High insulin levels are required for FAT/CD36 plasma membrane translocation and enhanced fatty acid uptake in obese Zucker rat hepatocytes

In myocytes and adipocytes, insulin increases fatty acid translocase (FAT)/CD36 translocation to the plasma membrane (PM), enhancing fatty acid (FA) uptake. Evidence links increased hepatic FAT/CD36 protein amount and gene expression with hyperinsulinemia in animal models and patients with fatty liver, but whether insulin regulates FAT/CD36 expression, amount, distribution, and function in hepatocytes is currently unknown. To investigate this, FAT/CD36 protein content in isolated hepatocytes, subfractions of organelles, and density-gradient isolated membrane subfractions was analyzed in obese and lean Zucker rats by Western blotting in liver sections by immunohistochemistry and in hepatocytes by immunocytochemistry. The uptake of oleate and oleate incorporation into lipids were assessed in hepatocytes at short time points (30-600 s). We found that FAT/CD36 protein amount at the PM was higher in hepatocytes from obese rats than from lean controls. In obese rat hepatocytes, decreased cytoplasmatic content of FAT/CD36 and redistribution from low- to middle- to middle- to high-density subfractions of microsomes were found. Hallmarks of obese Zucker rat hepatocytes were increased amount of FAT/CD36 protein at the PM and enhanced FA uptake and incorporation into triglycerides, which were maintained only when exposed to hyperinsulinemic conditions (80 mU/l). In conclusion, high insulin levels are required for FAT/CD36 translocation to the PM in obese rat hepatocytes to enhance FA uptake and triglyceride synthesis. These results suggest that the hyperinsulinemia found in animal models and patients with insulin resistance and fatty liver might contribute to liver fat accumulation by inducing FAT/CD36 functional presence at the PM of hepatocytes.     

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

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

Sarcolemmal FAT/CD36 in human skeletal muscle colocalizes with caveolin-3 and is more abundant in type 1 than in type 2 fibers

FAT/CD36 is a transmembrane protein that is thought to facilitate cellular long-chain fatty acid uptake. However, surprisingly little is known about the localization of FAT/CD36 in human skeletal muscle. By confocal immunofluorescence microscopy, we demonstrate high FAT/CD36 expression in endothelial cells and weaker but significant FAT/CD36 expression in sarcolemma in human skeletal muscle. No apparent intracellular staining was observed in the muscle cells. There are indications in the literature that caveolae may be involved in the uptake of fatty acids, possibly as regulators of FAT/CD36 or other fatty acid transporters. We show that in sarcolemma, FAT/CD36 colocalizes with the muscle-specific caveolae marker protein caveolin-3, suggesting that caveolae may regulate cellular fatty acid uptake by FAT/CD36. Furthermore, we provide evidence that FAT/CD36 expression is significantly higher in type 1 compared with type 2 fibers, whereas caveolin-3 expression is significantly higher in type 2 fibers than in type 1 fibers.     

Opposite regulation of CD36 ubiquitination by fatty acids and insulin: effects on fatty acid uptake

FAT/CD36 is a membrane scavenger receptor that facilitates long chain fatty acid uptake by muscle. Acute increases in membrane CD36 and fatty acid uptake have been reported in response to insulin and contraction. In this study we have explored protein ubiquitination as one potential mechanism for the regulation of CD36 level. CD36 expressed in Chinese hamster ovary (CHO) or HEK 293 cells was found to be polyubiquitinated via a process involving both lysines 48 and 63 of ubiquitin. Using CHO cells expressing the insulin receptor (CHO/hIR) and CD36, it is shown that addition of insulin (100 nm, 10 and 30 min) significantly reduced CD36 ubiquitination. In contrast, ubiquitination was strongly enhanced by fatty acids (200 microm palmitate or oleate, 2 h). Similarly, endogenous CD36 in C2C12 myotubes was ubiquitinated, and this was enhanced by oleic acid treatment, which also reduced total CD36 protein in cell lysates. Insulin reduced CD36 ubiquitination, increased CD36 protein, and inhibited the opposite effects of fatty acids on both parameters. These changes were paralleled by changes in fatty acid uptake, which could be blocked by the CD36 inhibitor sulfosuccinimidyl oleate. Mutation of the two lysine residues in the carboxyl-terminal tail of CD36 markedly attenuated ubiquitination of the protein expressed in CHO cells and was associated with increased CD36 level and enhanced oleate uptake and incorporation into triglycerides. In conclusion, fatty acids and insulin induce opposite alterations in CD36 ubiquitination, modulating CD36 level and fatty acid uptake. Altered CD36 turnover may contribute to abnormal fatty acid uptake in the insulin-resistant muscle.     

A novel function for fatty acid translocase (FAT)/CD36: involvement in long chain fatty acid transfer into the mitochondria

Fatty acid translocase (FAT)/CD36 is a long chain fatty acid transporter present at the plasma membrane, as well as in intracellular pools of skeletal muscle. In this study, we assessed the unexpected presence of FAT/CD36 in both subsarcolemmal and intermyofibril fractions of highly purified mitochondria. Functional assessments demonstrated that the mitochondria could bind (14)C-labeled palmitate, but could only oxidize it in the presence of carnitine. However, the addition of sulfo-N-succinimidyl oleate, a known inhibitor of FAT/CD36, resulted in an 87 and 85% reduction of palmitate oxidation in subsarcolemmal and intermyofibril fractions, respectively. Further studies revealed that maximal carnitine palmitoyltransferase I (CPTI) activity in vitro was inhibited by succinimidyl oleate (42 and 48% reduction). Interestingly, CPTI immunoprecipitated with FAT/CD36, indicating a physical pairing. Tissue differences in mitochondrial FAT/CD36 protein follow the same pattern as the capacity for fatty acid oxidation (heart > red muscle > white muscle). Additionally, chronic stimulation of hindlimb muscles (7 days) increased FAT/CD36 expression and also resulted in a concomitant increase in mitochondrial FAT/CD36 content (46 and 47% increase). Interestingly, with acute electrical stimulation of hindlimb muscles (30 min), FAT/CD36 expression was not altered, but there was an increase in the mitochondrial content of FAT/CD36 compared with the non-stimulated control limb (35 and 37% increase). Together, these data suggest a role for FAT/CD36 in mitochondrial long chain fatty acid uptake and demonstrate system flexibility to match FAT/CD36 mitochondrial content with an increased capacity for fatty acid oxidation, possibly involving translocation of FAT/CD36 to the mitochondria.     

Sulfo-N-succinimidyl Oleate (SSO) Inhibits Fatty Acid Uptake and Signaling for Intracellular Calcium via Binding CD36 Lysine 164: SSO ALSO INHIBITS OXIDIZED LOW DENSITY LIPOPROTEIN UPTAKE BY MACROPHAGES*

FAT/CD36 is a multifunctional glycoprotein that facilitates long-chain fatty acid (FA) uptake by cardiomyocytes and adipocytes and uptake of oxidized low density lipoproteins (oxLDL) by macrophages. CD36 also mediates FA-induced signaling to increase intracellular calcium in various cell types. The membrane-impermeable sulfo-N-hydroxysuccinimidyl (NHS) ester of oleate (SSO) irreversibly binds CD36 and has been widely used to inhibit CD36-dependent FA uptake and signaling to calcium. The inhibition mechanism and whether SSO modification of CD36 involves the FA-binding site remain unexplored. CHO cells expressing human CD36 were SSO-treated, and the protein was pulled down, deglycosylated, and resolved by electrophoresis. The CD36 band was extracted from the gel and digested for analysis by mass spectrometry. NHS derivatives react with primary or secondary amines on proteins to yield stable amide or imide bonds. Two oleoylated peptides, found only in SSO-treated samples, were identified with high contribution and confidence scores as carrying oleate modification of Lys-164. Lysine 164 lies within a predicted CD36 binding domain for FA and oxLDL. CHO cells expressing CD36 with mutated Lys-164 had impaired CD36 function in FA uptake and FA-induced calcium release from the endoplasmic reticulum, supporting the importance of Lys-164 for both FA effects. Furthermore, consistent with the importance of Lys-164 for oxLDL binding, SSO inhibited oxLDL uptake by macrophages. In conclusion, SSO accesses Lys-164 in the FA-binding site on CD36, and initial modeling of this site is presented. The data suggest competition between FA and oxLDL for access to the CD36 binding pocket.     

Acute regulation of fatty acid uptake involves the cellular redistribution of fatty acid translocase

We used muscle contraction, which increases fatty acid oxidation, as a model to determine whether fatty acid transport is acutely regulated by fatty acid translocase (FAT/CD36). Palmitate uptake by giant vesicles, obtained from skeletal muscle, was increased by muscle contraction. Kinetic studies indicated that muscle contraction increased V(max), but K(m) remained unaltered. Sulfo-N-succinimidyl oleate, a specific inhibitor of FAT/CD36, fully blocked the contraction-induced increase in palmitate uptake. In giant vesicles from contracting muscles, plasma membrane FAT/CD36 was also increased in parallel with the increase in long chain fatty acid uptake. Further studies showed that like GLUT-4, FAT/CD36 is located in both the plasma membrane and intracellularly (endosomally). With muscle contraction, FAT/CD36 at the surface of the muscle was increased, while concomitantly, FAT/CD36 in the intracellular pool was reduced. Similar responses were observed for GLUT-4. We conclude that fatty acid uptake is subject to short term regulation by muscle contraction and involves the translocation of FAT/CD36 from intracellular stores to the sarcolemma, analogous to the regulation of glucose uptake by GLUT-4.     

FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires plasma membrane rafts

We previously reported that lipid rafts are involved in long-chain fatty acid (LCFA) uptake in 3T3-L1 adipocytes. The present data show that LCFA uptake does not depend on caveolae endocytosis because expression of a dominant negative mutant of dynamin had no effect on uptake of [3H]oleic acid, whereas it effectively prevented endocytosis of cholera toxin. Isolation of detergent-resistant membranes (DRMs) from 3T3-L1 cell homogenates revealed that FAT/CD36 was expressed in both DRMs and detergent-soluble membranes (DSMs), whereas FATP1 and FATP4 were present only in DSMs but not DRMs. Disruption of lipid rafts by cyclodextrin and specific inhibition of FAT/CD36 by sulfo-N-succinimidyl oleate (SSO) significantly decreased uptake of [3H]oleic acid, but simultaneous treatment had no additional or synergistic effects, suggesting that both treatments target the same mechanism. Indeed, subcellular fractionation demonstrated that plasma membrane fatty acid translocase (FAT/CD36) is exclusively located in lipid rafts, whereas intracellular FAT/CD36 cofractionated with DSMs. Binding assays confirmed that [3H]SSO predominantly binds to FAT/CD36 within plasma membrane DRMs. In conclusion, our data strongly suggest that FAT/CD36 mediates raft-dependent LCFA uptake. Plasma membrane lipid rafts might control LCFA uptake by regulating surface availability of FAT/CD36.     

Gut expression and regulation of FAT/CD36: possible role in fatty acid transport in rat enterocytes

Fatty acid translocase (FAT)/CD36 is one of several putative plasma membrane long-chain fatty acid (LCFA) transport proteins; however, its role in intestinal absorption of LCFA is unknown. We hypothesized that FAT/CD36 would be differentially expressed along the longitudinal axis of the gut and during intestinal development, suggesting specificity of function. We found that intestinal mucosal FAT/CD36 mRNA levels varied by anatomic location along the longitudinal gut axis: stomach 45 +/- 7, duodenum 173 +/- 29, jejunum 238 +/- 17, ileum 117 +/- 14, and colon 9 +/- 1% (means +/- SE with 18S mRNA as control). FAT/CD36 protein levels were also higher in proximal compared with distal intestinal mucosa. Mucosal FAT/CD36 mRNA was also regulated during intestinal maturation, with a fourfold increase from neonatal to adult animals. In addition, FAT/CD36 mRNA levels and enterocyte LCFA uptake were rapidly downregulated by intraduodenal oleate infusion. These findings suggest that FAT/CD36 plays a role in the uptake of LCFA by small intestinal enterocytes. This may have important implications in understanding fatty acid absorption in human physiological and pathophysiological conditions.     

Ca2+/calmodulin-dependent protein kinase II inhibition reduces myocardial fatty acid uptake and oxidation after myocardial infarction

An AMP-activated kinase (AMPK) signaling pathway is activated during myocardial ischemia and promotes cardiac fatty acid (FA) uptake and oxidation. Similarly, the multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is also triggered by myocardial ischemia, but its function in FA metabolism remains unclear. Here, we explored the role of CaMKII in FA metabolism during myocardial ischemia by investigating the effects of cardiac CaMKII on AMPK-acetyl-CoA carboxylase (ACC), malonyl CoA decarboxylase (MCD), and FA translocase cluster of differentiation 36 (FAT/CD36), as well as cardiac FA uptake and oxidation. Moreover, we tested whether CaMKII and AMPK are binding partners. We demonstrated that diseased hearts from patients with terminal ischemic heart disease displayed increased phosphorylation of CaMKII, AMPK, and ACC and increased expression of MCD and FAT/CD36. AC3-I mice, which have a genetic myocardial inhibition of CaMKII, had reduced gene expression of cardiac AMPK. In post-MI (myocardial infarction) AC3-I hearts, AMPK-ACC phosphorylation, MCD and FAT/CD36 levels, cardiac FA uptake, and FA oxidation were significantly decreased. Notably, we demonstrated that CaMKII interacted with AMPK α1 and α2 subunits in the heart. Additionally, AC3-I mice displayed significantly less cardiac hypertrophy and apoptosis 2 weeks post-MI. Overall, these findings reveal a unique role for CaMKII inhibition in repressing FA metabolism by interacting with AMPK signaling pathways, which may represent a novel mechanism in ischemic heart disease.     

Selective transport of long-chain fatty acids by FAT/CD36 in skeletal muscle of broilers

Fatty acid translocase (FAT/CD36) is a membrane receptor that facilitates long-chain fatty acid uptake. To investigate its role in the regulation of long-chain fatty acid composition in muscle tissue, we studied and compared FAT/CD36 gene expression in muscle tissues of commercial broiler chickens and Chinese local Silky fowls. The results from gas chromatography–mass spectrometry analysis of muscle samples demonstrated that Chinese local Silky fowls had significantly higher (P < 0.05) proportions of linoleic acid (LA) and palmitic acid, lower proportions (P < 0.05) of arachidonic acid (AA) and oleic acid than the commercial broiler chickens. The mRNA expression levels of fatty acid (FA) transporters (FA transport protein-1, membrane FA-binding protein, FAT/CD36 and caveolin-1) in the m. ipsilateral pectoralis and biceps femoris were analyzed by Q-PCR, and FAT/CD36 expression levels showed significant differences between these types of chickens (P < 0.01). Interestingly, the levels of FAT/CD36 expression are positively correlated with LA content (r = 0.567, P < 0.01) but negatively correlated with palmitic acid content (r = −0.568, P < 0.01). Further experiments in the stably transfected Chinese hamster oocytes cells with chicken FAT/CD36 cDNA demonstrated that overexpression of FAT/CD36 improves total FA uptake with a significant increase in the proportion of LA and AA, and a decreased proportion of palmitic acid. These results suggest that chicken FAT/CD36 may selectively transport LA and AA, which may lead to the higher LA deposition in muscle tissue.     

Facilitation of fatty acid uptake by CD36 in insulin-producing cells reduces fatty-acid-induced insulin secretion and glucose regulation of fatty acid oxidation

Facilitation of fatty acid uptake in beta cells could potentially affect beta cell metabolism and secretory function; however such effects have not been clearly documented. CD36 facilitates uptake of fatty acids (FA) in muscle and adipose tissue and is likely to exert a similar effect in beta cells. We investigated the impact of over-expressing CD36 on fatty acid uptake and beta cell function by a Tet-on system in INS-1 cells. Doxycycline dose-dependently increased the CD36 protein with localization mainly in the cell membrane. Over-expression increased both specific uptake and efflux of oleate whereas intracellular glycerides were only marginally increased and incorporation of ¹⁴C-oleate or -palmitate into di- or triglycerides not affected. The normal potentiation of glucose-induced insulin secretion by acute addition of FA (50–100 μmol/l oleate and palmitate) was lost and the normal inhibitory effect of high glucose both on oleate oxidation and on the activity of carnitine palmitoyltransferase I was reduced. Over-expression did not induce apoptosis.     

Palmitate promotes monocyte atherogenicity via de novo ceramide synthesis

Elevated plasma free fatty acids (FAs) are associated with increased risk of cardiovascular disease. This study investigates the effects of the saturated FA palmitate and unsaturated FA oleate on monocyte phenotype and function. Incubation of human U937 and THP-1 monocytes with palmitate for 24h increased cell surface expression of integrin CD11b and scavenger receptor CD36 in a concentration-dependent manner with some decrease in mitochondrial reducing capacity at high concentration (300μM). Monocytes incubated with palmitate, but not oleate, showed increased uptake of oxidized LDL and increased adhesion to rat aortic endothelium, particularly at bifurcations. The palmitate-induced increase in CD11b and CD36 expression was associated with increased cellular C16 ceramide and sphingomyelin, loss of reduced glutathione, and increased reactive oxygen species (ROS). Increased monocyte surface CD11b and CD36 was inhibited by fumonisin B1, an inhibitor of de novo ceramide synthesis, but not by the superoxide dismutase mimetic MnTBap. In contrast, MnTBap prevented the mitochondrial ROS increase and metabolic inhibition due to 300μM palmitate. This study demonstrates that in viable monocytes, palmitate but not oleate increases expression of surface CD11b and CD36. Palmitate increases monocyte adhesion to the aortic wall and promotes uptake of oxidized LDL and this involves de novo ceramide synthesis.     

Identification of fatty acid translocase on human skeletal muscle mitochondrial membranes: essential role in fatty acid oxidation

Fatty acid translocase (FAT/CD36) is a transport protein with a high affinity for long-chain fatty acids (LCFA). It was recently identified on rat skeletal muscle mitochondrial membranes and found to be required for palmitate uptake and oxidation. Our aim was to identify the presence and elucidate the role of FAT/CD36 on human skeletal muscle mitochondrial membranes. We demonstrate that FAT/CD36 is present in highly purified human skeletal mitochondria. Blocking of human muscle mitochondrial FAT/CD36 with the specific inhibitor sulfo-N-succimidyl-oleate (SSO) decreased palmitate oxidation in a dose-dependent manner. At maximal SSO concentrations (200 muM) palmitate oxidation was decreased by 95% (P<0.01), suggesting an important role for FAT/CD36 in LCFA transport across the mitochondrial membranes. SSO treatment of mitochondria did not affect mitochondrial octanoate oxidation and had no effect on maximal and submaximal carnitine palmitoyltransferase I (CPT I) activity. However, SSO treatment did inhibit palmitoylcarnitine oxidation by 92% (P<0.001), suggesting that FAT/CD36 may be playing a role downstream of CPT I activity, possibly in the transfer of palmitoylcarnitine from CPT I to carnitine-acylcarnitine translocase. These data provide new insight regarding human skeletal muscle mitochondrial fatty acid (FA) transport, and suggest that FAT/CD36 could be involved in the cellular and mitochondrial adaptations resulting in improved and/or impaired states of FA oxidation.     

Munc18c provides stimulus-selective regulation of GLUT4 but not fatty acid transporter trafficking in skeletal muscle

Insulin-, and contraction-induced GLUT4 and fatty acid (FA) transporter translocation may share common trafficking mechanisms. Our objective was to examine the effects of partial Munc18c ablation on muscle glucose and FA transport, FA oxidation, GLUT4 and FA transporter (FAT/CD36, FABPpm, FATP1, FATP4) trafficking to the sarcolemma, and FAT/CD36 to mitochondria. In Munc18c(-/+) mice, insulin-stimulated glucose transport and GLUT4 sarcolemmal appearance were impaired, but were unaffected by contraction. Insulin- and contraction-stimulated FA transport, sarcolemmal FA transporter appearance, and contraction-mediated mitochondrial FAT/CD36 were increased normally in Munc18c(-/+) mice. Hence, Munc18c provides stimulus-specific regulation of GLUT4 trafficking, but not FA transporter trafficking.     

Free fatty acid transport across adipocytes is mediated by an unknown membrane protein pump

The role of cell membranes in regulating the flux of long chain free fatty acids (FFA) into and out of adipocytes is intensely debated. Four different membrane proteins including, FABPpm, CD36/FAT, caveolin-1, and FATP have been identified as facilitating FFA transport. Moreover, CD36 and caveolin-1 are also reported to mediate transport in conjunction with lipid rafts. The principal evidence for these findings is a correlation of the level of FFA uptake with the expression level of these proteins and with the integrity of lipid rafts. The 3T3-L1 and 3T3-F442A cell lines in their preadipocyte states reveal little or no expression of these proteins and correspondingly low levels of uptake. Here we have microinjected the adipocyte and preadipocyte cell lines with ADIFAB, the fluorescent indicator of FFA. The ADIFAB fluorescence allowed us to monitor the intracellular unbound FFA concentration during FFA influx and efflux. We show that these measurements of transport, in contrast to FFA uptake measurements, correlate neither with expression of these proteins nor with lipid raft integrity in preadipocytes and adipocytes. Transport characteristics, including the generation of an ATP-dependent FFA concentration gradient, are virtually identical in adipocytes and preadipocytes. We suggest that the origin of the discrepancy between uptake and our measurements is that most of the FFA transported into the cells is lost during the uptake but not in the transport protocols. We conclude that long chain fatty acid transport in adipocytes is very likely mediated by an as-yet-unidentified membrane protein pump.