UMEÅ UNIVERSITY MEDICAL DISSERTATIONS

New series 1352 ISSN 0346-6612 ISBN 978-91-7459-006-7

Novel factors affecting clearance of

triacylglycerol-rich lipoproteins from blood

Stefan K. Nilsson

Department of Medical Biosciences

Physiological Chemistry

901 87 Umeå

Umeå University 2010

Omslagsfoto: Ur kaos kommer ordning

av Frida Olofsson

Inlagebild: Signet Bygda Sochne

Av Walter Asplund

Copyright © Stefan K. Nilsson

ISBN: 978-91-7459-006-7

ISSN: 0346-6612

Tryck: Print & Media

Umeå, Sverige 2010

Tillägnad Anna och Hjalmar

Gloria Bygda Sochne ad Perpetuum

TABLE OF CONTENTS ABSTRACT 7 ABBREVIATIONS 8 LIST OF PUBLICATIONS 10 POPULÄRVETENSKAPLIG SAMMANFATTNING 11 INTRODUCTION 12 LIPOPROTEINS 14

Lipoprotein classes, structure and function 14 Apolipoproteins involved in lipoprotein metabolism 20 Apolipoprotein A 21 Apolipoprotein B 22 Apolipoprotein C 22 Apolipoprotein E 24

METABOLISM OF LIPOPROTEINS 26 Modulating enzymes 26 Lipases 27 Lipoprotein lipase 27 Hepatic lipase 29 Endothelial lipase 29

RECEPTORS INVOLVED IN LIPOPROTEIN METABOLISM 30 Low density lipoprotein receptor 30 Low density lipoprotein receptor-related protein 1 31 Very low density lipoprotein receptor 33 Sortilin-related receptor, LA repeats-containing 33 Sortilin 34 Heparan sulfate proteoglycans 34 Other receptors 36

LIPOPROTEINS IN DISEASE 37 Atherosclerosis 38 Lipoproteins in atherosclerosis 39

APOLIPOPROTEIN A-V 43 ApoA-V in disease 43 Expression patterns and regulation 44 Structure and molecular mechanisms 44 Proposed mode of action 45 Intracellular mechanisms 46 Extracellular mechanisms - LPL activator 46 Extracellular mechanisms - receptor ligand 48 Extracellular mechanisms - in vivo analyses 49 Final conclusion 50

ANGIOPOIETIN-LIKE PROTEINS 51 ANGPTLs in disease 51

Expression and regulation 52 Structure and molecular mechanism 53 Proposed mode of action 54

UNPUBLISHED RESULTS 56 Interaction of ApoA-V with LDLr 56 ApoA-V affects chylomicron clearance in rats 56

METHODS 58 Surface plasmon resonance 58 SPR in history 58 SPR in theory 58 Binding constant determination 59

AIMS OF THIS THESIS 61 RESULTS 62 DISCUSSION AND CONCLUSIONS 66

ApoA-V 66 ANGPTLs 68

REFERENCES 69 ACKNOWLEDGEMENTS 92

7

ABSTRACT

Apolipoprotein (apo) A-V is the most recently discovered member of a protein family responsible for the

structure and metabolic fate of plasma lipoproteins. While most of the apolipoproteins are well

characterized with regard to structure, interactions and function, the role of apoA-V is not well understood. ApoA-V is synthesized only in liver and is present in blood at much lower concentration than

the other apolipoproteins. Although apoA-V is firmly established as an important determinant for plasma

triacylglycerol (TG) metabolism, the mechanism is unclear. ApoA-V has been suggested to act through 1) an intracellular mechanism affecting lipoprotein assembly and secretion, 2) direct or indirect activation of

lipoprotein lipase (LPL), or 3) interaction with endocytotic lipoprotein receptors.

Two other novel players involved in the clearance of lipoproteins are angiopoietin-like protein

(ANGPTL) 3 and 4. Previous studies have shown that the coiled-coil domain (ccd) of ANGPTL3 and -4 can inactivate lipoprotein lipase (LPL). The functional site of action of LPL is at the capillary

endothelium, but the enzyme is synthesized mostly in adipocytes and myocytes and has to be transported

by trancytosis to the luminal side of endothelial cells. Both ANGPTLs are present in tissues and in the circulating blood, but it is not known were the inactivation of LPL normally takes place.

The aim of this thesis was to investigate the mechanism by which apoA-V exerts its effect on TG

metabolism and to investigate in further detail how ANGPTLs act on the LPL system.

Binding of apoA-V to receptors involved in lipoprotein metabolism was investigated by surface plasmon

resonance technique (SPR). ApoA-V was found to bind to the LDL receptor related protein 1 (LRP1) and to the mosaic type 1 receptor sorLA. Binding could be competed by receptor associated protein (RAP) or

by heparin, and was calcium dependent. We concluded that apoA-V binds to the LA-repeats of these

receptors. In further experiments apoA-V was shown to increase binding of TG-rich chylomicrons to the receptors. This demonstrated a possible mechanism for the TG-lowering effect of apoA-V in vivo.

A putative binding region in apoA-V for heparin and receptors was investigated by site-directed

mutagenesis. Two positively charged amino acid residues were changed (Arg210Glu/Lys211Gln),

resulting in decreased binding to heparin and to LRP1 and thus the localization of one important functional region in apoA-V.

Since the receptor sorLA also contains a Vsp10p domain, another Vsp10p domain family member,

sortilin, was investigated. ApoA-V was found to interact also with this receptor. In experiments with human embryonic kidney cells transfected with sorLA or sortilin, apoA-V was found to bind to cell

surfaces and to be rapidly internalized while co-localized with the receptors on the way to lysosomes for

degradation.

Additional apoA-V mutants, identified in patients with severe hypertriglyceridemia, were investigated

with regard to effects in vitro on LPL activity and receptor binding. The most severe mutants displayed

null binding to LRP1, whereas the effect on LPL activity was retained. These results suggest that lack of

receptor interaction mirrors the loss of biological function in a better way than the in vitro effect on LPL activity.

We noted that ccd-ANGPTL3 and -4 did not prevent the LPL-mediated uptake of chylomicron-like

lipoproteins in primary murine hepatocytes. Therefore LPL activity was measured after pre-incubation

with ccd-ANGPTL3 or 4 in the presence or absence of TG-rich lipoproteins. Physiological concentrations of lipoproteins were found to protect LPL from inactivation by ccd-ANGPTLs. Investigation by SPR

demonstrated that the ccd-ANGPTLs did not bind to the lipoproteins. Other experiments showed that less

than 1% of ANGPTL4 in human serum was bound to TG-rich lipoproteins. This implies that the known binding of LPL to TG-rich lipoproteins stabilizes the enzyme and protects it from inactivation by

ANGPTLs. We conclude that the normal levels of ANGPTLs in plasma are too low to affect the LPL-

system and that inactivation of the enzyme by ANGPTLs is more likely to occur locally in the extracellular interstitium of tissues where LPL is en route to its endothelial binding sites and where the

concentrations of the TG-rich lipoproteins are low.

8

ABBREVIATIONS ABCA1 – ATP binding cassette transporter A1 ABCG1 – ATP binding cassette transporter G1 A-FABP – adipocyte-fatty acid binding protein ANGPTL – angiopoietin-like protein AP – acute pancreatitis apo – apolipoprotein apoB48R – apoB48 receptor BSA – bovine serum albumin ccd – coiled-coil domain CD36 – cluster designation 36 CETP – cholesteryl ester transfer protein CVD – cardiovascular disease DMPC – dimyristoylphosphatidylcholine ECM – extracellular matrix EDTA – ethylenediaminetetraacetic acid EL – endothelial lipase ER – endoplasmic reticulum FA – fatty acid (non-esterified fatty acid, free fatty acid) fld – fibrinogen-like domain FRET – Försters resonance energy transfer HDL – high density lipoprotein Hek293 – human embryonic kidney cells 293 HL – hepatic lipase HSPG – heparan sulfate proteoglycan HTG – hypertriglyceridemia IDL – intermediate density lipoprotein KD – equilibrium dissociation constant kdiss – dissociation rate constant kass – association rate constant LA – ligand binding type A LCAT – lecithin-cholesterol acyltransferase LDL – low density lipoprotein LDLr – LDL receptor LDLR – LDL receptor (gene family) LPL – lipoprotein lipase LRP1 – low density lipoprotein receptor-related protein 1 LSR – lipolysis stimulated receptor LXR – liver X receptor MAM – micro- and macrovascular disease MTP – microsomal triglyceride transfer protein oxLDL – oxidized LDL PLTP – phospholipid transfer protein RAP – receptor associated protein RME – receptor-mediated endocytosis SMC – smooth muscle cell SR-A – scavenger receptor-class type A SR-BI – scavenger receptor-class type B I

9

SREBP – sterol regulatory element-binding protein T2D – type II diabetes mellitus TG – triacylglycerol (triglyceride) VCAM-1 – vascular adhesion molecule-1 VLDL – very low density lipoprotein VLDLr – very low density lipoprotein receptor

10

LIST OF PUBLICATIONS The publications will be referred to in the text by their roman numerals.

I Apolipoprotein A-V interaction with members of the low

density lipoprotein receptor gene family Nilsson SK, Lookene A,

Beckstead JA, Gliemann J, Ryan RO and Olivecrona G.

Biochemistry. (2007) 46:3896-3904.

II Endocytosis of apolipoprotein A-V by members of the low

density lipoprotein receptor and the VPS10p domain receptor

families Nilsson SK, Christensen S, Raarup MK, Ryan RO, Nielsen MS and

Olivecrona G.

J Biol Chem. (2008) 283:25920-25927

III Effects of six APOA5 variants, identified in patients with

severe hypertriglyceridemia, on in vitro lipoprotein lipase

activity and receptor binding Dorfmeister B, Zeng WW, Dichlberger A,

Nilsson SK, Schaap FG, Hubacek JA, Merkel M, Cooper JA, Lookene A, Putt

W, Whittall R, Lee PJ, Lins L, Delsaux N, Nierman M, Kuivenhoven JA,

Kastelein JJ, Vrablik M, Olivecrona G, Schneider WJ, Heeren J, Humphries

SE and Talmud PJ.

Arterioscler Thromb Vasc Biol. (2008) 28:1866-1871

IV Triacylglycerol-rich lipoproteins protect lipoprotein lipase

from inactivation by angptl 3 and angptl 4 Nilsson SK, Larsson M,

Lookene A, Sukonina V, Makoveichuk E, Heeren J and Olivecrona G.

Manuscript

All published papers are reproduced with permission from the copyright

holders.

11

POPULÄRVETENSKAPLIG SAMMANFATTNING Lipider, det som i vardagligt tal kallas fett, spelar en viktig roll i människokroppen. Förutom att de utgör vår viktigaste energikälla och vårt stora reservlager av energi så används de också som beståndsdelar i alla cellmembraner och för tillverkning av hormoner och andra viktiga signalsubstanser som kroppen behöver. Även om lipider är viktiga och nyttiga för oss, så finns det en baksida. När lipider transporteras som blodfetter så kan de orsaka sjukdomar om kroppen på något sätt är i obalans t.ex. på grund av att vi äter för mycket och rör oss för lite. De fetter som vi använder för att få energi finns i huvudsak i två olika former, triglycerider och fettsyror. Triglycerider är lagringsformen av fett eftersom de bildar fettdroppar som tar liten plats och som håller sig kvar tills de spjälkas. De kan också vara ett problem då fettet ibland lagras där vi inte vill ha det. Triglyceriderna kan inte passera genom cellmembran, men när triglyceriderna spjälkas bildas fettsyror. Dessa är mer vattenlösliga och kan enkelt passera både in och ut ur celler och de transporteras i blodet bundna till albumin. När vi äter en fettrik måltid packas triglyceriderna av celler i tunntarmen i små fettdroppar som kallas lipoproteiner. Dropparna täcks av ett stabiliserande lager av andra mera lösliga fetter och av speciella proteiner som kallas apolipoproteiner. Lipoproteinerna transporteras sedan med lymfan och släpps ut i blodet. När lipoproteinerna når de allra minsta grenarna av kärlträdet, kapillärerna, träffar de på fettspjälkande enzymer, lipaser, som spjälkar lipoproteinernas triglycerider till fettsyror. På så sätt kan fettet i lipoproteinerna tas upp i kroppens celler och organ och nyttjas till energi och för andra ändamål. Apolipoproteinerna spelar en viktig roll i lipoproteinernas omsättning. En del bromsar omsättningen, en del ansvarar för att aktivera spjälkningen och andra binder till receptorer som ansvarar för upptag av hela lipoproteiner i cellerna. Celler kan vilja ta upp hela lipoproteiner för att tillgodogöra sig inte bara energi utan även fettlösliga vitaminer och kolesterol som också finns i lipoproteinet. Min avhandling handlar om två proteiner som visat sig vara viktiga för lipoproteinernas omsättning. Apolipoprotein A-V tycks påverka hanteringen av triglycerider i blodet. Människor som har mutationer i proteinet har kraftigt förhöjda triglyceridnivåer. Man förstår ännu inte genom vilken mekanism apolipoprotein A-V påverkar triglyceridomsättningen. Detsamma gäller för de proteiner som kallas ANGPTL som också påverkar triglyceridnivåerna. ANGPTL har visats kunna inaktivera eller förstöra de enzymer som spjälkar lipoproteinerna. Eftersom förhöjda triglycerider, liksom förhöjt kolesterol, är en riskfaktor för kärlsjukdom så är det viktigt att förstå hur apolipoprotein A-V och ANGPTL egentligen fungerar. Vi hoppas att ökad kunskap om detta kan bidra till att nya och effektivare läkemedel kan utvecklas mot rubbade blodfetter. I avhandlingen beskrivs att apolipoprotein A-V binder till den viktiga lipoproteinreceptorn LRP1 (delarbete I). Vi tror att apolipoprotein A-V kan stimulera upptag av hela lipoproteiner genom att binda till receptorerna. Den tanken utvecklas vidare i delarbete II, där vi även studerar bindning till en annan receptor, Sortilin. Den är också spännande eftersom dess funktion har kunnat kopplas till utveckling av bl.a. stroke. Vi kunde visa att apolipoprotein A-V kan använda dessa receptorer för att ta sig in i celler och vi tror att det då kan ta med sig en last av lipoproteiner. I delarbete III visar vi att muterat apolipoprotein A-V, som man hittat hos individer med kraftigt förhöjda blodfetter, inte kan binda till LRP1. Dessa fynd stärker hypotesen att receptorbindningen är viktig för apolipoprotein A-Vs funktion. Ett snabbare cellulärt upptag av lipoproteiner kan vara en del av förklaringen till den triglyceridsänkande effekt man förknippar med apolipoprotein A-V. I delarbete IV studerar vi under vilka förhållanden ANGPTL kan förstöra lipoproteinlipas (LPL), det viktigaste enzymet för spjälkning av blodets triglycerider. Vi fann att bindning av LPL till lipoproteiner skyddar mot de mängder av ANGPTL som normalt finns i blodet så att enzymet kan behålla sin aktiva funktion. Detta förklarar att ANGPTL i blodet inte visar samband med triglyceridnivåerna, vilket stämmer med tidigare forskning inom fältet. Vi menar att ANGPTL inte utövar sin effekt på LPL i blodcirkulationen. Istället föreslår vi att kontrollen utövas på ett tidigare stadium genom att ANGPTL ligger i bakhåll på den väg som LPL måste ta för att nå ut till blodkärlens väggar. På den vägen saknas nämligen de skyddande lipoproteinerna och LPL blir därför ett lätt byte.

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INTRODUCTION

Lipids play an important role in biology as energy substrates, energy

storages, membrane components and in signaling. Triacylglycerol (a.k.a

triglycerides) (TG) and fatty acids (FA) are the energy substrates of choice

for eukaryotic cells. TG are non polar and insoluble in water making them

ideal for compact storage as lipid droplets, whereas FA are more polar, thus

somewhat soluble in water, and easily moved over biological membranes. FA

have low solubility in water; hence transport of both FA and TG requires

solubilizing proteins. FA are transported in the circulation bound to serum

albumin whereas TG are packed into the center of spherical particles,

lipoproteins, covered by an amphiphilic shell consisting of phospholipids,

cholesterol and apolipoproteins (Figure 1). Lipoproteins play an important

role in energy homeostasis and cholesterol transport, but also in transport of

lipid soluble vitamins.

As lipoproteins enter the circulation and reach their target tissue they are

immediately metabolized by lipases liberating FA through hydrolysis from

TG to the underlying tissue thus avoiding the difficult passage of TG across

the cell membrane. Lipoproteins can also be subject to cellular uptake by

receptor mediated mechanisms or remodeling by lipid transfer proteins.

Depending on need and lipoprotein status these factors work in concert.

There are good reasons to study the metabolism of lipoproteins. Lipoprotein

abnormalities are central in the development of cardiovascular disease

(CVD) and are manifested in many other metabolic diseases. Lipoprotein

abnormalities can also be secondary complications to other diseases.

Metabolic disorders are the most common cause of death in the world and an

increasing problem as developing countries copy the unhealthy life style of

the western world.

Although mature lipoproteins are easily assessable for scientific studies,

being present in blood, a few circulating actors directly involved in

lipoprotein metabolism have been discovered only during in the last decade,

e.g. endothelial lipase (EL), angiopoietin like proteins (ANGPTLs) and

apolipoprotein A-V (apoA-V). ApoA-V was found by two groups separately

through comparative genome sequencing between man and mouse (1), and

as a protein upregulated in liver regeneration (2). Through transgenic mouse

models and human population studies apoA-V was immediately recognized

as an important player in plasma TG metabolism.

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ANGPTLs consist of a gene family where two members, ANGPTL3 and -4,

have been found to affect lipoprotein metabolism through inhibition of

lipases (3, 4). Transgenic mouse models and human population studies

confirms that ANGPTL3 and -4 have important roles in lipoprotein

metabolism, and especially in TG-rich lipoprotein metabolism.

This thesis focuses on finding mechanisms by which apoA-V and ANGPTLs

exerts their roles in plasma TG metabolism.

Figure 1 Schematic view of a lipoprotein. The lipoprotein consists of an amphipatic shell consisting of phospholipids, apolipoproteins and cholesterol. The hydrophobic core cosists of cholesteryl esters and triglycerides. Reprinted with permission from Prentice Hall.

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LIPOPROTEINS

Lipoproteins are spherical particles consisting of a lipid part and a protein

part. The lipid part consists of TG and cholesteryl esters in the non-polar

particle core, whereas phospholipids and unesterified cholesterol forms an

amphiphilic surface layer together with apolipoproteins making the

lipoprotein soluble (Figure 1). This structure allows transport of

biomolecules not soluble in the water-based blood circulation. The biological

advantages of transporting lipids as TG instead of FA are several. The

physiological properties of these two lipids are distinctly different. Whereas

TG are biologically inert, FA are toxic at high concentrations giving rise to

lipotoxicity and thereby pathological conditions (5). TG cannot move across

biological membranes efficiently. Therefore fatty acids must be hydrolyzed

and re-esterified during membrane passages.

Lipoproteins are derived from two different pathways. Dietary lipids are

hydrolyzed in the gastro-intestinal tract and subsequently absorbed by

enterocytes in the small intestine. Here they are reesterified and packed as

chylomicrons (Figure 2). These huge lipoproteins are transported by the

lymphatic system and enter the blood stream via ductus lymphaticus dexter

at the bifurcation of vena jugularis interna and vena subclavia.

Chylomicrons are rapidly hydrolyzed in the circulation by lipoprotein lipase

(LPL) resulting in release of fatty acids and leaving remnant particles which

are mostly cleared by the liver through receptor mediated endocytosis

(Figure 2). This is known as the exogenous lipoprotein pathway.

The endogenous lipoprotein pathway starts in the liver which produces very

low density lipoproteins (VLDL) from lipids derived from de novo synthesis

of lipids, FA from plasma or reused chylomicron remnant-derived lipids

(Figure 2). Upon secretion VLDL are released into the circulation with

similar faith as chylomicrons.

Lipoprotein classes, structure and function

Chylomicrons are packed in the intestine by enterocytes and are destined to

carry dietary fat and lipid soluble vitamins for immediate utilization or

storage for later use. Intestinal lipid absorption is prepared in the stomach.

The digestion starts in the stomach where gastric lipases act on the

triglycerides (6); moreover, emulsification through gastric peristalsis occurs

here. When entering the duodenum the emulsions mix with bile salts and

pancreatic juice and subsequently change in physical and chemical form

making them accessible for various enzymes. During continuous hydrolysis,

15

mainly by pancreatic lipase supported by its co-factor colipase, the

hydrolysis products of the emulsified lipids will form mixed micelles

consisting of FA, monoglycerides and bile salts. The products from TG

hydrolysis are taken up from the intestinal lumen and transported across the

cell membrane of the enterocytes through diffusion and/or active transport

(reviewed in (7)). Once in the enterocytes the FA and monoglycerides are re-

esterified into TG and packed into chylomicrons via microsomal triglyceride

transport protein (MTP) dependent lipidation of apoB48 in the endoplasmic

reticulum (ER). During this process lipid soluble vitamins, e.g. vitamin E and

retinyl ester, are also added together with cholesteryl esters, apoAs and

phospholipids. The pre-chylomicrons are then transported via vesicles to the

Goli apparatus and subsequently after final preparation released to the

lymph for transportation to the blood circulation (reviewed in (8)).

The newly synthesized chylomicrons are huge lipoproteins, rich in TG and

low in density (Table 1.). Upon entering the blood at the bifurcation of the

subclavian veins chylomicrons exchange apolipoproteins with high density

lipoprotein (HDL) receiving apoCs, apoE and apoA-V (Figure 2). They are

quickly bound at the endothelium in blood vessels upon entrance in the

circulation, a process called margination. At the endothelial binding sites

chylomicrons are diminished through LPL-dependent lipolysis (9); released

free fatty acids are taken up by underlying tissue and utilized for energy

storage or immediate use (Figure 2). Excess of phospholipids are shedded off

together with intestinal apoAs. This process reshapes the chylomicrons into

remnant particles, still rich in TG, that are predominantly and swiftly cleared

by the liver (10-12). Thus, chylomicrons have a very short residence time

compared to other lipoprotein classes (Table 1.).

Very low-density lipoproteins (VLDL) are as previously mentioned

synthesized in the liver (Figure 2). The hepatocytes utilize TG droplets

derived from lipoprotein uptake, e.g. chylomicron remnants, or from re-

esterified extracellular FA carried from mainly the adipose tissue by serum

albumin or derived from plasma compartment lipolysis. TG is mobilized

from the TG droplet to the ER where it is hydrolyzed and re-esterified again;

the re-esterified product can now form a TG-rich precursor particle

dependent in MTP or through influence of insulin be recycled to the initial

TG droplet (reviewed in (21, 22)). Regulation via insulin helps avoid too high

lipoprotein levels during the postprandial phase.

During the formation of the TG-rich precursor particle an apoB100 containing

VLDL-precursor is formed. Synthesized on ribosomes bound to the ER the

VLDL precursor gets partly lipidated via MTP. MTP transfers TG,

phospholipids and cholesterol esters through direct interaction with apoB100

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(reviewed in (21)). The finished VLDL-precursor now called VLDL2 exits the

ER in an ER Golgi intermediate compartment. VLDL2 is then further

lipidated in the Golgi complex and subsequently transported to the plasma

membrane for secretion (reviewed in (23)). How VLDL2 and the TG-rich

precursor droplet fuse before they exit the ER and/or the Golgi compartment

is a poorly understood process.

Chylomicrons VLDL IDL LDL HDL

Molecular weight x10-6

>400 10-80 5-10 2-3 0.18-0.23

Density (g cm-3) <0.95 0.95-1.006

1.006-1.019

1.019-1.063

1.063-1.21

Diameter (nm) 75-1200 30-80 25-35 18-25 5-12

Chemical composition (%)

Protein 2 10 18 25 33

Triacylglycerol 85 50 31 10 8

Cholesterol 4 22 29 45 30

Phospholipid 9 18 22 20 29

Apolipoproteins

Major B48 B100 B100, E B100 AI, AII

Minor A, C, E A, C, E AV, C, E

Residence time 5-10 min 3.4 h 1 h 2.9 d 5.6 d

Table 1. Characteristics and composition of human plasma lipoproteins. Table data derived from (13-20).

VLDL secretion answers to the overall nutritional state; thus during the

postprandial state insulin inhibits VLDL secretion. In accordance, during the

fasting state adipose tissue derived FA is carried to the liver by albumin and

reconstituted into VLDL for delivery to extra hepatic tissues. There is also a

strong correlation between cytosolic lipid droplets and VLDL production

(24) as it is for insulin resistance (25).

17

Although the nascent VLDL is big and rich in TG it is not comparable to the

huge chylomicrons (Table 1). Once secreted the TG-rich VLDL particle

undergoes lipolysis, predominantly via LPL at the endothelial sites of

capillaries (9). FA are delivered to the underlying tissue and as the TG

content is hydrolyzed and released the VLDL particle changes lipoprotein

class, a process involving compositional changes in both the protein fraction

and the lipid fraction (Table 1.). VLDL can also undergo clearance by

receptors, e.g. by the very low-density lipoprotein receptor (VLDLr) (26), but

also by other receptors as described under the receptor sub chapter (Figure

2).

Figure 2 Overview of lipoprotein metabolism. Referred to in the text at several places where also all abbreviations are explained. In short, the endogenous and exogenous pathways are similar. TG-rich lipoproteins are lipolysed in capillaries making them more accessible for receptor uptake in the liver and to a minor degree in extrahepatic tissue. Newly synthesized and from recycled apoA-I collects PL via PLTP. The nascent HDL collects cholesterol from peripheral tissue which is delivered to the liver via SB-BI for secretion into the bile in a pathway called reverse cholesterol transport. PL; phospholipids, SM; skeletal muscle, AT; adipose tissue, CM; cardiac myocytes, CE; cholesteryl ester, FA; fatty acid, TG; triacylglycerol, HL; hepatic lipase, LPL; lipoprotein lipase, EL; endothelial lipase.

As the VLDL particle becomes hydrolyzed it gradually changes to an

intermediate density lipoprotein (IDL). Except for the partial loss of its TG

core the IDL might exchange apolipoproteins with e.g. HDL making it more

susceptible for liver-derived receptor clearance. By gaining apoEs and losing

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apoCs the IDL particle becomes more prone to be cleared via the liver (27).

In fact, 50 % of all IDLs are cleared by the liver (11) in a rather quick process

(Table 1.).

The remaining IDL get further hydrolyzed leaving mostly cholesterol esters

in the core and a surface enriched in free cholesterol. Thus the particle has

become a low-density lipoprotein (LDL). The LDL particle has a long

residence time due to the loss of apoE and it only carries one apoB100

molecule (28). If not cleared via the liver by the LDL-receptor (LDLr) it

delivers cholesterol to extra-hepatic tissues via the same receptor (29).

High-density lipoprotein (HDL) is mainly synthesized and secreted by the

liver and the intestine (Figure 2). The main function for HDL is in reverse

cholesterol transport i.e. the transport of peripheral cholesterol to the liver

for excretion as bile acids or neutral sterols, or for secretion as part of newly

synthesized lipoproteins (reviewed in (30).

HDL assembly primarily occurs through an ATP binding cassette transporter

A1 (ABCA1)-dependent mechanism where apoA-I is lipidated with cellular

lipids forming preβ-HDL (31) (Figure 3). ABCA1 is critical for HDL

formation, as seen in Tangiers disease and familial HDL deficiency (32),

although alternative pathways do exist as suggested by Kiss et al (31). Preβ-

HDL formation is also intimately linked to LPL activity and TG metabolism

(Figure 3). During LPL-dependent lipolysis of chylomicrons and VLDL,

lipids and apolipoproteins are shed to make part of preβ-HDL formation (33,

34). Preβ-HDL is then further loaded with cholesterol and phospholipids via

ABCA1 in extrahepatic tissues. Lecithin-cholesterol acyltransferase (LCAT)

present on the preβ-HDL particle esterifies the free cholesterol, sending it to

the particle core (Figure 3). During this process the particle changes to a

mature HDL particle. This particle can acquire other apolipoproteins than

apoAs, predominately apoCs and apoE. Thus HDL is a circulating pool of

apos for TG-rich lipoproteins. As seen in table 1 the protein content is high in

HDL.

On interactions with other lipoprotein classes HDL can be modulated via

phospholipid transfer protein (PLTP). As the name implies PLTP is

responsible for moving excess phospholipids from mainly chylomicrons and

VLDL to HDL (35). Mature HDL can also exchange cholesteryl esters with

apoB containing lipoproteins via Cholesteryl ester transfer protein (CETP).

This process enriches the HDL particle with TG and increases its turnover

rate (36).The turnover is driven by HDL lipid hydrolysis through EL activity

and hepatic lipase (HL) activity or by direct uptake of HDL cholesteryl esters

via the scavenger receptor-class B type I (SR-BI) (37-39). Processed HDL

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may reenter the HDL life cycle as lipid poor apoA-I disks or preβ-HDL-like

particles but can also be cleared from circulation via the liver or kidneys

(30).

Figure 3 Lipoprotein metabolism in extrahepatic tissues. Summary of events in e.g. adipose tissue where LPL is synthesized in parenchymal cells (adipocytes) and transported to the luminal side of the capillary endothelium where lipolysis of chylomicrons and VLDL occurs. This thesis suggests that ANGPTL inactivates LPL predominantly in the subendothelial space. GPIHBP1 and TG-rich lipoproteins protect LPL from inactivation by ANGPTLs. ApoC-I and C-III inhibits both lipolytic and receptor mediated events. All processes and abbreviations are explained in more detail in the text. ANG4; ANGPTL 4, RME; receptor-mediated endocytosis, mLPL; monomeric inactive LPL, -; negative charges of HSPG.

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Apolipoproteins involved in lipoprotein metabolism

Apolipoproteins are divided into exchangeable and non-exchangeable

apolipoproteins. Apolipoproteins such as apoA-I, -II, -IV, -V, apoC-I, -II, -III

and apoE are known as exchangeable due to their ability to move between

lipoproteins. They reside on almost every lipoprotein class with a few

exceptions and individual preferences (Table 2). In contrast to the

exchangeable apos the two apoB isoforms, apoB100 and apoB48, are

associated with the original particle until it is cleared from circulation.

MW (kDa)

Major component

Minor component

Function

ApoA-I 28.4 HDL Chylos, VLDL LCAT activator, ABCA1 and SR-BI interaction

ApoA-II 2 x 8.7 HDL Chylos, VLDL HDL remodeling, proatherogenic

ApoA-IV 46 Chylos, plasma HDL HDL remodeling, antiatherogenic

ApoA-V 39 HDL Chylos, VLDL TG metabolism activator

ApoB48 245 Chylos - Structural protein and HSPG binding

ApoB100 514 VLDL, IDL, LDL

- Structural protein, LDLr ligand, HSPG ligand

ApoC-I 6.6 Chylos, VLDL HDL LPL and receptor uptake inhibitor, CETP inhibitor

ApoC-II 8.9 Chylos, VLDL HDL, IDL LPL activator

ApoC-III 8.9 Chylos, VLDL HDL LPL and receptor uptake inhibitor

ApoE 35 IDL, (remnants)

VLDL, HDL LDLr family ligand and HSPG ligand

Table 2. Characteristics and function of human plasma apolipoproteins.

21

Apolipoprotein A

The apoA family consists of four members where apoA-I is the most

abundant one in circulation. ApoA-I is intimately associated with HDL

particles and is required for HDL formation. Thus it also plays an important

role in reverse cholesterol transport. On a molecular level apoA-I interacts

with SR-BI (39), ABCA1 (40) and most importantly activates LCAT (41) (see

Table 2). Transgenic mice overexpressing APOA1 provide evidence that

apoA-I is atheroprotective (42). In humans deficient for apoA-I, HDL

deficiency was recorded together with xanthomas and severe premature

coronary artery disease (43). On the other hand apoA-IMilano carriers, where

apoA-I forms disulfide-linked homo and heterodimers with apoA-II, show

low HDL cholesterol levels and still a decreased risk of cardiovascular

disease (44, 45). This is supported by animal experiments; infusion of apoA-

IMilano could improve fatty streaks in rabbits (44) and has been reported to

reverse atherosclerotic plaques in clinical trials (46).

ApoA-II is the second big component of HDL. It forms a homodimer via a

disulfide link (47). It has the ability to displace apoA-I from HDL, thus it is

considered proatherogenic (48). Moreover apoA-II has been implicated as an

activator of HL (49). ApoA-II has also been proposed to interact with lipid

transfer and modulating enzymes, e.g. CETP, PLTP and LCAT, as well as

with other factors affecting HDL structure-function and metabolism as

reviewed by Blanco-Vaca et al. (50). The evidence for apoA-II’s mechanism

and even role in vivo are week and inconsistent. A recent study by Julve et al.

implicated apoA-II in TG metabolism in both mice and men and suggested

that the effect seen was due to an LPL activity regulation due to HDL

remodeling and apolipoprotein displacement (51).

ApoA-IV was discovered in 1977 as a component in rat plasma (52). It is

expressed in the intestine and is secreted with lymph as a part of

chylomicrons. In plasma it is during fasting present as lipid poor particles

free of other apolipoproteins (53). As for apoA-II a physiological function has

not yet been determined. ApoA-IV has been reported to have a function in

feeding behavior, as a satiety factor and in intestinal lipid absorption

(54,55). Experiments with knockout animals have not supported these

findings (56). Studies on enzyme activity have shown an effect on LCAT, LPL

and CETP suggesting that apoA-IV has antiatherogenic properties (57-59).

Studies on humans support the role of apoA-IV as an antiatherogenic

protein. In men with coronary artery disease apoA-IV levels were found to

correlate independently with the disease (60). Mice transgenic for hAPOA4

22

on an apoE null background were protected against atherosclerotic lesions,

possible due to an antioxidative function of apoA-IV (61).

Apolipoprotein B

The human apoB gene gives rise to two distinct gene products found in

circulation, apoB48 and apoB100 (Figure 2). Human liver secretes the full

length gene product apoB100, an important structural protein required in

assembly and secretion of VLDL particles (21, 62). ApoB100 receives lipids

from the microsomal transfer protein (MTP) and is subsequently merged

with apoB100 free lipid droplets forming nascent VLDL particles.

ApoB100 is associated with VLDL particles and stays on as the particle

changes lipoprotein classes through IDL to LDL. Except for having an

important structural role, ApoB100 also binds negatively charged heparan

sulfate proteoglycans (HSPG) (63, 64) and the LDLr (65) via basic amino

acids in two different regions. Binding region B has mostly been implicated

in heparin interaction (Figure 3) whereas binding region A was shown to

bind the LDLr with increased affinity upon cholesterol enrichment of apoB-

containing LDL particles (65).

ApoB48 is synthesized in the intestine and has a crucial role in chylomicron

assembly. Only 48 % of the full gene product is expressed in the intestine,

hence apoB48. This is a due to mRNA editing which introduces a stop codon

in the full length mRNA (66). Like apoB100, ApoB48 possesses the ability to

bind HSPG (67) which is believed to be a requirement for functional lipase-

dependent hydrolysis in vivo (9) but lacks the ability to interact with LDL

family receptors. ApoB48 containing lipoproteins, however, contains apoE

which fulfills this function instead.

There has also been reports of expression of apoB100 in the intestine,

however the physiological role of this uncertain (68, 69). Since animal

models are essential in the study of lipoprotein metabolism it should be

noted that apoB48 is expressed in the liver of mice and rats.

Apolipoprotein C

The apoC protein family consists of three members C-I, C-II and C-III which

are similar in size (<10 kDa) and have a similar distribution among

lipoprotein classes (Table 2). The three apoCs are present on chylomicrons,

VLDL and HDL and are easily transferred between lipoproteins.

23

In TG metabolism apoC-II is absolutely central as an activator of LPL (70).

As chylomicrons enter the bloodstream via the lymphatic system apoC-II

immediately relocates from HDL to chylomicrons and subsequently starts

the LPL-dependent lipolytic process (Figure 3). ApoC-II is also expressed in

the intestine thus present on chylomicrons even before entering the blood

stream, although the expression level in the intestine compared to the liver is

more than 10-fold lower (71). The necessity of apoC-II for TG metabolism is

clearly shown by hypertriglyceridemia (HTG) in humans deficient in apoC-II

(72). Interestingly, apoC-II is only needed in minute levels since

overexpression of hAPOC2 in mice leads to HTG through delayed clearance

(73, 74).

The N-terminal part of apoC-II is necessary for lipid binding whereas the C-

terminal part, especially seven highly, among different species, conserved

amino acids activates LPL (75). In vivo and when using lipoproteins as

substrates in vitro both parts of apoC-II are needed for LPL activation (76).

ApoC-III is well studied and has been reported to inhibit lipoprotein

metabolism through impairment of LPL activity and receptor mediated

uptake (Figure 3). ApoC-III inhibits uptake via the important LDL-receptor

(LDLR) family through displacement of apoE from lipoproteins (77). A

similar displacement of apoC-II has been reported as a reason for its LPL

inhibiting mechanism (78). In accordance apoC-III transgenic mice displays

an increase of remnant and TG-rich lipoproteins in plasma (79).

In humans, apoC-III is the most abundant apolipoprotein. Genetic variation

in APOC3 has been linked to postprandial lipid metabolism. Moreover,

APOC3 deficiency is linked to a favorable plasma lipid profile and protects

against cardiovascular disease (80). ApoC-III’s structure has been solved

with NMR and displays 6 amphipatic helices with several highly conserved

motifs exposed towards the water surface suggesting possible interaction

sites for apoC-III’s mechanisms (81).

ApoC-I is present in lower plasma levels than both apoC-II and -CIII and is

functionally very similar to apoC-III (Figure 3). It has been implicated in

inhibition of receptor binding of VLDL (82); as an inhibitor of cholesteryl

ester transfer protein (CETP) (83); as an inhibitor, although less potent than

apoC-III, of LPL (84). Mice overexpressing apoC-I got HTG independently of

apoC-III, VLDLr, LDLr and low density lipoprotein receptor-related protein

1 (LRP1), suggesting a direct effect of apoC-I on TG metabolism (85). ApoC-I

has also recently been implicated in innate immunity (86).

24

Apolipoprotein E

ApoE is the most widely studied apolipoprotein and involved in many

physiological events and pathological conditions. ApoE was found as a

component of TG-rich lipoproteins 40 years ago and was subsequently

linked to lipoprotein metabolism. Naturally, apoE has been found to be an

important player in the development of CVD but has also been shown to

affect Alzheimer’s disease and to be involved in immunological diseases.

ApoE is predominantly expressed in the liver but also in other tissues, in

contrast to most other apos, such as skin, brain, tissue macrophages, adipose

tissue (87, 88).

ApoE is associated with VLDL which acquire the protein upon synthesis in

the liver and with chylomicrons which acquire apoE from HDL. As

lipoproteins are subject to lipolysis by lipases apoE will stay on chylomicron

remnant particles and IDL particles where it will have a major role in their

metabolism. ApoE mainly affects the receptor mediated clearance of TG-rich

lipoproteins (11, 89) and binds all members of the LDLR gene family (90).

ApoE has also a role in lipolysis; apoE inhibits LPL-mediated lipolysis

according to some publications (91, 92) but has also been suggested to

facilitate lipoprotein binding at the endothelium through its HSPG binding

capacity; as evidence a difference between apoE isoforms with respect to

LPL-dependent lipolysis has been observed (93). Moreover, apoE positively

affects lipid uptake through endocytic and LPL-mediated pathways in vitro

and in vivo (94). Ji et al. proposed the classical secretion capture pathway

where secreted apoE subsequently enrich TG-rich lipoproteins leading to

increased binding via HSPG (95). Interestingly, apoE can be recycled after

internalization and was associated with both newly synthesized and

exogenous lipoproteins in vivo (96).

The N-terminal structure of apoE was solved in 1991 showing that apoE

forms a four bundle helix (97). The amphipathic region residues 136-150 has

been shown to be important in lipid binding, receptor recognition and

heparin interaction (98, 99). The two latter interactions are due to this

region’s positively charged amino acids. In fact, this region can by itself

stimulate lipoprotein binding and uptake both in vivo and in vitro (100).

ApoE knockout mice are one of the most frequently used animal models for

studies of atherosclerosis. Since apoE is important in lipoprotein clearance

via receptors, these mice have a disturbed plasma lipid profile characterized

by abnormal cholesterol levels depending on diet, but even on standard chow

they develop atherosclerosis (101).

25

In humans, ApoE exists in three common isoforms named apoE2, apoE3 and

apoE4. These isoforms have different properties in lipid binding, HSPG

interaction and receptor binding due to two amino acid exchanges (87).

Thus, the different isoforms affects both lipid levels, CVD risk and other

pathological conditions (reviewed in (87)).

26

METABOLISM OF LIPOPROTEINS

Lipoproteins are subject to modulating enzymes responsible for transfer of

lipids, such as LCAT, CETP and PLTP, and catabolic enzymes responsible for

hydrolysis of lipids i.e. lipases (Figure 3). Moreover changes in lipoprotein

constitution and particle concentration affect exchangeable apolipoproteins

and their ability to move between lipoprotein particles. The movement of

apolipoproteins is extremely important since this regulates the metabolic

fate of the single lipoprotein. Holoparticle uptake of lipoproteins frequently

plays a final, and often very important, part in lipoprotein metabolism. This

is illustrated by pathological conditions seen in defective lipoprotein uptake

(29).

Modulating enzymes

LCAT binds to nascent HDL and is essential for the formation of mature

HDL. Since cholesterol loading of HDL consists of unesterified cholesterol,

LCAT plays an important role in esterification of surface bound cholesterol.

Upon transfer of the 2-acyl group from lecithin to cholesterol cholesteryl

ester is generated (102). Cholesteryl esters then move into the hydrophobic

HDL core. This process increases the size of the HDL particle and is part of

the HDL maturation. LCAT deficiency is associated with reduced HDL-

cholesterol (103). Since unesterified cholesterol can be directly transferred to

the liver and secreted in bile, LCAT’s role and importance for reverse

cholesterol transport has been questioned (104). Loss of LCAT function does

not always include an impaired reverse cholesterol transport.

CETP is a 74 kDa hydrophobic glycoprotein expressed in adipose tissue and

liver which circulates bound to lipoproteins (105). Expression is regulated

by, among other things, the cholesterol dependent nuclear receptor liver X

receptor (LXR) (106). CETP is responsible for transfer of lipoprotein core

contents i.e. TG and cholesteryl esters between HDL and apoB-containing

lipoproteins. The consequence of CETP action is smaller, more TG-rich HDL.

CETP prefers to transfer lipids between TG-rich lipoproteins and HDL but

not LDL (107). Inazu et al. showed that CETP deficient patients have

excessive HDL-cholesterol levels (108). The opposite is seen in hCETP-

transgenic mice (109). The role of CETP in pathological conditions has not

been fully understood thus the role of CETP inhibitors in HDL raising

therapy can be questioned. As a consequence CETP inhibitors have shown

diverging results in human clinical trials and in different animal models

when it comes to reducing atherosclerosis and its end points (reviewed in

(30, 110)).

27

PLTP is a glycoprotein expressed in adipose tissue responsible for transfer of

phospholipids from TG-rich lipoproteins to HDL as lipolysis via LPL occurs

(33, 35). HDL can through PLTP activity be remodeled by particle fusion

(35). PLTP knockout mice displays 60 % lower HDL phospholipid levels

because of enhanced clearance via liver and kidney (111). The

pathophysiological role is not yet elucidated but PLTP has been implicated in

obesity, type II diabetes and cardiovascular disease (reviewed in (112)).

Lipases

TG is a biologically inert substance that forms dense water free droplets.

Thus, when large amounts of lipids are stored or transported this is the

format chosen. The downside of using TG is when transport over biological

membranes is needed. For this the TG ester bonds needs to be hydrolyzed in

order to get more easily transported fatty acids or monoglycerides. Therefore

lipases are important in digestion of dietary lipids, for lipid mobilization in

adipose tissue and for transport in and out of the plasma compartment.

Extracellular lipolysis is carried out by a lipase gene family consisting of

lipoprotein lipase, hepatic lipase and endothelial lipase which differ

predominantly in substrate preference (9).

Lipoprotein lipase

LPL was originally described as a clearing factor in postprandial postheparin

plasma by Hahn (113). Further biochemical studies revealed LPL’s ability to

hydrolyze ester bonds in TG, diacylglycerol, monoacylglycerol, phospholipids

and other esters (114-116). LPL is the major lipase active in lipolysis of

plasma lipoproteins as humans and experimental animals deficient in the

LPL gene or APOC2 displays massive HTG due to abnormal lipoprotein

catabolism (72, 117, 118). LPL activity is intimately linked to its dimeric

integrity consisting of a non-covalent homodimer of two subunits arranged

in a head to tail fashion (9, 119, 120). The single subunit is a glycoprotein of

55 kDa where the N-terminal domain contains the active serine hydrolase

site responsible for TG hydrolysis and the C-terminal contains binding sites

for lipids, receptors and heparin (121).

In the in vivo situation active LPL is bound at the endothelium via HSPG

although this has been recently challenged by the discovery of GPIHBP1

which may be the most important LPL binding molecule at the endothelium

(Figure 3). Like LPL or APOC2 deficiency, lack of functional GPIHBP1 also

leads to massive chylomicronemia (122-124). From this binding site LPL

binds and hydrolyzes predominantly TG-rich lipoproteins. The role of LPL in

postprandial clearance is profound, it works as a factor in the rapid

28

chylomicron margination process, in the process of turning the chylomicron

into a remnant and in facilitating hepatic remnant internalization (10, 125-

127). The activity of LPL is ingeniously regulated depending on nutritional

status (Figure 3). Moreover, this regulation differs between different tissues.

In line with FA needs in the state of fasting, LPL activity in heart muscle and

skeletal muscle is high whereas it is low in adipose tissue and vice versa in

the fed state. The regulation is almost instantaneous as multi-fold changes

have been reported within hours in animal models (128, 129). This rapid

regulation is not fully understood, and it cannot be explained by mRNA

expression, thus it involves at least one post-transcriptional factor (130-132).

ANGPTL4 has been suggested as a candidate for the rapid regulation of LPL

activity but further evidence is needed in order to prove this hypothesis (3).

LPL is expressed in parenchymal cells. Thus there need to be a transport

mechanism to LPL’s site of action; the luminal side of the endothelium

(Figure 3). VLDLr, HSPG and GPIHBP1 have been suggested to facilitate

this transport that is incompletely understood (9).

Except for being the main TG hydrolyzing enzyme LPL also has a role in

receptor-mediated endocytosis of lipoproteins as shown by Beisiegel at al.

(127). There is a large body of evidence from in vitro and in vivo experiments

showing that LPL avidly binds members of the LDLR family, and HSPG as

well, and can facilitate lipoprotein binding to these receptors leading to an

increased lipoprotein uptake of both TG-rich lipoproteins and LDL (127, 133-

136). This interaction is independent of LPL’s catalytic activity (137). The

interaction between LPL and lipoproteins is solely dependent of LPL-lipid

interaction (138, 139). Heeren et al. proposed a model where TG-rich

lipoproteins carry LPL with them from the site of lipolysis to the liver where

hepatic uptake is facilitated (140).

LPL activity influences not only TG-rich lipoprotein metabolism, HDL

metabolism and formation is indirectly affected by LPL. CETP activity is

limited by VLDL presence and since increased LPL activity decreases VLDL-

TG levels HDL cholesterol exchange for VLDL-TG is restricted. Likewise, low

LPL activity resulting in high plasma TG levels allows HDL to be enriched in

TG making HDL easier to clear from plasma (141). Moreover, as already

mentioned in the lipoprotein sub-chapter, shedding of phospholipids and

apolipoproteins is part of nascent HDL formation (33, 34). In healthy

humans this is supported by the correlation between post-heparin LPL

activity and HDL levels in plasma (142).

There are many, more or less important, inhibitors and activators of LPL.

ApoC-II is absolutely crucial for LPL activation (70), GPIHBP1 seems to have

29

an important indirect role in LPL activity (122), the lipase maturation factor

1 (LMF1) also displays an important direct function in LPL mode of action

(143). Moreover, the ANGPTL family has been shown to have a direct effect

on LPL activity (144, 145). ApoA-V has been suggested to have an indirect

activating effect on LPL via facilitation of lipoprotein binding to HSPG (146),

similar to that suggested for apoE (93), although others have suggested an

opposite effect of apoE (91). The apoC-members -CI and -CIII have both

been shown to inhibit LPL-dependent lipolysis (147). Taken together, there

are many potential activators and inhibitors of LPL with sometimes partially

overlapping mode of actions. The complexity of the LPL system does make

studies of new factors and mechanisms challenging (Figure 3).

Hepatic lipase

HL is as the name implies associated with expression and also confinement

to the liver. It is attached to HSPG in the space of Disse and is thus

associated with hepatic parenchymal cells and sinusoidal endothelial cells

(148). As its relative LPL, HL hydrolyses TG and phospholipids of all

lipoprotein classes although HDL is the preferred substrate (34). Unlike LPL,

HL does not require apoC-II as an activator. Hydrolysis of HDL releases lipid

poor apoA-I which is needed for de novo HDL formation (148). HL has an

important role in lipoprotein metabolism as seen in patients deficient in HL

and in mice knockout models. Not only HDL levels are affected, but also TG-

rich lipoprotein classes (149). Remnant lipoproteins are further processed by

HL in the space of Disse which is an important step for hepatic uptake (see

Figure 5). Like LPL, HL also mediates uptake of lipoproteins, remnants and

LDL, via hepatic receptors such as HSPG, LRP1 and LDLr (11, 149, 150).

Endothelial lipase

EL is mainly synthesized by endothelial cells and part of the same lipase

family as LPL and HL. EL also binds sulfated glycans and is heparin

releasable but differs in substrate preference. Whereas LPL and HL

hydrolyses both TG and phospholipids EL predominantly has phospholipase

activity placing it at the other end of the lipase family compared to LPL (151).

Since HDL is the preferred substrate in vivo high EL activity is correlated

with low plasma HDL and vice versa in humans and animal models (37, 152-

154). Given its substrate preferences EL’s role in metabolism of apoB-

containing lipoprotein has been questioned although data from mice show

increased LDL phosholipid catabolism due to EL (155). In humans,

postheparin EL levels correlated with TG and apoB levels although

upregulation due to obesity was suggested as a reason (154-156).

30

RECEPTORS INVOLVED IN LIPOPROTEIN METABOLISM

Whole particle clearance is an important part of lipoprotein metabolism.

Both intestinal- and hepatic-derived lipoproteins are to a large extent cleared

through receptor mediated endocytosis by the liver (11). Central in this

process is the large LDLR gene family consisting of members that are

essential in lipoprotein clearance, among other functions. The LDLR gene

family and its relatives binds a variety of ligands and shows a significant

overlap when it comes to lipoprotein metabolism.

Receptor-ligand complexes of LDLR members and their ligands are taken up

by cells through a mechanism involving clathrin-coated pits. The lipoprotein

is released from the receptor in the acidic endosome whereas the receptor

itself recycles back to the cell surface (29). The delivered lipids may be used

for energy storage or immediate utilization.

Members of the LDLR gene family, as depicted in Figure 4, consist in most

cases of similar parts (157, 158). The LDLR class A binding (LA)

complement-type repeats are essential for ligand binding. The LA repeats

needs Ca2+ in order to bind its ligands. Solving the crystal structure revealed

that Ca2+ stabilizes the structure and promotes the correct folding (159, 160).

The LDLR gene family has a common ligand, the 39 kDa receptor associated

protein (RAP). RAP interacts with the LA repeats and work as a chaperone

for LDLR family members. As newly synthesized receptors are expressed

they risk binding of other ligands in the ER. RAP prevents this binding and

the subsequent risk of aggregation and degradation (161). The fact that RAP

is a potent inhibitor of ligand binding is well established and has been used

in many interaction studies in vivo and in vitro, e.g. paper I, paper II, (162).

Low density lipoprotein receptor

LDLr is the most widely studied LDLR family member. Discovered in 1974

and described by Goldstein and Brown, rendering them the Nobel Prize in

1985, it plays a crucial role in cholesterol homeostasis and the clearance of

apoB containing lipoproteins (29). LDLr is expressed widely in the body and

although the liver is the major organ for LDL uptake, adrenals, testis and

ovaries, being steroid-producing tissues, also has an increased LDL uptake

and LDLr presence (163). Except for apoB interaction the LDLr also interacts

with apoE. Since apoE is not a constituent of LDL this indicates that LDLr

31

also plays a role in TG-rich lipoprotein uptake, i.e. removing IDL and

chylomicron remnants (164).

Figure 4 Organization of the LDL receptor and Sortilin gene families. From T.

Willnow, reproduced by permission.

LPL facilitates uptake of LDL through LDLr and HSPG. The catalytic activity

of LPL and its bridging function was found to be responsible for this

mechanism. LPL did, however, not facilitate remnant uptake via LDLr (135).

LDLr expression is regulated by sterol regulatory element-binding proteins

(SREBP). Cellular cholesterol determines SREBP activity and when sterol

levels are low SREBP is transported from the ER lumen to the Golgi

apparatus where it is cleaved and sent for the nucleus to activate lipid

accumulation and synthesis genes (as reviewed in (165)). PSCK1 was

discovered in 2003 as a gene causing hypercholesterolemia (166). The

mature protein is involved in LDLr degradation and inhibits recycling (167).

Low density lipoprotein receptor-related protein 1

In 1988 Herz et al. discovered a huge 600 kDa receptor consisting of two

subunits of 515 kDa and 85 kDa showing homology with LDLr (168). At the

same time this receptor was recognized as the α2-macroglobulin receptor by

32

the group of J. Gliemann (169). The extracellular 515 kDa α-subunit contains

the ligand binding domain and the smaller β-subunit contains a small

extracellular part, the membrane spanning part and the cytoplasmic domain

responsible for intracellular signaling. The intracellular domain consists of

two NPxY motifs, whereas the distal motif overlaps with the YxxL motif.

These motifs have been implicated in transport and signaling function (170,

171). Gordts et al. recently showed that the intracellular distal NPxYxxL

motif is essential for normal control of LRP1 function (172).

Although already isolated as the α2-macroglobulin receptor, LRP1 was

through Herz and colleges implicated as a lipoprotein receptor which was

confirmed when apoE binding ability was shown (173). This was first thought

to be the longed for and expected remnant receptor since LDLr deficiency

had been shown not to affect remnant clearance to any higher extent in

humans or animals (174). The relationship between these two receptors is

quite complex due to overlap in function and upregulation of one receptor if

the other is not fully functional. LRP1 does not bind apoB100 or apoB48, thus

it is dependent on apoE to bind lipoproteins. This excludes LRP1 as an LDL

particle receptor since LDL only contains apoB100. Mice deficient in liver

LRP1 do not show any plasma lipid abnormalities in the fasting state due to

an effect of LDLr upregulation (175). Mice deficient in both LDLr and LRP1

in the liver, on the contrary, displays accumulation of remnant particles

(175). This is a strong indication that the receptors have overlapping

functions in vivo and that both are essential in TG-rich lipoprotein clearance

in the liver, but that there is a predominant role for LRP1 in remnant

clearance. As for LDLr, LRP1 binds LPL and HL which enhances lipoprotein

binding and endocytosis (Paper I) and has been shown to bind apoA-V (11,

127, 149, 150).

LRP1 has been shown to affect remnant uptake in bone and adipose tissue

(176, 177). In osteoblasts the remnant components are a necessity for cellular

function and in mice deficient for LRP1 in adipose tissue the fat pad mass

was reduced, possibly explained by locally impaired lipoprotein uptake. It

should be considered that adipose tissue endothelium is continuous and as

such impenetrable for large TG-rich lipoproteins unlike the fenestrated liver

and bone endothelium. The role for receptor uptake in adipose tissue is

therefore unclear (178).

Since LRP1 knockout mice are embryonic lethal, it was proposed that the

receptor has another role than being just a lipoprotein receptor. Indeed, as

reviewed by Lillis et al. (179) LRP1 has been implicated in many different

fields including neural development, immunology, cell signaling and more.

This is also displayed by the variety of ligands bound by LRP1.

33

LRP1 plays an important role in postprandial remnant clearance. This is an

insulin dependent mechanism as was elegantly shown by Laatsch et al. (12).

Insulin translocates LRP1 to the cell surface of hepatocytes and thus

increases LRP1-mediated ligand uptake. This finding suggests a partial

explanation to insulin resistant postprandial hyperlipemia.

Very low density lipoprotein receptor

VLDLr is expressed in endothelial cells in adipose tissue, heart and skeletal

muscle (Figure 2 and 3). Like its better known relatives it binds and

facilitates uptake of apoE containing lipoproteins by itself and even more so

when LPL assists lipoprotein binding by bridging (180-182). VLDLR

knockout mice displayed no significant changes in plasma lipoprotein levels

on normal diet (183), although when challenged by prolonged fasting, high

fat diet or when bred onto an already metabolically challenged background a

HTG phenotype was seen (184, 185). Goudriaan et al. show that VLDLr has

an important role in postprandial TG response (186). VLDLr has been

suggested to be involved in lipoprotein metabolism through other

mechanisms than just facilitating receptor mediated uptake. Obunike et al.

suggested that VLDLr is involved in transcytosis of LPL over endothelial

cells, a finding that supports the changes in LPL activity found by Goudriaan

et al. and others in VLDLr deficient mice (186-188).

Sortilin-related receptor, LA repeats-containing

SorLA is a 250 kDa Vps10p-domain Type-1 receptor and a relative to the

LDLR gene family (Figure 4). It consists of 11 LA repeats and one Vps10p

domain. SorLA is expressed in vascular smooth muscle cells (189-191). The

Vps10p-domain receptor family has an YxxL internalization motif and has

not been shown to recycle after endocytosis in contrast to the LDLR family

which has the NPxY internalization motif (158). SorLA was shown by Kaniki

et al. to be upregulated in atherosclerotic lesions (189) and to be involved in

formation of lipid-laden macrophages (192). SorLA binds the same ligands

associated with lipoprotein metabolism as the LDLR family members, i.e.

apoE containing lipoproteins, apoA-V and LPL, as well as RAP (Paper I)(134,

193). In transfected Human embryonic kidney 293 cells (Hek293) SorLA was

able to mediate uptake of apoA-V-dimyristoylphosphatidylcholine (-DMPC)-

disks (Paper II). Although implicated in lipoprotein metabolism, the

research concerning SorLA has mostly addressed neural function so far

(reviewed in (190)).

34

Sortilin

Sortilin is a 110 kDa Type-1 receptor of the Vps10p-domain gene family

highly expressed in neural cells, skeletal muscle and heart (194). It lacks LA-

repeats but interacts with its ligands via the Vps10p-domain (Figure 4).

Sortilin is present in a locus 1p13.3 that has been strongly connected to LDL-

cholesterol (195-197) and coronary artery disease (198). Just as SorLA,

Sortilin interacts with and mediates receptor mediated endocytosis of apoE,

LPL, RAP, apoA-V and the Sortilin specific Neurotensin polypeptide (Paper

II)(199). Recently, Linsel-Nitschke et al. showed that overexpression of

Sortilin in cells promoted cellular uptake of LDL (200). Sortilin has also

been shown to bind chylomicrons in vitro (Paper II). Although interesting

from a lipid metabolism perspective Sortilin, as SorLA, primarily has been

implicated with neural apoptosis and other neural events (190, 201);

although the physiological roles are far from fully elucidated.

SorLA and Sortilin differ from the LDLR gene family with regard to

intracellular trafficking; through their cytoplasmatic motifs they interact

with adaptor proteins GGAs, AP-1 and -2, and the retromer complexes, all

involved in endocytic and intracellular trafficking (202-204). This gives

SorLA and Sortilin ligands an unorthodox routing compared to when bound

to the LDLR gene family since they after endocytosis goes from early

endosomes followed by retrograde sorting into late endosomes and trans-

Golgi network (TGN). At the stage of late endosomes, a part of the ligand is

uncoupled and sent for degradation in the lysosome compartment (202,

203).

Heparan sulfate proteoglycans

HSPG are composed of a protein core part attached to the cell membrane

with one or several heparan sulfate glycosaminoglycan chains. These

glycosaminoglycan chains are negatively charged due to the sulfate groups

attached to them (205). HSPG are present on virtually all cell types and play

an important role in lipoprotein clearance, as depicted in figure 3 and 5.

Through the negatively charged chains HSPG interacts with apoE, apoB, LPL

and apoA-V (Paper I)(64, 93, 206). Hence HSPG mediates binding of

lipoproteins to cell surfaces which is a prerequisite for lipolytic processing,

primarily at the endothelium of capillaries, or through receptor mediated

endocytosis, primarily in the liver. HSPG plays an especially important role

in clearance of TG-rich remnant lipoproteins in the liver (207). In the

hepatic sinusoids and the space of Disse lipoproteins are processed with the

help of HSPG. Mahley and Huang suggested that remnant lipoproteins are

35

first sequestrated into the space of Disse, given that their size allows them to

enter through the endothelial fenestrations (Figure 5). After sequestration

follow additional lipolytic processing of the remnants by accompanying LPL

and/or local HL; as described above HSPG aid in this process (207). The

remnant particles are then transferred to LDLR family members (i.e. LDLr

or LRP1) for uptake (11, 140, 173, 175). The classical view has been

challenged by J. Esko’s lab who showed that liver specific reduction of Ndst1

expression in mice, reducing HSPG sulfation, leads to accumulation of both

intestinal and hepatic remnant lipoproteins. This suggests that a substantial

amount of remnants might be endocytosed directly via HSPG (208). Upon

cross-breeding Ndst1 liver knockout mice with LDLr deficient mice the LDL

accumulative effect was increased. They later showed that uronyl 2-0-

sulfation was essential for the function of HSPG in vivo and in vitro for TG-

rich lipoprotein clearance and that crossing of Ndst1 deficient mice with liver

specific LRP1 deficient mice does not cause additional dyslipidemia (209).

Nevertheless, with overlapping receptors there might still be a functional

role for both HSPG and LRP1 in particular. Further studies are needed here.

Figure 5 Processes involved in the hepatic receptor-mediated endocytosis (RME) of lipoprotein remnants. All events and abbreviations are discussed in the text. In short, lipoproteins that are small enough enter the space of Disse and binds to receptors and/or HSPG via apoE, apoBs, apoA-V, HL or LPL for subsequent receptor mediated endocytosis (RME) or further lipolytic processing. Hepatocytes secrete a number of stimulating factor enhancing this process while apoC-I or apoC-III inhibits these pathways. HDL delivers CE via SR-BI and HL to hepatocytes and is partly recycled as preβ-HDL in the pathway of reverse cholesterol transport.

36

Other receptors

There are a few more actors suggested to be involved in lipoprotein

metabolism; SR-BI facilitates selective cholesteryl ester uptake from HDL.

Moreover, SB-BI knockout mice have been shown to have elevated VLDL

levels and delayed VLDL clearance (210). By using mice deficient in LDLr,

VLDLr and LRP1 Hu et al. showed that inhibition of SR-BI impaired VLDL-

like emulsion clearance in vivo (211). Cluster designation 36 (CD36)

deficient mice were shown to have somewhat impaired chylomicron-like

emulsion clearance (212). Scavenger receptor class A (SR-A) is together with

CD36 responsible for uptake of modified lipoproteins and are therefore

central in foam cell formation and atherosclerosis. An LDLR family member

expressed in liver, LRP5 deficient mice showed delayed postprandial TG

clearance and displayed progressed atherosclerosis on an apoE null

background (213); the apoB48-receptor (apoB48R), primarily expressed by

reticuloendothelial cells, e.g. macrophages and endothelial cells, was found

to be involved in uptake of TG-rich lipoproteins in vitro and promote foam

cell formation (214, 215); the controversial lipolysis stimulated lipoprotein

receptor (LSR) discovered and thoroughly investigated by Bihain and

coworkers (216-223), and more recently by an independent group (Narvekar

et al. (224)) has been reported to have a significant effect in plasma TG

metabolism. LSR appears to have the classical traits of a lipoprotein receptor

binding apoB and apoE containing lipoproteins as well as RAP and being

inhibited by apoC-III. Adenoviral overexpression of LSR in apoE null and

ob/ob mice displayed decreased serum TG levels, whereas gene silencing did

the opposite in wild type mice (224). In LSR-/+ mice similar traits where

seen, with a predominate impairment in postprandial lipoprotein clearance

resulting in a more pronounced atherosclerotic progression under western

diet (223).

37

LIPOPROTEINS IN DISEASE

Lipoproteins are implicated in a variety of pathological conditions, both as a

consequence of disease and as a cause for disease; in some conditions

plasma lipid levels have been suggested to be both.

Hyperlipidemia and especially HTG has been found to affect renal disease

development and progression in animal studies. Glomerular and mesengial

cells express a wide battery of genes involved in lipoprotein metabolism such

as LPL and different lipoprotein receptors. Moreover oxidized LDL (oxLDL)

has been suggested to affect renal disease through toxic and pro-

inflammatory effects, e.g. through foam cell formation. Thus, lipoprotein

retention and lipotoxicity/lipid injury might have a role in

glomerulosclerosis. In humans the causal links are weaker and a potential

link to lipoprotein levels is not clearly established (225).

HTG in pancreatitis is a well established link between cause and disease

counting for up to 10 % of all acute pancreatitis (AP) cases and the majority

of cases of pregnancy induced AP (226, 227). Patients display a severe HTG

where plasma TG levels above 1000 mg/dl are considered to trigger AP. The

mechanism that HTG triggers leading to AP is not known. The most common

theory suggests that pancreatic lipase causes local hydrolysis of plasma TG

creating a FA overload in the pancreas giving lipotoxicity-related injury on

endothelial cells and on acinar cells. This leads to ischemia and

inflammatory response creating AP (226, 227).

Lipoproteins play their most profound role in metabolic diseases. In the

definition for the so called metabolic syndrome, two lipoprotein parameters

are recognized as independent criteria by all relevant organizations namely

HTG and low HDL-cholesterol, although exact values for each criteria differ.

The metabolic syndrome defines a set of disorders increasing the risk for

CVD and type II diabetes mellitus (T2D).

Although intimately connected to the metabolic syndrome the role of lipid

metabolism in the development of T2D remains unclear; it has, however,

been postulated that lipotoxicity has a major role in the pathogenesis (228).

Elevated delivery of FA to sensitive tissues has been suggested to increase

oxidative stress, although this mechanistic view has been challenged (229,

230), leading to β-cell failure and impaired insulin resistance in peripheral

tissue. It is however widely accepted that FA become toxic at chronically

elevated levels, inducing β-cell apoptosis (228). FA also inhibits gene

expression of insulin and insulin secretion mechanisms.

38

The mechanism of lipotoxicity is not fully understood but there are two

major hypothesis; 1) increased cellular intake and oxidation of FA leads to

decrease in glucose oxidation, chronic oxidative stress and impaired β-cell

function; 2) metabolites, especially long-chain fatty acyl CoAs, in the

esterification pathway of FA are proposed to be harmful when they

accumulate (228).

The role of lipoproteins in diabetes is however also unclear; using

compartmental modeling in rats it was shown by Teusink et al. that FA

uptake in heart derived from plasma lipoprotein TG mediated by LPL

hydrolysis can potentially be greater than FA released from adipose tissue

(231). Especially in the postprandial state dietary lipids are preferred as FA

substrate as shown in human studies (232). It is plausible that an increased

FA pressure plays a role in T2D pathogenesis, and even if not so, diabetes

strongly affects lipoprotein homeostasis resulting in VLDL overproduction,

impaired receptor mediated endocytosis and LPL-dependent hydrolysis

which all contribute to the increased risk of micro- and macrovascular events

in diabetic patients (12, 233-235).

When it comes to micro - and macrovasular (MAM) events lipoproteins are

a major risk factor. High LDL-cholesterol, low HDL-cholesterol and elevated

TG in plasma are all independent risk factors for the development of MAM

(235). Within MAM several diseases are accounted for; microvascular

disease complications include retinopathy, nephropathy, neuropathy;

macrovascular diease includes coronary heart disease, cerebrovascular

disease and peripheral vascular disease. Common for MAM diseases is that

they are manifestations of atherosclerosis as an underlying factor.

Atherosclerosis

Atherosclerosis is characterized by lesions (plaques) in the intima of the

arteries (Figure 6). These plaques consist of lipids, inflammatory cells,

connective tissue and debris from apoptotic or necrotic cells (236, 237). As

plaques progress they accumulate lipids and immune cells resulting in lipid-

laden macrophages (foam cells). Mature plaques or atheromas have a core

consisting of foam cells and extracellular lipids. This region is surrounded by

a smooth muscle cell cap and extensive extracellular matrix. Although

potentially causing stenosis, rupture of a plaque is the most severe clinical

event (236, 237).

The pathogenesis of atherosclerosis is multifactorial although inflammation

often is highlighted as central in the development (236). Low grade or

chronic inflammation in metabolic disorders, reviewed by Hotamisligil

39

(238), is commonly found and believed to be a key feature in metabolic

disease and thought to be regulated in part by lipids. Extensive reports on

the role of inflammation and the immune system in atherosclerosis

development has been published by among others Hansson and Libby (236,

237, 239).

With regard to this thesis, the proposed first step in development of

atherosclerosis involves accumulation of lipoproteins resulting in an

inflammatory response.

Lipoproteins in atherosclerosis

LDL and TG-rich lipoproteins are independent risk factors in the

pathogenesis of atherosclerosis. Contrary, HDL has a protective effect on the

development of disease. The role of plasma lipid levels is very well studied in

both humans and animal models. Considering the early onset of

atherosclerotic disease in humans with familial dysfunctions in lipoprotein

metabolism, e.g. LDLr deficiency as investigated by Brown and Goldstein

(29), the results from massive population studies and genome wide

associations (195-198, 200), and supported by the many animal models used

in atherosclerotic research, e.g. apoE null mice, LDLr knockouts, high fat

diet mice, it is clear that lipoproteins are central in the pathogenesis of

atherosclerosis. Thus statin treatment, affecting predominantly LDL, has

been the single most important clinical method in atherosclerotic related

disease management since it was established 1984 in clinical use by the first

large randomized double blinded primary intervention trial NIH Coronary

Primary Prevention (240, 241). Other statin drugs has since then proven

much more efficient in treating MAM. However, it has lately been

established that even though treatment goals are reached for LDL-

cholesterol, blood pressure and glycemic control there is a considerable

residual risk for MAM disease for many patients, likely due to high plasma

TG levels and low HDL-cholesterol (235). Hence, there is focus on

understanding the mechanisms involved in HDL and TG homeostasis and

the effect they have on atherosclerosis development.

40

Figure 6 Factors involved in development of atherosclerotic plaques in artery walls. All processes and abbreviations are discussed in the text. In short, LDL is retained in the intima and oxidized. OxLDL affect endothelial cells to express VCAM-1 which stimulates monocyte adhesion. OxLDL are also taken up via macrophage scavenger receptors. Lipid overload increases the inflammatory response in macrophages stimulating SMC migration, build-up of a fibrous cap and other inflammatory events. If the reverse cholesterol transport is inefficient, foam cells are formed. These cells finally go into apoptosis or necrosis forming a necrotic cholesterol rich core which may rupture causing thrombosis. SMC; smooth muscle cells.

The role of LDL in atherosclerosis is probably one of the most studied

mechanisms in any disease (Figure 6). High levels of LDL increase the risk,

but are not a prerequisite, for atherosclerosis. Arterial migration of LDL into

the intima of the vessel wall is considered the first step of atherosclerosis.

Here LDL is retained by interaction with HSPG. Borén and coworkers

showed in transgenic mice, expressing apoB unable to bind HSPG, that

retention is crucial for plaque formation, as these mice are being protected

from atherosclerotic lesion formation (242). During retention LDL is

modified through oxidation or by enzymatic activity forming oxLDL. This

leads to activation of endothelial cells possibly by oxidized phospholipids or

other proatherogenic or inflammatory stimuli, and the oxidation also

promotes the efficiency of uptake by macrophages (243, 244). Endothelial

cells express several adhesion molecules, where vascular cell adhesion

molecule-1 (VCAM-1) is the most important, by which monocytes and T-cells

41

are engaged (245). When monocytes adhere and migrate through the

endothelium they differentiate to macrophages upon exposure to

macrophage colony-stimulating factor which is expressed by the

inflammation affected intima. (246). The macrophages readily express

scavenger receptors, e.g. scavenger receptor-class type A (SR-A) and CD36,

which are responsible for oxLDL uptake. This leads to secretion of

inflammatory cytokines, proteases, and free radicals by the macrophages;

oxLDL also induces foam cell formation, which is not seen upon native LDL

uptake (236, 244). If cholesterol efflux is not sufficient from the

macrophages they ultimately form foam cells accelerating the inflammatory

response further. As a final mode of action the foam cells become necrotic

spilling their lipid content with cellular debris which forms the necrotic core

(Figure 6). This affects both the lesion stability and factors that, upon

rupture, will affect the coagulation cascade (236, 237, 239, 245, 247).

The increased inflammatory stimuli lead to proliferation and migration of

smooth muscle cells (SMC) from the vessel wall media to the intima and

endothelial cells. This has a protective effect since the process encircles the

content of the lesion. SMC produce collagen and other components of the

extracellular matrix thus form a fibrous cap over the lesion. In correlation

with the size of the necrotic core being more prone to rupture, a thicker cap

will stabilize the plaque. In coherence the balance between foam cell necrosis

and extracellular matrix breakdown, and SMC proliferation and extracellular

matrix buildup, determines the stability of an atherosclerotic plaque (236,

237, 239, 245, 247).

The role of HDL in the development of atherosclerosis is protective (30,

248). In fact, an increase in HDL or apoA-I levels can reverse plaque

development (44, 46). This is believed to depend on HDL’s capacity to do

reverse cholesterol transport as previously described. HDL or lipid-poor

apoA-I could work as an acceptor of cholesterol from foam cells, a process

believed to be driven by cholesteryl ester hydrolase, ABCA1 and ABCG1 (249,

250). HDL decreases the state of inflammation through inhibition of VCAM-

1 and interleukins, and also through antiapoptotic interaction with

endothelial cells (251). HDL is also associated with an antioxidative effect.

This counteracts the oxLDL mechanism as native LDL does not induce lipid-

laden foam cell macrophages (244).

HTG has been associated with the development of CVD for almost 30 years

(252). A role for TG-rich lipoproteins in atherosclerosis was proposed by

Zilversmit 1979 in ”Atherogenesis: a postprandial phenomenon” suggesting

chylomicrons or chylomicron remnants stimulate atherosclerosis

development (253). Since then, and especially during the last decade, it has

42

been established through huge epidemiological studies that plasma TG

levels, and even more clearly nonfasting TG levels, are strongly associated

with an increased CVD risk (235, 254-258). This is further supported by

premature CVD seen in familial type III hyperlipoproteinemia which is

characterized by impaired clearance of remnant particles (259). Karpe et al.

showed a relationship between remnant lipoproteins and carotoid intima-

media thickness (260). Moreover, remnant particles have been isolated from

atherosclerotic plaques in humans (261) and been seen to accumulate in the

intima of rabbits on cholesterol rich diet (262). However, as pointed out by

Stalenhoef and Graaf, severe HTG does not necessarily lead to premature

CVD (263, 264). They therefore suggest an upper threshold of TG

contribution to CVD risk. This could be due to the mere size of the

lipoproteins in familial HTG patients, since it is considered that lipoprotein

migration into the intima is limited to rather small lipoproteins, at least

when the endothelium is intact. It has also been reported that the relative

risk contribution of plasma TG levels is limited, about 2 times higher hazard

ratio was found comparing the highest and the lowest TG level groups (264).

The mechanism by which remnant lipoproteins exert their atherogenic effect

is not fully elucidated. It is however believed that remnant particles enter the

intima and are retained, as LDL, with subsequent receptor mediated uptake

in macrophages (Figure 6). However, remnants do not seem to need

oxidation to induce foam cell formation (244). In mouse macrophages 40 %

of chylomicron remnant uptake was due to the LDLr, at least 20 % was likely

due to LRP1 and the rest was unaccounted for (265). The apoB48R could

possibly explain the unknown uptake; interestingly when silenced in THP-1

cells, remnant dependent foam cell formation was inhibited (214, 266).

Except for inducing foam cell formation, remnant particles can also induce

inflammatory response in macrophages, further stimulating atherosclerotic

progress (267). Tanaka reviews a set of mechanisms implicating remnant

particles’ atherogenic properties; promoted platelet aggregation, increased

plasminogen activator inhibitor-1 activity, impaired endothelial function,

promoted proliferation of vascular SMC and promoting adhesion of

monocytes to endothelial cells (268).

43

APOLIPOPROTEIN A-V

ApoA-V was first described in 2001 as a result of comparative genome

sequencing between mouse and man, and by a separate group described as a

gene upregulated on liver regeneration (1, 2). Located on chromosome 11q23

and present in the APOA1-C3-A4 gene cluster APOA5 got its name from

homology with APOA4. Although the first impressive publication by

Pennacchio et al. (1) presented apoA-V as an important factor in plasma TG

metabolism the mechanism is still not clearly understood. Compared to

other apolipoproteins, apoA-V is present in human plasma in extremely low

concentrations with observations ranging from 24 to 406 ng/ml (19). On a

molar basis this is approximately a 1000-fold lower than the concentration

of apoB100, meaning that only one in 24 VLDL particles carries an apoA-V

molecule (269). In plasma apoA-V is found mainly as monomers distributed

on chylomicrons, VLDL and HDL (270). ApoA-V is positively correlated with

plasma TG levels in humans and in wild type mice. In contrast hAPOA5

transgenic mice show an inverse relationship (271). However, hAPOA5

transgenic mice lacking the murine APOA5 gene showed a positive

correlation with plasma TG levels, similar to humans (272). This is an

intriguing and yet unexplained finding which could suggest that apoA-V’s

action does not occur in the plasma compartment. Nevertheless, it has been

shown in humans over a large number of ethnicities that variation in the

APOA5 gene locus is associated with plasma TG levels (273-277).

Furthermore apoA-V deficiency in humans is related to severe HTG (278-

281).

ApoA-V in disease

Elevated TG is an independent risk factor for cardiovascular disease (235).

Therefore, apoA-V has been implicated in several pathological conditions

involving lipid metabolism and disorders of energy homeostasis e.g. in HTG

pancreatitis (282); coronary atherosclerosis (283-285); aortic

atherosclerosis (286); carotid atherosclerosis (287); diabetes mellitus type 2

(288); metabolic syndrome (287, 289); end stage renal disease (290) and

obesity (291).

In mouse models, apoA-V overexpression did not affect body weight gain or

glucose homeostasis which was hypothesized due to APOA5’s apparent link

to traits of the metabolic syndrome (292). An expected finding was that

overexpression of apoA-V had a positive effect on atherosclerosis

development. Both on an apoE-null background (293) and on an apoE2

knock-in background (294) there was a clear effect on atherosclerotic lesion

44

development and a more atheroprotective plasma lipid profile. Moreover,

transgenic mice expressing hAPOA5 crossed on an LDLr deficient

background got 50 % less aortic lesions compared to controls while fed a

high fat diet (295). These experiments show that lowering of plasma TG by

apoA-V has a significant role in protection against atherosclerosis in these

animal models.

Expression patterns and regulation

ApoA5 is mostly expressed in the liver (1, 2). Several nuclear receptors have

been implicated in upregulation of APOA5 expression including; peroxisome

proliforator-activated receptor α (PPARα) (296, 297); farnesoid X-activated

receptor (FXR) (297); retinoid acid receptor-related orphan receptor α

(RORα) (298); hepatocyte nuclear factor 4α (HNF4α) (299); and thyroid

receptor β (TRβ) (300). In contrast, LXR (301), insulin (302) and glucose via

the upstream stimulatory factor (USF)1/2 (303) have all been found to

downregulate APOA5 expression. Thus regulation in the liver occurs through

a number of different factors intimately linked to energy homeostasis.

Recently it has been reported that nuclear receptor Nur77 binds to a

response element in the human APOA5 promoter region (304) giving a role

in energy metabolism for this orphan receptor.

Structure and molecular mechanisms

APOA5 encodes a 366 amino acid protein in humans (1). The mature apoA-V

contains 343 amino acid residues and lacks the 23 amino acids signal

peptide (Figure 7). ApoA-V is a hydrophobic protein consisting of a large

amount of amphipathic α-helix secondary structure and the protein is

insoluble in aqueous solution at neutral pH (305). In the presence of lipids,

apoA-V shows good solubility due to the lipid binding properties of the C-

terminal end (residues 292-343), as shown by Beckstead et al. (306). The N-

terminal end (residues 1-146) is suggested to form a helix bundle

independently of lipid, similar to that seen in apoE (307). As suggested by

Lookene et al. (206) and later shown by Nilsson et al. (Paper I) apoA-V

interacts with heparin and with the LDLR gene family members LRP1 and

SorLA through an 42 amino acid residue long stretch (residues 186-227).

This part contains eight Arg/Lys residues and three His, but no negatively

charged residues. This resembles the interactions of apoB and apoE with

LDLR gene family members and heparin/HSPG, also involving positively

charged regions in the ligands and negatively charged regions in the

receptors. Using secondary structure prediction models, Sun et al. divided

apoA-V into six different regions (308). They concluded by doing whole

region deletion mutants, that residues 192-238 was absolutely necessary for

45

lipid binding and LPL activation (Figure 7). They also found that the C-

terminal residues 301-343 was important for lipid binding, a result

supported by others (306). Wong-Mauldin et al. showed that the N-terminal

part (residues 1-146) binds lipoproteins with a drift in preference towards

HDL instead for VLDL (309). ApoA-V has a single cysteine at residue 204.

Although unpaired, apoA-V is monomeric in human plasma (270). The

monomeric integrity of apoA-V is however subject to change in some

naturally occurring mutants, e.g. C185 and C271 for which a profound effect

on plasma TG levels can be seen in vivo (Paper IV).

Figure 7 Schematic representation of the structural organization of apoA-V. SP= signal peptide. Some proposed functional regions are indicated as well as the natural mutants studied in paper III.

The N-terminal end of apoA-V was recently shown to interact with midkine

and to be internalized in rat pancreatic β-cells, leading to increased insulin

secretion (310). Although this finding is not directly related to plasma TG

clearance, it open possibilities for a link to the metabolic syndrome and

general energy homeostasis (311). This could be part of the explanation why

metabolic syndrome patterns have been seen in apoA-V knockout animals

(312).

Proposed mode of action

Since the discovery of apoA-V and the confirmation that the apolipoprotein

is an important player in plasma TG metabolism the following hypothesis

explaining apoA-V’s mode of action have been proposed. Given the impact

apoA-V has on TG metabolism, a limited number of mechanisms have been

discussed. 1) ApoA-V acts intracellulary and is involved in VLDL synthesis

and/or secretion. 2) ApoA-V acts extracellulary and affects the metabolism

46

of TG-rich lipoproteins by acting on a) LPL-mediated lipolysis or b) receptor

mediated endocytosis of lipoproteins. There are many reports, from studies

both in vivo and in vitro, giving seemingly contradictionary results

supporting all three possible modes of action.

Intracellular mechanisms

An intracellular mechanism was suggested by Schaap et al. (313). They used

adenoviral overexpression of APOA5 and found reduced VLDL production.

An effect on VLDL size, but not on particle numbers, was observed which led

these authors to suggest that apoB lipidation might be affected. This finding

is however contradicted by results from apoA-V transgenic and knockout

mice, were no impact on VLDL synthesis could be seen (146, 312, 314). Chan

et al. modelled lipoprotein kinetics in human subjects and found no

correlation between plasma apoA-V and VLDL kinetics (315). Thus it seems

like transient overexpression of apoA-V and endogenous production or lack

of production involves partly different mechanisms.

Using hepatoma cell lines transfected with green fluorescent protein fused to

apoA-V, Shu and coworkers found that apoA-V did not colocalize with apoB

suggesting apoA-V should not affect apoB lipidation (316). In transfected

human and rat hepatoma cell lines apoA-V was localized to cytosolic lipid

droplets and was poorly secreted (316, 317). In liver sections apoA-V was

localized with intrahepatic lipid droplets (318). Data from Shu et al. displays

a discrepancy between cell associated apoA-V and secreted apoA-V in vivo

and in vitro (316, 318). In transgenic mice 80 % of apoA-V was found in

plasma whereas 20 % was present in the liver. In cell lines less than 50 % of

apoA-V was secreted. Interestingly, mice overexpressing apoA-V had higher

liver TG content than wild type and knockout controls (318). Taken together,

these data might suggest a role for apoA-V in hepatic TG retention although

future exploration of the exact mechanism and physiological role is needed.

Extracellular mechanisms - LPL activator

In contrast to the intracellular role of apoA-V, the extracellular role has been

more explored. There are a number of publications that report investigations

of different animal models from a hypothesis about an extracellular

mechanism for apoA-V. The most common hypothesis about apoA-V’s

mechanism has been as an activator for LPL (Figure 3 and 5). Some

laboratories have shown a stimulating effect of apoA-V in vitro using non-

physiological levels of apoA-V (600µg/ml) and lipid emulsion or VLDL as a

substrate (308, 313, 314). Lookene et al. were not able to show activation in

their system using three different lipid substrates for LPL, but they showed

47

that apoA-V binds to heparin and is able to mediate binding of TG-rich

lipoproteins to heparin (206). Interestingly, GPIHBP1 has been shown to

interact with apoA-V, although the physiological role for this interaction is

yet unexplained (122). Clearly, interaction between the highly acidic

GPIHBP1 and basic apoA-V is not surprising.

Merkel et al. found a stimulatory effect of apoA-V in a micro titer plate assay

for LPL activity with immobilized HSPG and lipoprotein substrates (146).

These authors suggested that apoA-V facilitates lipoprotein hydrolysis by

guiding lipoproteins to the site of lipolysis through direct interaction

between LPL and apoA-V. Using the same lipase assay several severe

naturally occurring apoA-V mutants were investigated (Paper III). Wild type

apoA-V increased the lipolysis by about 45 %, but the reported loss of

function mutants Q139X and Q148X (281, 319) also stimulated lipolysis by

about 15 to 20 %. This leaves the assay system and the effect of adding

recombinant apoA-V protein under question (Paper III).

Animal models with apoA-V overexpression propose that apoA-V stimulates

LPL activity leading to reduced plasma TG levels (146, 313, 314) or low LPL

activity due to lack of apoA-V expression (146). Merkel et al. showed by a

series of experiments that hepatic VLDL production rate was unaffected by

hAPOA5 expression. In addition intestinal lipid absorption was unchanged.

VLDL and chylomicron metabolism was accelerated by overexpression of

apoA-V. Moreover, apoA-V could be transferred from HDL to TG-rich

lipoproteins and stimulate lipolysis in vitro. Although this effect could be

due to transfer also of other apos to/from HDL, these results favor a role for

apoA-V in stimulating lipolysis. In apoA-V knockout mice Grosskopf et al.

observed that inhibited LPL-dependent hydrolysis was due to that apoA-V

deficient VLDL were poor substrates for LPL. There was also a markedly

lower post-heparin LPL activity in these animals (312). The authors

speculated that the reduced amount of LPL activity could be due to a

metabolic syndrome like pattern, which is supported by the insulin

resistance found in the apoA-V null mice. Although there is partly

compelling in vivo evidence from different animal models that apoA-V

accelerates plasma TG metabolism through increased LPL activity, several

issues need further exploration in order to make a convincing case.

Using stable isotopes and multi-compartmental modeling Chan et al.

investigated the role of apoA-V in VLDL metabolism in obese and non-obese

men, but could not find a correlation for plasma apoA-V with VLDL kinetics

(315). ApoC-III on the other hand was found to be a major determinant of

plasma TG levels (315, 320). Others have found a positive correlation

between plasma apoA-V and plasma TG levels (271). To put this in

48

perspective, apoC-III is strongly associated with a delayed catabolism of

plasma TG (315) and correlates positively with plasma TG in diabetic men

(147), in mice (321) and in healthy humans (322-324). Since apoA-V and

ApoC-III both have been suggested to affect LPL activity, this discrepancy

remains to be resolved. In addition, the low levels of apoA-V in plasma need

further exploration if the plasma compartment is where apoA-V exerts its

effect.

Extracellular mechanisms - receptor ligand

Lookene et al. suggested that apoA-V might serve as a ligand for endocytotic

receptors responsible for lipoprotein uptake (206). The highly basic 42

amino acid residue long stretch of apoA-V is similar to regions in apoE and

apoB responsible for interaction with sulfated proteoglycans and with LDLR

family members. This stimulated Nilsson et al. to study interaction between

apoA-V and two receptors of the LDLR-family, LRP1 and SorLA (Paper I).

ApoA-V was found to interact with the receptors both in free form and in

complex with lipids. The interaction was Ca2+-dependent. Furthermore the

interaction could be competed with the receptor ligand RAP and also with

heparin, suggesting a specific interaction via the putative heparin binding

domain of apoA-V and the LA repeats of LRP1 and SorLA. In agreement with

these findings Dichlberger et al. confirmed interaction of apoA-V with the

major LDL receptor family member called LR8 in the laying hen (325). We

showed that apoA-V-DMPC disks interact with the Sortilin receptor via the

Vps10p domain and, most importantly, that the complexes are endocytosed

in stably transfected human embryonic kidney 293 cells (Hek293)

overexpressing Sortilin and SorLA (Paper II). This provides evidence for

apoA-V as a receptor ligand for both receptors from the LDLR family and

from the Sortilin family (Paper II). In support of this Grosskopf et al. showed

that removal of chylomicron remnants from plasma was impaired in apoA-V

deficient mice, and uptake of remnants in liver perfusion experiments was

weakened (312). The slower particle clearance was attributed to a lower

affinity for the LDLr for apoA-V deficient lipoproteins (312). In further

support of a receptor mediated mode of action for apoA-V is the fact that

apoA-V binds to HSPG (206). This has recently become in focus with regard

to numerous convincing reports regarding the role of HSPG for TG-rich

lipoprotein uptake in the liver (208, 209, 326). It is not unlikely that the

strong heparin binder apoA-V could affect lipoprotein uptake via a direct

HSPG-dependent mechanism (208, 326), or an indirect mechanism similar

to that of apoE (95) as seen in figure 3 and 5.

49

Extracellular mechanisms - in vivo analyses

There are a few interesting notes to be taken with regard to the plasma lipid

profiles in hAPOA5 transgenic mice. Pennacchio et al. found differences only

in plasma TG compared to controls for both hAPOA5 transgene and

mAPOA5 knockout mice (1). Grosskopf et al. found elevated TG and lower

LDL in knockout animals (312). In the study by Pamir et al. only plasma TG

differed between transgenic and wild type mice (292). When knockout and

transgenic animals were compared, there was a massive difference in VLDL

particle concentration and in TG concentration, partly due to larger VLDL

particles in knockout animals (272). There was also a significant difference

in the LDL levels. Knockout animals had 50 % lower LDL levels than

hAPOA5 transgenes. Acute overexpression via adenoviral delivery reduced

serum TG in the VLDL fraction and total serum cholesterol mainly in the

HDL fraction (313, 327). Thus the expected pattern of increased LPL activity

that normally correlates with increased HDL levels is not seen in apoA-V

overexpressing mice. Instead clearance of TG-rich lipoproteins and their

remnants seems to be accelerated (146, 312). Most studies in humans and

mice do not find apoA-V on IDL or LDL. Therefore it is possible that TG-rich

lipoproteins containing apoA-V are cleared before they are fully lipolysed,

and before excess surface material is transferred to HDL. The low apoA-V

levels in plasma might thus be explained by rapid clearance.

In order to address the proposed opposing effect of apoA-V and apoC-III,

double knockout and double transgenic mice were generated (328). These

mice showed plasma TG levels similar to those in corresponding wild type

controls. The apolipoprotein levels were kept at a ratio similar to that

normally seen in vivo, i.e. with 500-fold higher levels of apoC-III than of

apoA-V. The authors concluded that APOA5 and APOC3 independently

influence plasma TG levels in an opposite manner (328). Using adenoviral

gene expression Qu et al. studied apoA-V overexpression on an APOC3

transgenic background (329). Hepatic overexpression of apoA-V reduced

plasma TG levels by more than 60 %, without affecting total plasma

cholesterol levels. Adenoviral expression of apoA-V resulted in a four-fold

increase in plasma apoA-V levels, and also a significant reduction of apoC-III

in plasma, possibly explaining some of the effects on plasma TG levels (329).

In a very interesting paper by Gerritsen et al. apoA-V was studied in mice on

an APOE2 background (330). ApoE2 knock-in causes hyperlipidemia due to

impaired remnant clearance and defective lipoprotein lipolysis (331). These

effects are due to a high equilibrium dissociation constant (KD) for the LDLr

(332, 333), and also a higher KD for HSPG binding for the apoE2 variant

(93). ApoA-V and LPL adenoviral expression decreased plasma TG and total

50

cholesterol levels significantly, whereas APOC3 knockout did not alter

plasma lipid levels in this model (330). These results were supported by the

study of Mansouri et al. who showed that permanent overexpression of

apoA-V in apoE2 knock-in mice reduced plasma TG and cholesterol levels

(294). Since APOC3 deficiency did not alter plasma lipid levels in this model,

apoA-V dependent displacement of apoC-III should not be considered when

explaining the effect seen for apoA-V. These results suggest that apoC-III

inhibits lipolysis and receptor mediated endocytosis via an apoE dependent

mechanism, e.g. by displacing apoE from the lipoprotein surface (77). In

contrast apoA-V lowers plasma lipid levels independently of apoE and apoC-

III (329, 330).

What could be the final chapter in the book on possible mechanisms for of

apoA-V was presented Merkel and Heeren in 2009. By crossing APOA5

transgenic mice onto a liver specific LRP1 deficient background they showed

that the presence of hepatic LRP1 is absolutely necessary for the TG lowering

effect of apoA-V. On the other hand LDLr deficiency did not matter for the

effect of apoA-V (295). Whether the role of LRP1 for apoA-V’s mode of action

is due to a direct interaction between LRP1 and apoA-V needs further

exploration.

Final conclusion

There is evidence supporting all three major hypotheses for the role of apoA-

V. Likewise, there is evidence to discard all three of them. ApoA-V is a player

in a complex system where overlapping functions and secondary effects are

hard to distinguish, especially in in vivo systems with single or multiple

transgenic or knockout traits. Systematic work in well controlled models may

shed more light over the role of apoA-V in the future.

51

ANGIOPOIETIN-LIKE PROTEINS

Changes in LPL activity appears quickly and in a tissue specific manner due

to changes in nutritional status. Neither changes in LPL mass nor LPL

expression can explain this fully. Therefore at least one post-transcriptional

LPL regulator has been implied (9, 132, 334). The ANGPTL family may prove

the most interesting candidates for swift effects on LPL activity. ANGPTLs

are soluble proteins and they are found extracellularly, supposedly close to

where LPL exerts its function or maybe en route where LPL is transported to

its functional luminal position in capillaries (Figure 3).

The ANGPTL gene family consists of several members encoding secreted

proteins with homology to angiopoietins. Three of them have been found to

influence metabolism (ANGPTL3, -4 and -6), whereas ANGPTL3 and -4 have

been shown to influence on lipase activity both in vivo and in vitro (reviewed

in (335)). ANGPTL 4 was discovered in 2000 by three separate groups (336-

338). A mutation in the gene for ANGPTL3 was found to explain the

unusually low plasma TG levels in KK/San mice (339). ANGPTL3 and -4 are

both found in the circulating blood. In humans average concentrations of

368 ng/ml and 23 ng/ml, respectively, were found (340).

ANGPTLs in disease

Although displaying a powerful effect in animal models the role of ANGPTL4

in humans is not as clear. ANGPTL4 levels in plasma do not correlate with

plasma TG levels as would be expected if circulating ANGPTL4 had a major

effect on endothelial LPL (341). PPAR agonists increase ANGPTL4 in blood,

and diabetic patients were found to have increased levels of ANGPTL 4 in

plasma while no correlations seem to exist with body mass index (342, 343).

The common E40K variant is associated with low plasma TG levels and high

HDL-cholesterol, but surprisingly also with an increased risk for coronary

heart disease (344-346). Romeo et al. identified a number of non-

synonymous mutations in ANGPTL4 in the Dallas Heart study. Most of them

were found in the lowest percentile of plasma TG levels (347).

In the well established apoE knockout mouse model for atherosclerosis

ANGPTL4 deficiency improved plasma lipid levels, thus protecting from

atherosclerotic lesion formation. In vitro findings also suggest that

ANGPTL4 knockout strongly suppress foam cell formation (348).

ANGPTL3 knockout mice crossed to an apoE knockout background also

displayed markedly lower levels of atherosclerosis (349). In humans genomic

52

and clinical data are limited. A common SNP studied in a genome-wide scan

was associated with plasma TG (195). Stejskal et al. showed a correlation

with LDL-cholesterol, systolic blood pressured and the metabolic syndrome

marker plasma adipocyte-fatty acid binding protein (A-FABP), but not with

plasma TG levels (350). Non-synonymous mutations in the Dallas Heart

Study displayed a non-significant distribution in the upper and lower

quartile of plasma TG levels (14 against 5, P=0.064) (347). Hatsuda et al.

found a positive correlation between plasma ANGPTL3 levels and intima-

media thickness in healthy humans, indicating a role in atherosclerotic

development (351).

Expression and regulation

Yoon et al. identified ANGPTL4 as a PPARγ target in adipose tissue whereas

Kersten et al. initially found ANGPTL 4 to be regulated by PPARα in mouse

liver (337, 338). PPAR response elements were accordingly found in a highly

conserved region of ANGPTL4. ANGPTL4 is upregulated under fasting, and

this protein was initially called fasting-induced adipose factor (3, 338, 341).

Fasting increases FA levels in circulation, which may activate PPARs.

Activation could also be due to LPL-dependent lipolysis as in figure 3.

ANGPTL4 expression is inversely correlated with LPL activity in rat adipose

tissue during metabolic transitions (3). In contrast, fasting also induced

ANGPTL4 expression in skeletal muscle which is opposite to the effect on

LPL activity (352, 353). ANGPTL4 was found to be upregulated by hypoxia

in 3T3-L1 adipocytes and in endothelial cells suggesting a role for ANGPTL4

in angiogenesis (354, 355).

The tissue expression pattern for ANGPTL4 differs in mice and men. Mice

have high expression in adipose tissue and a little less in ovary, liver, heart

and lung, while men have the highest expression in liver followed by adipose

tissue (337, 338).

ANGPTL4 was recently shown to be a positive acute phase protein which

could help to explain part of the marked HTG associated with the acute

phase response (356).

ANGPTL3 is almost exclusively expressed in the liver under regulation of

cholesterol sensing LXR. Activation in vitro using an LXR agonists markedly

increased ANGPTL3 mRNA (145, 357). Cholesterol feeding also increased

mRNA levels (358). Insulin, leptin and PPARβ/δ suppresses ANGPTL3

expression (359-361).

53

Structure and molecular mechanism

ANGPTL4 consists of an N-terminal coiled-coil domain (ccd) and a C-

terminal fibrinogen-like domain (fld) making up a 50 kDa protein consisting

of 410 amino acid residues. The domains can exist separately since the

protein can be proteolytically cleaved in serum (362). ANGPTL4 is found as

full length protein in adipose tissue and as a truncated protein in liver. In

circulation both full length and cleaved forms are found (342). ANGPTL4 is

glycosylated in the fld which, but glycosylation does not influence on its

effect on lipases (362). Cysteines are found both in the ccd and the fld parts

which engage in forming covalent higher oligomeric structures of various

sizes (337, 342, 357, 362, 363). Oligomerization was shown to be important

for inhibition of lipases by ANGPTL4 but not for secretion (344, 364). The

ccd contains structures needed for lipase inhibition and is also responsible

for the interaction with heparins and HSPG. This interaction protects

ANGPTL4 from proteolysis (3, 365). ANGPTL4 is also reported to interact

with the extra cellular matrix (366). Heparin seems to influence LPL

inactivation by ANGPTL4, although there is some uncertainty (3, 367). If

anything, the protective effect on LPL inactivation is rather weak. When LPL

is bound to GPIHBP1, it is almost fully protected from ANGPTL4-dependent

inactivation (367). In humans ANGPTL4 has been found to be physically

associated with HDL. There is a correlation between plasma levels of

ANGPTL4 with HDL, but not with plasma TG levels (341). In mice ANGPTL4

and LPL monomers were shown to interact and both were shown to bind to

LDL (352). We found, using SPR technology that VLDL and apoC-II

deficient TG-rich lipoproteins do not interact with recombinant ccd-

ANGPTL 4 and that only <1% of ANGPTL4 in human serum is associated

with VLDL (Paper IV).

We have recently found that LPL is protected from inactivation by

recombinant ccd-ANGPTL 4 by the presence of TG-rich lipoproteins (Paper

IV). Furthermore, ccd-ANGPTL 4 did not influence the LPL-dependent

bridging of receptor mediated endocytosis of TG-rich lipoproteins in primary

hepatocytes (Paper IV).

The fld domain has been suggested to be involved in a variety of processes

such as angiogenesis, endothelial cell function, cell adhesion, vascular

permeability (366, 368, 369). Sukonina et al. showed that ccd-ANGPTL4

binds to LPL and acts as an extracellular unfolding chaperone turning active

LPL dimers into inactive monomers without being consumed itself (3).

Lichtenstein et al. demonstrated that ANGPTL4 also inhibits HL (352).

54

ANGPTL3 consists of 460 amino acid residues and is also processed into a

ccd part and an fld part (370). It is highly homologous with ANGPTL4 and

forms multimeric structures (370). ANGPTL3 needs proteolytic cleavage to

exert its full lipase inhibiting effect (370). In fact, residues 17-165 are enough

to inhibit LPL efficiently.

ANGPTL3 inhibits EL in vivo and in vitro (361). HL was marginally

inhibited in vitro but ANGPTL3 knockout mice displayed an increase in

post-heparin plasma HL activity (349, 371).

We found, similar to ANGPTL4, that recombinant ccd-ANGPTL3 did not

interact with VLDL and apoC-II deficient TG-rich lipoproteins. Furthermore,

LPL was protected from recombinant ccd-ANGPTL3 dependent inactivation

in vitro already at minute concentrations of TG-rich lipoproteins. Ccd-

ANGPTL3 did not inhibit the LPL-dependent bridging of TG-rich

lipoproteins to receptors mediating endocytosis in primary murine

hepatocytes (Paper IV).

Proposed mode of action

Several mouse models have been generated with overexpression of human or

endogenous knockout of ANGPTL4. Transgenic or adenoviral

overexpression led to increased levels of TG, FA and non-HDL cholesterol.

Plasma lipid levels were found to be affected in the opposite direction

compared to the transgenes in ANGPTL4 knockout animals and in

transgenic animals treated with a monoclonal ANGPTL4 antibody (144, 341,

343, 364, 372, 373). These studies made it clear that ANGPTL4 primarily

works through LPL in regulating plasma TG levels.

It has been debated whether ANGPTL4 exerts an endocrine or paracrine

effect in vivo. When Yu et al. overexpressed ANGPTL4 locally in heart of

mice, only cardiac LPL activity was inhibited, and when fasted for 6 h these

animals exhibited HTG (374). Stejskal et al. measured human plasma

ANGPTL4 levels and found a negative correlation with HDL-cholesterol and

positive correlations with plasma glucose and TG, supporting a paracrine

role of ANGPTL4 outside the plasma compartment (375). When

overexpression was localized to the liver there was a systemic effect on LPL

activity, although the liver does not express LPL (343). It can therefore be

speculated that ANGPTL4 under certain conditions may serve as a

circulating LPL inhibitor (376).

55

ANGPTL4 might also stimulate lipolysis in adipose tissue, since plasma FA

and glycerol levels are elevated in transgenic mice and injections of

recombinant ANGPTL4 also raises FA concentrations in blood (144).

ANGPTL3 knockout mice have reduced FA, TG and cholesterol associated

with an increased level of post-heparin plasma LPL activity (339, 371, 377),

whereas recombinant ANGPTL3 injections and adenoviral overexpression

displayed the opposite effects (4, 339). As for ANGPTL4, ANGPTL3 seems to

have an effect on adipose tissue lipolysis and release of FA, but the

mechanism involved is unknown.

In summary, ANGPTL3 and -4 are both potent inhibitors of LPL. Thus they

have the ability to control plasma TG levels. There is possibly a difference in

the preference for EL and HL between the ANGPTLs which can be seen in

effects on LDL and HDL levels. Except for regulation of lipases, an endocrine

role has been proposed for both proteins. Since FA and glycerol increase

with ANGPTL plasma levels, they could have an important role in regulating

adipose tissue lipolysis.

56

UNPUBLISHED RESULTS

Interaction of ApoA-V with LDLr

As described under the LDLr sub chapter, this receptor recognizes apoE and

is responsible for some clearance of remnants. Since the LDLr gene family

shares a common ligand binding domain in the form of LA-repeats, they also

share many ligands. ApoE interacts with both LRP1 and LDLr via positively

charged regions. The putative heparin binding and receptor binding domain

of apoA-V consists of a 42 amino acid residue long stretch with no negative

residues but 5 Arg, 3 Lys and 3 His.

Using SPR under the same conditions as in previous work (Paper I-III), the

specific interaction between apoA-V-DMPC disks and LDLr was seen (Figure

8). The interaction was compared with that for LRP1 and the KD was

determined to be similar.

Figure 8 ApoA-V binds LRP1 and LDLr with similar affinity. LRP1 and LDLr were immobilized to a CM5 sensor chip at a concentration of 3,0 ng/mm2 and 1,5 ng/mm2 respectively. BSA was used as a control surface. ApoA-V-DMPC disks were injected at increasing concentrations and steady state kinetics were calculated. KD was close to 100 nM for both receptors. Nilsson SK, Heeren J and Olivecrona G, unpublished.

ApoA-V affects chylomicron clearance in rats

Intestinal chylomicrons were isolated from rats given 3H-labelled retinol and 14C-labelled oleic acid. Thus the chylomicrons had both a core label of retinyl

esters and a label in TG (10). Sprague-Dawley rats were anesthetized and

injected with chylomicrons corresponding to 5 mg TG with (n=5) or without

(n=4) addition of recombinant apoA-V. The calculated concentration was

16.5 µg apoA-V/ml blood. Blood was drawn at minute(s) 1, 3, 7, 13 after

injection and a final bleeding was made at 20 min.

57

Analysis of blood samples indicated a significantly faster clearance of 3H-

labelled retinyl ester and 14C-labelled TG in animals injected with

chylomicrons containing added recombinant apoA-V (Figure 9). Hydrolysis

of the chylomicrons was also investigated in vitro by incubation with LPL. In

concert with what has been shown by others (146, 206), addition of apoA-V

did not affect the LPL activity (data not shown).

Figure 9 ApoA-V accelerates clearance of chylomicron core and TG label from the blood in rats. *P<0.05 **P<0.01 ***P< 0.001 Nilsson SK, Olivecrona T, and Olivecrona G, unpublished.

Our results indicate a faster turnover of chylomicrons in rats injected with

added apoA-V. This could be due to an effect on receptor mediated

endocytosis, to a quicker margination process due to apoA-V’s HSPG binding

properties, or to a quicker receptor clearance secondary to increased particle

hydrolysis. Since there were no differences in levels of radioactivity in FA in

plasma (data not shown), this indicates that apoA-V does not increase the

hydrolysis rate of chylomicrons in vivo. In such case, margination and/or

uptake must account for the differences observed.

58

METHODS

Surface plasmon resonance

SPR is a powerful in vitro technique that enables interaction studies of

virtually all kinds of biomolecules or even of whole cells. SPR allows label

free qualitative and quantitative interaction studies in real time.

SPR in history

In 1902 R.M. Wood published a paper which is the theoretical embryo for

SPR (378). He noticed that polarized light shone on a reversed metal

diffraction grating gave an unexpected white and dark band reflection

pattern. This phenomenon was not explained until 1957 by R.H. Ritchie as

surface plasmons (379).

Surface plasmons are electron density waves, oscillations, which propagate

along a metal-dielectric interface. Surface plasmons can be excited by both

electrons and photons, i.e. visable light is enough to excite surface plasmons.

The surface plasmons propagate along the metal specimen but are sensitive

to contaminants on the surface. Contaminants results in excitation of

evanescence waves at the metal specimen surface and loss of surface

plasmons. Surface plasmons and the evenescene waves created by physical

hindrances implicated the use of the phenomenon in studies of ultra-thin

surfaces.

Liedberg et al. developed the technique for using prism coupled excitation of

surface plasmons which opened the door for biosensing (380). Later on, in

1984, Pharmacia Biosensor AB was founded to develop a commercial SPR

instrument. The first Biacore was sold in 1990 and has since then developed

into many different instruments for different applications.

SPR in theory

In Biacore instruments a gold covered dextran coated sensor chip is used.

These sensor chips contain flow channels which are continuously rinsed with

a running buffer. Different biomolecules can be immobilized on the sensor

chip surface e.g. by amine coupling. With the ligand immobilized to the

sensor chip surface and a continuous buffer flow passing through the flow

cell, time and quantity specific injections of analytes can be made. As the

analyte reaches the flow cell the interaction can be studied in the

sensorgram.

59

In short, when polarized light shines through the prism covering the sensor

chip gold surface, surface plasmons will excite in the gold layer. This will

cause a change in the reflected light (Figure 10). Binding of the injected

analyte to the immobilized ligand (molecule) at the sensor chip surface will

cause a change in refractive index at the gold surface buffer interface.

Consequently this will change the angle needed for total internal refraction

and excitement of surface plasmons. This change is measured in the

reflected light by a photo detection unit and displayed as a sensorgram

measured in response units (RU) where 1 pg of protein mass bound per mm2

is equivalent to 1 RU.

Figure 10 Schematic view of the Biacore SPR technology system. Ligands (displayed as receptors) are immobilized to the gold surface of the sensor chip via, most often, covalent interaction. A continuous buffer flow passes the surface and upon injection free analytes are allowed to pass. Interaction between analytes and ligands changes the refractive index which is recorded by the photo detector.

Binding constant determination

When studying the interaction of the ligand and the analyte under controlled

conditions the equilibrium dissociation constant KD [M], often expressed as

affinity, can be determined. In the simplest case this can be done by

determining the dissociation- and association rate constants, kdiss [ s-1] or kass

[M-1s-1].

This model does only allow one-step binding without conformational change

or other complex binding. If such interactions are studied there are

enhanced models that can be used. If unspecific binding or multistep binding

60

does not allow calculation of the dissociation- and association rate constants

KD can be calculated with steady state kinetics;

where y is the response in RU, x the concentration of the analyte, b is KD and

a the number of binding sites. This single rectangular two parameter

equation can be expanded to a double rectangular four parameter equation if

two binding reactions occur simultaneously.

It should be noted that a low KD can be due to different combinations of rate

constants, e.g. high association- and dissociation rate constants can display

the same KD as a combination of low association- and dissociation rate

constants.

61

AIMS OF THIS THESIS

The overall aims of the thesis work were to investigate new mechanisms

involved in plasma TG regulation. More specifically;

Investigate if apoA-V binds to and undergoes endocytosis together with LDLR gene family members and members of the Sortilin family

Characterize which region(s) in apoA-V that is/are involved in binding to heparin and receptors

Elucidate the role of TG-rich lipoproteins for the effects of ANGPTLs on LPL activity

62

RESULTS

Paper I examines the interaction between apoA-V and the LDLR gene

family member LRP1 and the Sortilin gene family receptor SorLA.

ApoA-V binds to LRP1 and to SorLA Using SPR, apoA-V-DMPC disks were

shown to interact specifically with both LRP1 and SorLA. As evidence for

this, the binding was Ca2+ dependent and was competed by the established

receptor ligand RAP. ApoA-V also interacted with the receptors in lipid free

form although this binding was not fully competed by the Ca2+ chelator

EDTA.

ApoA-V mediates binding of TG-rich lipoproteins to LRP1 and to SorLA In

order to have an effect on plasma TG clearance via endocytotic receptors,

apoA-V must be able to facilitate lipoprotein binding to these receptors.

Since binding of apoA-V cannot be distinguished from lipoprotein binding to

receptors using SPR, we chose to pre-inject lipid free apoA-V in the active

flow cells containing receptors. Human chylomicron-like lipoproteins from

an apoC-II deficient patient were subsequently injected. In flow cells

containing apoA-V a powerful bridging effect was recorded. ApoA-V

increased binding of chylomicrons-like lipoproteins up to 15-fold with

relatively small amount of apoA-V present. Interestingly, compared to the

well known bridging molecule LPL, a very slow dissociation of the TG-rich

lipoproteins was seen from the receptor-apoA-V complex.

Heparin and receptor interaction domains in apoA-V partly overlap

Binding of apoA-V-DMPC disks to LRP1 or SorLA was competed by co-

injection with heparin, suggesting at least a partial overlap of the heparin-

and receptor binding domains. By doing site-directed mutagenesis of the 42

amino acid residue long positive stretch in apoA-V we could affect the

binding affinity to heparin and LRP1. The double mutant R210E/K211Q

eluted prior the wild type protein from heparin Sepharose. When SPR was

used to study heparin interaction, steady state kinetics revealed an unaltered

KD but a remarkable 9-fold change in binding capacity, i.e. loss of binding

sites. Similar results were seen in the interaction with LRP1; KD was

somewhat lower for the double mutant but the binding sites were also fewer,

giving a three-fold higher response for the wild type apoA-V. These data

suggest that the sites for receptor- and heparin-binding at least partially

overlap.

63

Paper II investigates the interaction between apoA-V and the Vps10p-

domain receptor Sortilin, which shares similarity with SorLA and has been

implicated as an important player in CVD and LDL-cholesterol homeostasis

(195-198). Furthermore, internalization of apoA-V in cell cultures

overexpressing Sortilin and SorLA was investigated quantitatively via 125I-

labeled ligand and qualitatively via direct immuno-fluorescence and Försters

Resonance Energy Transfer (FRET). The endocytic route of apoA-V was

explored with markers for different cellular compartments.

Sortilin specifically binds ApoA-V and chylomicrons via the Vps10p-domain

Using SPR we investigated if apoA-V-DMPC disks interact with the Sortilin

receptor which lacks LA repeats in contrast to SorLA. Indeed, apoA-V-DMPC

disks were found to bind Sortilin. Co-injection with the small peptide

neurotensin (NT), that binds the Vps10p domain, abolished all binding. This

was also the case for co-injection with heparin, or pre-injections of the

multibinding ligand RAP. Injection of NT resulted in a markedly increased

dissociation of apoA-V from Sortilin. We concluded that the apoA-V

interaction with Sortilin occurs between the Vps10p domain and the

heparin/receptor domain in apoA-V. We also showed that human

chylomicron-like lipoproteins bind to Sortilin using the SPR system and that

co-injection with NT inhibits this interaction.

ApoA-V is internalized in cell cultures overexpressing SorLA and Sortilin

ApoA-V-DMPC disks were labeled with an Alexa fluorochrome. Using stably

transfected Hek293 cells internalization was studied. Cells overexpressing

SorLA or Sortilin displayed increased surface binding of the ligand when

incubated a 4ºC compared to the non-transfected control cells. Upon

incubation at 37ºC a quick internalization of apoA-V-DMPC disks was

observed in the transfected cells. Moreover, apoA-V co-localized with SorLA

and Sortilin as determined by confocal microscopy.

Using a few well characterized mutant receptors we further determined the

specificity of the interaction. ProSortilin is unable to bind ligands. This

variant did not bind or internalize apoA-V-DMPC disks. SorLA and Sortilin

mutated in the internalization motif display slow endocytosis but have the

ability to bind ligands. This was confirmed with apoA-V-DMPC disks which

did not internalize but was markedly enriched on the cell surface compared

to non-transfected cells. In order to strengthen the case that apoA-V and the

receptors co-localize upon endocytosis we performed FRET experiments.

FRET analyses displayed a strong co-localization of apoA-V with SorLA or

Sortilin. FRET data indicated that the receptors and apoA-V-DMPC disks

clustered together, rather than being randomly distributed.

64

Finally apoA-V-DMPC disks were labeled with 125I in order to quantitate the

receptor mediated uptake and degradation. Cells overexpressing SorLA and

Sortilin were found to bind and internalize higher amounts of 125I-labeled

apoA-V-DMPC disks than control cells. As expected, SorLA- and Sortilin-

expressing cells degraded a higher amount of apoA-V compared to the non-

transfected control cells.

ApoA-V follows the endocytotic route of the Sortilin family members Using

markers for intracellular compartments we studied the route of apoA-V after

endocytosis by SorLA and Sortilin. We found that after a rapid

internalization and transport through early endosomes, apoA-V was via the

retromer complex sorted to the trans-Golgi network (TGN) and to late

endosomes. In the late endosomes apoA-V was uncoupled and sent to the

lysosomes for degradation. Neither apoA-V, nor the receptors were seen, or

have previously been seen, to recycle. This is in contrast to e.g. LRP1 and

apoE.

Paper III looked into the function of a few natural human mutants, and

variants of apoA-V, connected to HTG. The mutants were investigated with

the same micro-titer plate based lipase assay, as previously used by Merkel et

al. to establish apoA-V as an LPL activator (146). The mutants were also

investigated with regard to interaction with LDLR receptor family members

by ligand blotting and SPR.

In vitro lipolysis is markedly unaffected by severe apoA-V mutations In the

in vitro lipolysis experiments both human VLDL and lipid emulsions were

used as substrate. Wild type apoA-V stimulated lipolysis by about 45 % with

both substrates. The mutants displayed a weak to medium strong effect on

lipolysis compared to wild type apoA-V. Surprisingly, the truncated stop

mutants X138 and X148 showed about 15 % stimulation. The multimer

forming C271 mutant displayed a clear LPL stimulating effect.

Severe natural mutants do not interact with LDLR gene family members

Using SPR the stop mutants X139 and X148 were found not to interact with

LRP1 at all. The same was the case for the multimer forming cysteine mutant

C271. Mutant C185 did not display major differences compared to wild type

apoA-V, whereas G255, L321 and Δ139-147 showed up to three-fold higher

KD values then wild type apoA-V.

All SPR data was confirmed and supported by ligand blotting using the avian

LDLR homologue LR8.

65

Paper IV studies the role of TG-rich lipoproteins for the LPL-ANGPTL

system. As previously shown, heparin and GPIHBP1, protects LPL from

ANGPTL dependent inhibition (3, 367). Since LPL avidly binds TG-rich

lipoproteins and ANGPTL4 is only found in the LDL and HDL fractions we

wanted to investigate if TG-rich lipoproteins could protect LPL from

inactivation by ANGPTLs.

Ccd-ANGPTL3 and -4 do not prevent LPL-mediated internalization of TG-

rich lipoproteins in hepatocytes Cellular uptake of human chylomicron-like

lipoproteins mediated by LPL was studied with and without addition of

recombinant ccd-ANGPTL3 and -4 in primary murine hepatocytes.

Surprisingly, ccd-ANGPTL3 or -4 in concentrations known to inactivate LPL

did not affect uptake. When LPL was pre-incubated with ccd-ANGPTL3 or -4

before addition of the lipoproteins there was an almost complete

abolishment of the effect of LPL on the uptake.

Using the active site inhibitor tetrahydrolipstatin (THL) we could

demonstrate that pre-incubation of LPL with ccd-ANGPTL3 or -4 blocked

both lipolysis and the bridging function.

TG-rich lipoproteins protect LPL from inactivation by ccd-ANGPTL3 and -4

Since TG-rich lipoproteins appeared to have a protective effect on LPL

against inhibition by ccd-ANGPTL3 and -4, we investigated this further in an

in vitro lipolysis system. VLDL and chylomicron-like TG-rich lipoproteins

protected LPL against ccd-ANGPTL3 and -4 in a dose dependent manner. At

physiological levels of TG and ccd-ANGPTL3 or -4, inhibition was shown to

be limited, indicating that circulating ANGPTLs in blood may not be able to

inactivate endothelial-bound LPL.

Ccd-ANGPTL3 and -4 are unable to bind to TG-rich lipoproteins Ccd-

ANGPTL3 and -4 were immobilized on Biacore sensor chips with bovine

serum albumin (BSA) as a negative control and apoC-III as a positive

control. Injections of VLDL and chylomicron like lipoproteins were made.

Both classes of lipoproteins bound to the apoC-III covered surfaces, whereas

no binding was detected to ccd-ANGPTL3 or -4 or to BSA.

66

DISCUSSION AND CONCLUSIONS

This thesis aimed to investigate new mechanisms involved in plasma TG

metabolism. Others had demonstrated that apoA-V and ANGPTL3 and -4

are strongly connected to plasma TG clearance in vivo and lipolysis in vitro,

although the mechanisms behind their effects had not been fully elucidated.

ApoA-V

ApoA-V had been considered as an LPL activator in the literature (see

reviews (269, 381, 382)). Recent findings had questioned this, or at least

suggested alternative or parallel mechanisms by which apoA-V exerts its TG

lowering effect (Paper I-III)(206, 295, 312, 325). We have shown that apoA-

V interacts with endocytotic receptors in vitro and undergoes internalization

with these receptors in cell cultures (Paper I, paper II). Interaction with

SorLA and Sortilin routes apoA-V for degradation and could to some extent

explain the low levels of apoA-V seen in plasma. The fact that apoA-V is

expressed solely in the liver, and the low plasma levels, might suggest that

apoA-V exerts its effect in the hepatic compartment. Moreover, the positive

correlation between apoA-V and plasma TG levels in both humans and mice

suggests that plasma apoA-V may not contribute to TG lowering. Nelbach et

al. found a significant increase of liver TG in mice transgenic for APOA5

(272). If the major effect of apoA-V would be to stimulate LPL, the liver

would not be expected to accomodate lipids.

We found that severe natural mutants connected to HTG do not bind LRP1

or to the avian receptor homologue LR8. Other mutants that have been

implicated in HTG exhibited normal or somewhat impaired interaction with

LRP1. In vitro lipolysis experiments showed that some of the severe mutants

were not significantly different compared to wild type apoA-V with regard to

stimulation of LPL. Given the limited effects seen in this system, and the

difficulties in finding differences even with severe, in vivo non-functional,

mutants one could suggest that apoA-V does not play a major role in

facilitating lipolysis in vivo.

Although most animal studies suggest a role for apoA-V in the lipolytic

processing of TG-rich lipoproteins, recent findings by Heeren at al. reveals

very interesting insight into the role of apoA-V (295). APOA5 overexpression

in mice lacking LRP1 abolishes the TG lowering effect of apoA-V. These

findings clearly favor a role for apoA-V in receptor mediated endocytosis of

TG-rich remnant lipoproteins. Whether this is because of a direct effect on

apoA-V function or if LRP1 deficiency affects LPL in an adverse way remains

67

to be determined. In an older study by Merkel et al., heterozygote LPL

deficiency was shown to modulate the effect of APOA5 transgenic

overexpression (146). If apoA-V only mediates receptor mediated clearance,

predominantly in the liver, it would still be dependent on LPL forming

remnants that are small enough to enter the space of Disse. Nelbach et al.

found that APOA5 transgenic liver contained more TG than littermate

controls (272). Since a negative effect on VLDL secretion has not been seen,

this indicates an increased lipid uptake. Compartmental modeling might

unravel if this lipid is derived from FA from adipose tissue or derived from

lipoprotein remnant lipids.

Recent findings from the Esko lab has highlighted the role of HSPG in

remnant clearance (205, 326). ApoA-V binds to HSPG and mediates binding

of lipoproteins to HSPG (206) and could thus contribute to receptor

mediated endocytosis via HSPG. This thesis confirms the role of a region

(residues 186-227) in apoA-V with only basic and neutral residues involved

in receptor and HSPG interaction. Although mutations at position

R210/K211 do not abolish binding, there was an effect on binding capacity.

Additional mutations of positive residues in this region cause an increased

loss of binding to both HSPG and LRP1 (383).

A factor in TG metabolism which most often has not been given proper

attention, is the effect of lipoprotein remodeling and apolipoprotein

displacement due to addition of recombinant apolipoproteins or

endogenously overexpressed apolipoproteins. There is a risk of non-

physiological effects which do not concur with the study object in vivo. This

can be seen in the case of apoC-II overexpression in vivo where the activator

of LPL actually inhibits the enzyme (73, 74) or in the ambiguous case of apoE

in lipolysis (91, 93) or when apoA-V overexpression locates the protein to

LDL particles (272). In many studies of apoA-II, most efforts to study the

role of this apolipoprotein caused apoA-I displacement (51). In contrast,

specific functions of apolipoproteins in vivo might act through other

apolipoproteins.

Although this thesis does not reach the goal to find the mechanism of apoA-

V, it describes an area that might be of great importance. Given the new

players and some old, e.g. GPIHBP1, ANGPTLs, VLDLr, SorLA, Sortilin and

even the controversial LSR, which could be added to the mix of factors

affecting plasma TG metabolism, apoA-V should perhaps be considered from

yet other perspectives.

68

ANGPTLs

In vitro and in vivo ANGPTL3 and -4 are potent inhibitors of lipases.

Intriguingly, plasma concentrations of the ANGPTLs do not correlate as

expected with plasma TG levels, implying that the plasma compartment is

not where ANGPTL3 and -4 have their major site of action, at least not on

LPL.

We have shown that at normal levels of TG-rich lipoproteins and ccd-

ANGPTL3 and -4, the inhibiting effect on LPL is low. Therefore we suggest

that it is more likely that ANGPTL4 interacts with LPL when it is en route to

its site of action on the luminal side of the endothelium. ANGPTL4 is

expressed in heart and adipose tissue which are also the major LPL

producing tissues. ANGPTL4 binding to components of ECM and to HSPG

allows binding and retention in the intima of arteries or in the

subendothelial space around capillaries where lipoprotein levels are low and

where LPL has to pass on its way to the luminal side of the endothelium.

Although there is ambiguity concerning the ability of heparin to protect LPL

from inactivation by ANGPTLs, this protective effect appears much weaker

than that seen with TG-lipoproteins. Furthermore, the concentrations of

ANGPTLs could be much higher at these sites than in the circulating blood.

When LPL reaches the endothelial cells it will bind to GPIHBP1 which has

been shown to efficiently protect LPL from inhibition by ANGPTL 4.

In the liver the role of the ANGPTLs might be different because here LPL is

normally not expressed. Furthermore, in the liver sinusoids and in the space

of Disse lipoproteins are present. If LPL enters carried on lipoprotein

remnant particles they should help in stabilizing LPL in active dimeric form

which is needed for its assistance in receptor-mediated uptake of

lipoproteins.

69

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ACKNOWLEDGEMENTS

Jag hoppas att du tyckte att avhandlingen var läsvärd nu när du har jobbat

dig ända fram till mina tackord.

Gunilla, jag ser tacksamt tillbaka på den här tiden som präglats av ditt

tillåtande, uppmuntrande och utvecklande ledarskap. Det är i sanning en

gåva att kunna se möjligheterna i all galenskap som du fått lyssna på. Jag ser

framtiden an och de kommande 3 åren kommer att ge ett mycket spännande

och givande samarbete.

Aivar, du har lärt mig väldigt mycket och inspirerat mig att tänka i andra

banor. Det är till stor del din förtjänst att jag blev intresserad av AV-

projektet. Jag önskar dig all lycka och ser fram emot fortsatt samarbete.

Ett stort tack till Lipasgruppen; Solveig, för att du har koll på allt i labbet

och alltid hjälper till när jag behöver det. Nestor Thomas, för att du vet allt

som gjorts före min tid. Jag hoppas du är sugen på att jobba med

kylomikroner igen. Olessia, för att du har varit en trevlig rumskamrat.

Nästa gång är det din tur! Det kommer att bli kanonbra. All lycka till! Lasse,

för trevligt sällskap och för att du alltid tar dig tid och hjälper till. Det

uppskattas. Elena, för att du läser noga till JC och för att du delar med dig

av kunskap och material i labbet. Rakel, för att du alltid är på bra humör,

det smittar av sig. Jag håller räkningen på hur länge du varit här så du inte

slipper igenom för lätt ;) Micke, för att du kom och höjde medelvikten. Vi

har mycket att göra tillsammans nu men framtiden har aldrig varit mer

spännande. Fettryck! Tack också till Liyan och Valja för den tid ni var här.

Collaboration partners all over the world; in Århus, thank you Morten for a

fruitful collaboration and a wonderful time in your lab. I wish you the best of

luck and I hope that other projects will come in the future. Stine for helping

out with the JBC paper and for being a good host. Good luck with your own

PhD. Merete for being the FRET genius. In San Francisco; thank you

Robert Ryan for good cooperation regarding apoA-V and for willingly

providing us with material. Jennifer Beckstead for all those liters of

samples you prepared for me. In Hamburg; thank you Jörg for a great time

in your lab, your kindness and willingness to help. You are truly a guy who

deserves success. To all my friends Alex, Alexander, Annika, Birgit,

Ditte, Doro, Dorte, Ludger, Olli, Sandra, Svenja, Walter and the rest

of the past and present members of the lab, for helping me out with

experiments and for making my time in Hamburg so very nice. A special

thank you to Ulrike Beisiegel. In Leuwen; Philip, the time we had in

93

Hamburg was great, lousy results for me but a really good time. Cuisine

extraordinaire, the HSV football match (never has stadium seats been so

comfy) and going to the jazz boat, just to mention a few of my memories.

Good luck in the States! In London; Philippa Talmud, thanks for the

opportunity to be on the Dorfmeister paper. Thank you Birgit

Dorfmeister for at nice collaboration.

Ett stort tack till damerna i barngruppen; ni bidrar alla till en trevlig

stämning vid fikabordet. Sussie, det är alltid lika trevligt med en pratstund

och nu får jag följa dina barns utveckling 3 år till! Yvonne, det känns bra

med en sockenbo på våningen. Lotta, för din humor och för att du gillar

innebandy, en ständig källa till munterhet. Carina, för att du alltid håller

med mig. Anna M, jag hoppas vi får se dig oftare. Man kan ju inte plugga

hela tiden? När stugan är klar ska ni få komma ut på grillning.

Tack till Clara, för allt jobb i det osynliga. Åsa, för att du bedriver en

toppklassig serviceinrättning. Terry, för alla papper, betalningar och intyg

som du fixar fram och har koll på. Jag säger bara, - åh, choklad!

Tack alla ni på 3 trappor; Elin, du är som en av grabbarna. På ett bra sätt.

Angelika för att du är så rolig och är vegan. Tomas för att du är så

eftertänksam och inte längre vegan. Urban, min Bruichladdichbroder! Må

din malt aldrig sina. Nina G, tack för Illuminahjälp och hårda

Vetgirigmatcher. Nina N, dansgurun! Lycka till med din egen forsk. Åsa, du

är som en av gubbarna. På ett bra sätt. Malin, för att du är min

bakåtsträvarkompis. Maria B, för samsande kring instrument och

apparater. Monika, det är alltid trevligt med dig som bordsgranne. Resten

av 11:00-lunchgänget, nuvarande och före detta; Marie, Lisbeth, Heidi,

Dan, Sofia, Linda, Susann, Maria L tack för trevligt lunchsällskap.

På våning 4, UCMM, ett speciellt tack till Fredrik och Pär för all hjälp och

gott samarbete.

Lars, du är mitt substitut för Pasi, fast tvärtom - lätt och med ofta

motsträviga åsikter ;) Låt oss nu skriva den där debattartikeln.

Bäste bror Pasi, du var under en alltför kort tid min livboj i ett hav av

galenskap. På sätt och vis är du min idol.

Kompanjon Martin, bäste Martin, 10 år senare är vi inte längre S&M utan

Dr&Dr. Låter det inte bättre, så säg? Den som vet sitt eget värde, bestämmer

själv kursen. - Sixten Landby

94

Tack Mor och Far för att ni gav mig förutsättningarna och tack för all hjälp

under doktorandåren.

TILL MIN FAMILJ Allt jag gör, gör jag för er. Tack älskade Anna för att du

varit tillåtande och positiv till alla utlandsvistelser, måsten och sena kvällar,

och tack för all markservice. Speciellt nu på slutet av doktorandtiden. Tack

lille Hjalmar för att du fått mig att inse vad som är viktigt i livet. Ni båda är

mitt allt.

Listan över er övriga utanför jobbet som är viktiga i mitt liv blevo allt för lång

om jag skrev den här men ni skall veta att ni är uppskattade.

Först ett ukast, sedan ett manuskript och slutligen

ett fulländat verk. – Sixten Landby