CHAPTER 1: GENERAL INTRODUCTION

CHAPTER 1:

1. INTRODUCTION

The metabolic syndromes of insulin resistance, type 2 diabetes, visceral obesity and

dyslipidemia contribute up to 40% of the risk of premature coronary heart disease in the

Western society (1). A common feature of these conditions is an increased production of

apolipoprotein B-containing lipoproteins, i.e., very-low-density lipoproteins (VLDL), by the

liver (2,3). These lipoproteins are relatively large particles consisting of a hydrophobic

core of triglycerides (TG) and cholesteryl esters surrounded by a phospholipid monolayer

containing free cholesterol and proteins. VLDL provides extrahepatic tissues with fatty

acids to cover their energy demands during fasting. An inappropriately high VLDL

secretion by the liver may lead to elevated plasma LDL-cholesterol levels after partial

lipolysis of VLDL-TG. LDL can be taken up by macrophages, a process that is accelerated

by modification of the LDL particles, leading to development of lipid-laden foam cells.

Accumulation of these foam cells in the blood vessel wall results in the formation of

atherosclerotic plaques, eventually causing atherosclerosis. It is essential therefore to

efficiently remove remnant VLDL from the circulation. The liver is very important in this

respect: it is involved in the production of TG-rich lipoproteins as well as in the clearance

of remnant lipoproteins. Deviations in production as well as in clearance can cause

hyperlipidemia. Apolipoprotein E appears to be involved in both production and

clearance of lipoproteins. Its “anti-atherogenic” role as a ligand involved in

receptor-mediated uptake of remnant lipoproteins is well-established. More recently, a role for

apoE in control of the hepatic secretion of VLDL has been proposed (4). This would

imply a more “pro-atherogenic” action of apoE, and also underscores the notion that

regulation of VLDL metabolism is a subtle process controlled at multiple levels. Studies

described in this thesis were carried out to unravel the role of apoE in the regulation of

hepatic secretion of VLDL particles.

2. THE LIVER

of bile and provide the major force for bile formation. Bile acids are formed by the

hepatocyte from cholesterol. Other major components of bile are cholesterol,

phospholipids, glutathione, some proteins, bilirubin, electrolytes and some trace

elements (5,6). Cholesterol can only be removed from the body via the bile, either as

bile acid or as free cholesterol (7). Bile formation is therefore essential for maintenance

of cholesterol homeostasis. In the intestine, bile acids play an important role in the

absorption of dietary lipids. Bile acids are effectively reabsorbed by the enterocytes and

transported back to the liver. This cycling of bile acids between liver and intestine is

called the enterohepatic circulation (8).

Endothelial cells are situated adjacent to hepatocytes at their basolateral

(sinusoidal) side, lining the sinusoids. In the sinusoidal space, two other important cell

types are located: fat-storing (Ito) cells and Kuppfer cells. The fat-storing cells have a

prominent physiological role in storage of fat and vitamin A and are key players in

hepatic fibrogenesis. Kuppfer cells are the macrophages of the liver, being responsible

for immune response, phagocytosis, and for the uptake and catabolism of certain lipid

particles and proteins (9-12).

3. TRIGLYCERIDE AND CHOLESTEROL TRANSPORT

3.1 E

P

Dietary TG is hydrolyzed to monoacylglycerol and fatty acids in the intestine and

subsequently taken up by the enterocytes. Monoacylglycerol and fatty acids are

reesterified in the enterocyte to form TG and transported towards the endoplasmic

reticulum (ER) where TG is packed into apoB-48 containing chylomicrons. This last step,

the formation of chylomicrons, is mediated by the microsomal triglyceride transfer

protein (MTP), with a role comparable to that in VLDL assembly in the liver as detailed in

paragraph 4 (18). Chylomicrons are large buoyant TG-rich particles containing

cholesteryl esters and TG surrounded by a phospholipid monolayer containing

cholesterol, apolipoprotein B-48 and apolipoprotein A-I and A-IV. Chylomicrons are

secreted into the lymph to be transported to the blood circulation. In the blood,

apolipoprotein E and apolipoproteins C-1, C-2, and C-3 are added to the particle.

Lipoprotein lipase hydrolyzes TG to release fatty acids for uptake by adipose tissue and

muscle (19). The chylomicron remnants formed after the hydrolysis of TG are rapidly

taken up by the liver after binding of apolipoprotein E to the LDL-receptor (LDLR) or to

the LDL-receptor Related Protein (LRP) followed by endocytosis and degradation of the

particle.

Figure 1: Illustration of the liver lobule. The lobule is hexagonal in shape. The central vein (CV) is localized at the centre of the lobule. Hepatocytes (H) extend radially from the CV to the periphery. The hepatic artery (HA), portal vein (PV) and bile ductulus (BD) are located at the angles of the hexagon.

Table 1: Physical properties and composition of human lipoproteins (20,21)

Chylomicrons VLDL IDL LDL HDL

Origin Intestine Liver VLDL VLDL Liver/intestine

Diameter (nm) 75-1200 30-80 25-35 18-25 5-12 Density (g/mL) <0.96 0.96-1.006 1.006-1.019 1.019-1.063 1.063-1.210 Composition (weight%) Triglycerides 88 56 29 13 15 Cholesterol esters 3 15 34 48 30 Free cholesterol 1 8 9 10 10 Phospholipids 8 20 26 28 45 Protein 1-2 6-10 11 21 45-55

Apolipoproteins B48, A-I, A-IV, C1, C2, C3, E B100, C1, C2, C3, E B100, C1, C2, C3, E B100 A-I, A-II, E

Physical properties and composition of very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL).

3.2 E

The endogenous pathway maintains TG and cholesterol supply from the liver to the

periphery via the secretion of Very Low Density Lipoproteins (VLDL). VLDL is the

obligatory precursor of atherogenic LDL particles.

3.3 R

Cholesterol accumulation in macrophages is considered to represent an early event in

the development of atherosclerosis. Transport of cholesterol from the macrophages back

to the liver followed by removal into bile is therefore considered to be an

anti-atherogenic process (24,25). This pathway is called the reverse cholesterol pathway. In

the liver, cholesterol can be either re-secreted into the circulation as VLDL or secreted

into bile as bile acid or cholesterol. The apoA-I containing lipoprotein HDL (High Density

Lipoprotein) is the key particle in this process. ApoA-I is formed in the intestine and liver

(figure 2). Cholesterol and phospholipids are delivered to nascent HDL by macrophages

in a process controlled by the ATP-binding cassette protein ABCA1 (26). The gene

encoding this protein is mutated in patients with Tangiers’ Disease, that lack HDL in their

plasma (27-29). ApoE from macrophages is also suggested to play a role in the transfer of

cholesterol to HDL (30), probably independent from ABCA1 (31). Free cholesterol in

HDL can be esterified to form cholesteryl ester by action of the enzyme

lecithin:cholesterol acyltransferase (LCAT) and subsequently stored in the core of the

HDL particle, leading to generation of a larger α-HDL particle (32).

Esterification of cholesterol enables the particle to continue accepting free

cholesterol from peripheral tissues (32). One way of removal of cholesterol from HDL is

by transfer of cholesteryl esters and phospholipids to LDL and VLDL in exchange for TG

by cholesteryl ester transferase (CETP) (33) and phospholipid transfer protein (PLTP) (34),

respectively. In rodents, however, CETP is not present which contributes to the high

HDL/LDL cholesterol ratio in these animals. HDL-cholesterol is cleared by the liver by at

least two ways: i) specific uptake of cholesteryl esters via the Scavenger Receptor B type

I (SR-BI) (35) or ii) clearance of apoE-containing HDL particles via the LDL receptor or

the LRP.

Table 2: Properties and major functions of the major apolipoproteins (36-40)

Apolipoprotein Molecular weight (kD) Plasma concentration (mg/dL) Function

apoA-I 28.3 100-120 LCAT cofactor, HDL formation

apoA-II 17.4 30-50 Hepatic lipase inhibitor

apoA-IV 44.5 12-20 LCAT activator

apoB48 264.0 3-5 Chylomicron formation

apoB100 549.0 70-100 VLDL formation, LDLR ligand

apoC1 6.6 4-6 Inhibitor lipoprotein clearance

apoC2 8.9 3-5 LPL activator

apoC3 8.8 12-14 LPL inhibitor

apoE 34.2 3-5 Ligand for LDLR and LRP, VLDL assembly, LPL

LDL IDL VLDL Liver LDLR LRP CM CMR Extrahepatic tissue LDLR FFA FFA LPL LPL LPL ABCA1 CE, PL α-HDL CETP CETP α-HDL SR-BI FFA Intestine apoA-I β-HDL LCAT CD36 LDLR Extrahepatic tissue

Figure 2: Lipoprotein metabolism. Continuous line: Endogenous pathway, Large dots: Exogenous pathway, small dots: Reversed cholesterol pathway. CM=chylomicron, CMR=chylomicron remnant, FFA=free fatty acids, CE=cholesteryl esters, PL=phospholipids

4. VLDL ASSEMBLY AND SECRETION

Currently, the most accepted model for the assembly of VLDL particles by hepatocytes

comprises two steps: a first step in which apoB is synthesized with concordant primary

lipidation of the protein and a second step in which addition of bulk lipid to this

primordial particle takes place (see figure 3) (41) .

4.1 A

B

stop codon (43-45). ApoB-48 is the only form synthesized in the intestine and is

therefore found in chylomicrons and their remnants. In rodents, apoB-48 is also

synthesized in the liver and packed into VLDL particles. As ApoB-48 misses the

receptor-binding domain, apoB48-containing particles can only be cleared by the LDLR after

acquiring apoE. ApoB-48 secretion has been shown to be less dependent from MTP

activity that of apoB-100 (46,47).

The secondary structure of apoB-containing lipoproteins is largely known. At

amino acid 4050 of apoB-100, a bow starts folding the peptide back to the main ribbon

where it crosses between amino acid 3000 and 3500 forming a loop. This loop probably

acts as an negative regulator for the LDL receptor to prevent early binding of apoB-VLDL

to this receptor (48). Conformational changes during lipolysis of VLDL to IDL and LDL

will overcome this effect by permitting Arg-3500 to interact with the C-terminal loop

allowing normal binding of LDL to its receptor (49). A mutation at this position may

cause familial defective apoB100 (FDB), characterized by hypercholesterolemia that

results from defective binding of LDL to the receptor (50).

before they have left the “unstirred water layer” in the space of Disse, and are therefore

not actually released into the circulation.

4.2 T

M

T

P

Lipidation is the essential step in the assembly of VLDL. Humans with

abetalipoproteinemia show very low levels or absence of apoB in plasma due to

microsomal triglyceride transfer protein (MTP)-deficiency (64-66).

MTP is a heterodimer and consists of a 97 kD catalytic subunit and a 58 kD

subunit known as protein disulfide isomerase (PDI). PDI is an ER-resident protein and is

involved as a chaperone in protein folding and in the formation, isomerization, and

reduction of disulfide bonds (67). However, in absence of this enzymatic activity, PDI

still facilitates MTP activity (68,69). The function of PDI in MTP is not completely known,

although it is essential for MTP activity (69,70). MTP is only found in the ER, while it does

not contain a ER-specific KDEL sequence. PDI does have a KDEL sequence (71) and may

therefore act as an anchor for MTP to keep it inside the ER. The development of

liver-specific MTP knock-out mice has recently confirmed the crucial role of MTP in VLDL

assembly. VLDL-TG and apoB-100 levels in plasma are severely reduced in these mice.

Strikingly, hepatic apoB-48 secretion is hardly affected (47). Inhibition of MTP action

through novel inhibitors leads to a decrease in apoB secretion in vivo and in human

hepatoma HepG2 cells (72).

4.3 T

VLDL

The folding of apoB is assisted by several chaperones like GRP94, calrectulin, and Erp72

that bind the growing apoB peptide even before any lipidation has occurred (79). Also,

evidence has been provided that apoB is first membrane-bound inside the lumen of the

ER after translocation (80). There, lipid is added to form the primordial apoB-lipoprotein

particle that buds off the membrane, which, according to some investigators, represents

the rate-limiting step in VLDL assembly (81).

The bulk apoB-free lipid particle required for the second step of VLDL formation

is formed within the smooth ER (SER). The formation of this particle also requires MTP

activity (82), although some controversy still exists in this respect (83). In the enterocyte,

a similar process takes place during the formation of chylomicrons. This apoB-free lipid

particle could be visualized by electron microscopy in enterocytes from apoB-deficient

mice (84), and at the junction of smooth and rough ER in rat liver (85). At a certain time

point, this lipid droplet fuses with the primordial apoB-containing particle. Some

chaperone proteins, like BiP, GRP94, CaBP2, Calreticulin, and, importantly, PDI have

been shown to interact with this nascent, VLDL-particle inside the ER (86).

TG in VLDL originates primarily from the cytosolic lipid compartment (87). TG

first has to be hydrolyzed to fatty acyl coenzyme A (FA-CoA), diacylglycerol (DAG),

monoacylglycerol (MAG) or even to glycerol (88). The last two molecules are not very

likely to be generated as the enzyme activity needed for this process is relatively low in

the liver (89). Hydrolysis of TG to DAG is achieved by the enzyme triglyceride hydrolase

(TGH) (90,91) and the reverse, i.e., reesterification, is mediated by the enzyme

diacylglycerol acyltransferase (DGAT I) (92,93). This hydrolysis-reesterification cycle

constitutes a continuous process, providing the cell with a relatively constant DAG

concentration (94). DAG can diffuse into the ER, while free fatty acids have to be

transported into the ER in a similar way as in mitochondria, requiring a carnitine

palmitoyl transferase (CPT)-like activity. A CPT-I protein similar or identical to the

mitochondrial protein has been found in the ER (95,96). Inside the ER lumen, DAG can

be reesterified to TG by DGAT II. At his moment, only a single DGAT gene has been

cloned (97) although two groups have shown independently that DGAT activity is

localized at the cytosolic as well as at the lumenal side of the ER (93,98).

A Dgat knock-out mouse model, based on the cloned sequence, still showed TG

synthesis although these mice were very lean and were resistant to diet-induced obesity

(99). Female Dgat

_{mice did not show lactation. TG levels in plasma, however, were not}

changed, indicating that a second DGAT gene indeed may exist.

common phospholipid phosphatidyl choline (PC). Choline-deficiency causes a reduction

in VLDL secretion by reducing the number of particles processed towards the Golgi

apparatus. No change was observed in the number of apoB-containing particles inside

the ER, strongly suggesting that de novo synthesis of PL is not needed for the first step in

the assembly of VLDL (101,102). Recently, it has been shown that inhibition of

phospholipase A

(iPLA

), the enzyme that catalyzes the deacylation of PL a the

sn2-position, reduced the bulk TG incorporation into VLDL. ApoB synthesis and translocation

were not affected, indicating that PLA

acts at the level of the second step of the VLDL

assembly without affecting TG synthesis, as also suggested before by others (103).

Probably, the changes in ER membrane structure and composition by inhibition of PLA

has a significant effect on VLDL assembly (104).

Not much is known yet about the role of cholesterol and cholesteryl esters in the

assembly and secretion of VLDL. Cholesterol is stored in the cell in lipid droplets and is

continuously hydrolyzed and reesterified (comparable to TG) to form a dynamic

cholesterol pool for secretion into bile (as free cholesterol or as bile acids) or secretion

by VLDL as free cholesterol in the PL monolayer or as cholesteryl ester (CE) in the core.

The esterification of cholesterol is mediated by the enzyme acyl-coenzyme A:cholesterol

acyltransferase (ACAT) (see for review (105)). At least two isoforms exist, called ACAT-1

and ACAT-2. Whereas ACAT-1 is present in most tissues, ACAT-2 is localized in liver and

intestine only, i.e., the organs involved in lipoprotein secretion. ACAT-1 deficient mice

show normal hepatic cholesterol concentrations, cholesterol synthesis rates, and ACAT

activity (105,106), whereas ACAT-2-deficient mice have almost no hepatic and intestinal

microsomal ACAT activity and display a dramatically reduced amount of cholesteryl

esters in these tissues. ApoB-containing lipoproteins appeared to be smaller in these

knockouts than in wild-type mice and plasma cholesterol levels were reduced (107),

providing evidence for a role for ACAT-2 in control of VLDL secretion. Overexpression of

ACAT-1 in the liver of ldlr

_{mice resulted in accumulation of hepatic CE and an increased}

Lumen

Cytosol

TG

TG

apoB

TG

CE

DAG

DGAT

II

I

TG

_TG

MTP

TG

CE

DAG

AcylCarn.

AcylCarn.

+

Acyl CoA

Acyl CoA

+

TGH

MTP

apoB

CPT-I

PDI

PDI

apoE

apoCs

ACAT

Figure 3: Second step in the VLDL assembly

5. APOLIPOPROTEIN E

Apolipoprotein E (apoE) is a 34 kD apolipoprotein and a constituent of chylomicrons,

VLDL, IDL, and HDL (see for review ref 39). In addition, free apoE is secreted by the liver

into the space of Disse, where it interacts with lipoproteins to promote their

internalization, the so-called “secretion-capture” process (113). Recently, the role of apoE

has been extended to intracellular lipid transport, e.g., in VLDL assembly. This is the topic

of research described in this thesis (see section 5.6). In addition, apoE has also been

connected to the development of Alzheimer’s’ Disease. ApoE is synthesized in the

astrocytes (114) and secreted into the cerebrospinal fluid in association with small

discoidal particles. The function of apoE in brain may be related to neural cholesterol

metabolism (115).

5.1 G

E

to form a propeptide of 317 amino acids that is post-translationally cleaved to yield the

mature apoE protein of 299 amino acids with a molecular mass of 34 kD (120).

The three major isoforms of human apoE can be distinguished by isoelectric

focusing and are referred to as apoE2 (Cys112; Cys158), apoE3 (Cys112; Arg158), and

apoE4 (Arg112;Arg158). ApoE3 is the most common variant and considered to be the

wild-type protein. ApoE2 and ApoE4 differ only at a single position, resulting in a more

acidic (apoE2) and a more basic (apoE4) protein, respectively, compared to the wild-type

apoE3 (118,121,122). APOE is expressed ubiquitously throughout the body, but most

prominently in the hepatocytes (123), especially in the periportal area of the liver (124),

and in astrocytes in the brain (123).

APOE3 has an allele frequency of 0.75 in Caucasian populations, compared to

0.10 for E2 and 0.15 for E4, resulting in 6 common genotypes, i.e., E2/E2, E2/E3, E2/E4,

E3/E3, E3/E4, and E4/E4. APOE2 and some very rare mutations (including APOE3Leiden)

are related to risk for development of cardiovascular diseases (CVD). ApoE2 shows

defective receptor binding due to the Arg158 -->Cys mutation just outside the receptor

binding site, which, at first sight, might be expected to lead to increased LDL-cholesterol

levels. This is, however, not always the case. The majority of subjects with E2/E2 have

high levels of apoE and low levels of apoB, plasma cholesterol, and LDL-cholesterol

(125,126). This is probably caused by upregulation of the LDLR due to decreased

cholesterol delivery in the liver (127). Approximately 5% of all subjects with APOE2/E2

do develop type III hyperlipoproteinemia (128), which is characterized by high levels of

VLDL remnants in plasma. These subjects have xanthomatous lesions of the skin and

develop atherosclerosis. The pathogenesis of type III hyperlipoproteinemia has

excellently been reviewed by Mahley et al. (128). The APOE4 gene is associated with a

higher risk for development of Alzheimer’s Disease. This is maybe the result of an

impaired cholesterol homeostasis causing a poor dendritic remodeling and

synaptogenesis, resulting in plaque formation in the brain (115).

5.2

P

E

The receptor-binding domain is characterized by a basic area that interacts directly with

the receptor. In the APOE2 protein, the arginine at position 158 is replaced by a

cysteine, just outside the receptor-binding domain. This causes a change in confirmation

of the 136-150 region. Normally, Arg158 forms a salt bridge with Asp154. In the APOE2

protein, however, a salt bridge is formed between Asp154 and Arg150 which disrupts

the LDLR domain and results in a diminished receptor binding (134).

5.3 R

APOE

Knowledge about the regulation of APOE gene expression is still limited (135).

Expression of apoE and apoC-I in the liver is directed by the cis-acting regulatory Hepatic

Control Region (HCR-1) located 15 kb downstream of the APOE gene (136,137). A

second HCR (HCR-2) has been identified at 27 kb of the 3’ end of the APOE gene and is

possibly a duplicate of HCR-1. In the absence of HCR-1, presence of HCR-2 may take

over the regulation of hepatic APOE expression (138,139). Upstream of APOE, between

nucleotides –161 and -141 in the APOE gene promoter, a proximal enhancer element is

located which is required for APOE expression in the liver. This region, the Positive

Element for Transcription (PET) is able to bind at least the transcription factor Sp-1

(136,140). Other transcription sites were identified to bind HNF-3, C/EBP and nuclear

hormone receptors (135,137,141).

5.4 E

E

Intake of sucrose-rich diets have been shown to affect plasma lipid levels in subjects with

one or two APOE2 alleles more than in subjects expressing APOE3 or APOE4 (142).

Especially TG concentrations were elevated in these subjects, which might be due to the

interactions of dietary carbohydrates with VLDL metabolism. It has been shown that

both sucrose and fructose feeding induce VLDL-TG secretion in rats (143,144). This

effect was associated with increased fatty acid synthase activity (145). Insulin levels and

mitochondrial β-oxidation were hardly changed under these dietary conditions. In some

reports (144,146), but not all (145), an increase in liver TG content was observed.

Development of a fatty liver by excess carbohydrates in rats is probably dependent on

age, as shown by Nassir et al. (147). These authors did not observe development of a

fatty liver after fructose-feeding in young rats, whereas it did occur in adult animals. In

both groups, however, VLDL secretion was markedly increased after fructose feeding. In

vitro data clearly showed an increase in apoE gene transcription and synthesis in

which a binding site is present in the APOE gene promoter (137), is involved in glucose

homeostasis (150,151).

5.5 R

E

The role of apoE in lipoprotein clearance has been recognized since the early 1980’s.

More recently, evidence has become available that apoE might also play a role in

intracellular cholesterol and TG metabolism. ApoE appears to redirect TG intracellularly

in macrophages, possibly by acting as a chaperone for lipids (152). As a consequence,

TG accumulates inside apoE-deficient macrophages due to a lower utilization of TG.

Intracellular cholesteryl ester content appeared to be decreased in the presence of apoE,

probably due to an increased secretion of cholesterol from the macrophage (152). This

was confirmed by the observation that apoE has a function in the secretion of cholesterol

from macrophages independent from the presence of cholesterol acceptors such as

apoA-I (30,153) and ABCA1 (31). In addition, apoE is associated with lipid transport from

astrocytes (115). Disturbances herein may lead to Alzheimer’s Disease.

In 1996, it was shown by our laboratory that apoE-deficient mice develop a fatty

liver when fed a normal chow diet (154). This was remarkable, as these mice show a very

low clearance of lipoproteins and therefore develop elevated plasma cholesterol levels.

On a chow diet, hepatic cholesterol metabolism is also altered in apoE-deficient mice.

Probably related to the overload of hepatic cholesterol, cholesterol synthesis is reduced.

Bile flow and bile acid output, however, are not changed. Challenging cholesterol

metabolism in mice by a high-cholesterol diet normally results in increased biliary

cholesterol secretion. In apoE-deficient mice, however, the increase in cholesterol

secretion into bile is strongly attenuated (155,156). The underlying mechanism is not yet

known.

5.6 K

APOE

ApoE-deficiency in humans is very rare and leads to development of type III

hyperlipoproteinemia (160). ApoE-deficient mice were independently constructed by

Plump et al. (161), Maeda et al. (162) and by van Ree et al. (163). The latter model, used

in experiments in this thesis, was created by replacement of exons 1, 2, and a part of

exon 3 by a hygromicine B resistance cassette, resulting in disruption of the whole Apoe

gene. The mouse is characterized by strongly increased plasma cholesterol levels,

already when fed a low fat chow diet. TG levels on the other hand are hardly affected.

As expected, these mice develop severe atherosclerotic lesions (163). As noted before,

deficient mice also show a 60% decreased VLDL-TG secretion (4). The

apoE-deficient mouse is probably the most widely used animal model for atherosclerosis

research (see for instance ref. 164-168).

A rare mutation in the APOE gene is the Dutch APOE3Leiden variant, which is

characterized by a tandem repeat of codons 120-126 yielding a protein of 306 amino

acids (169,170). It is associated with a dominantly inherited form of familial

dysbetalipoproteinemia due to defective clearance of lipoprotein remnants (171). The

first transgenic mouse model carrying the human APOE3Leiden gene was developed by

van Maagdenberg et al. (172). At the time this mouse was developed, the APOE/APOC1

cluster was not yet completely identified. Therefore, the complete cluster, including

APOC1 and the hepatic control region (HCR), was used (172). These mice develop

atherosclerotic lesions when fed high-cholesterol diets (173) due to a decreased

lipoprotein lipolysis and clearance (174). APOE3Leiden transgenic mice are used as a

model for atherosclerosis, as they show a similar sensitivity to dietary induction of

atherosclerosis as humans. It is a relative “mild” model compared to the apoE-deficient

mouse, making the APOE3Leiden mouse probably more clinically relevant. These mice

are therefore frequently used for determining dietary effects on lipoprotein metabolism

(e.g., 175-177).

6. OBJECTIVE AND OUTLINE OF THIS THESIS

(dys)regulation of VLDL secretion. The discovery of MTP as regulatory protein in the

VLDL secretion in the early nineties has notably accelerated research on basic aspects of

VLDL assembly. The growing availability of transgenic and knockout mouse models has

also greatly contributed to our current knowledge of lipoprotein formation. In particular,

the apoE-knockout mouse has been used extensively in lipoprotein research. Historically,

apoE was considered only as a ligand for lipoprotein receptors. The findings that

apoE-deficient mice develop a fatty liver and show a decreased VLDL-TG secretion when fed

normal chow (4,154) were the first indications that apoE might play a specific role in

hepatic lipid metabolism, independent from its role in lipoprotein uptake. Specifically,

these data suggested a role of apoE in the secretion of VLDL particles.

The aim of this thesis was to characterize the role of apoE in the assembly and

secretion of VLDL and to gain insight in the underlying mechanism(s). In work described

in chapter 2 the regulatory role of apoE in VLDL secretion was firmly established, using

transgenic APOE3/Apoe

_{and Apoe}

_{mice. To address the role of apoE in VLDL}

secretion, a transgenic mouse was created expressing low levels of the human APOE3 in

the absence of murine Apoe. Localization of intrahepatic APOE3 to specific sites in the

hepatocyte by immuno electron microscopy established the model. Effects of the

transgene on plasma lipid levels, VLDL secretion and composition and hepatic lipid

accumulation were determined. Adenoviral overexpression of APOE3 in apoE-deficient

mice was used to directly determine the relationship between the level of APOE3

expression, production of the protein, and VLDL secretion.

The possible regulatory role of apoE in mediating the reported effects of dietary

carbohydrates on VLDL secretion was investigated in chapter 3. It is known that

carbohydrate-rich diets lead to an increased VLDL secretion in rodents and in humans

and, in some animal models, to development of a fatty liver. This has been attributed to

an increased de novo lipogenesis (DNL). Dietary carbohydrates have also been

suggested to induce Apoe gene expression in the liver. The hypothesis was that apoE

might, in part, mediate the effect of carbohydrates in VLDL production, and that

apoE-deficiency therefore would lead to an even stronger lipid accumulation in the liver

without stimulation of VLDL secretion. To address this issue, hepatic lipid accumulation,

plasma lipid levels and VLDL-TG secretion were determined in wild-type C57BL/6J and

apoE-deficient mice fed a high-carbohydrate diet for two weeks. VLDL composition and

size were also determined. Effects of carbohydrates on de novo lipogenesis was

estimated by determining the expression of the lipogenic genes fatty acid synthase (Fas)

and acylCoA carboxylase (Acc).

and development of liver abnormalities. The mice used in this study expressed, in

addition to APOE3Leiden, also human APOCI and murine Apoe. In chapter 5, mice

expressing APOE3Leiden without APOC1 were used, in the presence or absence of

murine apoe, to study specifically the effect of APOE3Leiden on VLDL metabolism and

hepatic lipid accumulation.

In Chapter 6, a more mechanistic approach was used to assess the role of apoE

in TG incorporation into nascent VLDL particles. Subsequent steps in hepatocytic TG

metabolism were quantified in presence and absence of apoE. TG turnover was

determined in primary hepatocytes from wild-type and apoE-deficient mice and

microsomal activity of DGAT, the enzyme responsible for esterification of diacylglycerol

to form TG, was measured. The microsomal triglyceride transfer protein (MTP) is

considered to be the rate-controlling enzyme in the VLDL assembly by transferring lipid

to the growing primordial particle and by assisting the folding of apoB. MTP activity was

determined in hepatic microsomes isolated from livers from both strains of mice. In

addition, the effect of MTP inhibitor BMS-197636-02 on VLDL-TG secretion was

determined in primary hepatocytes. To visualize the outcome of apoE-deficiency on

VLDL assembly, hepatic lipids were stained by osmium tetroxide and analyzed by

electron microscopy. The combined data were used to propose a possible mechanism

for the apoE-mediated assembly of TG into VLDL.

Finally, Chapter 7 gives an overview of the current knowledge of the role of apoE

in hepatic lipid metabolism and provides an integrated discussion.

7. REFERENCES

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