Intestinal CD36 and Other Key Proteins of Lipid Utilization: Role in Absorption and Gut Homeostasis

Abstract

Several proteins have been implicated in fatty acid (FA) transport by enterocytes including the scavenger receptor CD36 (SR-B2), the scavenger receptor B1 (SR-B1) a member of the CD36 family and the FA transport protein 4 (FATP4). Here, we review the regulation of enterocyte FA uptake and its function in lipid absorption including prechylomicron formation, assembly and transport. Emphasis is given to CD36, which is abundantly expressed along the digestive tract of rodents and humans and has been the most studied. We also address the pleiotropic functions of CD36 that go beyond lipid absorption and metabolism to include recent evidence of its impact on intestinal homeostasis and barrier maintenance. Areas of progress involving contribution of membrane phospholipid remodeling and of cytosolic FA-binding proteins, FABP1 and FABP2 to fat absorption will be covered.

Introduction

Dietary fats are an important source of energy, essential fatty acids (EFAs) and fat-soluble vitamins (146). In humans, the absorption of dietary fat is highly quantitative ensuring uptake of most of the ingested lipid (95% of a 500 g fat load) (76). The poor solubility of triacylglycerols (TAGs) in the aqueous environment of the digestive tract requires involvement of specialized chaperones and enzymes with amphipathic properties, which contribute to efficiency but also to the complexity of fat absorption.

Dietary TAGs are hydrolyzed in the intestinal lumen primarily by pancreatic TAG lipase to 2 FAs and sn-2-monoacylglycerol (MAG). These lipolytic products are absorbed at the apical (i.e., luminal) membrane of enterocytes (Fig. 1A, step 1), and directed to the endoplasmic reticulum (ER) where they are converted back to TAG (Fig. 1A, step 2) destined for production of intestinal lipoproteins (chylomicrons and very low density lipoproteins, VLDL). TAGs are transported from the ER in a specialized vesicle, the prechylomicron transport vesicle (PCTV) that is directed to the Golgi for further processing (Fig. 1A, step 3). The mature chylomicron packaged into another vesicle is transported to the basolateral membrane where it is exocytosed (Fig. 1A, step 4) and enters the lymphatic vessels or lacteals located within intestinal villi (Fig. 1A, step 5). The chylomicronrich lymph runs through the collecting lymphatic vessels and lymph nodes ultimately reaching the thoracic duct which empties the transported chylomicrons into the bloodstream at the left subclavian vein (Fig. 1B).

Figure 1.

Lipid absorption from epithelial cells (enterocytes) and chylomicron secretion in lymphatic vessels (lacteals). (A) During absorption of dietary lipids, the FA and MAGs are released from micelles in the lumen (indicated by 1, top panel) and both enter at the apical side of intestinal epithelial cells, also called enterocytes that line the luminal side of the small intestine. TAGs are synthesized in the ER of enterocytes (2, middle panel) from the absorbed FA and MAG and exit the ER en route to the Golgi in the PCTVs. The PCTVs mature in the Golgi (3, middle panel) and are then released by the enterocytes as TAG-enriched chylomicron particles (4, bottom panel) that enter the lymphatic vessels located inside intestinal villi, called lacteals (5 bottom panel). (B) Once in the lacteals, chylomicrons are transported via the lymph through mesenteric lymph nodes and collecting lymphatic vessels ultimately reaching the thoracic duct, which drains into the venous circulation at the level of the left subclavian vein.

FA Uptake and Processing by Enterocytes

Absorption of FA in the small intestine is likely mediated by both diffusion and protein-mediated transfer (3). Several mechanisms cooperate to ensure delivery of FA generated from TAG hydrolysis in the intestinal lumen across the enterocyte membrane and then to the ER to be reincorporated into TAG.

FA uptake in the intestine differs in one major aspect from FA uptake by peripheral tissues. FA are carried in the circulation quantitatively bound to albumin and the unbound FA concentration in equilibrium with albumin-bound FA determines uptake by cells (15, 153). In the intestine, the FA released from TAG digestion by lipases is incorporated into bile salt micelles. Luminal micelles, like albumin in the serum can solubilize millimolar concentrations of FA. In both systems, the monomeric free FA dissociated in the aqueous phase is low relative to the total FA concentration. However, an important difference is that the free FA concentration in the intestinal lumen is estimated to be in the low micromolar (μM) (65, 131, 161) as compared to the low nanomolar in the circulation (124). Involvement of protein-facilitated FA transport by peripheral tissues is supported by evidence of process saturability, sensitivity to protein reactive agents, specificity for long-chain FA and a transport Km in the low nanomolar range appropriate for circulating unbound FA concentrations (Fig. 2A) [for detailed reviews, refer to (1,2,13,48,56)].

Figure 2.

FA uptake is reduced in enterocytes from the proximal intestine of Cd36^−/− mice but its contribution to net intestinal FA uptake is small. (A) FA uptake includes saturable and nonsaturable components. The graph illustrates the high affinity saturable FA uptake component that is mediated by CD36 (1). The blue bar shows the range of unbound FA concentrations (<10 nmol/L) present in the blood during feeding and fasting. At this range of concentrations, the saturable CD36-mediated component (dashed orange line) contributes the major part of FA uptake from the circulation to peripheral tissues such as muscle and adipose tissues. In the intestine, the relative contribution of FA transport to total uptake by enterocytes is likely to be small since the concentration of free FA released from micelles (green bar) is estimated to be 1000-fold higher as compared to that in the blood; FA transfer by diffusion or other mechanisms would constitute the major route of FA uptake by enterocytes (see section “Role of phospholipid remodeling in intestinal FA absorption”). (B) Time course of OA uptake by enterocytes from WT (filled squares) and Cd36-null mice (open squares). Cells were incubated with [³H]-oleate for the indicated times (0–30 min) and uptake was stopped using cold Krebs-Ringer-Hepes bicarbonate (KRH) buffer. Cells collected by centrifugation through a Ficoll layer were analyzed for associated radioactivity. In these experiments, the FA was used bound to bovine serum albumin at a FA:BSA ratio of 2 (107), which associates with nanomole per liter concentrations of unbound FA. In the intestine, the saturable component of FA uptake would function in FA sensing early during absorption and exerts a regulatory role in initiating chylomicron production (see section “Chylomicron formation”) and enteroendocrine secretion of CCK and secretin to facilitate absorption (see section “Other function of CD36”).

Early evidence indicated that FA processing by enterocytes depends on the entry side, apical versus basolateral (150). Presence of saturable versus nonsaturable FA uptake was reported using either albumin FA or micellar FA delivery systems (65) in enterocyte cell models as well as in primary enterocytes isolated from murine proximal intestine (107) (Fig. 2B). However, the kinetic measures obtained indicate that at the concentrations of FA likely to be present in the intestinal lumen, the relative contribution of FA transporters to total uptake by enterocytes in vivo is likely to be small as compared to cell types such as heart, muscle or adipose cells where the saturable process is important (Fig. 2A). In enterocytes, although the contribution of protein facilitated FA transport to uptake is quantitatively marginal (1) (Fig. 2A), it is likely to play an important regulatory function by transducing signals that facilitate fat absorption as discussed in section “CD36 (SR-B2 or FAT).”

Role of phospholipid remodeling in intestinal FA absorption

Recent data suggested that remodeling of membrane phosphor-lipids is important for passive diffusion of FA into enterocytes (62,164). FAs composition of cell membrane phospholipids exhibits considerable diversity. Saturated and monounsaturated FA are usually esterified at the sn-1 position, whereas polyunsaturated FA (PUFAs) are esterified at the sn-2 position (96). The asymmetrical distribution of FA at the sn-1 and sn-2 positions is maintained in part by a deacylationreacylation process known as Lands cycle (84). The deacylation step catalyzed by calcium-independent phospholipase A2 removes saturated or monounsaturated FA from the sn-2 position of phosphatidylcholine (PC) which represents 70% of phospholipids. The reacylation step is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT), which adds the PUFA at the sn-2 position of PC. Lpcat3 knockout mice die shortly after birth and when rescued by supplementing their diet with phosphatidylcholine, they display reduced lipid absorption and lower expression levels of Nieman-Pick C1-like 1 (NPC1L1), CD36, and FA transport protein 4 (FATP4) (88). Intestine specific Lpcat3 deficiency leads to a selective defect in the incorporation of the EFAs linoleate and arachidonate into phospholipids changing the biophysical properties of the enterocyte membranes (62,164). EFA deficiency has been known to associate with fat malabsorption for decades (29, 86) although the underlying mechanisms were not defined. It is now thought that arachidonate-enrichment of membrane phospholipids is important for promoting FA targeting to lipoprotein formation, preventing over-accumulation of cytosolic lipid droplets in enterocytes (62,164). LPCAT3 enriches membranes in PUFA-containing phospholipids.TAG synthesis in the proximity of these PUFA-enriched domains favors formation of blister-like structures with high curvature that facilitate lipid transfer to the microsomal triglyceride transfer protein (MTTP) for initiating lipoprotein formation (62). In the setting of a TG-rich diet, intestine-specific LPCAT3 deficiency led to cessation of food intake, despite starvation, which was thought to reflect increased production of the prosatiety gut hormones glucagon-like peptide 1 (GLP-1) and oleoylethanolamide (164).

Proteins Involved in Intestinal FA Uptake and Trafficking

Enterocytes are equipped at the level of the apical brush border with several proteins that facilitate FA uptake and targeting to lipoproteins. The following membrane proteins will be discussed; the scavenger receptor CD36 (SR-B2), the scavenger receptor B1 (SR-B1) a member of the CD36 family and the FA transport protein 4 (FATP4). In addition, the role of cytosolic FA binding proteins in intracellular FA trafficking and movement of lipid vesicles between ER and Golgi will be reviewed. The plasma membrane FA binding protein (FABPpm), a membrane associated form of the mitochondrial enzyme aspartate aminotransferase (14), was isolated from intestinal as well as hepatic membranes and early studies showed that an antibody against the protein reduced FA uptake into isolated enterocytes (151). However, since there was no additional work that examined its role in intestinal FA uptake, it will not be discussed in this review.

CD36 (SR-B2 or FAT)

CD36/FAT (FA translocase), is a 75- to 88-kDa (depending on extent of glycosylation), 472-amino acid heavily glycosylated transmembrane protein expressed in several tissues including muscle, adipose tissue, intestine and the capillary endothelium (4,54). CD36 binds a range of ligands including long chain FA, native or modified lipoproteins, thrombospondin-1, collagen, apoptotic cells, amyloid B, and malaria-infected erythrocytes (39). CD36 is a scavenger receptor, a type of membrane protein characterized by recognizing similar molecular patterns rather than specific epitopes in their ligands. Scavenger receptors interact with a broad repertoire of ligands and as a result are often multifunctional. In addition to its role in lipid transport, CD36 is involved in events such as phagocytosis, antigen presentation and the clearance of apoptotic cells, involving a range of signaling pathways (21). Extensive in vivo evidence supports CD36’s role in facilitating high affinity tissue FA uptake and metabolism in rodents (49,58) and humans (59,64,171).

CD36 crystal structure and potential mechanism for FA transport

The crystal structure for the ectodomain of lysosomal LIMP-2 (109), a CD36 family member, and that of Drosophila CD36 (50) provided insight into the potential mechanism of CD36-mediated FA transport. A large internal tunnel spanning the protein and ending in the membrane vicinity that would function in lipid transfer was identified(Fig. 3).CrystallizedCD36 was also found in complex with long chain FA (palmitic and stearic acids) (67). The FA is thought to dock within a surface hydrophobic cavity that leads to the internal tunnel. In this cavity, the carboxylic tail of the FA is in proximity to lysine 164 (K164) (80). Interaction with K164 could position the FA to favor its access to the tunnel(118)(Fig. 3).Surface plasmon resonance demonstrated similar binding properties of different long chain FAs to the CD36 ectodomain, including oleic acid (OA), docosahexaenoic acid, elaidic acid, and palmitic acid (72). Interestingly, K164 in the CD36 ectodomain is also involved in binding oxidized LDL (80). The Drosophila CD36 homologue named sensory neuron membrane protein (SNMP), as well as its orthologs in other insects, is expressed in the olfactory sensory neurons that detect lipid-derived pheromones (12). The SNMP1 ectodomain was shown to be required for detection and binding of pheromones and the internal tunnel funnels these molecules to their cognate receptors (50).

Figure 3.

Model of CD36-mediated FA transport. Proposed mechanism of FA transfer by CD36 modeled based on CD36’s crystal structure (67,109). CD36 is a transmembrane receptor with a large ectodomain and two transmembrane segments ending in two short cytoplasmic tails (orange cylinders). Crystallized CD36 was found in complex with long chain FA (palmitic and stearic acids) (67). The FA (FA skeleton shown) is thought to dock within a surface hydrophobic cavity where the carboxylic tail of the FA is in proximity of lysine 164 (K164 is highlighted in green) (80). The cavity would lead the FA to an internal tunnel inside the protein (translucent cylinder) that empties at the membrane bilayer (80). Interaction with K164 could position the FA to favor its access to the tunnel (80,118). Green: K164 residue; Red: hydrophobic residues; Blue: hydrophilic residues; cylinder indicates the location of the internal tunnel inside the CD36 receptor. The figure shows the structure of OA interacting with K164 residue.

CD36 contributes to FA uptake by enterocytes but does not impact net fat absorption

CD36 is highly expressed on the apical membrane of villi enterocytes (90, 121) in the proximal small intestine (duodenum-jejunum) of rodents where most fat absorption occurs; it is also present, although less abundantly, in the distal segments (107). The same expression pattern applies to human intestines (90, 101). CD36 mediates the high affinity saturable FA uptake component that plays an important role at the range of unbound FA concentrations (<10 nmol/L) present in the blood during feeding or fasting (Fig. 2A) (1). The CD36-mediated component contributes the major part of FA uptake by peripheral tissues such as muscle and adipose tissues. In the intestine, the relative contribution of FA transport to total uptake by enterocytes is likely to be small since the concentration of free FA released from micelles is estimated to be 1000fold higher (low micromolar concentration) (Fig. 2A). However, the CD36-mediated component would function early during absorption and exerts a regulatory role in initiating chylomicron production, inducing enteroendocrine secretions and facilitating absorption(for more details see section “Role of CD36 in fat-induced satiety OEA, CCK, and secretin release”). Quantitative contribution of CD36 to net lipid absorption is small as shown by studies in Cd36^−/− mice where 24 h fecal lipid recovery showed no evidence of lipid malabsorption (108) except for very long chain FA (35). Administration of a lipid load also resulted in similar blood appearance of intestinally derived TAGs (34,52). The lack of net impact on lipid absorption is observed despite findings of defective FA uptake by primary enterocytes isolated from the proximal intestine of Cd36^−/− mice (Fig. 2B). In addition, in vivo intragastric administration of triolein associates with reduced OA enrichment of mucosal lipids in the proximal intestine of these mice (107) and with reduced OA uptake into the duodenum and jejunum of mice refed for 30 min after a 6 h fast (135). Feeding of a high-fat diet results in lipid accumulation in the proximal segment with evidence for an absorption delay with more of the lipid being absorbed distally (34,108). Together these findings indicate that distal segments compensate for proximal defects in FA uptake in Cd36^−/− mice consistent with efficiency and redundancy of intestinal transport systems.

CLA-1/SR-B1

The Scavenger Receptor Class B Type I (SR-B1) belongs to the CD36 family. SR-B1 and CD36 share several properties including binding of anionic phospholipids, native and oxidized lipoproteins and apoptotic cells (5, 79). SR-BI binds high-density lipoproteins (HDLs) more efficiently than CD36 which can also bind these particles (30) and facilitates selective cellular uptake of cholesteryl ester (CE) in mammalian steroidogenic tissues (i.e., adrenals and ovary) where it is highly expressed (5). Like CD36, SR-B1 structure includes a large ectodomain that is important for ligand binding in this case HDL and a non-aqueous internal tunnel able to accommodate CE to mediate its selective transfer into the cell (109,125,175).

SR-B1 is abundantly expressed in the small intestine (17, 20, 90, 107). Global deletion of SR-B1 in mice did not affect intestinal cholesterol (9,99) or FA absorption (35). On the other hand, transgenic mice that overexpress SR-B1 in the gut show enhanced absorption of ¹⁴C-cholesterol and ³Htriolein (17). The effect on triolein cannot be explained by SR-B1-mediated uptake of whole triglycerides since these are quantitatively hydrolyzed by lipases in the intestinal lumen. Thus SR-B1 might mediate uptake of FA released from core triglyceride hydrolysis (17). Consistent with this, SRB1 knockout mice have increased plasma FA, similar to those observedinCd36^−/− mice and mice double knockouts for both receptors show additive effects on increasing plasma FA (35).

SR-B1 has been recently shown to activate kinases in endothelial cells after HDL binding and in the intestine in response to postprandial micelles. Therefore, SR-B1 might function as a sensor of plasma membrane cholesterol inducing apolipoprotein B cellular trafficking in response to postprandial micelles (129). Variants in the human CLA-1/SR-BI gene have been identified in healthy men and associated with increased level of HDL cholesterol (18,163,176).

Cytosolic FA binding proteins: FABP1 and FABP2

Inside the enterocyte, small (∼15 kDa) cytosolic FA binding proteins (FABP) that bind nanomolar concentrations of FA might help FA trafficking to specific metabolic sites. Two FABP proteins are expressed in enterocytes FABP1 (or liver FABP) and FABP2 (or intestinal FABP). FABP1, first isolated from liver tissue, plays a central role in β-oxidation, both through FA trafficking and peroxisome proliferatoractivated receptor (PPAR)-α mediated regulation of gene expression (11, 149, 158). Mechanisms of FA delivery are different for the two FABPs with passive diffusion involved in the case of FABP1 and membrane collision in the case of FABP2 (69, 159). FABP2 has positive charges and an amphipathic peri-portal area that favor membrane interaction (148). Other important differences include the specificity of FABP2 for binding a single FA and essentially no other lipid while FABP1 binds two FA molecules and recognizes a broad range of ligands including bile salts, lyso-phospholipids, cholesterol, acyl-CoA, MAG, and endocannabinoids (60, 70, 75, 83, 100, 134). Genetic deletion of either Fabp1 or Fabp2 did not impact absorption of dietary lipid, even during chronic high-fat feeding. In the case of Fabp1-null mice on a Western diet, fat was normally absorbed despite a modestly reduced chylomicron output (112). This reduction is likely to reflect the role of FABP1 in ER generation of PCTVs (110) as discussed in section “Role of FABP1 and CD36 in PCTV and chylomicron formation.”

Fatty acid transporter protein 4

The FA transport protein (FATP) family is composed of six highly conserved members (FATP1–6) (46,147) that are differentially expressed in various tissues (46,133). In general, FA uptake enhancement by FATP reflects the FA acylation activity of these proteins which traps FA inside the cells (36,40,165) similar to the vectorial FA uptake pathway proposed in bacteria (33).

FATP4, the only member of the FATP family present in the apical brush border of intestinal epithelial cells (also expressed in brain, adipose tissue, and muscle) was initially proposed to play a role in FA uptake by enterocytes (147). However, later studies indicated that FATP4 localizes to the ER and there is evidence that its expression at the ER where FA esterification occurs can drive more FA uptake (104). FATP4 deletion in mice was either embryonically lethal (47), or mice die early after birth with features of restrictive dermopathy (63) making it difficult to investigate the role of the protein in lipid metabolism. Rescue of skin FATP4 expression in Fatp4 null mice allowed mice survival and the examination of FATP4’s role in intestinal absorption (138); no major changes were observed in Fatp4 null mice fed a high fat diet as food intake, weight gain, intestinal cholesterol, and TAG absorption or fecal fat losses were similar. However, small but significant increases in enterocyte TAG and FA content were observed with feeding fat-rich diets (138). Therefore, available data do not support a primary role of FATP4 in dietary TAG absorption. Interestingly, the protein was suggested to function in intestinal cholesterol handling since its expression is decreased by the cholesterol absorption inhibitor, ezetimibe (82).

Chylomicron Formation

Chylomicrons are the intestine specific lipoproteins highly enriched in TAG and cholesterol esters that are generated and exported by enterocytes. Chylomicrons are secreted into the lymph reaching the circulation at the level of the left subclavian vein (Fig. 1B). In the circulation, chylomicrons are rapidly hydrolyzed by lipoprotein lipase to release FA for uptake by various tissues (49).

Prechylomicron

Several apolipoproteins are involved in formation of the prechylomicron: apolipoprotein B (apoB), apolipoprotein AI (apoAI) and apolipoprotein AIV (apoIV). ApoB is the quintessential lipoprotein in chylomicrons. There are 2 apoB gene products in mice and humans, ApoB48 and ApoB100. Unlike the apoB-48 and apoB-100 structural equivalents in human, which are synthesized in the gut and liver, respectively, in the mouse apoB-48 is also found in the liver.ApoB48 is expressed in the intestine as ApoB and is translated from an apoB100 mRNA transcript that has been edited at cytidine 6666 by the cytidine deaminase apobec-1 (apolipoprotein B mRNA editing enzyme) resulting in a stop codon at this position (26). In Apobec-1 null mice, only apoB100 is produced and intestinal TAG secretion is slower probably due to increased degradation of apoB100 (89,168). ApoB48 regulates efficiency of intestinal TAG output; Apobec-1 null mice have increased TAG retention in their mucosa and decreased serum TAG levels compared to control mice. Genetic ablation of apoB48 in mice also causes a reduction of plasma HDL level (174).

ApoAI is synthesized in the ER but becomes part of the pre-chylomicron in the Golgi (141, 144) and is transported from ER to Golgi in COPII vesicles, a route separate from the transported lipid (Fig. 4). During catabolism of chylomicrons by lipoprotein lipase, apoAI elutes off the surface of the remnant leading to formation of HDL upon addition of phospholipid and subsequently cholesterol (reverse cholesterol transport) (123). The C-terminal portion of apoAI (specifically amino acids344–354) is important for chylomicron-TAG output into the lymph (94).

Figure 4.

Prechylomicron formation and budding. Assembly of prechylomicrons occurs in the lumen of the ER. Prechylomicrons are packaged into specialized PCTVs that bud off the ER membrane and move to the cis-Golgi where they fuse with the Golgi membrane to deliver the prechylomicron cargo into the Golgi lumen. Note that PCTVs bud from the ER membrane in the absence of COPII, which are required for vesicular transport of nascent protein such as apoproteins from ER to Golgi. After processing in the Golgi, a separate vesicular system transports mature chylomicrons to the basolateral membrane.

ApoAIV is part of the prechylomicron particle in the ER (141) and its overexpression greatly increases chylomicron-TAG output into the lymph by increasing chylomicron size (94).

Formation of the prechylomicron transport vesicle in the ER and its budding and transfer to the Golgi

Prechylomicron or primordial particle formation requires the presence of surface lipoproteins apoB48 and apoAIV. These proteins together with cholesterol and phospholipids, mainly phosphatidylcholine (PC), stabilize the core of the particle. The monolayer surface accommodates a 5-fold change in particle diameter that is controlled by the mass of core lipids composed of TAG and cholesterol ester. There is one apoB48 per chylomicron (120).

To avoid apoB48 degradation by the ubiquitin-proteasome (130), apoB48 passage through its translocon (170) must be controlled by chaperones and lipid must be present. Otherwise, partially synthesized apoB48 is directed to degradation by Hsp70 and proteasomes while still attached to ribosomes (116).Microsomal triglyceride transport protein (MTTP), BiP and HSP110 are the main chaperones that assist apoB48 during this translocation (66,166). In addition to its chaperone function, MTTP binds phospholipids providing an additional stabilizing surface for apoB48 (122) and increasing interaction with phospholipids (73).

The newly formed primordial particle, containing apoB48, chaperones, cholesterol, phospholipid, and some TAG, detaches from the ER membrane into the ER lumen. Simultaneous to the production of this particle (similar in density to HDL), neutral lipids accumulate in a large, non-apoB containing lipid droplet within the ER lumen (51). TAG accretion of this droplet from the cytosol is controlled in part by MTTP, which has an M protein subunit, which belongs to a family of large lipid transfer proteins (LLTP) and a protein disulfide isomerase component. In the absence of the M protein, the large lipid droplet in the ER lumen does not form while cytosolic lipid droplets accumulate (169). In the absence of apoB48 synthesis, large lipid droplets accumulate in the ER lumen with no chylomicron formation. The “two-step process” involving formation of an apoB containing dense particle and that of a neutral lipid-containing droplet is followed by a merge of these structures to form the pre-chylomicron (3,23,61).

Role of FABP1 and CD36 in PCTV and chylomicron formation

Once formed, the prechylomicron with an average size of 250 nm (177) is ready to exit the ER (Fig. 4). This process involves a specialized ER to Golgi PCTV, which transport lipid, differ from ER to Golgi vesicles that transport newly synthesized proteins such as apoAI (Fig. 4). The PCTV are bigger given the size of the chylomicron cargo and their formation is intermittent to coincide with dietary fat intake whereas protein vesicle formation is continuous ferrying new proteins to the Golgi for cellular distribution and use (Fig. 4). ATP and not GTP is required for prechylomicron transport (141).

Formation of a PCTV is initiated by FABP1 (liver FABP) and three additional proteins: apoB48, CD36, and vesicle-associated membrane protein 7 (VAMP7) (98,140) (Fig. 5). Absence or antibody inhibition of any of the 4 proteins in the budding complex greatly attenuates ER PCTV formation (140) and coimmuno-precipitation studies suggested physical interaction between apoB48, CD36, VAMP7, and FABP1. PCTV are efficiently sealed to protect their cargo from lipases (97,110,113). Deletion of either L-FABP or CD36 results in nearly complete cessation of PCTV budding activity. VAMP7 is usually found in the post-Golgi fraction of eukaryotic cells and mediates vesicular transport from endosomes to lysosomes (7). During PCTV formation, VAMP7 functions as an ER-derived vesicle-associated SNARE (N-ethylmaleimidesensitive attachment receptors) directing docking and fusion of PCTV with the Golgi (142).

Figure 5.

Role of CD36 and FABP1 in budding of the ER prechylomicron particle for transfer to the Golgi. Newly absorbed FA and MAG are endocytosed at the enterocyte apical membrane in caveolin-1 containing endocytic vesicles (CEV) that also carry CD36 (step 1). The FA and MAG are reesterified into TAG to generate lipid droplets in the ER lumen and in the cytosol and both merge to form the prechylomicron particle in the ER (see section on prechylomicron formation). Budding of the prechylomicron is thought to require FABP1 binding to the ER, which is induced by the following sequence of events. The CEV contains protein kinase zeta (PKC-ζ) and lyso-phosphatidylcholine (LPC) on its surface and PKC-ζ is activated by LPC. Upon activation, PKC-ζ elutes from the CEV into the cytosol where it phosphorylates its substrate Sar1b (step 2). Phosphorylation of Sarb1 releases FABP1 from a cytosolic heteroquatromeric protein complex where it is sequestered together with other proteins (Sec13, and SVIP) (step 3). Released FABP1 binds to the ER and together with CD36, apoB48, and VAMP7 promotes budding from the ER of the PCTVs (step 4) for its transfer to the Golgi (step 5). Once the PCTV reaches the Golgi, it becomes tethered and subsequently fuses with the Golgi membrane delivering its chylomicron cargo into the lumen. The chylomicron matures in the Golgi where additional apoproteins are added (ApoA-1 and ApoA-IV) and then is released across the basolateral membrane of the enterocyte to be transported into the lymph (see Fig. 1).

PCTV is an ER to Golgi transport vesicle and its budding requires binding of cytosolic FABP1 to the ER (Fig. 5). Cytosolic FABP1 is normally sequestered in a heteroquatromeric complex that includes the proteins Sar1b, Sec13, and SVIP. Protein kinase C (PKC) has also been to be required for the budding step (98,143). The PKC family is comprised of 3 isoforms, the conventional cPKC activated by DAG and Ca²⁺, the novel nPKC requiring only DAG and the atypical aPKC (includes PKCzeta, PKCζ) that requires neither. Sarb1 is a PKCζ substrate and its phosphorylation by PKCζ dissociates the heteroquatromeric complex releasing its components including monomeric FABP1 (Fig. 5 step 3). This allows budding of PCTV as the released FABP1 binds to the ER (Fig. 5 step 4) (98, 139). Interestingly humans with a mutation in Sar1b retain chylomicrons in their intestines (32,74). Once the PCTV reaches the Golgi, it becomes tethered and subsequently fuses with the Golgi membrane delivering its chylomicron cargo into the lumen (Fig. 5, step 5). The fusion process requires formation of a complex between soluble SNAREs, the v-SNAREs of the PCTV and those of the target membrane T-SNAREs (77). In the Golgi, the chylomicron acquires additional apoproteins ApoA-1 and ApoA-IV and then the chylomicron is released at the basolateral membrane of the enterocyte into the lymphatic vessels or lacteals.

CD36 is important for chylomicron production and the intestinal lipoproteins produced in Cd36^−/− mice are 35% smaller and exhibit delayed blood clearance (34, 53, 102). CD36 also mediates the effect of glucagon-like peptide 2 (GLP-2) to enhance intestinal FA absorption and chylomicron production in hamsters and mice as the GLP-2 effect is absent in Cd36^−/− mice (68,111). CD36-mediated signaling is important for inducing expression of key chylomicron proteins such as apoB48 and MTTP (19,160).

Internalization of CD36 from the plasma membrane can be initiated by ligand binding to the receptor and is highly regulated (153). During the postprandial period, CD36 is internalized and undergoes proteolysis by the ubiquitin-proteasome pathway, causing its disappearance from the luminal side of intestinal villi (160). This event is triggered by long chain fatty acid and/or diglycerides derived from early digestion of dietary TAG. CD36 internalization and degradation appears to be required for the induction of key proteins of chylomicron formation (19,160). In a mouse model of diet-induced metabolic syndrome, production of smaller chylomicrons that are slowly cleared from the circulation is observed and correlates with dysfunctional trafficking of intestinal CD36 most likely as a result of higher insulin, which inhibits CD36 ubiquitination (19,153). A defect in CD36 function in rodents fed high fat diets was proposed to explain the secretion of a substantial fraction of smaller intestinal triglyceride-rich particle. Intestinal chylomicron secretion into the lymph, measured by cannulating the mesenteric duct, is decreased by 50% in Cd36^−/− mice (34, 108) but the mechanistic events involved in chylomicron output to the lymph and why it is impaired in CD36 deficiency remain to be elucidated.

In addition to FA, CD36 may also facilitate intestinal cholesterol absorption for optimal chylomicron production. Enterocytes isolated from Cd36^−/− mice exhibit reduced cholesterol uptake (107) and in vivo cholesterol output into the lymph is reduced by 50% (108). Consistent with this, ezetimibe treatment in mice (82) and phytosterol (plantsterol) intake in humans (127) which inhibit cholesterol absorption, decrease CD36 expression. Ezetimibe also inhibits CD36 facilitated cholesterol uptake in COS-7 cells (162).

Maturation of the prechylomicron in the Golgi

In the lumen of the Golgi, the prechylomicrons undergo two maturation events in preparation for export into the intestinal lymphatic vessels called lacteals. Prechylomicrons acquire apoAI in an energy independent process (144). Although ApoAI is present in the ER lumen it does not bind the ER prechylomicron while it binds to chylomicrons in the Golgi (98). The second maturation step involves apoB48 which undergoes glycosylation becoming endoglycosidase Hresistant (16). The prechylomicron might undergo additional lipidation in the Golgi. MTTP, which performs the lipidation step in the ER, has been identified in the Golgi (87). However, there is evidence arguing against further lipidation including demonstration that the average diameter of PCTV is similar to that of mature chylomicrons, suggesting lack of particle expansion by added lipids (98,177). The mature chylomicron exits the Golgi into the cytosol in a large vesicle containing additional chylomicrons by an unknown mechanism. These vesicles can be seen at the basolateral membrane where they are exocytosed into the interstitium to enter the lymphatic lacteals (128).

CD36 genetic variants influence chylomicron formation and postprandial lipids in humans

Single nucleotide polymorphisms (SNPs) in the CD36 gene have been linked to abnormal lipid metabolism(91,92,95)and to metabolic syndrome risk in humans (93). Total CD36 protein deficiency is relatively common in people of Asian and African descent (3–9%) (8,31,64) and noninvasive scintigraphy showed absence of myocardial FA uptake in these subjects (42,155,157). Asian individuals with CD36 deficiency have abnormalities of fasting and postprandial plasma lipid levels (44,81,105,172). In African Americans, SNPs in the CD36 gene associate with higher plasma FA (137) and altered TAG and cholesterol (92) and many influence metabolic syndrome risk (38,93). In Caucasians, common polymorphisms (40%−50% incidence) associate with high plasma FA and increased risk of diabetes-linked cardiovascular disease (95).

The contribution of CD36 to defective lipid processing by the small intestine in humans is supported by findings in CD36 deficient subjects of postprandial lipemia with higher levels of smaller chylomicron remnants (102) showing similarities with the altered intestinal metabolism of Cd36^−/− mice. A recent study involving close to a thousand Caucasian participants found that CD36 SNPs, that reduce CD36 expression, increase levels of postprandial lipids after ingestion of a high fat meal (91). Many of the associated SNPs localize near previously validated binding sites for PPARγ, a major CD36 transcriptional regulator. In addition, DNA methylation sites that strongly reduced CD36 expression were identified and correlated with high plasma TAG and LDLs suggesting a potential role of epigenetic regulation of CD36 in abnormalities of lipid metabolism (91). The altered postprandial lipid profile in humans with partial or total CD36 deficiency is likely to contribute to the associations previously reported between CD36 SNPs and diabetes-linked cardiovascular disease (95) and risk of metabolic syndrome (91).

Other Functions of CD36 in the Digestive Tract

CD36 has additional functions in the gastrointestinal tract and although these functions are not directly related to intestinal fat absorption they impact lipid metabolism.

Orosensory fat perception and the cephalic phase of digestion

Recent evidence supports existence of orosensory fat taste perception mediated by FA receptors expressed on taste buds in the tongue. Two main classes of FA taste receptors have been proposed, CD36 (85) and the G-protein-coupled receptors (GPRs) (22). CD36 is abundantly expressed on the apical surface of taste bud cells in the tongue of rodents, pigs, and humans (43,85) and its deletion reduces spontaneous FA preference in mice (25,85). Consequent to long chain FA binding to CD36 on taste bud cells, a signaling cascade is triggered ultimately resulting in the release of neurotransmitters that relay signals to the central nervous system (37). These events mediate fat taste perception, as well as the early preabsorptive (also called cephalic) phase of digestion, which is characterized by secretion of small amounts of insulin and bile acids and serves to prime the organism for fat absorption (85,156). GPRs involvement in fat taste perception is less clear. Among the GPRs, GPR120 is detected in gustatory epithelia (22,45), however, unlike CD36, GPR120 is unresponsive to the low FA concentrations likely to be present in the mouth during fat intake and might function at high concentrations of FA and other tastants, consistent with its expression in a variety of taste cells responsive to various stimuli (115).

In humans, there is support for role of CD36 as a fat taste receptor. Several studies investigated the impact of a common CD36 SNP, rs1761667 involving A/G substitution, which reduces CD36 protein level on oral sensory fat perception. African American individuals with obesity carrying the A allele of the SNP have decreased OA orosensory detection thresholds compared to non-carriers (119). The effect of this SNP on sensory fat perception was replicated in other populations (103,132). Subjects homozygous for the A allele were insensitive discriminators of fat content and displayed higher mean acceptance of added fat (78). Together these data suggest that carriers of the CD36 SNP have low FA sensitivity might be prone to increase fat intake since they need higher FA concentrations to reach taste “saturation”. Additional studies are needed to test this hypothesis.

Role of CD36 in fat-induced satiety; OEA, CCK, and secretin release

In the small intestine, CD36 is important for fat-induced satiety by delivering OA for production in the jejunum of the satiety messenger oleylethanolamide (OEA) which prolongs the intermeal interval (135). OEA, in turn, increases intestinal CD36 expression (41, 173) and FA uptake (173). Although the above findings might suggest thatCD36absence would enhance food intake, intake is decreased in Cd36 null mice (57) likely reflecting postingestive effects of the delayed intestinal lipid absorption in these mice (136).

CD36 is expressed in enteroendocrine cells (EECs) (154), which are specialized secretory cells that release several peptides, including cholecystokinin (CCK) and secretin in response to FA. CCK helps optimize fat digestion by inhibiting gastric emptying while stimulating intestinal motility and gallbladder contraction (24). Secretin inhibits gastric emptying and synergizes with CCK to induce pancreatic secretions (27). The Cd36 null mouse displays a 50% reduction in basal release of CCK and secretin and in response to gastric administration of oil. Diminished release of these peptides in response to FA is also observed with CD36 deficient intestinal segments in vitro. In EEC expressing CD36, release of CCK and secretin involves FA-induced and CD36-mediated increases in calcium and the second messenger cAMP (154). A dysfunction in CD36 may affect secretion of these hormones resulting in a reduction of fat digestion capacity, which could contribute to more overflow of dietary lipids reaching the distal intestine.

Role of CD36 in gallstone formation

Cd36^−/− mice have been recently reported to be protected from developing gallstones induced by feeding a lithogenic diet (LD) high in cholesterol (167), suggesting a regulatory role of CD36 in canalicular and biliary cholesterol transport and secretion. Hepatic lipid content in LD-fed mice did not identify major differences in total or free cholesterol content, but revealed increased bile acid content in the liver ofCd36^−/− mice. Biliary lipid secretion was altered in Cd36^−/− mice with increased concentration of taurobetamuricholic acid, which decreased the hydrophobicity index. Dysregulated gallbladder contractility in Cd36^−/− mice that could result in more frequent bile emptying together with lower bile hydrophobicity would explain the protective effect of CD36 deletion. Interestingly, CD36 deletion reversed the effect of L-FABP deletion to promote gallstone formation as the double knockout mouse was protected. Neither intestine- nor liver-specific CD36 deletion showed protection against LD induced gallstone formation (167).

Role of CD36 in intestinal homeostasis and inflammation

Recent findings have revealed an unsuspected role of CD36 in maintaining integrity of the epithelial barrier (28). This role appears separate from that in lipid uptake and metabolism, however, it could suggest that CD36 dysfunction in the intestine might increase susceptibility to inflammation consequent to abnormalities of gut fat handling. Global deletion of CD36 in mice results at the level of the proximal small intestine in abnormal extracellular matrix remodeling and in a leaky epithelial barrier with neutrophil infiltration and inflammation. Lower levels of interleukin (IL)-22 which is important for epithelial barrier maintenance together with the higher level of the proinflammatory IL-6 in the intestines of Cd36^−/− mice are likely to play a role in the observed disruption of barrier integrity. Systemically, the deletion associates with signs of chronic subclinical inflammation and loss of the antiinflammatory Ly6C^low monocytes that help maintain the vasculature. Endotoxemia is observed after an intragastric bolus of triolein consistent with presence of a leaky epithelium. CD36 deletion specific to endothelial cells (EC-Cd36^−/−) recapitulated many aspects of the abnormal gut phenotype of germline Cd36^−/− mice suggesting that CD36 is important for vascular health and highlighting potential role of the endothelium in initiating gut inflammation (28). The inflammation might be related to trans-epithelial migration of neutrophils which often associates with disruption of barrier function and correlates with disease flares and severity of inflammatory bowel disease (106, 117). The findings also support importance of CD36 in phagocytic clearance of apoptotic neutrophils for inflammation resolution and tissue healing (10,71).

High intake of dietary fat has been associated with gut inflammation that ultimately can result in systemic inflammation. The findings related to CD36 regulation of the intestinal barrier suggest novel mechanisms related to how CD36-mediated fat intake can result in intestinal inflammation. CD36 recognizes bacteria and is important for mounting an acute immune response against pathogens (152) but whether it plays a role in determining the profile of the gut microbiota and how it is impacted by fat diets remains unknown. The microbiota can shape the intestinal environment driving homeostasis or inflammation (114) and how CD36 function affects the cross-talk between intestinal cells and microbiota requires investigation.

Conclusion

There is strong evidence to support the concept that intestinal lipid metabolism plays an important role in energy homeostasis and health of the overall organism. Postprandial lipemia, a common complication of obesity or insulin-resistance, increases risk of developing atherosclerosis and cardiovascular disease (6) and could be alleviated by targeting gut FA transporters. However, further understanding of transporter regulation and structure-function relationships is needed. For example, FAs induce ubiquitination and internalization of CD36 (145) and this event is important to initiate chylomicron formation (160). Control of the ubiquitination process could have metabolic benefits. More insight is needed into the mechanisms by which CD36 facilitates FA signaling and the implications of both processes as related to gut function and homeostasis (55,135,136). The role of FA signaling at the start of a meal in mediating the early preabsorptive secretions (by pancreas, stomach, gallbladder, and intestines) that prime the organism for processing nutrients and help maintain insulin sensitivity, also needs to be examined (126). The associations identified between DNA methylation sites or common CD36 SNPs (minor allele 10%−45%) (92,93,95) and alteration of plasma lipids (91) suggest that CD36 function contributes significantly to individual variability in lipid metabolism. Additional studies of the functional impact of epigenetic regulation by overnutrition of CD36 and potentially of other intestinal transporters on lipid metabolism would be informative and likely to have clinical relevance. The findings of CD36 importance for gut homeostasis and barrier integrity require additional work that defines the cell types and factors responsible for these phenotypes and that examines the general role of endothelial damage in intestinal inflammation. Finally the microbiota can shape the intestinal environment driving homeostasis or inflammation (114) and how CD36 function affects the cross-talk between intestinal cells and the microbiota requires investigation.

Didactic Synopsis.

The information provided in this review should facilitate teaching students about our current knowledge related to key proteins involved in uptake and absorption of dietary fatty acids in the small intestine. The following specific topics might be suitable for graduate or advanced undergraduate levels:

• Absorption of dietary fatty acid (FA) across the brush border membrane of enterocyte in the proximal small intestine can occur through passive diffusion and/or can be mediated by specific transport proteins.

• Remodeling of membrane phospholipid might be important for diffusion of FA in enterocytes.

• Scavenger receptor CD36 (SR-B2) is a brush-border membrane protein that facilitates FA uptake and FA processing in enterocytes of rodents and human.

• The component of uptake mediated by CD36 is a small fraction of net FA uptake by the small intestine but it plays a regulatory role by priming the organ for packaging the absorbed lipid and secreting it as lipoproteins.

• Absorbed lipids are packaged into lipoprotein particles called chylomicrons in the enterocyte. Generation of chylomicrons requires several steps including prechylomicron formation and assembly in the endoplasmic reticulum(ER), enter the lymphatic system.

• CD36 and liver FA binding protein 1, FABP1, play a crucial role in chylomicron formation, assembly and trafficking from ER to Golgi.

• The FA receptor CD36 has pleiotropic functions. In addition to its role in absorption and chylomicron formation, it mediates secretion of intestinal peptides and is important for maintenance of intestinal homeostasis and epithelial barrier integrity.