Intestinal lipid absorption

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Intestinal Lipid Absorption

Jahangir Iqbal and M. Mahmood Hussain

Department of Anatomy and Cell Biology, State University of New York Downstate

Medical Center, Brooklyn, New York

Running title: Intestinal Lipid Absorption

Correspondence/Reprint request:

Department of Anatomy and Cell Biology,

450 Clarkson Avenue

State University of New York Downstate Medical Center,

Brooklyn, New York 11203

718-270-1443/718-270-4790

[email protected] or [email protected]

Articles in PresS. Am J Physiol Endocrinol Metab (January 21, 2009). doi:10.1152/ajpendo.90899.2008

Copyright © 2009 by the American Physiological Society.

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ABSTRACT

Our knowledge of the uptake and transport of dietary fat and fat-soluble vitamins

has advanced considerably. Researchers have identified several new mechanisms by

which lipids are taken up by enterocytes and packaged as chylomicrons for export into

the lymphatic system or clarified the actions of mechanisms previously known to

participate in these processes. Fatty acids are taken up by enterocytes involving protein

mediated as well as protein independent processes. Net cholesterol uptake depends on the

competing activities of NPC1L1, ABCG5, and ABCG8 present in the apical membrane.

We have considerably more detailed information about the uptake of products of lipid

hydrolysis, the active transport systems by which they reach the endoplasmic reticulum,

the mechanisms by which they are re-synthesized into neutral lipids and utilized within

the endoplasmic reticulum to form lipoproteins, and the mechanisms by which

lipoproteins are secreted from the basolateral side of the enterocyte. ApoB and MTP are

known to be central to the efficient assembly and secretion of lipoproteins. In recent

studies, investigators found that cholesterol, phospholipids, and vitamin E can also be

secreted from enterocytes as components of high-density ApoB-free/ApoAI-containing

lipoproteins. Several of these advances will probably be investigated further for their

potential as targets for the development of drugs that can suppress cholesterol absorption,

thereby reducing the risk of hypercholesterolemia and cardiovascular disease.

KEY WORDS

Absorption, intestine, dietary fat, triacylglycerol, cholesterol, phospholipids, fat-

soluble vitamins, microsomal triglyceride transfer protein.

ABBREVIATIONS

TAG, triacylglycerol; DAG, diacylglycerol; MAG, monoacylglycerol; PL,

phospholipid; PC, phosphatidylcholine; FATP, fatty acid transport protein; FABP, fatty

acid binding protein; CRBP, cellular retinol-binding protein; DGAT, diacylglycerol

acyltransferase; ACAT, acyl-CoA:cholesterol acyltransferase; LRAT, lecithin:retinol

acyltransferases; MTP, microsomal triglyceride transport protein; PPAR, peroxisome

proliferator-activated receptor; LXR, liver X receptor; NPC1L1, Niemann-Pick C1 like 1;

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SR-BI, scavenger receptor class B type I; CM, chylomicron; VLDL, very low density

lipoprotein.

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INTRODUCTION

Dietary fats consist of a wide array of polar and nonpolar lipids (32; 33).

Triacylglycerol (TAG) is the dominant fat in the diet, contributing 90% to 95% of the

total energy derived from dietary fat. Dietary fats also include phospholipids (PLs),

sterols (eg, cholesterol), and many other lipids (eg, fat-soluble vitamins). The

predominant PL in the intestinal lumen is phosphatidylcholine (PC), which is derived

mostly from bile (10–20 g/d in humans), but also from the diet (approximately 1–2 g/d).

The predominant dietary sterols are cholesterol (mostly of animal origin) and β-sitosterol

(the major plant sterol). Although β-sitosterol accounts for 25% of dietary sterols, it is not

absorbed by humans under physiological conditions.

Researchers have been investigating the various steps involved in lipid digestion,

absorption, and metabolism to identify factors that could serve as targets for the

development of drugs capable of reducing the risk of lipid-associated disorders, including

dyslipidemias and cardiovascular disease. In so doing, they have explored key aspects of

lipid digestion—hydrolysis, emulsification, and micelle formation. They have also

explored key issues of absorption, eg, the uptake of the products of lipid hydrolysis by

enterocytes (the epithelial cells lining the walls of the intestinal lumen) and their transport

to intracellular compartments, where fatty acids and sterols are transformed into neutral

lipids. Efficient absorption ensures that dietary fat is available to be used as a source of

energy that supports various cellular functions or to be stored and serve as reservoirs for

lipoprotein trafficking, bile acid synthesis, steroidogenesis, membrane formation and

maintenance, and epidermal integrity in various mammalian cells. Excess fat is stored in

cytosolic lipid droplets until it is needed to support intracellular processes.

In this review, we will concentrate on the biochemical processes involved in the

digestion and absorption of the most common dietary lipids: TAGs, PLs, cholesterol, and

fat-soluble vitamins.

DIGESTION AND ABSORPTION OF LIPIDS: A BRIEF OVERVIEW

The digestion of lipids begins in the oral cavity through exposure to lingual

lipases, which are secreted by glands in the tongue to begin the process of digesting

triglycerides. Digestion continues in the stomach through the effects of both lingual and

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gastric enzymes. The stomach is also the major site for the emulsification of dietary fat

and fat-soluble vitamins, with peristalsis a major contributing factor. Crude emulsions of

lipids enter the duodenum as fine lipid droplets then mix with bile and pancreatic juice to

undergo marked changes in chemical and physical form. Emulsification continues in the

duodenum along with hydrolysis and micellization in preparation for absorption across

the intestinal wall. [For details about the emulsification, hydrolysis, and micellization of

fats, see References (131) and (144).]

Bile and pancreatic juice provide pancreatic lipase, bile salts, and colipase, which

function cooperatively to ensure the efficiency of lipid digestion and absorption. The

importance of bile to the efficiency of these processes is indicated by the decreased rate

of lipid absorption in humans with bile fistulas. The greatly reduced concentration of bile

acid in the duodenum of such individuals suggests that bile salts, although possibly not

absolutely necessary for digestion, are essential for the complete absorption of dietary

fats (150). Elevated concentrations of bile salts have been shown to inhibit pancreatic

lipase activity in the duodenum (190). Such inhibition is offset, however, by colipase,

which has been shown in vitro to restore pancreatic lipase activity under such

circumstances (112; 128). The importance of colipase in the digestion of fat was

indicated in a clinical report of steatorrhea in two brothers with a congenital absence of

colipase, which indicated that the steatorrhea decreased with the administration of

colipase (76). It has also been demonstrated in colipase-deficient mice (41), which, when

placed on a high-fat diet, developed steatorrhea that was so severe that undigested lipids

could be seen in their feces. When these mice were placed on a low-fat diet, their ability

to digest fat returned to normal. These findings suggest that colipase plays a critical, but

not essential, role in the digestion of dietary lipids by pancreatic lipases.

Triacylglycerides

Digestion and absorption of TAGs. TAG is digested primarily by pancreatic lipase

in the upper segment of the jejunum. This process generates a liquid:crystalline interface

at the surface of the emulsion particles (13; 161). The activity of pancreatic lipase on the

sn-1 and sn-3 positions of the TAG molecule results in the release of 2-monoglycerol (2-

MAG) and free fatty acids (FFAs) (122-124); 2-MAG is the predominant form in which

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MAG is absorbed from the small intestine. The formation of 2-MAG (and 1-MAG)

through isomerization in an aqueous medium occurs more slowly than the uptake of 2-

MAG from the small intestine (20). Further hydrolysis of 1- or 2-MAG by pancreatic

lipase results in the formation of glycerol and FFAs (78); cholesterol esterase can also

hydrolyze the acyl group at the sn-2 position to form glycerol and FFAs (111).

Free fatty acids are taken up from the intestinal lumen into the enterocytes and

used for the biosynthesis of neutral fats. A protein-independent diffusion model and

protein-dependent mechanisms have been proposed for the uptake and transport of fatty

acids (FAs) across the apical membrane of the enterocyte [For details, see Reference

(119).] A number of candidate proteins have been proposed to take part in protein-

dependent uptake mechanisms. FAT/CD36 plays a key role in the uptake of FAs (1; 19).

FAT/CD36 is highly expressed in the intestine (37), and its expression is up-regulated by

the presence of dietary fat (148), genetic obesity, and diabetes mellitus (64). Studies in

CD36 null mice suggest that it is intimately involved in the uptake and transport of FAs

targeted for transport to the lymphatic system through their use in the assembly of

chylomicrons (45). This relationship is suggested by the finding that the uptake of FAs is

not impaired in CD36-null animals. However, lipids tend to accumulate in the proximal

small intestine of these animals primarily because of decreased transport of FAs to the

lymphatic system (45). Fatty acid transport proteins (FATPs) are well represented in the

small intestine by their FATP4 isoform, which is thought to help facilitate the uptake of

FAs by the enterocytes (162).

TAG synthesis. Once inside the enterocyte, the products of TAG hydrolysis must

traverse the cytoplasm to reach the ER, where they are used to synthesize complex lipids.

Specific binding proteins carry FAs and monoacylglycerol (MAG) to the intracellular site

where they will be used for TAG biosynthesis. The two major fatty-acid binding proteins

(FABPs) found in enterocytes are liver FABP (L-FABP) and intestinal FABP (I-FABP)

(2; 16; 69). Most TAG biosynthesis in the enterocyte occurs along the MAG pathway, in

which MAG and fatty acyl-CoA are covalently joined to form diacylglycerol (DAG) in a

reaction catalyzed by monoacylglycerol acyltransferases (MGATs) (40; 197). Further

acylation of DAG by diacylglycerol acyltransferase (DGAT) leads to the synthesis of

TAG. Two DGATs have been identified and characterized: DGAT1 and DGAT2. [For

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additional information about DGATs, refer to the recent review (197).] DGAT2 is

expressed mainly in the liver and intestine. Its importance in the absorption of fat has

been demonstrated by a reduction in fat absorption in DGAT2 knockout animals.

DGAT1, which is expressed in several tissues (including liver, intestine, and skin), differs

from DGAT2, in that defective fat absorption is not seen in DGAT1 KO mice. In recent

studies, investigators have found that MGAT2 (29; 31) and MGAT3 (30) possess DGAT

activity (26; 170). Some TAG synthesis also occurs through the dephosphorylation of

phosphatidic acid and acylation of the resultant DAG. Thus, several enzymes take part in

the biosynthesis of TAG in intestinal cells.

Use of TAG in lipoprotein assembly. The existence of multiple pools of DAG

(159; 160) and TAG (51; 55; 60; 104; 175; 183; 195) has been known for some time.

Using differentiated human colon carcinoma (CaCo-2) cells, Luchoomun and Hussain

(115) showed that nascent TAG is used preferentially for chylomicron assembly. TAG is

also synthesized de novo from fatty acyl chains and glycerol phosphate. When generated

through this pathway, however, TAG is only partly used to assemble nascent ApoB-

containing lipoproteins. Most of the TAG synthesized through this pathway enters the

cytosolic pool to be used to generate a distinct pool of DAG. For a review of this topic,

see Reference (202).] DAG esterification also occurs in the ER lumen (142), where the

resultant TAG binds to the microsomal triglyceride transport protein (MTP), which

participates in the assembly and secretion of neutral lipids in chylomicrons. This process

is described further in a subsequent section in this review. [For additional information,

also see References (18) and (119).]

Phospholipids

The predominant PL in the lumen of the small intestine is phosphatidylcholine

(PC), which is found in mixed micelles that also contain cholesterol and bile salts. The

digestion of PLs is carried out primarily by pancreatic phospholipase A2 (pPLA2) and

other lipases secreted by the pancreas in response to food intake. These lipases interact

with PLs at the sn-2 position to yield FFAs and lysophosphatidylcholine (21; 182). These

products of lipolysis are removed from the water-oil interface when they are incorporated

into the mixed micelles that form spontaneously when they interact with bile salts.

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Having both hydrophilic and hydrophobic components, bile salts are able to facilitate

micelle formation; MAG and PL enhance their ability to form mixed micelles. pPLA2

knockout mice are indistinguishable from wild-type controls when fed regular chow,

except for their resistance to diet-induced obesity (84). Increased excretion of TAG, on a

high-fat diet, in the feces of pPLA2 knockout mice indicates that pPLA2 deficiency has a

greater effect on the digestion of TAG than that of PL hydrolysis (84). It does not affect

PL hydrolysis and absorption, possibly because its activity is compensated for by other

PLA2 enzymes (158).

Cholesterol

Cholesterol in the body represents both endogenous sources (produced in the liver

and peripheral tissues) and dietary sources absorbed from the intestine (186). The human

diet provides approximately 400 mg of cholesterol daily, and the liver secretes

approximately 1 g daily (66; 194). Approximately 50% of the cholesterol in the intestine

is absorbed; the remainder is excreted in feces (10; 39).

Digestion. Only nonesterified cholesterol can be incorporated into bile acid

micelles and absorbed by enterocytes. Most dietary cholesterol exists in the form of the

free sterol, with only 10% to 15% existing as the cholesteryl ester. The latter must be

hydrolyzed by cholesterol esterase to release free cholesterol for absorption. Cholesterol

is only minimally soluble in an aqueous environment (82; 177) and, thus, must be

partitioned into bile salt micelles prior to absorption. It usually enters these micelles

along with TAGs and PLs, ionized and nonionized FAs, MAGs, and lysophospholipids to

form mixed micelles (74; 196). These micelles are transported to the brush border of the

enterocyte, where cholesterol is absorbed. Its absorption depends on the presence of bile

acids in the intestinal lumen (191) and correlates directly with the total bile acid pool

(149).

Absorption: Cholesterol uptake. Cholesterol must pass through a diffusion barrier

at the intestinal lumen-enterocyte membrane interface before it can interact with

transporter proteins responsible for its uptake and subsequent transport across the cellular

brush border. Bile salt micelles facilitate the transfer of cholesterol across the unstirred

water layer. The mechanism by which the cholesterol in micelles is taken up by the cell

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and crosses the brush-border membrane is still under investigation. Cholesterol

absorption had long been considered an energy-independent, simple passive diffusion

process. However, it is taken up by the enterocyte with relatively high efficiency

compared with structurally similar phytosterols (130). New evidence strongly suggests

that a transporter-facilitated mechanism is involved. Inter-individual differences and

inter-strain variations in the efficiency of intestinal cholesterol absorption (195; 196)

support this suggestion, as does the discovery that multiple genes (4; 14; 106; 113)

participate in the regulation of cholesterol absorption and several molecules appear to

inhibit it (59).

Absorption: Cholesterol transport. Several proteins have been investigated for

their potential roles as intestinal cholesterol transporters, but evidence for their direct role

in the uptake of cholesterol remains elusive. Studies in genetically modified animal

models have helped investigators gain important insights into the mechanisms of

transport and identity of transporter proteins. Through these studies, investigators have

identified Niemann-Pick C1 like 1 (NPC1L1) as a cholesterol uptake transporter (4) and

the ATP-binding cassette (ABC) proteins ABCG5 and ABCG8 as cholesterol efflux

transporters (14; 106; 113). These 3 molecules appear to be key players in the control of

the cholesterol absorption from the intestinal lumen.

ABCG5 and ABCG8, which function as a heterodimer (62), are critical for the

control of sterol absorption. Mutations in the genes encoding human ABCG5 and

ABCG8 transporters cause β-sitosterolemia (14; 106; 113), which is characterized by the

accumulation of plant sterols in blood and other tissues as a result of their enhanced

absorption from the intestines and decreased removal in bile. These proteins are localized

at the canalicular membrane of hepatocytes and at the brush border of enterocytes.

ABCG5/G8 deficiency in mice results in reduced biliary cholesterol secretion (199) and

enhanced phytosterol absorption (102; 146; 199), but has only minimal effects on the

efficiency of cholesterol absorption (146; 199). The pharmacological induction or

overexpression of ABCG5 and ABCG8 in mice (199-201) results in a reduction in

fractional cholesterol absorption (ie, the percentage of cholesterol absorbed from the

intestine, which is determined using a dual-isotope feeding technique) and indicates that

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ABCG5 and ABCG8 play a role in the control of cholesterol absorption under certain

conditions.

The identification of NPC1L1 as a putative cholesterol transporter in the

enterocytes (4) was facilitated by the discovery of the cholesterol absorption inhibitor

ezetimibe (4; 59), which reduces diet-induced hypercholesterolemia (49; 56; 103; 187;

203). NPC1L1 is a glycosylated protein localized at the brush border membrane of the

enterocyte (95). The deletion of Npc1l1 in mice results in a reduction in fractional

cholesterol absorption (4). Ezetimibe has been shown to bind to NPC1L1-expressing cells

and to the intestinal brush border (59). Deletion of Npc1l1 also results in the elimination

of the binding capacity of the brush border (59), which indicates that NPC1L1 is a target

of ezetimibe.

A sterol-regulatory element in the promoter and a sterol-sensing domain of

NPC1L1 appear to regulate cholesterol absorption in response to cholesterol intake.

Expression of Npc1l1 is enhanced in the cholesterol-depleted porcine intestine and

suppressed in mice placed on a cholesterol-rich diet (83). Most of the NPC1L1 in the

body is found in intracellular membranes. However, cholesterol deprivation induces its

translocation to the plasma membrane, where it can pick up cholesterol and transport it to

the ER for esterification and packaging into nascent lipoproteins (50; 198).

Reducing the expression of NPC1L1 at the level of transcription may reduce

cholesterol absorption. Activation of the nuclear receptor peroxisome proliferator-

activated receptor (PPAR) δ/β by the synthetic agonist GW610742 has been shown to

reduce cholesterol absorption by decreasing NPC1L1 expression without altering the

expression of ABCG5 and ABCG8 (185). A decrease in NPC1L1 expression has also been

observed following treatment of human colon-derived Caco-2 cells with ligands for

PPARδ/β, but not for PPARγ or PPARα (185).

Absorption: Other regulatory factors. The nuclear liver X receptors (LXRs),

LXRα (mainly expressed in the liver, kidney, intestine, spleen, and adrenals) and LXRβ

(expressed ubiquitously), regulate pathways involved in the metabolism of cholesterol

and in lipid biosynthesis. LXR target genes have been shown to be involved in

cholesterol and lipid homeostasis. [For a list of target genes and their regulation, see

Reference (174).] After activation by natural ligands (eg, oxysterols), the LXR forms a

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heterodimer with the retinoid X receptor (96; 97) and binds to specific LXR response

elements (LXREs) in the promoter regions of their target genes to activate gene

transcription. LXR target genes include those that express proteins involved in the efflux

of cholesterol from the cell (155; 157; 174; 189), as well as bile acid synthesis (174) and

lipogenesis (174). Thus, global LXR activation by synthetic agonists has a plethora of

effects, including elevated high-density lipoprotein (HDL) levels (25; 28; 98; 126; 147;

163; 178), hypertriglyceridemia (156; 163), hepatic steatosis (65), increased excretion of

cholesterol in bile (147; 201), reduced efficiency of cholesterol absorption from the

intestine (103; 157; 159; 191; 206), and increased loss of neutral sterols in feces (201).

Other transporters, such as scavenger receptor class B type 1 (SR-B1)—which is

localized both at the apical and basolateral membranes of enterocytes (27)—have also

been suspected of having a role in the control of cholesterol absorption. The possibility

that SR-B1 is a cholesterol transporter is suggested by the observation that intestine-

specific overexpression of SR-BI in mice leads to an increase in cholesterol and TAG

absorption in short-term absorption experiments (17). The importance of the FA

translocase CD36 (which is also expressed in epithelial cells lining the small intestine) in

FA absorption is well established. CD36 has also been implicated as the cholesterol

transporter in brush border membranes. Additionally, overexpression of CD36 in COS-7

cells has been shown to enhance cholesterol uptake from micellar substrates (184). In

another study, CD36-null mice showed a significant reduction in cholesterol transport

from the intestinal lumen to the lymphatic system (133). Decreased cholesterol uptake

has been shown in brush border membrane vesicles prepared from the proximal (but not

distal) intestine of SR-B1 knockout mice (184) and in brush border membrane vesicles

and Caco-2 cells pre-incubated with antibodies to SR-B1 (71). However, targeted

disruption of SR-B1 in mice has little effect on in vivo intestinal cholesterol absorption (3;

120; 192), which suggests that SR-B1 might not be essential for absorption of cholesterol

from the intestine.

Another potential step in the regulation of cholesterol absorption involves two

ER-membrane–localized enzymes, acyl-CoA:cholesterol acyltransferase 1 (ACAT1) and

ACAT2, which catalyze the esterification of intracellular cholesterol (107). ACAT1 is

expressed in many tissues (36; 61), but its level of expression in the mouse small intestine

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is very low (125). By contrast, ACAT2 expression is restricted to the small intestine and

liver (6; 35; 137). ACAT2 is highly specific for cholesterol and does not esterify plant

sterols. It is the predominant enzyme responsible for the synthesis of cholesterol esters

and their subsequent secretion with lipoproteins. ACAT2 deficiency results in a

considerable decrease in the rate of cholesterol absorption (26). The rate of cholesterol

esterification in the presence of these enzymes is notably enhanced by substrate

availability, but, as we demonstrated recently, it is inhibited by product accumulation.

This inhibition is relieved by MTP (94), which transfers cholesterol esters from the ER

membranes to nascent ApoB-lipoproteins. The importance of MTP in cholesterol

absorption has been well documented (85; 86; 89).

Approximately 70% to 80% of cholesterol entering the lymphatic system is

esterified, which suggests that esterification is important for bulk entry of cholesterol into

nascent chylomicrons. Re-esterification of the absorbed cholesterol within the enterocyte

enhances the diffusion gradient to favor the entry of intraluminal cholesterol into the cell

and, therefore, is an important regulator of cholesterol absorption from the intestine.

Thus, inhibition of ACAT by pharmacological intervention (38; 73) or deletion of ACAT2

(26) significantly reduces the rate of cholesterol absorption.

It has also been suggested that ABCA1 plays a role in the control of cholesterol

absorption. ABCA1 was initially thought to be localized to the apical membrane (157);

more recent studies have provided evidence suggesting that it is present in the basolateral

membrane of chicken enterocytes (132) and human Caco-2 cells (138). Our studies in

Caco-2 cells and in the ApoA1 knockout mouse model showed for the first time that

basolateral efflux (secretion) of cholesterol occurs in high-density ApoB-free/ApoA-I–

containing lipoproteins (Figure) (91; 93). The importance of ABCA1 in the biogenesis of

HDL in the intestine was demonstrated by deleting intestinal ABCA1, which resulted in a

30% decrease in plasma HDL cholesterol levels (24). Enterocytes deficient in ABCA1

absorb smaller amounts of cholesterol (24). Thus, HDL contributes considerably to

cholesterol absorption from the intestine. The origin of HDL particles in mesenteric

lymph fluid has been a subject of considerable controversy (11; 53; 117; 139; 152; 169;

188). The measurement of cholesterol in lymph led to the hypothesis that HDL is not

important in cholesterol transport. However, Brunham et al (24) recently demonstrated

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that the HDL in lymph is dependent on the activity of hepatic ABCA1 and enters the

lymph from plasma. Thus, quantification of HDL in lymph may not provide an accurate

assessment of the extent of cholesterol absorption via this pathway.

Vitamin E

Vitamin E is one of the most abundant lipid-soluble antioxidants found in the

plasma and somatic cells of higher mammals. Vitamin E exists in 2 forms—as

tocopherols and tocotrienols—which vary dramatically in terms of biological activity due

to variations in bioavailability and in intrinsic antioxidant properties. The hydrophobic

nature of vitamin E creates a major challenge to living organisms in maintaining adequate

uptake, transport, and delivery of this vitamin to various tissues.

Vitamin E absorption. Micelle formation is required for the absorption of vitamin

E (57; 171). Pancreatic enzymes may also aid in its absorption (121); this is suggested by

the finding of vitamin E deficiency in patients with cystic fibrosis, who do not secrete

pancreatic enzymes (12; 48; 70; 172; 193).

The mechanism of absorption of vitamin E from the intestine is similar to the

mechanism involved in the transport of other lipid molecules in vivo and involves

molecular, biochemical, and cellular mechanisms closely related to overall lipid and

lipoprotein homeostasis (22; 67; 75; 100; 101; 179-181). The uptake of vitamin E from

the intestine has traditionally been assumed to be a simple process of passive diffusion.

However, in a study in CaCo-2 cells, it was shown to be a rapid, saturable, temperature-

dependent process (7). Recent studies suggest that SR-BI plays a role in vitamin E

absorption (154). Vitamin E absorption from the intestine is thought to occur

predominantly through the secretion of chylomicrons into the lymphatic system.

Consistent with this hypothesis is the observation that vitamin E absorption is dependent

on the availability of oleic acid for triglyceride synthesis and chylomicron assembly and

is inhibited by MTP inhibitors (7). The importance of alternative pathways for vitamin E

absorption has also been suggested by the observation that symptoms of vitamin E

deficiency are ameliorated after treatment with high doses of vitamin E in patients

deficient in chylomicrons. Indeed, intestinal vitamin E absorption may also occur via

direct secretion from epithelial cells into the portal venous circulation by HDL efflux.

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This HDL-dependent mechanism was recently characterized in Caco2 cells (7). Whether

intestinal ABCA1 or other ABC transporters are also critical for the absorption of vitamin

E from the intestine and for steady-state plasma α-tocopherol levels remains to be seen.

Vitamin A

The de novo synthesis of compounds with vitamin A activity is limited to plants

and microorganisms. Higher animals must obtain vitamin A from the diet. The main

dietary forms of preformed vitamin A are carotenoids in fruits and vegetables and long-

chain FA esters of retinal in foods of animal origin (145). Carotenoids are either cleaved

to generate retinol or absorbed intact, whereas retinyl esters must be completely

hydrolyzed within the intestinal lumen to release free retinol before retinol can be taken

up by enterocytes. Hydrolysis of the retinyl esters requires lipase activity. The products

of TAG hydrolysis provide a better milieu for the solubilization of vitamin E in mixed

micelles.

Free retinol is taken up by intestinal mucosal cells (44). In studies conducted in

Caco-2 cells (151) and intestinal segments, investigators have found that saturable

carrier-mediated processes at physiologic concentrations (81) and nonsaturable diffusion-

dependent processes at pharmacologic concentrations (80) of free retinol are involved in

retinol uptake. After cellular uptake, the intercellular retinol-binding proteins (CRBPs)

CRBP-I and CRBP-II immediately sequester free retinol. CRBP-I is expressed in many

tissues, whereas CRBP-II is expressed primarily in the absorptive cells of the small

intestine. CRBP-II is one of the most abundant soluble proteins in the jejunal mucosa,

which indicates that it may be uniquely suited for the absorption of retinol from the

intestine (109; 136; 140; 141). In studies conducted in retinoid-deficient rats (153) and in

rats placed on diets containing long-chain FAs (176), investigators found that CRBP-II

mRNA expression increased in the small intestine. In studies carried out in Caco-2 cells,

investigators found that overexpression of CRBP-II or treatment with retinoic acid

(which is associated with increased CRBP-II mRNA expression) resulted in increased

absorption and intracellular esterification of retinol. In CRBP-II knockout mice,

investigators found that CRBP-II plays an important (albeit not essential) role in the

absorption of vitamin A from the intestine (47). Some of the free retinol in the

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enterocytes remains associated with CRBP, but much of the retinol is usually esterified

by lecithin:retinol acyltransferases (LRATs) and stored within the cell (116). The recent

characterization of the LRAT knockout mouse (in which no detectable tissue retinyl

esters were found (9)) has largely resolved the question of whether enzymes such as

ARAT are physiologically involved in retinol esterification in the intestine.

Metabolic studies have revealed that most of the retinyl esters in plasma are

present in small chylomicrons; significant amounts are found in large chylomicrons, and

smaller amounts are found in very–low-density lipoproteins (108). Studies have been

carried out in differentiated Caco-2 cells under postprandial conditions to determine the

mechanism of retinol secretion by the intestine. In these studies, investigators found that

a significant amount of retinyl ester was secreted mainly with chylomicrons independent

of the rate of retinol uptake and intracellular levels of free or esterified retinol (134).

Pluronic L81, which inhibits the secretion of chylomicrons, decreased the secretion of

retinyl ester and did not result in their increased secretion of smaller lipoproteins. This

suggests that intestinal retinyl ester secretion is a highly specific and regulated process

that is dependent on the assembly and secretion of chylomicrons. A significant amount of

retinol is also secreted into the portal circulation, probably as free retinol; this is expected

to be physiologically significant in pathologic conditions that affect the secretion of

chylomicrons (79). However, very little is known about the regulation of the retinol

secretion by this pathway. Under fasting conditions, Caco-2 cells secrete mainly free

retinol unassociated with lipoproteins (134). The secretion of free retinol may require

facilitated transport. This notion is supported by the marked inhibition of the free retinol

efflux into the basolateral medium by glyburide, a known inhibitor of the ABCA1

transporter (46). Mechanisms regulating retinol output through these pathways are not

well understood.

ASSEMBLY, SECRETION, AND REGULATION OF INTESTINAL

LIPOPROTEINS

The major lipoproteins secreted by the intestine are very–low-density lipoproteins

(VLDL) and chylomicrons. Of these, the chylomicrons are synthesized exclusively in the

intestine to transport dietary fat and fat-soluble vitamins into the blood (Figure).

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Chylomicrons are primarily very large, spherical TAG-rich particles (5; 58) that also

contain PLs, cholesterol, vitamin E, vitamin A, and protein. The lipoprotein core contains

TAG, cholesteryl esters, and fat-soluble vitamins, whereas the surface contains a

monolayer of PLs (mainly phosphatidylcholine), free cholesterol, and protein (127; 204;

205). A key structural component of the chylomicron is the huge, hydrophobic,

nonexchangeable protein ApoB48. Other proteins associated with nascent chylomicrons

(which are synthesized in enterocytes) are ApoA-I, ApoA-IV, and ApoCs. ApoB48

synthesis is generally believed to be constitutive (88). However, the amount of lipids

transported during the postprandial state is several-fold greater than that transported

during the fasting state. Increased transport of fat with similar amounts of ApoB48 occurs

because of an increase in the size of the particles during the postprandial state (42; 43; 54;

63; 72; 173). Fat feeding also increases the expression of ApoA-IV, which serves as a

surface component for ApoB48 particles in the enterocyte (77; 135).

It has been proposed that chylomicron assembly involves the synthesis of

“primordial lipoproteins” followed by their core expansion, which results in the

formation of “nascent lipoproteins” (88). This basic proposal was later expanded to

suggest that chylomicron assembly may involve 3 independent steps: assembly of

primordial lipoproteins, formation of lipid droplets, and core expansion (85).

Subsequently, different biochemical signposts for various biosynthetic milestones were

articulated (89). The association of a preformed PL with nascent ApoB was proposed to

determine the assembly of primordial lipoproteins. An increase in the amount of nascent

TAG would serve as an indicator of the synthesis of larger lipoproteins, and retinyl esters

were proposed as markers of the completion of chylomicron assembly.

Chylomicron assembly occurs mainly in the ER. These particles are then

transported to the cis-Golgi in pre-chylomicron transport vesicles (PCTVs) (18; 105;

119). Budding of the PCTVs has been shown to require L-FABP (135). Pre-chylomicrons

are very large and carry a unique cargo compared with vesicles that carry nascent

proteins (205). Siddiqi et al (165) showed that chylomicrons are exported from the ER in

large vesicles (250 nm) by a coat protein complex II (COPII)-independent mechanism,

although COPII proteins are found on PCTVs. These investigators demonstrated further

that COPII-interacting proteins (Sar1, Sec23/24, and Sec 13/31) are needed to form lipid

17

vesicles that can fuse with the Golgi complex. The unique presence of vesicle-associated

membrane protein 7 (VAMP7) in the intestinal ER and on PCTVs has been suggested to

play a role in the export of chylomicrons from the ER to the cis-Golgi (166). Recently, it

was shown that an isoform of protein kinase C (PKC)—PKCζ—is required for PCTV

budding (167). Thus, chylomicrons are transported on unique particles, different than

those used for protein transport, by intestinal cells. Similarly, unique particles have been

shown to transport hepatic lipoproteins in the liver (164).

Various mechanisms have been shown to regulate the secretion of lipoproteins

from the intestine. Intestinal lipoprotein production is affected by the transcription of

ApoB, which has generally been believed to be constitutive. Previously, it was known

that ApoB levels change primarily through co- and posttranslational mechanisms;

however, Singh et al showed that ApoB secretion is affected by a modest change in its

transcription (168). In recent studies, investigators have found that ApoA-IV levels also

affect intestinal lipoprotein assembly and secretion. Increased secretion of nascent TAG

and PLs with chylomicrons has been shown in IPEC-1 cells (a newborn swine enterocyte

cell line) overexpressing ApoA-IV (114). Similarly, apical supplementation of Caco-2

cells with lipid micelles has been shown to increase ApoA-IV mRNA and protein levels,

which, in turn, facilitates lipid secretion (34).

Lipoprotein assembly and secretion is also regulated by MTP, which transfers

several lipids and assists in the formation of primordial ApoB-lipoproteins (87; 90). MTP

is modulated by various factors and is typically regulated at the transcriptional level (68).

The initial incorporation of lipids into ApoB by MTP prevents it from proteosome-

mediated degradation. Genetic deficiency or pharmacological inhibition of MTP allows

continued synthesis of ApoB, but the protein is misfolded and, thus, is destroyed by ER-

associated degradation (52; 110). Changes in MTP activity affect plasma ApoB

lipoprotein levels significantly. In an in vitro cell-free system, investigators found that

MTP plays a pivotal role in facilitating lipid recruitment by acting as a chaperone to

assist in ApoB folding (99). They also found evidence suggesting that PL recruitment is

intrinsic in the N-terminal domain of ApoB during the translational process and may

facilitate protein folding. Recently, we showed that a deficiency in inositol-requiring

enzyme 1 (IRE1)-β in mice placed on a high-fat, high-cholesterol diet enhances intestinal

18

MTP expression, which leads to increased lipid absorption and chylomicron secretion

(92). IRE1β was shown to cause endolytic cleavage of MTP mRNA between exons 2 and

7. The cleaved products are subsequently degraded by 3'-5' Ski2 nuclease and 5'-3' XRN1

and XRN2 exonucleases. Thus, IRE1β decreases MTP mRNA levels by enhancing its

posttranscriptional degradation. Three potential mechanisms of MTP mRNA degradation

have been proposed: (1) Because IRE1β and MTP mRNA translation are both translated

within the ER membrane, it is possible that under the conditions that increase MTP

mRNA synthesis (eg, high cholesterol and lipid levels), IRE1β and MTP become

juxtaposed and, thus facilitate the specific degradation of MTP mRNA; (2) IRE1β may

activate or recruit another nuclease that initiates MTP mRNA degradation; or (3) under

the conditions of increased chylomicron assembly, the protein translational machinery is

temporarily stalled, leading to the recognition of the MTP mRNA by endoribonucleases

and its subsequent degradation. IRE1β is a mammalian ER stress sensor protein with

prominent expression within intestinal epithelial cells. Within intestine, IRE1β protein is

detectable in the stomach, small intestine and colon. Furthermore, it is mainly expressed

in the intestinal epithelial cells (15). By modulating MTP mRNA levels and,

consequently, the extent of ApoB lipidation, IRE1β may (1) protect the ER from the

consequences of rapid fluctuations in membrane PL, neutral lipids, or vitamin E levels;

or (2) may mediate down-regulation of chylomicron secretion in response to satiety

signals or respond to endocrine factors released by the liver and adipose tissue in the

presence of excess accumulation of lipid throughout the body. Thus, modulation of

IRE1β activity can provide a way to modulate intestine-specific regulation of lipoprotein

assembly and lipid absorption. In this mechanism, up-regulation of IRE1β may be useful

for avoiding a diet-induced hyperlipidemia.

Intestinal lipid absorption has been shown to exhibit diurnal variations in rodents

and humans (8; 23; 118; 129; 143). Although plasma lipid concentrations are maintained

within a narrow physiological range, several factors (eg, plasma clearance, cholesterol

biosynthesis, and hormonal changes) can lead to changes in plasma lipids. It was recently

demonstrated that diurnal variations in plasma lipid levels in rodents are due to changes

in MTP levels (143). Lipid absorption was higher at 24:00 hours than at 12:00 hours

because of the rodents’ nocturnal feeding behavior. Using in situ loops and isolated

19

enterocytes, it was demonstrated that circadian variations were due to changes in

intestinal activities and not because of variations in gastric functions. Further studies have

revealed that intestinal expression of MTP also has diurnal variations. Consistent with

this finding, the transcription rate for mttp was high at 24:00 hours compared with 12:00

hours. Thus, it appears that MTP expression and lipid absorption are maximized to

absorb more lipids at mealtime. The diurnal variations in MTP, in turn, may be

responsible for the change in total plasma lipids and ApoB lipoproteins (143).

CONCLUSIONS

The absorption of lipids from the intestinal lumen into the enterocytes and their

subsequent secretion into circulation is a complex process. Membrane transporters

regulate lipid uptake on the apical surface of the brush border of the enterocyte. An

essential role for NPC1L1 in cholesterol absorption and for the coordinated activities of

ABCG5 and ABCG8 in limiting excess cholesterol uptake is well established. Several

proteins involved in FA uptake have also been identified. Transport of lipids from the

plasma membrane to the ER involves the activity of intracellular trafficking proteins.

Lipids that have been absorbed into the ER are resynthesized and packaged into

chylomicrons; this process is dependent on ApoB and MTP activity. Specialized vesicles

carry these chylomicrons to the basolateral membrane of the cell for secretion.

Posttranscriptional degradation of MTP by IRE1β provides a way to modulate intestine-

specific regulation of lipoprotein assembly and absorption and may be useful for avoiding

diet-induced hyperlipidemias. Recent findings concerning the basolateral efflux of

cholesterol as high-density ApoA-I–containing lipoproteins will help us identify

additional targets for the development of drugs that may suppress cholesterol absorption

to reduce hypercholesterolemia and the risk for cardiovascular disease.

Acknowledgements

This work was supported in part by NIH grant DK-46700 and a Grant-in-Aid

from the American Heart Association to MMH.

20

FIGURE LEGEND

Figure. Intestinal lipid absorption. Products of lipid hydrolysis are solubilized in micelles and

presented to the apical membranes of enterocytes. This membrane harbors several transport

proteins that participate in the uptake of various types of lipids. NPC1L1 is a protein involved in

cholesterol uptake. CD36 and FATP have been shown to participate in FA transport, whereas SR-

B1 is involved in vitamin E uptake. In the cytosol, FABP and CRBP transport FAs and retinol,

respectively. ACAT, DGAT, and LRAT are found in the ER membrane, where they facilitate the

esterification of cholesterol, monoacylglycerols, and retinol, respectively. These esterified

products are incorporated into ApoB48-containing chylomicrons in an MTP-dependent manner.

The newly synthesized prechylomicrons are transported in specialized vesicles to the Golgi

apparatus for further processing and for secretion. In addition, enterocytes express ABCA1 on the

basolateral membrane to facilitate the efflux of cholesterol.

21

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