a greatly enhanced ability to oxidize fatty acids in response to stimulation/contraction. This increased oxidative ability was not associated with significant alterations in histological appearance of muscle fibers. Transgenic mice had lower blood levels of triglycerides and fatty acids and a reduced triglyceride content of very low density lipoproteins. Blood cholesterol levels were slightly lower, but no significant decrease in the cholesterol content of major lipoprotein fractions was measured. Blood glucose was significantly increased, while insulin levels were similar in the fed state and higher in the fasted state. However, glucose tolerance curves, determined at 20 weeks of age, were similar in control and transgenic mice. In summary, the study documented, in vivo, the role of CD36 to facilitate cellular FA uptake. It also illustrated importance of the uptake process in muscle to overall FA metabolism and glucose utilization.

A null mutation in murine CD36 reveals an important role in fatty acid and lipoprotein metabolism.

A null mutation in the scavenger receptor gene CD36 was created in mice by targeted homologous recombination. These mice produced no detectable CD36 protein, were viable, and bred normally. A significant decrease in binding and uptake of oxidized low density lipoprotein was observed in peritoneal macrophages of null mice as compared with those from control mice. CD36 null animals had a significant increase in fasting levels of cholesterol, nonesterified free fatty acids, and triacylglycerol. The increase in cholesterol was mainly within the high density lipoprotein fraction, while the increase in triacylglycerol was within the very low density lipoprotein fraction. Null animals had lower fasting serum glucose levels when compared with wild type controls. Uptake of 3H-labeled oleate was significantly reduced in adipocytes from null mice. However, the decrease was limited to the low ratios of fatty acid:bovine serum albumin, suggesting that CD36 was necessary for the high affinity component of the uptake process. The data provide evidence for a functional role for CD36 in lipoprotein/fatty acid metabolism that was previously underappreciated.

Muscle contractile activity increases fatty acid metabolism and transport and FAT/CD36.

We have examined whether 1) fatty acid (FA) uptake, 2) FA transporter expression, and 3) FA metabolism are increased when the oxidative capacity of skeletal muscle is increased. The oxidative capacities of red and white tibialis anterior and extensor digitorum longus muscles were increased via chronic stimulation (10 Hz, 24 h/day for 7 days). The contralateral muscles served as controls. After 7 days of increased muscle activity 1) palmitate uptake by giant sarcolemmal vesicles was increased twofold (P < 0.05), 2) the expression of FA translocase (FAT)/CD36 was increased at both the mRNA (3.2- to 10-fold) and protein (3.4-fold) levels, and 3) palmitate oxidation and esterification into triacylglycerols and phospholipids were increased 1.5-, 2.7-, and 1.7-fold, respectively (P < 0.05). These data show that when the oxidative capacity of muscle is increased, there is a parallel increase in the rate of FA transport and FA transporters at the sarcolemmal membrane, which is associated with the enhanced expression of the membrane transporter FAT/CD36.

Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats.

The human insulin-resistance syndromes, type 2 diabetes, obesity, combined hyperlipidaemia and essential hypertension, are complex disorders whose genetic basis is unknown. The spontaneously hypertensive rat (SHR) is insulin resistant and a model of these human syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridaemia and hypertension map to a single locus on rat chromosome 4. Here we combine use of cDNA microarrays, congenic mapping and radiation hybrid (RH) mapping to identify a defective SHR gene, Cd36 (also known as Fat, as it encodes fatty acid translocase), at the peak of linkage to these QTLs. SHR Cd36 cDNA contains multiple sequence variants, caused by unequal genomic recombination of a duplicated ancestral gene. The encoded protein product is undetectable in SHR adipocyte plasma membrane. Transgenic mice overexpressing Cd36 have reduced blood lipids. We conclude that Cd36 deficiency underlies insulin resistance, defective fatty acid metabolism and hypertriglyceridaemia in SHR and may be important in the pathogenesis of human insulin-resistance syndromes.

Two antiatherogenic effects of progesterone on human macrophages; inhibition of cholesteryl ester synthesis and block of its enhancement by glucocorticoids.

The effects of progesterone and estradiol on cholesteryl ester (CE) formation by monocyte-derived human macrophages were examined. Formation was assessed from incorporation of 14C-cholesterol during a 20-h incubation with hormone and from that of 3H-oleate (3 h) after hormone removal. Progesterone inhibited cholesterol into CE and decreased CE cellular levels. Inhibition: 1) was reversed by progesterone removal; 2) was independent of the progesterone receptor (not blocked by the receptor antagonist RU40555); and 3) exhibited specific structural requirements; 11alpha-OH-progesterone was inhibitory, whereas its stereoisomer 11beta-OH-progesterone was not. In contrast to progesterone, estradiol was ineffective. We had reported that dexamethasone enhanced CE accumulation by human macrophages (1). In this study, we describe similar effects of the endogenous steroid, cortisol, and of the most widely prescribed glucocorticoid, prednisolone. Both steroids increased CE formation from two folds, in the presence of cholesterol-liposomes, to five folds, in the presence of modified low-density lipoprotein. Progesterone (0.1-1 micromol/L), added during glucocorticoid treatment, blocked this increase. The progesterone block: 1) was duplicated by the steroid receptor inhibitor RU40555; 2) was not reversed by hormone removal; and 3) reflected inhibition of glucocorticoid-induced increases in messenger RNA for acyl-CoA-cholesterol:acyl transferase. Thus, progesterone exerted two effects on macrophages: it acutely inhibited CE formation, and it prevented glucocorticoid-induced increases in acyl-CoA-cholesterol-acyl transferase gene expression and CE synthesis.

Comparison of class B scavenger receptors, CD36 and scavenger receptor BI (SR-BI), shows that both receptors mediate high density lipoprotein-cholesteryl ester selective uptake but SR-BI exhibits a unique enhancement of cholesteryl ester uptake.

Scavenger receptor BI (SR-BI) mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE), a process by which HDL CE is taken into the cell without internalization and degradation of the HDL particle. The biochemical mechanism by which SR-BI mediates the selective uptake of HDL CE is poorly understood. Given that CE transfer will occur to some extent from HDL to protein-free synthetic membranes, one hypothesis is that the role of SR-BI is primarily to tether HDL close to the cell surface to facilitate CE transfer from the particle to the plasma membrane. In the present study, this hypothesis was tested by comparing the selective uptake of HDL CE mediated by mouse SR-BI (mSR-BI) with that mediated by rat CD36 (rCD36), a closely related class B scavenger receptor. Both mSR-BI and rCD36 bind HDL with high affinity, and both receptors mediate HDL CE selective uptake. However, SR-BI mediates selective uptake of HDL CE with a 7-fold greater efficiency than rCD36. HDL CE selective uptake mediated by rCD36 is dependent on HDL binding to the receptor, since a mutation that blocks HDL binding also blocks HDL CE selective uptake. These data lead us to hypothesize that one component of HDL CE selective uptake is the tethering of HDL particles to the cell surface. To explore the molecular domains responsible for the greater efficiency of selective uptake by mSR-BI, we compared binding and selective uptake among mSR-BI, scavenger receptor BII, and various chimeric receptors formed from mSR-BI and rCD36. The results show that the extracellular domain of mSR-BI is essential for efficient HDL CE uptake, but the C-terminal cytoplasmic tail also has a major influence on the selective uptake process.

CD36 expression is altered in retinal pigment epithelial cells of the RCS rat.

The retinal pigment epithelial cell has several important functions, one of which is the phagocytosis of photoreceptor outer segments which are discarded diurnally. We previously provided evidence in human retinal pigment epithelium that CD36, an 88 kDa integral membrane glycoprotein, participates in the phagocytosis of photoreceptor outer segments. Since in the Royal College of Surgeons dystrophic rat, retinal pigment epithelial cells fail to perform this function and as a result the photoreceptor cells degenerate, the expression of CD36 has now been examined by retinal pigment epithelial cells of the dystrophic rat. Consistent with earlier work using human retinal pigment epithelial cells, expression of CD36 by freshly isolated retinal pigment epithelial cells of Long Evans rats was confirmed by immunoblotting and immunocytochemistry with antibody to rat CD36. The protein was also present in lysates of cultured retinal pigment epithelium. Furthermore, with an in vitro phagocytosis assay using 125I-labeled outer segments, it was demonstrated that the binding and ingestion of outer segments by rat retinal pigment epithelial cells was reduced by 64% in the presence of antibodies to rat CD36. In contrast to observations in the Long Evans rat, immunoblotting of retinal pigment epithelial cells isolated from the adult Royal College of Surgeons rat revealed that CD36 protein was not present. This appeared to be a tissue-specific absence since CD36 protein was present in peritoneal macrophages harvested from the adult Royal College of Surgeons rat. A developmental study of CD36 expression also demonstrated an absence of the protein on the day of birth and at 1 and 2 weeks postnatally. By reverse transcriptase-polymerase chain reaction, CD36 mRNA was detected in freshly harvested retinal pigment epithelial cells of the Royal College of Surgeons rat at only PN1, 1 week and 10 days. Significantly, at 2 weeks of age and in the adult Royal College of Surgeons rat. CD36 transcripts were no longer present. Nevertheless, by Northern blot analysis CD36 mRNA was detected in various other tissues shown previously to express CD36. We conclude that in RPE cells of the Royal College of Surgeons rat, CD36 protein is not expressed while CD36 mRNA is present only transiently during postnatal development.

Reversible binding of long-chain fatty acids to purified FAT, the adipose CD36 homolog.

Transport of long-chain fatty acids into rat adipocytes was previously shown to be inhibited by the reactive derivative sulfosuccinimidyl oleate consequent to its binding to a membrane protein FAT, which is homologous to CD36. In this report, the ability of the purified protein to bind native fatty acids was investigated. CD36 was isolated from rat adipocytes by phase partitioning into Triton X-114 followed by chromatography on DEAE and then on wheat germ agglutinin. Fatty acid binding was determined by incubating CD36, solubilized in buffer containing 0.1 Triton X-100, with fatty acids at 37 degrees C, and then by adsorbing the unbound ligand with Lipidex 1,000 at 0 degrees C. Bovine serum albumin was used as a positive control and gelatin, a protein that does not bind fatty acids, as a negative control. Measurements with albumin yielded reproducible binding values which were not altered by the presence of 0.1% Triton X-100. Under the same conditions, gelatin yielded reproducibly negative measurements that did not differ significantly from zero. CD36 bound various long-chain fatty acids at low ligand to protein ratios. Warming the protein-FA-Lipidex mixture to 37 degrees C removed the FA off the protein. Thus, binding was reversible and distinct from the palmitoylation of the protein known to occur on an extracellular domain. Comparison of the predicted secondary sequence of CD36 with that of human muscle fatty acid binding protein suggested that a potential binding site for the fatty acid on CD36 may exist in its extracellular segment between residues 127 and 279.

Expression of the CD36 homolog (FAT) in fibroblast cells: effects on fatty acid transport.

An adipocyte membrane glycoprotein, (FAT), homologous to human CD36, has been previously implicated in the binding/transport of long-chain fatty acids. It bound reactive derivatives of long-chain fatty acids and binding was specific and associated with significant inhibition of fatty acid uptake. Tissue distribution of the protein and regulation of its expression were also consistent with its postulated role. In this report, we have examined the effects of FAT expression on rates and properties of fatty acid uptake by Ob17PY fibroblasts lacking the protein. Three clones (P21, P22, and P25) were selected based on FAT mRNA and protein levels. Cell surface labeling could be demonstrated with the anti-CD36 antibody FITC-OKM5. In line with this, the major fraction of immunoreactive FAT was associated with the plasma membrane fraction. Assays of oleate and/or palmitate uptake demonstrated higher rates in the three FAT-expressing clones, compared to cells transfected with the empty vector. Clone P21, which had the highest protein levels on Western blots, exhibited the largest increase in transport rates. Fatty acid uptake in FAT-expressing P21 cells reflected two components, a phloretin-sensitive high-affinity saturable component with a Km of 0.004 microM and a basal phloretin-insensitive component that was a linear function of unbound fatty acid. P21 cells incorporated more exogenous fatty acid into phospholipids, indicating that binding of fatty acids was followed by their transfer into the cell and that both processes were increased by FAT expression. The data support the interpretation that FAT/CD36 functions as a high-affinity membrane receptor/transporter for long-chain fatty acids.