Intestinal Fatty acid Absorption - IngentaConnect

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Immun., Endoc. & Metab. Agents in Med. Chem., 2009, 9, 60-73

Intestinal Fatty acid Absorption Yong Ji, Xiaoming Li and Patrick Tso* Department of Pathology, University of Cincinnati College of Medicine, 2120 East Galbraith Road, Cincinnati, OH 45237, USA Abstract: Dietary lipids and many lipophilic vitamins and compounds are transported in lymph by chylomicrons which are secreted by the enterocytes of the small intestine. This article reviews our current understanding of the digestion, uptake, intracellular metabolism, and packaging of dietary lipids into chylomicrons. An overview of the digestive enzymes involved in the digestion of triacylglyerol is provided. We also discuss the relative importance of vesicles and micelles in delivering the lipid digestion products for uptake by the small intestine. While there is certainly evidence for the involvement of transporters in the uptake of cholesterol by enterocytes, we believe the uptake of fatty acids is predominantly passive. The active uptake of fatty acids is only important when the luminal fatty acid concentration is very low (probably to ensure that the essential fatty acids are not lost). We also describe the roles of the various lipid esterifying enzymes in the metabolism of the absorbed partial glycerides and fatty acids. In addition, the present review illustrates the mechanism by which intracellular assembly and modification occur during the formation of chylomicrons and very low density lipoprotein. Furthermore, clinical disorders related to intestinal lipid transport are discussed. The authors’ basic and clinical insights into the processes of intestinal lipid absorption provide valuable information about effective, safe ways to prevent obesity and hyperlipidemia.

Key Words: Lipase, enterocytes, fatty acid binding protein, transporter, pluronic-81, Acyl-CoA cholesterol acyltransferase. 1. TYPES OF DIETARY LIPIDS Dietary lipids, especially fatty acids, are carried by chylomicrons in lymph. The intestinal digestion, uptake, intracellular metabolism, and packaging of dietary lipids into chylomicrons are complex but we have learned a great deal about the processes involved by employing both cellular and molecular approaches. The major purpose of this review is to provide a succinct and yet thorough account of the digestion, uptake, and transport of dietary lipids. Dietary fat is defined as that part of the diet which can be extracted by organic solvents [1]. Consequently, dietary fat is comprised of a wide array of compounds, from the highly non-polar hydrocarbons to the highly polar phospholipids (PL) and glycolipids. The classification of these various lipids and their respective behaviors in the aqueous medium has already been ably reviewed by Carey and Small [2]. Whether a lipid is classified as polar or non-polar depends on its interaction with water. Non-polar lipids are insoluble in the bulk water phase and, therefore, will not interact with water (Fig. 1). Examples of non-polar lipids include cholesteryl ester (CE), hydrocarbons, and carotene. Polar lipids, on the other hand, fall into one of three classes: I) insolube non-swelling amphiphiles; II) insoluble swelling amphiphiles; and III) soluble amphiphiles. The insoluble non-swelling amphiphiles include triacylglycerol (TG), diacylglycerol (DG), non-ionized long-chain fatty acids (FA), cholesterol, and fat-soluble vitamins. When these water insoluble amphiphiles are added to water, a thin monolayer film of the lipid molecules is formed. Non-swelling amphi*Address correspondence to this author at the Department of Pathology, University of Cincinnati, Genome Research Institute, 2120 East Galbraith Road, Bldg. A, Room 271, Cincinnati, OH 45237, USA; Tel: (513) 5582151; Fax: (513) 558-1006; E-mail: [email protected] 1871-5222/09 $55.00+.00

philes are so named because they interact very little with water in the bulk phase. The second group of polar lipids, the insoluble swelling amphiphiles, includes monoacylglycerols (MG), ionized FAs, and PLs. In addition to forming a monolayer on the surface of water, this group of lipids has the unique ability to interact with water to form a laminated lipid-water structure referred to as liquid crystal. In the liquid crystal state, non-polar lipid molecules face one another, sandwiching water between the polar groups. This unique lipid property is referred to as "swelling,” hence the name swelling amphiphiles. The third group of polar lipid molecules consists of the swelling amphiphiles, which possess strong polar groups, thus enabling the molecules to become soluble in water at low concentrations. This group of lipids can be sub-divided further into soluble amphiphiles with lyotropic mesomorphism and those without. Sodium salts of long-chain fatty acids (FAs) are a good example of soluble amphiphiles with lyotropic mesomorphism. When the concentration of these lipids reaches the critical micellar concentration, the monomers aggregate to form micelles. In the micelle, the polar group of monomers faces the aqueous medium outside, and the non-polar group of monomers faces inside. Before sodium oleate molecules form micelles, however, they pass through an intermediate liquid crystal phase, a phenomenon referred to as lyotropic mesomorphism. In contrast, soluble amphiphiles without lyotropic mesomorphism do not undergo the intermediate liquid crystal phase before micelles form. Bile salt, secreted by the liver, is a good example of a soluble amphiphile without mesomorphism. This review will focus primarily on the digestion, absorption, and transport of TG, the predominant dietary lipid. Readers interested in cholesterol absorption should also refer to the excellent review published by Dawson and Rudel [3] as well as those by other investigators [4, 5]. © 2009 Bentham Science Publishers Ltd.

Intestinal Fatty acid Absorption

Immun., Endoc. & Metab. Agents in Med. Chem., 2009, Vol. 9, No. 1

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Fig. (1). Classification of lipids based on their ability to interact with water. Non-polar Lipids: octadecane, carotene, squalene, cholesteryl oleate, cholesteryl linoleate, and paraffin oil. Polar Lipids: I. - triacylglycerols, diacylglycerols, long-chain protonated fatty acids, and fat-soluble vitamins. II. - phospholipids, monoacylglycerols, monoethers, and alphahydroxy fatty acids. IIIA. - sodium salts of long-chain fatty acids, many anionic, cationic and nonionic detergents, and lysophosphatidylcholine. IIIB. - bile salts, sulfated bile alcohols, and saponins [2].

Dietary fat constitutes a significant source of calories in the Western diet—as much as 30 percent of total caloric intake, or 90 - 100 grams per day. The dietary intake of fat has received considerable attention over the last few decades since diets which are high in fat have been linked to high blood lipids, in particular cholesterol, as well as increased risk of coronary heart disease [6-8]. It has generally been accepted that diets rich in saturated FAs are more cholesterolemic (raise blood cholesterol) than diets rich in polyunsaturated FAs [9]. Trans-fatty acids have double bonds, yet they behave more like saturated FAs and are considered to be just as atherogenic as saturated fat [10, 11]. The Surgeon General of the United States currently recommends that overall fat consumption be reduced to an average of 30 percent or less of total caloric intake, with saturated fat consisting of 10 percent or less, and that consumption of cholesterol be limited to no more than 300 mg per day. This standard has also been adopted by the American Heart Association. In the past two decades, there has been increased effort to either chemically or biologically modify the FA profile of naturally occurring TGs in order to obtain a particular physical property or physiological function. STGs are produced by the chemical interesterification of both medium- and long-chain FAs incorporated on the same glycerol backbone by hydrolysis and random reesterification

[12, 13]. These TG molecules are chemically distinct and offer unique advantages due to their constituent physical mixture of medium-chain (MCT) and long-chain (LCT) TGs. For example, STGs that contain medium-chain FAs may serve as useful vehicles for rapid hydrolysis and absorption due to their smaller molecular size and greater water solubility relative to LCTs. Although STGs retain some characteristics of MCTs and LCTs, they may also prove to be an alternative lipid source capable of overcoming gastrointestinal intolerance related to the sole use of MCT or LCT in patients with malabsorptive diseases. The clinical application of STGs has already been skillfully reviewed by Heird et al. [14], Bell et al. [15] and Mu and Porsgaard [16]. Beneficial effects include improved absorption of linoleic acid in cystic fibrosis patients [17, 18] and a protein sparing effect in STG based safflower oil [19] and fish oil [20]. Studies from our laboratory have demonstrated that STG promotes the lymphatic absorption of both vitamin A and vitamin E better than the constituent physical mixture with similar FA composition. Interestingly, the enhanced delivery of vitamins A and E into lymph by STG was not limited solely to normal animals but could also be detected in animals with impaired gastrointestinal absorptive functions resulting from ischemia reperfusion induced injury [21, 22]. Whether or not STGs are also capable of enhancing lymphatic delivery of lipid soluble drugs is an area which merits further investigation in the future.

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2. LUMINAL DIGESTION OF DIETARY LIPIDS 2.1. Gastric Lumen The digestion of dietary lipids in humans begins in the stomach. The enzyme involved in lipid digestion in the stomach is called acid lipase and is secreted into the stomach by the gastric mucosa. Acid lipase has its highest activity level in the acidic medium. The highest activity level of gastric lipase can be detected in the fundus of the stomach, as confirmed by an independent study conducted during the post-mortem examination of two healthy human organ donors [23]. Human gastric lipase has a pH optimum ranging from 3 to 6.0 [24, 25], and hydrolyzes medium-chain TGs better than long-chain TGs [26]. Acid lipase does not hydrolyze PL or cholesterol esterase. The main hydrolytic products of gastric lipase are DGs and FAs [24, 25, 27]. The digestion of TG by gastric lipase in the stomach plays a particularly important role in neonates. Milk fat, the primary source of nourishment for neonates, contains a considerable amount of medium-chain TG, and acid lipases work more efficiently with medium-chain TG than longchain TG. Furthermore, the pancreatic lipase system is not fully developed in the neonate [28]; thus, gastric lipases consequently play a crucial role in the normal digestion of milk fat. Gastric lipases also play an important role in lipid digestion in adults. This is particularly evident in cystic fibrosis patients, who maintain the ability to absorb dietary lipid despite the fact that pancreatic lipase secretion in these patients is markedly or completely inhibited [29]. Gastric lipase activity in the stomach is not compromised in patients with cystic fibrosis [30]. The stomach is also the major site for the emulsification of dietary fat, an important prerequisite for efficient hydrolysis by pancreatic lipase. Gastric chyme is propelled forward through the antrum to the pylorus via the peristaltic waves of the corpus (Fig. 2). Liquid is squirted into the duodenum along with any small solid particles present in the chyme. The pylorus closes, and the antrum contracts forcefully, grinding the solid particles. As result of these contractions, the antral contents are retropelled from the terminal antrum back into the corpus. The squirting of antral contents into the duodenum, the grinding action of the antrum, and the retropulsion of antral contents back to the corpus represent most of the mechanical action involved in the initial emulsification of dietary TG. The DG and FA resulting from the action of acid lipases in the stomach as well as the PL normally present in the diet further aid the emulsification of dietary fat.

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34]. Desnuelle [35] expanded on these observations as well as those made by Schonheyder and Volqvartiz in 1946 [36]. The monolayer technique, so eloquently detailed by Verger [37], has greatly advanced our understanding of how the oil/water interface affects lipolysis. Spreading the lipid as a monomolecular film at the air/water interface has allowed researchers to study the effects of the surface area and physicochemical properties of the interface on the rate of lipolysis. Only a small amount of lipid is required in the monolayer technique, an advantage when using rare synthetic lipids. One potential problem with the monolayer technique, however, is that enzymes may become denatured at the lipid/water interface [38, 39]. The velocity of lipolysis is dependent on both the surface area of the interface and also those factors that modify the physicochemical properties of the interface [40-42]. Based on the landmark paper by DiMagno et al. [43], it has been generally assumed by gastroenterologists that the pancreas secretes more lipase than needed to digest the fat ingested by humans. This concept, however, has been challenged by the research of Carriere et al., who found that human pancreatic lipasespecific activity on dietary TG is three orders of magnitude lower than the very high specific activity measured under the usual in vitro experimental conditions [44]. They proposed that the lipase activity in the human small intestine is not as high as we had previously thought, which explains why such a large amount of lipase is normally secreted by the human pancreas and why fat absorption takes a relatively long period of time.

2.2. Intestinal Lumen

Fig. (2). Diagrammatic representation of the consequences of antral peristalsis [174].

The emulsification process in the stomach ensures that the lipid emulsion entering the small intestine is in the form of fine lipid droplets which are less than 0.5 micrometer in diameter [31, 32]. The combined action of bile plus pancreatic juice brings about a marked change in the chemical and physical form of the ingested lipid emulsion. Most of the digestion of TG is brought about by pancreatic lipase in the upper part of the intestinal lumen. The pancreatic lipase works at the interface between oil and aqueous phases [33,

Pancreatic lipase acts mainly on the sn-1 and sn-3 positions of the TG molecule to release 2-monoacylglycerol (2MG) and free FAs [41, 45-47]. Although 1-monoacylglycerol (1-MG) is formed from 2-MG through isomerization in an aqueous medium, 2-MG is probably the predominant form in which MG is absorbed by the small intestine since the formation of 1-MG occurs at a slower rate than uptake of 2-MG by the small intestine [48]. Further hydrolysis of the 1- or 2-MG by pancreatic lipase results in the formation of



glycerol and FA [49]. The enzyme (pancreatic lipase) works more efficiently with 1-MG than with 2-MG; however, since the absorptive rate is so fast, most of the 2-MG is absorbed before degrading or isomerizing to form 1-MG.

3. MICELLAR SOLUBILIZATION AND UPTAKE OF DIETARY LIPIDS BY ENTEROCYTES

Pancreatic lipase (EC 3.1.1.3) is abundant in pancreatic juice, representing 2 - 3 % of the total protein present [50]. Its high concentration in pancreatic secretions and its high catalytic efficiency ensure the efficient digestion of dietary fat. Most humans absorb between 90 - 95 % of the fat ingested. Consequently, only very severe pancreatic deficiencies result in the malabsorption of fat. Pancreatic lipase has been purified from a number of species, including humans [51-54]. Porcine lipase has been sequenced and has 449 amino acid residues [55]. Porcine pancreatic lipase is a glycoprotein with a carbohydrate chain of approximately 2000 attached to asparagine at position 166 of the protein [56, 57]. For pancreatic lipase to work, it must anchor itself at the interface. The serine residue at the 152 position of the peptide plays an important role in the binding site [58]. Scientists have long been fascinated by the observation that while pure pancreatic lipase works extremely inefficiently in a bile salt-lipid mixture, lipase present in pancreatic juice hydrolyzes TG extremely efficiently. This interesting observation led to the discovery of the cofactor called colipase. In 1969, Morgan et al. made the crucial observation that colipase is only necessary for lipase activity if bile salt is present [59]. The laboratories of Professor Borgstrom in Lund and Professor Desnuelle in Marseille have provided most of the information which informs our current understanding of the biochemistry of colipase and its interaction with pancreatic lipase. Colipase has been purified from a number of animal species [60-63]. The mechanism by which lipase works has been discussed in detail by ErlansonAlbertsson [64]. TG lipid droplets covered with bile salts are inaccessible to pancreatic lipase; however, the binding of the colipase to the TG/aqueous interface allows the lipase molecule to bind to the lipid/aqueous interface. Using site directed mutagenesis, Freie et al. [65] have demonstrated recently that valine at position 407 and leucine at 412 are important in the interaction of lipase with colipase and bile salt micelles. Thus the bile salt micelle, in addition to aiding in the transport of digested lipids to the enterocytes, also plays a role in the lipolysis of TG by pancreatic lipase. Pancreatic lipase binds with colipase with a 1:1 molar ratio [66]. The inhibition of lipase has been used clinically as a strategy to combat obesity. Orlistat, a semisynthetic derivative of lipstatin, is a potent and selective inhibitor of these enzymes, showing little or no inhibitory effects against amylase, trypsin, chymotrypsin and phospholipases. Lipstatin is a natural, irreversable inhibitor of pancreatic lipase first isolated from the Actinobacterium Streptomyces toxytricini. Orlistat acts by binding covalently to the serine residue of the active site of gastric and pancreatic lipases. By inhibiting lipase activity, the amounts of monoaclglycerides and free fatty acids available for uptake are greatly reduced. Although lipase activity is greatly reduced by Orlistat, other important gastrointestinal functions such as gastric emptying and acidity and gallbladder motility remain unaffected.

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The concept that micellar solubilization is critical to the uptake of dietary lipids comes from the work of Hofmann and Borgstrom [67, 68]. This concept was later challenged by Carey and his associates, who discovered the co-existence of unilamellar liposomes with bile salt-lipid mixed micelles in the small intestine [32]. This, then, begs the question: which is the more important (micelle or vesicle) vehicle in the promoting the uptake of lipid digestion products? 3.1. Importance of Micellar Solubilization To evaluate the importance of micellar solubilization of 2-MG and FA in their uptake by enterocytes requires an understanding of the role of the unstirred water layer, a concept introduced by Dietschy and his colleagues [69-71]. As shown in Fig. (3), the brush-border membrane of enterocytes is separated from the bulk fluid phase in the intestinal lumen by an unstirred water layer. The unstirred water layer, as the name implies, mixes poorly with the bulk phase in the intestinal lumen. Consequently, solute molecules in the bulk phase can only gain access to the brush border membrane by first diffusing across the unstirred water layer. Because the solubility of FA and MG in an aqueous medium is extremely low, very few molecules gain access to the brush-border membrane (arrow 1). In contrast, the micellar solubilization of MG and FA greatly enhances the number of molecules available for uptake by the enterocytes (arrow 2). Micellar solubilization increases the aqueous concentration of FA and MG many-fold. Despite the slower rate of diffusion of the micelle relative to the monomolecular FA molecule (due to size), micellar solubilization still markedly enhances the diffusion of FA and MG molecules across the unstirred water layer. While micellar solubilization plays a crucial role in the absorption of lipid soluble molecules such as fatty acids and cholesterol, the absorption of lipid soluble molecules can, by no means, be explained purely on the basis of degree of solubilization by micelles. Several investigators have demonstrated that trihydroxy bile acids are more effective in promoting cholesterol absorption than dihydroxy bile acids [72-74]; however, the degree of solubilization was not measured in these experiments. Watt and Simmonds [74] showed a linear relationship between the amount of cholesterol taken up by the small intestine and the micellar cholesterol concentration, thus demonstrating the importance of micellar solubilization in the uptake of cholesterol. Through a series of elegant studies using Pluronic F-68, a non-ionic surfactant capable of promoting the micellar solubilization of cholesterol but not its uptake by enterocytes, Watt and Simmonds demonstrated that planar structure is important in promoting the uptake of cholesterol by the enterocytes [74]. The function of bile salt in intestinal cholesterol uptake thus extends beyond its role in solubilizing cholesterol. This is further illustrated by the fact that chenodeoxycholyl taurine (CDC-tau) is a better micellar solubilizer of cholesterol than cholyl taurine (C-tau), even though cholesterol uptake is significantly greater with C-tau than it is with CDC-tau [75-77].


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Fig. (3). Diagrammatic representation of the effect of bile salt micelles (or vesicles) in overcoming the diffusion barrier resistance by the unstirred water layer. In the absence of bile acids, individual lipid molecules must diffuse across the barriers overlying the microvillus border of the intestinal epithelial cells (arrow 1). Hence, uptake of these molecules is largely diffusion limited. In the presence of bile acids (arrow 2), large amounts of these lipid molecules are delivered directly to the aqueous-membrane interface so that the rate of uptake is greatly enhanced [71].

3.2. Importance of Unilamellar Vesicles When the jejunal contents of humans digesting a lipid meal were spun using an ultracentrifuge, there was a solid particulate layer on the bottom of the tube followed by a clear micellar layer and finally an oily phase on top [68]. The oily phase consisted primarily of TG, partial glycerides, and FAs. The clear micellar phase consisted of bile salts, FAs, and MGs. When Porter and Saunders [78] carefully analyzed the aqueous phase present after ultracentrifugation, however, by passing the aqueous phase through a series of filters with progressively smaller pores (the smallest being 100 nm in diameter), they found that the final filtrate still did not display a clear micellar phase. Instead, a slightly turbid phase was observed. Furthermore, a concentration gradient of lipids could be detected in the micellar phase. The full implication of these findings were not appreciated by the investigators until much later, when Patton and Carey observed the digestion of fat in vitro using light microscopy [79]. They found that the presence of at least three phases during fat digestion: 1) the oil phase (mainly TG, partial glycerides and FA); 2) the calcium soap phase (Ca++ ions and protonated long-chain FA), and 3) the viscous isotropic phase (MG and FA). Based on these results, they concluded that it is probably an oversimplification to divide the intestinal contents into only two phases, oil and micellar. Carey et al. [32] proposed that when the concentration of bile salt in the lumen exceeds the critical micellar concentration, the lipid in the intestinal lumen is incorporated into mixed micelles which probably take the form of large mixed disc-like micelles more or less saturated with lipids with a hydrodynamic radius of approximately 200 Å. When the

amount of lipid in the aqueous phase increases further, the formation of liquid crystalline vesicles (liposomes) with a hydrodynamic radii of 400 - 600 Å results [32, 80]. These findings have important clinical implications. Because patients with low intraluminal bile salt concentration [81], or those with bile fistulae [82], seem to be able to absorb fat fairly effectively, Carey et al. proposed that the liquid crystalline vesicles likely play an important role in the uptake of FA and MG by enterocytes [32] in these diseased states. This question was addressed recently in studies conducted by Setchell and Heubi [83]. In these studies, subjects with inborn bile acid synthesis defects were placed on supplemental bile acid therapy. The luminal contents were collected from experimental subjects given bile salt supplements and from control subjects not given these supplements. The luminal contents consisted of either mostly vesicles or mostly micelles. Using these luminal samples, they showed that FA can be taken up by the small intestine from micelles as well as vesicles whereas cholesterol can only be taken up when solubilized in micelles. 4. MUCOSAL BRUSH BORDER MEMBRANE LIPID TRANSPORTERS Until very recently, it was generally believed that FA and MG are absorbed by the enterocytes through simple diffusion [84]. Strauss simply and elegantly demonstrated that the uptake of FA is passive and not temperature dependent [85]. MG and FA enter the enterocytes as monomers [84]. Studies by Stremmel have suggested the existence of an FA binding protein associated with the brush border membrane which plays a role in the uptake of FA by enterocytes [86]. The idea that FA may be taken up by



enterocytes via a carrier-mediated process is also supported by an earlier study by Chow and Hollander [87]. They demonstrated that the uptake of linoleate by the small intestine points toward a concentration-dependent dual mechanism of FA transport. At low linoleate concentrations, FA is taken up via a carrier-dependent process, whereas at a higher lineolate concentration, FA is taken up predominantly by means of passive diffusion. Stremmel’s work has also raised the possibility that some lipids, especially FA, may be taken up by enterocytes via carrier-mediated processes [86, 88]. Although Stremmel focused mainly on the FA binding protein involved in FA uptake, he found that this protein was also capable of transporting cholesterol, but not cholesterol ester. This discoverycame after Stremmel found evidence that when a jejunal loop was treated with the antibody against the FA binding protein, cholesterol uptake in the loop was also significantly reduced. Stremmel et al. [88] demonstrated that the protein is mainly present in the apical and lateral of the villus (in the region of the tight junction) and in the crypt. Consequently, it is quite plausible that the FA binding protein may be a transporter for a whole array of lipid molecules, including cholesterol. This conclusion, however, is of concern for two reasons. First, this transporter has more recently been demonstrated to be the mitochondrial glutamic oxaloacetic transaminase, an intracellular enzyme which is not involved in lipid absorption [89]. Second, crypt cells express this protein despite the fact that they are not involved in fat absorption.

observation was confirmed in a subsequent report by Herrmann et al. [100] who showed that the lack of FATP in the skin epidermis resulted in the same phenotype. Hall et al. [101] showed that FATP4, in addition to playing a role in the uptake of FA, may also have acyl-CoA synthetase activity. The role of FATP4 in the uptake of FA in vivo has remained unclear until lately. Recently, Shim and colleagues examined this question in considerable detail and concluded that there is no evidence that ntestinal FATP4 has physiological role i in dietary lipid absorption in mice [102].

A membrane transporter for FA has been identified in adipocytes. This FA transporter is inhibited by sulfo-Nsuccinimidyl derivatives of long-chain FAs [90-94] and by 4,4'-diisothiocyanostilbene-2-2'-sulfonate (DIDS). This protein has an apparent molecular weight of 88 kD, an isolectric point of 6.9, and a strong sequence homology to CD36 and PAS IV. PAS IV is a protein enriched in the apical membranes of lipid-secreting mammary cells during lactation. Cloning this binding protein revealed that it bears 85% homology to CD36, which is present in human platelets and lactating mammary epithelia [91]. Northern blot analysis revealed that the message is abundant in the heart, intestine, fat, muscle, and testis [92-94]. Fatty-acid transporters have been localized to the brush border membrane, and their expression is highest in the jejunum, lower in the duodenum, and lowest in the ileum [94]. These same investigators reported that a high-fat diet rich in long-chain FA but low in medium chain FAs resulted in increased fatty-acid transporter expression [95]. Using the lymph fistula mouse model, Nauli et al. [96] have recently demonstrated that CD36 is involved in promoting the uptake of cholesterol by the small intestine. While CD36 does not appear to be involved in the uptake of fatty acids by the small intestine, it does play an important role in the packaging of the absorbed fatty acids such as triglycerides into chylomicrons. Other potential binding proteins have been identified as well. One protein which binds with long-chain FA has been cloned in adipocytes by Schaffer and Lodish [97]. Recently, Stahl et al. [98] identified the presence of FATP4, a member of the large family of FA transport proteins expressed in the small intestine. Moulson et al. [99] reported that a wrinklefree, thick skin phenotype is associated with the spontaneous autosomal recessive mutation of the FATP4 gene. This

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In summary, we do not believe sufficient evidence exists to support the view that normal physiological intestinal fatty acid absorption requires transporters. Rather, we believe that these transporters are present in order to ensure that the body does not lose any essential fatty acids when luminal concentration is very low. This concept is well supported by the experimental evidence published by Chow and Hollander [103]. The absorption of fatty acids described in this scenario is very different from that of cholesterol, which clearly involves transporters in intestinal uptake, a subject ably reviewed by Wang [5]. 5. INTRACELLULAR METABOLISM OF DIETARY LIPIDS To date, investigators have been unable to explain how the various absorbed lipids migrate from the site of absorption to the endoplasmic reticulum, where biosynthesis of complex lipids takes place. An FA binding protein (FABP) present in the small intestine has been isolated and characterized by Ockner and Manning [104]. It has been suggested that this FABP plays an important role in the intracellular transport of absorbed FA. This is supported, to some degree, by the finding that FABP concentration is greater in villi than in crypts, in the jejunum than the ileum, and in the intestinal mucosa of animals fed a high-fat diet than the intesting mucosa of those fed a low-fat diet [104, 105]. We know there are at least two types of FABPs in enterocytes— the I-FABP and L-FABP. The letter preceding “FABP” indicates the organ from which it was first isolated (I = intestine and L = liver). These two FABPs differ in their binding specificity. I-FABP binds strongly with FA, whereas L-FABP binds with long-chain FAs as well as LPC, retinoids, bilirubin, carcinogens, and even selenium [106108]. Based on NMR binding studies, Cistola et al. [109] speculated that the binding of I-FABP may involved in the intracellular transport of FA, while L-FABP may involved in the intracellular transport of MG and LPC. None of these functions of FABP, however, has been tested rigorously in in vivo animals. Binding proteins of FAs have been ably reviewed by Storch and Thumser [110]. Demonstrating in vivo functions of the FABPs has been very difficult. One strategy to do so has been to knockout the gene for a particular FABP. This, however, has not yielded definitive results since other FABPs are often capable of compensating for the deficit in function which the knockedout FABP once assumed. For example, when Shaughnessy et al. [111] used the adipocyte FABP knockout mouse to study its function, the keratinocyte FABP functionally compensated for the absence of the adipocyte FABP. Using the I-FABP knockout mouse model, investigators found that


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I-FABP knockout males have higher plasma TG and greater body weight than wild types [112], implying that fat absorption is probably normal in these genetically modified animals. The female I-FABP knockouts weighed more than wild type females, but less than male knockouts. When the investigators who generated the knockout mouse analyzed whether or not fat absorption is compromised in I-FABP knockout mice, they found that I-FABP null animals could absorb lipid as well as the wild type animals [113]. As noted earlier, however, one should not conclude from this data that I-FABP is not physiologically important for fat absorption since other intracellular FABPs may be compensating for the lack of I-FABP. Recently, Newberry et al. reported that LFABP knockout animals did not exhibit fat malabsorption but that the kinetics of intestinal lipid secretion occurred more slowly in the null animals than in the wild type animals [114]. How these FABPs work together in a concerted manner to facilitate fat absorption in the intestine is still under investigation in a number of laboratories and we can hope to gain a better understanding of the process in coming years.

tuted to form TG, a process which occurs mainly via the MG pathway. As shown in Fig. (4), 2-MG is reacylated into TG by the consecutive action of MG acyl transferase (MGAT) and DG acyltransferase (DGAT) [115, 116]. The enzymes involved in this MG pathway are present in a complex called "triglyceride synthetase" [115, 117], which has been purified by Lehner and Kuksis [118]. The synthesis of TG from DG is thought to be catalyzed by the enzyme acyl CoA:DGAT. The gene for this enzyme has been isolated, and a knockout mouse has been generated. Interestingly, this knockout mouse can synthesize normal amounts of TG in the intestinal mucosa [119], suggesting that another enzyme(s) may be involved in the formation of TG from DG.

5.1. Intracellular Metabolism of Monoglycerides and Fatty Acid Thanks to the contributions of two laboratories, directed by Fareses and Shi respectively, our understanding of the enzymes involved in the intracellular metabolism of monoacylglycerol, diacylglycerol, and fatty acids has progressed considerably in the last few years. Before we discuss their work, we will first review of our understanding of the intracellular metabolism of lipid digestion products, 2monoacylglycerol and fatty acid. 2-MG and FA are reconsti-

Several studies have demonstrated that the enzymes involved in the MG pathway are located on the cytoplasmic surface of the endoplasmic reticulum [120]. This finding has important implications for our understanding of the intracellular packaging of lipoproteins. The data seems to indicate that TG is formed at the cytoplasmic surface of the endoplasmic reticulum and that TG enters the cisternae of the endoplasmic reticulum. Because TG has a low solubility in PL bilayers (~ 3 mol %), Small [121] postulated that TG molecules saturate the membrane rapidly. Once the solubility of TG has been exceeded, the TG splits the bilayer and forms a small lens. As the lens grows, it bulges into the cytoplasmic side or the cisternal side of the endoplasmic reticulum. Finally, this protrusion pinches off the membrane together with newly synthesized apolipoprotein B48 (apo B48) and forms either lipid droplets in the cytoplasm or precursors of lipoproteins in the cisternae of the endoplasmic reticulum. The formation of apo B containing lipoproteins is a process mediated by the protein called microsomal triglycerdide

Fig. (4). Pathways of triacylglycerol biosynthesis in the intestinal mucosa [116].



transfer, originally isolated and characterized by Wetterau and Zilversmit [122-124] who demonstrated that a protein in the liver, small intestine, and several other organs promotes the transfer of TG and cholesterol esterase between membranes. This is an active research area not only because of its biological Interest but also because of its potential as a target for drug development to reduce fat absorption. There have been a number of excellent reviews written on this subject and interested readers should refer to those reviews for additional information.

Thus far, there have been three MGATs identified, MGAT1, 2, and 3. Although all three MGATs are localized in the endoplasmic reticulum and require strong enzymatic activity to convert MG into DG [127], their tissue distributions are unique. MGAT1 mRNA has been detected mostly in the stomach, kidney, and adipose tissue, while MGAT2 and MGAT3 mRNAs have been found mostly in the small intestine. The MGATs are also unique in that MGAT3 can be found in higher mammals and humans but not in rodents [130] and shares higher sequence homology with DGAT2 than with other MGATs. Thus, it is not surprising that when presented with MG, MGAT3 has the ability to catalyze the formation of TAG, functioning like a TAG synthase. It has recently been reported that although the overall amount of fat absorption in MGAT2 knockout animals is the same relative to the wild type controls, the MGAT2 knockout animals showed a significant delay in the intestinal absorption of fat. Due to the long delay in lipid transport in the MGAT2 knockout animals, it is possible that these animals employ more of the small intestine to transport the dietary lipid absorbed. This may have metabolic consequence as Wu and Bennett-Clark have demonstrated that the lower small intestine is less efficient in packaging and transporting the absorbed lipid than the upper intestine [131, 132]. Thus, we propose that the presence of multiple MGAT and DGAT enzymes functions to ensure that the absorbed lipid is rapidly packaged to form chylomicrons and is transported out of the enterocytes by exocytosis. While the lack of one enzyme (as in the case of MGAT2 knockout animals) may not affect the total volume of dietary lipids absorbed, it may affect the metabolism of the dietary lipids by requiring different parts of the small intestine (e.g. ileum) to be involved in absorbing and transporting the lipids.

A second pathway in intestinal mucosa that forms TG is the alpha-glycerophosphate pathway [115, 116]. This pathway involves the stepwise acylation of glycerol-3-phosphate to form phosphatidic acid (Fig. 4). In the presence of phosphatidate phosphohydrolase, phosphatidic acid is hydrolyzed to form DG, which is then converted to TG. The alphaglycerophosphate pathway, unlike the monoglyceride pathway, is extremely sensitive to inhibition caused by the intake of omega rich fatty acids (such as fish oil). The Caco-2 cells, a common cell line used as a surrogate of the small intestine epithelial cells, use the alpha-glycerophosphate pathway instead of the monoglyceride pathway for the formation of triacylglycerol. Thus, the secretion of TG-rich lipoproteins is greatly inhibited by omega-3 fatty acids in Caco-2; however, the normal human or rodent small intestine is not sensitive to the inhibition of TG-rich lipoprotein secretion by omega-3 fatty acids. The relative importance of the MG-pathway and the alpha-glycerophosphate pathway depends on the supply of 2-MG and FA. During normal lipid absorption, when 2MG is present in sufficient quantities, the 2-MG pathway facilitates the conversion of 2-MG and FA to form TG and also aids in inhibiting the alpha-glycerophosphate pathway [115, 116, 125]. Conversely, when the supply of 2-MG is lacking or insufficient, the alpha-glycerophosphate pathway becomes the major pathway for the formation of TG. As mentioned earlier, considerable progress has been made in understanding acyl-coenzyme A: monoacylglycerol acyltransferase (MGAT) and acyl-coenzyme A: diacylglycerol acyltransferase (DGAT); two excellent reviews have recently been published on the subject [126, 127]. We now know that there are at least three MGATs and two DGATs. The three MGATs are known as MGAT1, 2, and 3 and the three DGATs are known as DGAT1 and 2. DGAT1 knockout animals can absorb lipids quite well, even when fed a high fat diet [128]. The only major difference between a DGAT1 knockout animal and a wild type animal is that the DGAT1 knockout exhibits delayed transport of chylomicrons into the circulation. In the same study, the authors have provided evidence that DGAT2 and diacylglycerol transacylase can also participate in the conversion of diacylglycerols into triacylglycerols. DGAT 1 has also been demonstrated to catalyze the synthesis of diacylglycerols, waxes, and retinyl esters [129]. New information on the the relative roles of the DGAT1, DGAT2, and diacylglycerol transacylase in the formation of triacylglycerols under different physiological conditions can be expected in the near future in view of the active research in this area of investigation, which can be partly attributed to the potential therapeutic applications for enzyme inhibitors and activators involved in triacylglycerol metabolism.

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6. FORMATION OF INTESTINAL CHYLOMICRONS AND VERY LOW DENSITY LIPOPROTEINS (VLDL) Despite the wealth of information gathered over the last thirty years regarding intestinal lipid absorption, the mechanisms involved involved the intracellular assembly, modification, and secretion of lipoproteins from the small intestinal epithelial cells is still not fully understood. This is due, in part, to the complexity of the processes involved and the lack of good experimental models to study the various steps. Intestinal cell culture systems (e.g. CaCo-2 cells) have been used extensively to study the formation and secretion of lipoproteins [133-137] and the genetic expression and posttranslational modification of apolipoproteins [138]. During fasting, very low density lipoproteins (VLDL) are the only lipoproteins produced by the small intestine [139, 104, 140]. After a meal, however, the small intestine produces predominantly CMs [140, 141]. Currently, the separation of intestinal CMs and VLDLs is based on operational criteria. Lipoproteins that have a Svedberg flotation (Sf) rate exceeding 400 are classified as CMs; those with a Sf rate of 20-400 are defined as VLDLs [142]. Numerous studies support the idea that intestinal CMs and VLDLs represent lipoproteins produced by two separate pathways. Ockner et al. [139] demonstrated that the intraduodenal infusion of palmitate causes a marked increase in VLDL transport, but that VLDL output remains unchanged when oleate and linoleate are infused. In contrast, CM output is markedly


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increased when oleate and linoleate are infused. The FA composition of the VLDL-TG is also different from the FA composition of the CM-TG, suggesting the presence of a different pathway for VLDL and CM assembly. An ultrastructural and biochemical study conducted by Mahley et al. [143] showed that intestinal Golgi vesicles contain either CMs or VLDL particles and that little mixing of particle sizes occurs, which led them to conclude that separate biosynthetic pathways must exist for these lipoproteins. Further evidence for this argument is provided by Vahouny et al. [144], who demonstrated that puromycin had no significant effect on the incorporation of radioactive leucine into VLDL peptides in male rats. In contrast, the incorporation of radioactive leucine into CM peptides was markedly inhibited.

information about this model are referred to an excellent recent review by Hussain [133]. It should be emphasized that Caco-2 cells differ from the enterocytes in the small intestine in a number of important ways and the following are just a few examples. First, the Caco-2 cells are derived from human colon carcinoma. Second, the Caco-2 cells use the glycerol 3-phosphate pathway for the formation of TG from fatty acids and glycerol while the intestinal epithelial cells use the monoacylglycerol pathway for the formation of TG [150]. Third, the intestinal epithelial cells synthesize only apo B48 while Caco-2 cells synthesize both apo B-48 and apo B-100. Lastly, the intestinal epithelial cells produce mainly CM particles during active fat absorption; however, CM particles are not the major lipoproteins produced by the Caco-2 cells. Thus, while the Caco-2 cells have certainly been used successfully as surrogates for intestinal epithelial cells, one should interpret the data generated by the Caco-2 cells cautiously.

Studies conducted by Tso et al. [145, 146] have shown that rats fed as little as 0.5 mg/hour of a hydrophobic surfactant Pluronic L-81 (L-81), display markedly impaired lymphatic transport of TG and cholesterol. They further demonstrated that L-81 inhibited lipid transport by blocking the formation of intestinal CMs, but not VLDL particles. Using this unique tool, Tso et al. further showed that intraduodenal infusion of egg PC in rats primarily resulted in the lymphatic transport of VLDLs, and that this transport was not affected by L-81 [147]. When triolein was infused, however, the lymphatic transport of lipid was inhibited because triolein injestion results primarily in the formation of CMs [145, 146]. These results led Tso et al. to propose two pathways for the formation of intestinal lipoproteins— one that forms predominantly CMs, and one that forms predominantly VLDLs (Fig. 5) [147]. Moreover, Tso et al. proposed that the pathway forming predominantly CMs is inhibited by L-81, but that the pathway forming predominately VLDLs is not affected by L-81, and that pre-CM and pre-VLDL particles are packaged in the Golgi apparatus into either pre-CM or pre-VLDL-containing vesicles. The Golgi-derived vesicles containing either pre-CMs or preVLDLs probably correspond to those vesicles observed by Mahley et al. [143]. This hypothesis is supported by the study conducted by Nutting et al. [148], who measured the appearance time for CMs in control rats and for VLDLs in L-81 treated rats. The appearance time, defined as the time between placement of radioactive FA into the intestinal lumen and the appearance of radioactive lipid in lymph, was 10.8 min in control rats—significantly shorter than the 16.2 min in the L-81-treated rats. This difference in appearance time further supports the hypothesis that CMs and VLDLs are packaged separately in enterocytes. Finally, it seems that this in vivo observation can also be duplicated in cell culture systems. Using Caco-2 cells, Luchoomun and Hussain [149] also demonstrated that L-81 inhibits CM but not VLDL formation by these cells. Caco-2 cells have been used extensively to study the uptake of lipids as well as the formation and secretion of TG rich lipoproteins. Based on their Caco-2 studies, Hussain and his colleagues have proposed a sequential assembly model to study VLDL and CM formation [149]. This model is expected to provide information on the assembly of primordial lipoprotein particles, the synthesis of TG-rich lipid droplets, and the core expansion involving the fusion of primordial lipoproteins with lipid droplets. Readers interested in further

Fig. (5). Packaging and secretion of intestinal chylomicrons (CM) and very low-density lipoproteins (VLDL). The diagram depicts Golgi derived vesicles containing either pre-CM or pre-VLDL particles. Little mixing of pre-CM and pre-VLDL particles in these vesicles occurs [140].

7. PACKAGING AND EXOCYTOSIS OF CHYLOMICRONS Due to the size of chylomicron, specific vesicles large enough to accommodate the CM are required for transport, vesicles which have been isolated by Mansbach and his colleagues from the small intestine [151]. The pre-chylomicron transport vesicles (PCTV) are large, with diameter of 250 nm. Because of the diameter of the PCTV, one would predict that the vesicles could contain no more thana few CM particles. The PCTV has the following characteristics: 1) contains pre-chylomicrons with apo B and apo AIV incorporated; 2) sealed with a membrane; 3) fuses with cisGolgi. The PCTV are distinct from the COPII containing vesicles involved in protein trafficking between endoplasmic reticulum and Golgi apparatus. Another major difference between PCTV and the protein vesicles is the presence of the v-soluble N-ethylmaleimide-sensitive factor (v-SNARE) and vesicle associated membrane protein 7 (VAMP7) with PCTV only [151]. These proteins are involved in the fusion with the membranes of the Golgi apparatus. Mansbach and



his colleagues have devoted much attention to the question ofwhich protein or proteins are involved in the budding of the PCTV from the endoplasmic reticulum, given the absence of COPII with PCTV. A recent paper from Mansbach and his colleagues provided convincing evidence that liver fatty acid binding protein is involved in this process [152].

Anderson’s disease, named after the investigator who first reported it, is another disorder related to the formation of CMs by the small intestine [171-173]. Anderson’s disease is also known as chylomicron retention disorder because although observable CM particles can be found in the enterocytes in patients with this disorder, none exist in the intercellular space. This suggests that the secretion of CMs is defective among patients who have this disease. In a recent study, Dannoura et al. carefully demonstrated that patients with Anderson’s disease do not have a defect in those genes which carry known apoproteins or microsomal TG transfer protein, indicating that this diseased condition is most likely caused by a an unknown factor crucial for the secretion of chylomicrons [174]. Unfortunately, there is no animal model is available to study this perplexing disorder. There has been a recent report showing that the patients with chylomicron retention disorder exhibit the five nmutation of the Sar 1b gene [175]. It is interesting that the work of Mansbach and colleagues showed that the budding of PCTV from the ER does not involve the COPII proteins and yet chylomicron retention disorder clearly involves the mutations of the Sar1b gene [151]. Obviously, we have a lot more to learn about the various proteins involved in the intracellular packaging and trafficking of pre-chylomicrons and can expect new information to emerge from ongoing studies using both cellular and molecular approaches.

When we examine carefully the PCTV, it becomes quite apparent that there are probably only a very small number of pre-chylomicrons contained in each of these vesicles. In contrast, when we examined the Golgi derived vesicles, there are many more pre-chylomicron particles present in them. Thus, the Golgi apparatus is not only involved in the processing of the pre-chylomicron and its components, but it is also responsible for transferring the pre-chylomicron particles in large numbers to the much larger Golgi vesicles for transport outside the enterocytes by exocytosis. 8. CLINICAL DISORDERS OF INTESTINAL LIPID TRANSPORT In humans, the liver secretes only apolipoprotein B-100 (apo B-100) and the small intestine secretes only apolipoprotein B-48 (apo B-48) [153-155]. Both apo B-100 and apo B-48 are encoded by the same gene [156, 157]. The biogenesis of apo B-48 involves a unique mechanism by which the CAA codon encoding Gln at 2153 of the apo B-100 mRNA is changed to UAA (stop codon) and thus forms apo B-48. Rat apo B-48 has a molecular weight of approximately 240,000 kDa [158] and is an extremely hydrophobic protein [159]. Each CM or VLDL particle has one apo B-48 [160]. The absolute requirement of apo B for the formation of CM is well illustrated by the apo B knockout animals. While we do not know whether the production of apo B is physiologically rate-limiting in the production of CM by the enterocytes, data from several laboratories indicate that the supply of apo B is probably not the rate-limiting step for forming CM. Hayashi et al. [161] demonstrated that apo B output in lymph, an indication of the number of CM produced by the small intestine, does not change after intraduodenal infusion of lipid, despite the fact that lymphatic TG output increases seven- to eightfold. These data support the finding by Davidson et al. [162] that no increase in apo B synthesis results from lipid absorption. Abetalipoproteinemia is a rare genetic disorder involving the complete failure of the liver and gut to make TG-rich lipoproteins [163-165]. It has been generally assumed that abetalipoproteinemic patients have a problem synthesizing apo B. Glickman et al. [155] reported that apo B synthesis, as determined by 3H leucine incorporation, was decreased but not abolished in abetalipoproteinemic patients, suggesting that failure by the gut and liver to synthesize apo B may not be the only reason that abetalipoproteinemic patients do not produce CM and VLDL. This was later confirmed by the finding that abetalipoproteinemia results from the mutation of the microsomal TG transfer protein gene [165-170]. The role of microsomal TG transfer protein in the formation of chylomicrons by the small intestine is well covered in the reviews by Berriot-Varoqueaux et al. [168] and Hussain [133].

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9. CONCLUDING REMARKS As we have already discussed at the beginning of this review, many dietary lipids and lipophilic compounds are carried by chylomicrons and VLDL and are then transported in lymph; therefore, it is important to have a good understanding of the processes involved in the digestion, uptake, intracellular metabolism, and packaging of dietary lipids into chylomicrons. Hopefully, this review has achieved its mission by providing the readers a comprehensive review of the subject. More importantly, the authors hope that this review will serve as a stimulus to many investigators working on both the basic and clinical aspects of intestinal fat absorption. Given the prevalence of obesity and hyperlipidemia, the inhibition of gastrointestinal absorption of lipids without significant side effects may prove to be an effective and safe way of dealing with these problems. ACKNOWLEDGEMENTS This work was supported by the National Institutes of Health, grants DK-32288, DK-54504, and DK-56910. REFERENCES [1] [2] [3] [4]

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Revised: March 11, 2009

Accepted: March 12, 2009