CD36 as a target to prevent cardiac lipotoxicity and insulin resistance

The fatty acid transporter and scavenger receptor CD36 is increasingly being implicated in the pathogenesis of insulin resistance and its progression towards type 2 diabetes and associated cardiovascular complications. The redistribution of CD36 from intracellular stores to the plasma membrane is one of the earliest changes occurring in the heart during diet induced obesity and insulin resistance. This elicits an increased rate of fatty acid uptake and enhanced incorporation into triacylglycerol stores and lipid intermediates to subsequently interfere with insulin-induced GLUT4 recruitment (i.e., insulin resistance). In the present paper we discuss the potential of CD36 to serve as a target to rectify abnormal myocardial fatty acid uptake rates in cardiac lipotoxic diseases. Two approaches are described: (i) immunochemical inhibition of CD36 present at the sarcolemma and (ii) interference with the subcellular recycling of CD36. Using in vitro model systems of high-fat diet induced insulin resistance, the results indicate the feasibility of using CD36 as a target for adaptation of cardiac metabolic substrate utilization. In conclusion, CD36 deserves further attention as a promising therapeutic target to redirect fatty acid fluxes in the body.     

Insight into the mechanism of lipids binding and uptake by CD36 receptor

The membrane protein CD36 is a member of the class B scavenger receptor family. It plays a crucial role in some cardiovascular pathologies and metabolic diseases. Studying the mechanism of action of CD36 receptor is limited due to the absence of its tridimensional crystallized structure. The molecular docking method has allowed us to perform various simulation of the CD36 receptor interaction with their ligands involved in the development of some diseases. In this work, we predicted a tridimensional structure model of CD36 extracellular domain. In addition, we have achieved several tests of rigid and flexible docking by acting on residues proposed in previous experimental researches as essential in fixing of LFCAs. Furthermore, we have acted on regions that appear a key binding site of LFCAs. The physicoc hemical evaluation indicated the reliability of the proposed CD36 structure used for different molecular docking tests. Based on the docking outcome, we were able to propose the different steps of the mechanism allowing the interaction of fatty acids on CD36 receptor and their penetration into the cell cytoplasm. The obtained results and taking in consideration CD36 receptor as a therapeutic target will help us to suggest the mechanism by which an antagonist may inhibit this receptor by acting on its extracellular domain.     

CD36 and atherosclerosis

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.     

CD36 mediates both cellular uptake of very long chain fatty acids and their intestinal absorption in mice

The intestine has an extraordinary capacity for fatty acid (FA) absorption. Numerous candidates for a protein-mediated mechanism of dietary FA absorption have been proposed, but firm evidence for this process has remained elusive. Here we show that the scavenger receptor CD36 is required both for the uptake of very long chain FAs (VLCFAs) in cultured cells and the absorption of dietary VLCFAs in mice. We found that the fraction of CD36-dependent saturated fatty acid association/absorption in these model systems is proportional to the FA chain length and specific for fatty acids and fatty alcohols containing very long saturated acyl chains. Moreover, intestinal VLCFA absorption is completely abolished in CD36-null mice fed a high fat diet, illustrating that the predominant mechanism for VLCFA absorption is CD36-dependent. Together, these findings represent the first direct evidence for protein-facilitated FA absorption in the intestine and identify a novel therapeutic target for the treatment of diseases characterized by elevated VLCFA levels.     

CD36调控脂质代谢作用及机制

脂质代谢是机体的重要代谢过程,其紊乱会导致众多疾病的发生。人类白细胞分化抗原36(cluster of differentiation 36,CD36)是一种在单核细胞、巨噬细胞、平滑肌细胞以及脂肪细胞高度表达的清道夫受体,是识别氧化低密度脂蛋白及长链脂肪酸的主要受体和转运蛋白,在脂质代谢过程中发挥着重要作用。本文综述了CD36基因及蛋白的结构和生理功能,阐述了清道夫受体CD36在脂质代谢过程中发挥的作用,并系统地总结了其级联AMPK、mTOR和MAPK信号通路参与脂质代谢过程的分子机制,为相关生物学研究提供了理论基础。     

Caveolin-1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts

Several lines of evidence suggest that lipid rafts are involved in cellular fatty acid uptake and influence fatty acid translocase (FAT/CD36) function. However, it remains unknown whether caveolae, a specialized raft type, are required for this mechanism. Here, we show that wild-type (WT) mouse embryonic fibroblasts (MEFs) and caveolin-1 knockout (KO) MEFs, which are devoid of caveolae, have comparable overall expression of FAT/CD36 protein but altered subcellular FAT/CD36 localization and function. In WT MEFs, FAT/CD36 was isolated with both lipid raft enriched detergent-resistant membranes (DRMs) and detergent-soluble membranes (DSMs), whereas in cav-1 KO cells it was exclusively associated with DSMs. Subcellular fractionation demonstrated that FAT/CD36 in WT MEFs was localized intracellularly and at the plasma membrane level while in cav-1 KO MEFs it was absent from the plasma membrane. This mistargeting of FAT/CD36 in cav-1 KO cells resulted in reduced fatty acid uptake compared to WT controls. Adenoviral expression of caveolin-1 in KO MEFs induced caveolae formation, redirection of FAT/CD36 to the plasma membrane and rescue of fatty acid uptake. In conclusion, our data provide evidence that caveolin-1 is necessary to target FAT/CD36 to the plasma membrane. Caveolin-1 may influence fatty acid uptake by regulating surface availability of FAT/CD36.     

Scavenger receptor class B,type 1 facilitates cellular fatty acid uptake

SR-B1 belongs to the class B scavenger receptor, or CD36 super family. SR-B1 and CD36 share an affinity for a wide array of ligands. Although they exhibit similar ligand binding specificity, SR-B1 and CD36 have some very specific lipid transport functions. Whereas SR-B1 primarily facilitates the selective delivery of cholesteryl esters (CEs) and cholesterol from HDL particles to the liver and non-placental steroidogenic tissues, as well as participating in cholesterol efflux from cells, CD36 primarily mediates the uptake of long-chain fatty acids in high fatty acid-requiring organs such as the heart, skeletal muscle and adipose tissue. However, CD36 also mediates cholesterol efflux and facilitates selective lipoprotein-CE delivery, although less efficiently than SR-B1. Interestingly, the ability or efficiency of SR-B1 to mediate fatty acid uptake has not been reported. In this paper, using overexpression and siRNA-mediated knockdown of SR-B1, we show that SR-B1 possesses the ability to facilitate fatty acid uptake. Moreover, this function is not blocked by BLT-1, a specific chemical inhibitor of HDL-CE uptake activity of SR-B1, nor by sulfo-N-succinimidyl oleate, which inhibits fatty acid uptake by CD36. Attenuated fatty acid uptake was also observed in primary adipocytes isolated from SR-B1 knockout mice. In conclusion, facilitation of fatty acid uptake is an additional function that is mediated by SR-B1.     

Amyloid-β inhibits No-cGMP signaling in a CD36- and CD47-dependent manner

Amyloid-β interacts with two cell surface receptors, CD36 and CD47, through which the matricellular protein thrombospondin-1 inhibits soluble guanylate cyclase activation. Here we examine whether amyloid-β shares this inhibitory activity. Amyloid-β inhibited both drug and nitric oxide-mediated activation of soluble guanylate cyclase in several cell types. Known cGMP-dependent functional responses to nitric oxide in platelets and vascular smooth muscle cells were correspondingly inhibited by amyloid-β. Functional interaction of amyloid-β with the scavenger receptor CD36 was indicated by inhibition of free fatty acid uptake via this receptor. Both soluble oligomer and fibrillar forms of amyloid-β were active. In contrast, amyloid-β did not compete with the known ligand SIRPα for binding to CD47. However, both receptors were necessary for amyloid-β to inhibit cGMP accumulation. These data suggest that amyloid-β interaction with CD36 induces a CD47-dependent signal that inhibits soluble guanylate cyclase activation. Combined with the pleiotropic effects of inhibiting free fatty acid transport via CD36, these data provides a molecular mechanism through which amyloid-β can contribute to the nitric oxide signaling deficiencies associated with Alzheimer's disease.     

Molecular basis of human CD36 gene mutations

CD36 is a transmembrane glycoprotein of the class B scavenger receptor family. The CD36 gene is located on chromosome 7 q11.2 and is encoded by 15 exons. Defective CD36 is a likely candidate gene for impaired fatty acid metabolism, glucose intolerance, atherosclerosis, arterial hypertension, diabetes, cardiomyopathy, Alzheimer disease, and modification of the clinical course of malaria. Contradictory data concerning the effects of antiatherosclerotic drugs on CD36 expression indicate that further investigation of the role of CD36 in the development of atherosclerosis may be important for the prevention and treatment of this disease. This review summarizes current knowledge of CD36 gene structure, splicing, and mutations and the molecular, metabolic, and clinical consequences of these phenomena.     

The lipid whisker model of the structure of oxidized cell membranes

An essential feature of the innate immune system is maintaining cellular homeostasis by identifying and removing senescent and apoptotic cells and modified lipoproteins. Identification is achieved through the recognition of molecular patterns, including structurally distinct oxidized phospholipids, on target cells by macrophage receptors. Both the structural nature of the molecular patterns recognized and their orientation within membranes has remained elusive. We recently described the membrane conformation of an endogenous oxidized phospholipid ligand for macrophage scavenger receptor CD36, where the truncated oxidized sn-2 fatty acid moiety protrudes into the aqueous phase, rendering it accessible for recognition. Herein we examine the generality of this conformational motif for peroxidized glycerophospholipids within membranes. Our data reveal that the addition of a polar oxygen atom on numerous peroxidized fatty acids reorients the acyl chain, whereby it no longer remains buried within the membrane interior but rather protrudes into the aqueous compartment. Moreover, we show that neither a conformational change in the head group relative to the membrane surface nor the presence of a polar head group is essential for CD36 recognition of free oxidized phospholipid ligands within membranes. Rather, our results suggest the following global phenomenon. As cellular membranes undergo lipid peroxidation, such as during senescence or apoptosis, previously hydrophobic portions of fatty acids will move from the interior of the lipid bilayer to the aqueous exterior. This enables physical contact between pattern recognition receptor and molecular pattern ligand. Cell membranes thus "grow whiskers" as phospholipids undergo peroxidation, and many of their oxidized fatty acids protrude at the surface.     

Myristic acid stimulates endothelial nitric-oxide synthase in a CD36- and an AMP kinase-dependent manner

Dietary free fatty acids have been reported to have various effects on the endothelium including the generation of nitric oxide. The goal of the current study was to determine the mechanism whereby free fatty acid causes an increase in nitric oxide synthesis. The specific hypothesis tested was that free fatty acid association with CD36, a class B scavenger receptor, induces the activation of endothelial nitric-oxide synthase (eNOS). A human microvascular endothelial cell line and a transfected Chinese hamster ovary cell system were used to determine which free fatty acids stimulate eNOS. Surprisingly, only myristic acid, and to a lesser extent palmitic acid, stimulated eNOS. The stimulation of eNOS was dose- and time-dependent. Competition experiments with other free fatty acids and with a CD36-blocking antibody demonstrated that the effects of myristic acid on eNOS required association with CD36. Further mechanistic studies demonstrated that the effects of myristic acid on eNOS function were not dependent on PI 3-kinase, Akt kinase, or calcium. Pharmacological studies and dominant negative constructs were used to demonstrate that myristic acid/CD36 stimulation of eNOS activity was dependent on the activation of AMP kinase. These data demonstrate an unexpected link among myristic acid, CD36, AMP kinase, and eNOS activity.     

Regulation of fatty acid transport and membrane transporters in health and disease

Long chain fatty acid uptake across the plasma membrane occurs, in part, via a protein-mediated process involving a number of fatty acid binding proteins known as fatty acid transporters. A critical step in furthering the understandings of fatty acid transport was the discovery that giant vesicles, prepared from tissues such as muscle and heart, provided a suitable system for measuring fatty acid uptake. These vesicles are large (10–15 m diameter), are oriented fully right side out, and contain cytosolic FABP in the lumen, which acts as a fatty acid sink, while none of the fatty acid taken up is metabolized or associated with the plasma membrane. The key fatty acid transporters FAT/CD36 and FABPpm are expressed in muscle and heart and their plasma membrane content is positively correlated with rates of fatty acid transport. These transporters are regulated acutely (within minutes) and chronically (days). For instance, both muscle contraction and insulin can translocate FAT/CD36 from an intracellular pool to the plasma membrane, thereby increasing fatty acid transport. With obesity, fatty acid transport is increased along with a concomitant increase in plasmalemmal FAT/CD36 (heart, muscle) and FABPpm (heart only), but without change in the expression of these transporters. This latter observation suggests that some of the fatty acid transporters are permanently relocated to the plasma membrane. In other studies it also appears that fatty acid transport rates are altered in a reciprocal manner to glucose transport. Since disorders in lipid metabolism appear to be an important factor contributing to the etiology of a number of common human diseases such as diabetes and obesity, our evidence that protein-mediated fatty acid transport is a key step in lipid metabolism allows the speculation that malfunctioning of the fatty acid transport process could be a common critical factor in the pathogenesis of these diseases.     

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.     

Thrombospondin-1 inhibits nitric oxide signaling via CD36 by inhibiting myristic acid uptake

Although CD36 is generally recognized to be an inhibitory signaling receptor for thrombospondin-1 (TSP1), the molecular mechanism for transduction of this signal remains unclear. Based on evidence that myristic acid and TSP1 each modulate endothelial cell nitric oxide signaling in a CD36-dependent manner, we examined the ability of TSP1 to modulate the fatty acid translocase activity of CD36. TSP1 and a CD36 antibody that mimics the activity of TSP1 inhibited myristate uptake. Recombinant TSP1 type 1 repeats were weakly inhibitory, but an anti-angiogenic peptide derived from this domain potently inhibited myristate uptake. This peptide also inhibited membrane translocation of the myristoylated CD36 signaling target Fyn and activation of Src family kinases. Myristate uptake stimulated cGMP synthesis via endothelial nitric-oxide synthase and soluble guanylyl cyclase. CD36 ligands blocked myristate-stimulated cGMP accumulation in proportion to their ability to inhibit myristate uptake. TSP1 also inhibited myristate-stimulated cGMP synthesis by engaging its receptor CD47. Myristate stimulated endothelial and vascular smooth muscle cell adhesion on type I collagen via the NO/cGMP pathway, and CD36 ligands that inhibit myristate uptake blocked this response. Therefore, the fatty acid translocase activity of CD36 elicits proangiogenic signaling in vascular cells, and TSP1 inhibits this response by simultaneously inhibiting fatty acid uptake via CD36 and downstream cGMP signaling via CD47.     

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- 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.     

Cholesterol depletion inhibits fatty acid uptake without affecting CD36 or caveolin-1 distribution in adipocytes

Caveolin-1 and CD36 are plasma membrane fatty acid binding proteins that participate in adipocyte fatty acid uptake and metabolism. Both are associated with cholesterol-enriched caveolae/lipid rafts in the plasma membrane that are important for long chain fatty acid uptake. Depletion of plasma membrane cholesterol reversibly inhibited oleate uptake by adipocytes without altering the amount or the cell surface distribution of either caveolin-1 or CD36. Cholesterol levels thus regulate fatty acid uptake by adipocytes via a pathway that does not involve altered cell surface localization of caveolin-1 or CD36.     

Lipid‐induced Insulin Resistance Is Associated With Increased Monocyte Expression of Scavenger Receptor CD36 and Internalization of Oxidized LDL

Elevated free fatty acids (FFAs) contribute to the development of insulin resistance, type 2 diabetes mellitus (T2DM), and may be atherogenic. We tested the relationship among lipid‐induced insulin resistance, endothelial dysfunction, and monocyte capacity to form foam cells through scavenger receptor A (SRA) and CD36. Ten healthy subjects underwent 24‐h infusion of Intralipid/heparin and saline (0.5 ml/min) on two separate occasions followed by brachial artery reactivity testing and a euglycemic hyperinsulinemic (80 mU/(kg·min)) clamp study to determine insulin sensitivity. Isolation of blood monocytes was performed 24 h after infusion. Surface expression and function of CD36 and SRA to take up oxidized low‐density lipoprotein (oxLDL) was determined by flow cytometry and quantitative confocal imaging. Lipid infusion resulted in a twofold increase in serum FFA levels, reduced whole‐body glucose disposal by ～20% (P < 0.05), and possibly impaired endothelial‐dependent vasodilation (P = 0.1). Blood monocytes obtained during lipid infusion demonstrated a ～25% increase in cell surface expression of CD36 (P < 0.05) but no change in SRA expression. Enhanced CD36 expression was associated with a 50% increase in internalization of oxLDL (P < 0.05). The increase in CD36 surface expression during lipid infusion correlated inversely with glucose disposal (P < 0.05) and not with FFA levels or brachial artery dilation. These data support a role for FFAs in induction of insulin resistance and provide a link to atherogenic mechanisms mediated by expression of scavenger receptor CD36.     

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.