Umeå University Medical Dissertations, New Series No. 1669
Endogenous and exogenous factors affecting
lipoprotein lipase activity
Mikael Larsson
Department of Medical Biosciences, Physiological Chemistry
Umeå 2014
Copyright © Mikael Larsson 2014
Responsible publisher under Swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7601-115-7 ISSN: 0346-6612 E-version available at http:// http://umu.diva-portal.org/ Printed by: Cityprint I Norr AB Umeå, Sweden 2014
i
Abstract
Individuals with high levels of plasma triglycerides are at high risk to develop cardiovascular
disease (CVD), currently one of the major causes of death worldwide. Recent epidemiological
studies show that loss-of-function mutations in the APOC3 gene lower plasma triglyceride levels
and reduce the incidence of coronary artery disease. The APOC3 gene encodes for
apolipoprotein (APO) C3, known as an inhibitor of lipoprotein lipase (LPL) activity. Similarly, a
common gain-of-function mutation in the LPL gene is associated with reduced risk for CVD.
LPL is central for the metabolism of lipids in blood. The enzyme acts at the endothelial surface of
the capillary bed where it hydrolyzes triglycerides in circulating triglyceride-rich lipoproteins
(TRLs) and thereby allows uptake of fatty acids in adjacent tissues. LPL activity has to be rapidly
modulated to adapt to the metabolic demands of different tissues. The current view is that LPL is
constitutively expressed and that the rapid modulation of the enzymatic activity occurs by some
different controller proteins. Angiopoietin-like protein 4 (ANGPTL4) is one of the main
candidates for control of LPL activity. ANGPTL4 causes irreversible inactivation through
dissociation of the active LPL dimer to inactive monomers. Other proteins that have effects on
LPL activity are the APOCs which are surface components of the substrate TRLs. APOC2 is a well-
known LPL co-factor, whereas APOC1 and APOC3 independently inhibit LPL activity.
Given the important role of LPL for triglyceride homeostasis in blood, the aim of this thesis was
to find small molecules that could increase LPL activity and serve as lead compounds in future
drug discovery efforts. Another aim was to investigate the molecular mechanisms for how
APOC1 and APOC3 inhibit LPL activity.
Using a small molecule screening library we have identified small molecules that can protect LPL
from inactivation by ANGPTL4 during incubations in vitro. Following a structure-activity
relationship study we have synthesized lead compounds that more efficiently protect LPL from
inactivation by ANGPTL4 in vitro and also have dramatic triglyceride-lowering properties in vivo.
In a separate study we show that low concentrations of fatty acids possess the ability to prevent
inactivation of LPL by ANGPTL4 under in vitro conditions.
With regard to APOC1 and APOC3 we demonstrate that when bound to TRLs, these
apolipoproteins prevent binding of LPL to the lipid/water interface. This results in decreased
lipolysis and in an increased susceptibility of LPL to inactivation by ANGPTL4. We demonstrate
that hydrophobic amino acid residues that are centrally located in the APOC3 molecule are
critical for attachment of this protein to lipid emulsion particles and consequently for inhibition
of LPL activity.
In summary, this work has identified a lead compound that protects LPL from inactivation by
ANGPTL4 in vitro and lowers triglycerides in vivo. In addition, we propose a molecular
mechanism for inhibition of LPL activity by APOC1 and APOC3.
ii
Table of Contents
Abbreviations iii
List of papers iv
Introduction 1
Transport and metabolism of exogenous lipids 2 Transport and metabolism of endogenous lipids 3 Reverse cholesterol transport 4
Dyslipidemia and cardiovascular disease 5
Determinants of plasma triglyceride metabolism 6
Lipoprotein lipase 6 Regulation of lipoprotein lipase activity 10 Lipoprotein lipase in atherosclerosis 11
Angiopoietin-like proteins 13
GPI-anchored HDL-binding protein 1 16
Apolipoproteins 17 Apolipoprotein A1 18 Apolipoprotein B 18 Apolipoprotein C1 19 Apolipoprotein C2 20 Apolipoprotein C3 22 Apolipoprotein E 24 Apolipoprotein A5 26
Aims of the thesis 27
Results and discussion 28 Paper I 28 Paper II 32 Paper III and IV 33
Conclusions 39
References 40
Acknowledgements 55
iii
Abbreviations
ABCA1 – ATP-binding cassette transporter A1
ANGPTL – angiopoietin-like protein
APO – apolipoprotein
ATP – adenosine triphosphate
CETP – cholesteryl ester transfer protein
CHD – coronary heart disease
CVD – cardiovascular disease
ER – endoplasmic reticulum
GPI – glycosylphosphatidylinositol
GPIHBP1 – GPI-anchored HDL-binding protein 1
HDL – high-density lipoprotein
HTS – high-throughput screening
IDL – intermediate-density lipoprotein
LCAT – lecithin-cholesterol acyltransferase
LDL – low-density lipoprotein
LDLR – low-density lipoprotein receptor
LMF1 – lipase maturation factor 1
LPL – lipoprotein lipase
LRP1 – low-density lipoprotein receptor-related protein 1
LXR – liver X receptor
LY6 – lymphocyte antigen 6
MTP – microsomal triglyceride transfer protein
PPARs – peroxisome proliferator-activated receptors
PTLP – phospholipid transfer protein
SAR – structure–activity relationship
SR-B1 – scavenger receptor class B member 1
TG – triglyceride
TRL – triglyceride-rich lipoproteins
VLDL – very low-density lipoprotein
iv
Paper I
Apolipoproteins C-I and C-III inhibit lipoprotein lipase activity by displacement of the enzyme
from lipid droplets
Larsson, M., Vorrsjö, E., Talmud, P., Lookene, A., and Olivecrona, G. (2013) The Journal of
Biological Chemistry 288(47):33997-4008
Paper II
Fatty acids bind tightly to the N-terminal domain of angiopoietin-like protein 4 and modulate
its interaction with lipoprotein lipase
Robal, T., Larsson, M., Martin, M., Olivecrona, G., and Lookene, A. (2012) The Journal of
Biological Chemistry 287(35):29739-52
Paper III
Identification of a small molecule that stabilizes lipoprotein lipase in vitro and lowers triglycerides in vivo
Larsson, M., Caraballo, R., Ericsson, M., Lookene, A., Enquist, P. A., Elofsson, M., Nilsson, S.
K., and Olivecrona, G. (2014) Biochemical and Biophysical Research Communications
25;450(2):1063-9
Paper IV
Structure-activity relationships of small molecules lowering plasma triglycerides
Caraballo, R., Larsson, M., Nilsson, S.K., Ericsson, M., Qian, W., Nguyen, N.P., Kindahl, T.,
Svensson, R., Mastej, M., Artursson, P., Olivecrona, G., Enquist, P.A., and Elofsson, M.
Manuscript
1
Introduction
Triglycerides (or triacylglycerols) are the most abundant dietary lipids. They are used as
source of energy in most tissues or for storage in adipose tissue. Triglycerides are non-polar
esters made up of glycerol and long-chain fatty acids that are incapable of entering cells on
their own. Hydrolysis of the ester bonds, catalysed by enzymes called lipases is therefore
needed. By the action of lipases fatty acids are released from the glycerol backbone so that
the polar lipolysis products (fatty acids and monoglycerides) can cross the plasma membrane
of cells and be used for metabolic purposes. Besides serving as a source of energy, fatty
acids are active substances and function as signal molecules. When in excess, fatty acids may
lead to cellular dysfunction and even cell death. In contrast triglycerides are inert. Therefore
fatty acids in excess are re-esterified to form triglycerides that in turn enable safe storage in
intracellular lipid droplets and/or transport in lipoproteins in blood.
Lipoproteins are macromolecular assemblies of lipids and proteins composed of
phospholipids and cholesterol that form spherical monolayers covering a core of
triglycerides and cholesteryl esters. The polar headgroups of the phospholipids and the
hydroxyl groups of cholesterol compose a hydration shell that surrounds the hydrophobic
core. Apolipoproteins (APOs) are specific protein components of the surface layer of
lipoproteins. They regulate lipoprotein metabolism by serving as receptor ligands and
cofactors/inhibitors of enzymes. Lipoproteins are divided into subclasses based on their
density (Table 1). The different lipoprotein classes have distinct origins and functions and
compose a dynamic system that maintains lipid homeostasis in blood. Chylomicrons and
VLDL are the largest lipoproteins. They are responsible for the transport of triglycerides in
blood and are therefore important carriers of energy to cells, while LDL and HDL mainly
serve as carriers of cholesterol.
Disturbances in lipid homeostasis are associated with common human diseases such as
obesity, insulin resistance and diabetes. Ultimately, lipid disorders may lead to
cardiovascular disease with fatal outcomes.
2
Chylomicrons VLDL IDL LDL HDL
Density (g/cm3) <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 (% dry weight)
Protein 1-2 10 18 25 33
Triglyceride 83 50 31 10 8
Cholesterol and cholesteryl ester
8 22 29 46 30
Phospholipid 7 18 22 22 29
Apolipoproteins B48, A, C, E B100, C, E B100, C, E B100 A, C, E
Table 1. Characteristics and composition of human lipoprotein classes [1].
Transport and metabolism of exogenous lipids
After a meal, dietary triglycerides enter the gut in large insoluble lipid droplets. Bile salts
help to emulsify these lipid droplets into smaller entities and thereby increase the accessible
surface. The protein colipase binds and promotes hydrolysis of the triglycerides by
pancreatic lipase [2]. The lipolysis products (fatty acids and monoglycerides) are taken up by
intestinal absorptive cells (enterocytes) and subsequently resynthesized to triglycerides.
With the aid of microsomal transfer protein (MTP) and APOB48 triglycerides are incorporated
into chylomicrons [3]. Chylomicrons are the largest of the lipoproteins and transport dietary
triglycerides, fat-soluble vitamins, cholesterol and cholesteryl esters (Table 1). Compared to
other lipoproteins, chylomicrons are large in size because of their massive triglyceride core.
Due to their size chylomicrons cannot pass into the fenestrated capillaries of the intestinal
mucosa. Consequently, chylomicrons are directed to the lymphatic system before entering
the bloodstream via the left subclavian vein [4]. In blood, chylomicrons acquire additional
apolipoproteins (APOCs and APOE) from high-density lipoproteins (HDL), which function as
an apolipoprotein reservoir [5,6]. In capillaries chylomicrons bind avidly to a membrane-
bound protein complex composed of lipoprotein lipase (LPL) and GPI-anchored HDL-binding
protein 1 (GPIHBP1) [7]. LPL in turn is activated by APOC2 on the chylomicron surface and
triglycerides from the core are readily hydrolyzed allowing delivery of lipolysis products to
the underlying parenchymal cells [6,7]. During intravascular lipolysis the chylomicron particle
decreases in size and is transformed into a chylomicron remnant. The chylomicron remnant
particle is remodeled by release of excess surface material to HDL while mainly APOB48 and
APOE remain bound to the remnant surface [8,9]. The small size of the chylomicron
remnants allow them to pass the fenestrated capillary endothelium in the liver followed by
cellular uptake via binding of APOE to members of the LDL (low-density lipoprotein) receptor
3
family [9]. A brief summary and schematic representation of chylomicron metabolism is
shown in Figure 1.
Figure 1. Chylomicron metabolism.
Transport and metabolism of endogenous lipids
The chylomicron is taken up in the liver by receptor-mediated endocytosis after the majority
of its core triglycerides have been depleted by intravascular lipolysis. Constituents from the
chylomicron remnant such as remaining triglycerides and dietary cholesterol, together with
lipids endogenously synthesized in the liver, are utilized in the hepatocytes for formation of
very low-density lipoprotein (VLDL). VLDL is formed by the stepwise lipidation of APOB100
with the aid of MTP [10]. After secretion from the liver the VLDL particle is rich in
triglycerides and is hydrolyzed by LPL in capillaries [7]. During lipolysis, VLDL is transformed
to an intermediate-density lipoprotein (IDL). Consequently surface rearrangements must
occur and APOCs will detach from the surface while APOE is less affected and remain on the
IDL particle [5,11]. IDL can be removed from the circulation by APOE/APOB100-mediated
uptake in the liver [12]. Alternatively, IDL can be furthered depleted of its triglyceride
content by the enzyme hepatic lipase [13]. When core triglycerides in IDL are depleted, APOE
will detach and the remaining particle is referred to as low-density lipoprotein (LDL) [14]. In
contrast to the parental VLDL particle, LDL has a high cholesteryl ester to triglyceride ratio.
LDL delivers cholesterol to extrahepatic tissues via endocytosis mediated via LDL receptors
4
[15], followed by hydrolysis of all constituents in the lysosomes. Uptake of LDL also occurs in
the liver where the cholesterol content is incorporated in VLDL or excreted to the gut via bile
[16,17]. A brief summary and schematic representation of the endogenous lipoprotein
metabolism is shown in Figure 2.
Figure 2. Endogenous lipoprotein metabolism.
Reverse cholesterol transport
HDL functions as a reservoir for APOCs and APOE in both the exogenous and the endogenous
pathways for lipoprotein metabolism. Another important role for HDL is to accept excess
cholesterol from extrahepatic tissues for transport to the liver where the cholesterol can be
excreted in bile. This HDL-mediated process is called reverse cholesterol transport. Nascent
HDL is formed in enterocytes and hepatocytes by the addition of phospholipids and free
cholesterol to APOA1 via the ATP-binding cassette transporter A1 (ABCA1) [3,18]. In the
circulation, nascent HDL is remodeled by phospholipid transfer protein (PTLP) which
transfers phospholipids to the maturing HDL particle from excess surface coats of
chylomicron remnants [19]. The phospholipid-rich nascent HDL becomes fully matured by
the acquisition of free cholesterol from extrahepatic tissues by ATP-binding cassette
transporters [20]. The majority of the mobilized cholesterol is esterified to cholesteryl esters
by the action of HDL-bound lecithin-cholesterol acyltransferase (LCAT) and thereby
relocalized from the surface of the HDL particle to the core [21]. Mature HDL release free
5
cholesterol and cholesteryl esters in the liver via binding to scavenger receptor class B1 (SR-
B1) [22]. Alternatively, HDL can be relieved of cholesteryl esters by cholesteryl ester transfer
protein (CETP) which exchange HDL-cholesteryl esters for triglycerides from other
lipoprotein classes [23].
Dyslipidemia and cardiovascular disease
Cardiovascular disease (CVD) includes conditions that narrows and block blood vessels, often
as a consequence of atherosclerosis and/or high blood pressure, leading to life-threatening
events such as heart attacks or strokes [24]. Inflammation and impaired lipid metabolism
(dyslipidemia) severely increases the risk of developing atherosclerosis. The onset of
atherosclerosis is believed to be due to discrete areas of chronic inflammation in large and
medium sized arteries due to infiltration and retention of LDL particles inside the blood
vessel wall. A cascade of events follows that ultimately lead to the recruitment of monocytes
that differentiates into macrophages which internalize lipoproteins and are transformed into
lipid-loaded foam cells – the archetypical cell in atherosclerosis [reviewed [25,26]].
Patients at increased cardiovascular risk commonly display high levels of plasma
triglycerides, elevated LDL cholesterol, small dense LDL particles and low levels of HDL-
cholesterol. Statin therapy effectively reduces CVD events in patients with elevated LDL-
cholesterol. However, many patients remain at high cardiovascular risk even after optimal
reductions in LDL-cholesterol [27]. Numerous case-control studies have established positive
correlations between plasma triglyceride levels and CVD, even after adjustment for LDL-
cholesterol and HDL-cholesterol [reviewed [28]]. However, whether triglycerides have a
causal role in the development of CVD, or serve as a predictive biomarker, has been debated
for decades. Similarly, a discussion is ongoing on the benefit of HDL-cholesterol which since
long has been shown to correlate inversely with CVD risk [reviewed [29,30]].
The level of triglycerides in blood is the result of environmental factors in combination with
common and rare variants of multiple genes that govern lipoprotein metabolism. Some
genes known to be involved in lipoprotein catabolism have a large impact on the levels of
plasma triglycerides, such as LPL or genes that have been proven essential for proper LPL
functionality. Individuals that are homozygous or compound heterozygous for loss-of-
function mutations in these genes are incapable of hydrolyzing triglyceride-rich lipoproteins
(TRLs), i.e. chylomicrons and VLDLs. Consequently these patients display severe
hypertriglyceridemia, often accompanied by low levels of HDL-cholesterol due to impaired
intravascular lipolysis. Interestingly, from a disease perspective, these patients may develop
pancreatitis due to chylomicronemia but they do not experience increased CVD risk. Instead
evidence point toward an increased CVD risk for individuals with moderate
hypertriglyceridemia [31,32]. The etiology of non-severe hypertriglyceridemia is complex
and may involve heterozygous mutations in genes affecting LPL functionality in combination
with other determinants that each may have small effects on plasma triglyceride levels
[reviewed [33]]. The current belief is that elevated fasting plasma triglyceride levels are a
6
consequence of an accumulation of deleterious alleles in genes regulating lipoprotein
catabolism. Conversely, reduced plasma triglyceride levels are believed to be due to an
accumulation of loss-of-function mutations in genes regulating lipoprotein assembly or
secretion, or genes responsible for inhibition of lipoprotein catabolism like APOC3. Thus,
plasma triglyceride levels and CVD risks are presumed to be due to the combined effects of
such deleterious and protective alleles [31].
Even though much is known about determinants for plasma triglyceride and HDL-cholesterol
levels, there are still gaps in our knowledge. This is well exemplified by the recent outcomes
from randomized control trials regarding CETP-inhibition and triglyceride lowering therapy.
CETP-inhibition resulting solely in increased plasma HDL-cholesterol was shown to be
ineffective in reducing recurrent cardiovascular events [34]. Similarly, administration of
niacin as an add-on therapy to statins failed to reduce the incidence of CVD despite
reductions in plasma triglyceride and LDL-cholesterol levels in combination with increased
levels of HDL-cholesterol [35]. It should be mentioned that there are ongoing trials with
other CETP-inhibitors, and that randomized control trials with fibrates as lipid lowering
therapy have shown beneficial effects in the prevention of coronary events [29,36].
Thus, assessing CVD risk solely based on plasma triglyceride and HDL levels may have its
caveats. However, numerous epidemiological studies infer an associated CVD risk for both
elevated triglycerides and low levels of HDL-cholesterol. Presumably future discoveries will
enable us to distinguish between benign and at-risk individuals with similar lipid profiles.
Determinants of plasma triglyceride metabolism
The catabolism of circulating TRLs is important in maintaining plasma triglyceride
homeostasis. LPL is central for this function and acts as a gatekeeper by directing fatty acids
from the circulation to underlying tissues. The demands of fatty acids differ between tissues
and depend on nutrient status as well as the relative energy consumption. As mentioned in
the beginning, excessive fatty acid delivery may lead to cellular dysfunction and death
because fatty acids at high concentrations are toxic to cells. Thus, LPL must be carefully
regulated to meet up with the tissue-specific metabolic demand. Below follows a summary
of the currently known main determinants of plasma triglyceride metabolism.
Lipoprotein lipase
The effect of LPL was first described by Hahn who noticed that administration of heparin to
dogs abolished the turbidity in blood associated with postprandial lipemia [37]. Later, Korn
concluded that the “clearing factor” was a heparin-releasable lipase that hydrolyzed core
triglycerides of large lipoproteins and consequently reduced the light-scattering in lipemic
plasma, and hence the enzyme was later named lipoprotein lipase [38].
Homozygous loss-of-function mutations in the gene for LPL are rare and result in creamy
blood plasma due to accumulated TRLs. Individuals that are heterozygous for these
7
mutations display diverse phenotypes ranging from normal to high levels of plasma
triglycerides, suggesting an increased predisposition to hypertriglyceridemia when
confounding factors are present [39]. There are numerous identified human mutations in the
LPL gene [reviewed [40]]. Loss-of-function mutations include splice site mutations,
frameshift mutations, and missense mutations affecting secretion, endothelial transport or
the catalytic function of LPL [7]. Also, other mutations may be accompanied by increased
plasma triglyceride levels and reduced levels of HDL-cholesterol [40]. In contrast, a common
gain-of-function mutation, LPL-S447X, has been associated with increased pre- and post-
heparin LPL activity, reduced levels of plasma triglycerides and increased levels of HDL-
cholesterol [41,42]. Numerous epidemiological studies have suggested that S447X-carriers
have a lower risk for CVD compared to non-carriers, while other investigations have not
been able to confirm these observations [42].
LPL is a 448 amino acid residue glycoprotein that is mainly synthesized in heart, skeletal
muscle and adipose tissue. Other tissues also produce LPL. Of special interest is LPL in
macrophages which may promote atherosclerosis (discussed below). LPL catalyzes the ester
hydrolysis of triglycerides in TRLs, resulting in the release of fatty acids and monoglycerides.
LPL is a serine hydrolase and a member of the lipase gene family with highest sequence
homology to endothelial lipase followed by hepatic lipase and pancreatic lipase [43]. Due to
the lack of available 3-D structure for LPL or any of its closest relatives an in silico modelling
was performed based on the crystal structure of pancreatic lipase [44]. By analogy with
pancreatic lipase it is assumed that LPL is composed of two domains; a large N-terminal
domain (residues 1-312) which contains the active site and a smaller C-terminal domain
(residues 313-448) that is required for binding to lipid substrates [45]. Unlike pancreatic
lipase, LPL is catalytically active only as a non-covalent homodimer [46]. It was suggested
that the two LPL monomers are oriented in a head-to-tail fashion where the C-terminal
domains bind to the surface of lipid particles and allow the opposing catalytic N-terminal
domains to act on individual lipid molecules in the surface layer of lipoproteins (Figure 3)
[45,47]. Evidence for this is that co-expression of two inactive forms of LPL, with loss-of-
function mutations in the C-terminal and N-terminal domains, respectively, resulted in
catalytically active LPL [48]. LPL has high affinity for heparin. This was early indicated by the
release of lipolytic activity to blood upon intravenous heparin injections [37,38], and this
property was used for the initial large-scale purifications of LPL from bovine milk [49]. Later
it has been demonstrated that surface exposed clusters of positive charges located in both
the N- and C-terminal domains of LPL interact with negatively charged sulphate groups in
heparin [44]. By using heparin-Sepharose chromatography it was shown that LPL protein
elutes at two distinct salt concentrations, approximately at 0.5 M and 1 M of NaCl
respectively, and that catalytic activity was only associated with the 1 M NaCl fraction [50].
Analytical ultracentrifugation and sedimentation studies in sucrose gradients have shown
that native LPL has a molecular mass of about 110 kDa, corresponding to a non-covalent
homodimer of 55 kDa subunits [46].
8
Figure 3. Homology model of LPL based on the crystal structure of pancreatic lipase using SWISS-MODEL [51,52,53]. The N-terminal (orange) of one LPL monomer is presumed to interact with the opposing C-terminal (gray) of another LPL monomer, forming a head-to-tail homodimer.
LPL can hydrolyze triglycerides and phospholipids in all lipoprotein classes. However, when
different substrates are presented in a mixture, such as in the plasma, triglycerides of TRLs
are almost exclusively hydrolyzed, presumably due to the preferred binding of LPL to TRLs
and to that triglycerides are a preferred substrate over phospholipids [54]. Cleavage of LPL
by chymotrypsin at residues 390-391 and 392-393 abolished binding and enzymatic activity
towards TRLs, while the activity towards the more water-soluble substrates tributyrin and
para-nitrophenyl butyrate remained unaffected. These findings suggested a anchoring
function of the C-terminal domain of LPL to the surface of the lipid droplets [55]. The
chymotrypsin-truncated form of LPL was capable of binding to synthetic lipid emulsions
although with severely impaired lipolytic capability and the authors proposed that the C-
terminal domain is also crucial for a correct interfacial orientation of the enzyme. APOC2
9
increases the LPL activity on synthetic lipid emulsions composed of long-chain triglycerides,
but has little or no effect with short-chain triglycerides (e.g. tributyrin) [56]. By the use of
chimeras (hepatic lipase and lipoprotein lipase) it was suggested that APOC2 interacts
directly both with the LPL C-terminal domain after residue 388 and with the N-terminal
domain, presumably each on different subunits [57]. Other studies showed, however, that
chymotrypsin-cleaved LPL was dependent on APOC2 and had a maximal activity of about
25% compared to that of intact LPL for lipid emulsions of long-chain triglycerides. Soluble
ester substrates like tributyrin were hydrolyzed just as well with the chymotrypsin-cleaved
LPL as they were with the intact enzyme [55].
When LPL hydrolyzes long-chain triglyceride substrates, the activity becomes markedly
reduced with time unless a fatty acid acceptor is present (e.g. albumin) due to product
inhibition. In the absence of fatty acid acceptors, the LPL reaction may seemingly come to a
stop, but on addition of albumin the inhibition is immediately relieved [58]. The authors
suggested that LPL forms a complex with fatty acids that prevents further hydrolysis of
triglycerides, and that this could serve as a feed-back mechanism to prevent excessive fatty
acid delivery to cells and tissues in vivo.
LPL contains two conserved N-linked glycosylation sites. In human LPL these are located at
Asn 43 and Asn 359. Expression studies with mutated LPL in COS cells demonstrated
intracellular accumulation within the endoplasmic reticulum (ER) and complete loss of
enzymatic activity when Asn 43 was substituted for Ala. In contrast, the LPL mutant N359A
was secreted, and showed only a minor loss in enzymatic activity compared to the wild-type
protein [59,60]. The ability of cells to properly secrete active LPL depends on the unique ER
membrane protein called lipase maturation factor 1 (LMF1). Recently it was shown that
homozygous loss-of-function mutations in the LMF1 gene cause severe hypertriglyceridemia
due extremely low LPL activity, combined with moderate reductions in hepatic lipase activity
in post-heparin plasma [61]. It was suggested that LMF1 functions as a chaperone for the
assembly of functional homodimers for LPL, endothelial lipase and hepatic lipase [62]. Unlike
humans with LPL deficiency, Lpl-knockout mice die soon after birth. They can be rescued by
transient expression of LPL from adenovirus during the neonatal stage [63,64]. Lpl-knockout
pups are mildly hypertriglyceridemic immediately after birth but on suckling they manifest
severe hypertriglyceridemia with markedly reduced levels of LDL- and HDL-cholesterol
compared to wild-type pups. Heterozygous Lpl-knockout pups display only mild
hypertriglyceridemia during suckling [63]. In contrast, transgenic mice that overexpress LPL
display hypotriglyceridemia with increased LDL-cholesterol levels and a concomitant
decrease in VLDL-cholesterol. These observations were suggested to reflect a more rapid
conversion of VLDL to LDL [65].
Chylomicrons and large VLDLs do not readily move across the capillary endothelium. Thus,
lipid delivery to cells depends on the activity of LPL that generates more polar lipolysis
products, fatty acids and monoglycerides, which can cross cellular membranes. LPL also
10
promotes cholesterol delivery to cells by initiating the formation of LDL particles from
parental VLDLs [66]. Released lipolysis products are predominantly taken up by parenchymal
cells and used as energy source or stored as triglycerides in lipid droplets for later use.
Tissues with a high energy demand, such as striated muscle tissues (heart and skeletal
muscle), primarily use fatty acids as an energy source. Adipose tissue instead stores lipolysis
products for later mobilization when dietary lipids are no longer available. To accommodate
for efficient and appropriate fatty acid delivery, the activity of LPL is rapidly modulated in
different tissues to readily adapt to their metabolic needs [54]. The rapid regulation of LPL
activity is believed to mainly occur at the post-transcriptional level. The current knowledge
regarding the tissue-specific regulation of LPL suggests two conceptually different
mechanisms. In adipose tissue it appears that regulation occurs by irreversible inactivation
of the enzyme, while in striated muscle the amount of active LPL that is located on the
vascular side of the endothelium in contact with blood seems to be regulated. A summary of
the tissue-specific regulation of LPL in rats follow below.
Regulation of lipoprotein lipase activity
In adipose tissue the nutrient status is presumed to be the main determinant of the level of
LPL activity. In the fed state LPL activity is high to promote lipid storage. In contrast, the
enzymatic activity is markedly reduced in the fasted state. By comparing fed and fasted rats,
Olivecrona and coworkers showed that fasting had modest effects on LPL mass while the
ratio of active LPL dimers over inactive monomers was substantially decreased with a
concomitant loss of LPL activity in adipose tissue of the fasted animals [67]. Subsequent
studies showed that administration of the transcription blocker actinomycin D to fasted rats
rapidly reverted the fasting-induced reduction in LPL activity and this was accompanied by
an increased ratio of active LPL dimers over inactive monomers [68]. These observations
could not be explained by changes in LPL mRNA or mass. It was suggested that another
protein, with a more rapid turnover, causes inactivation of LPL in adipose tissue on fasting.
Later studies showed that angiopoietin-like protein 4 (ANGPTL4) could inactivate LPL by
converting active dimers to inactive monomers. In addition, the expression of ANGPTL4 in
adipose tissue correlated with LPL activity during both fed-to-fasted and fasted-to-fed
transitions [69]. It now seems likely that ANGPTL4 is a key determinant for regulation of LPL
activity in adipose tissue [70].
In contrast to adipose tissue, LPL activity in homogenates of rat hearts does not change
much on fasting. More detailed studies have shown that the amount of heparin-releasable
LPL activity increases during fasting [71,72]. An advantage with the heart is that it allows for
extracorporeal perfusion experiments in which LPL bound to the endothelium facing the
lumen can be released by a heparin-containing medium and then quantified [73]. Due to
technical difficulties to perfuse adipose tissue, an alternative was to compare the LPL activity
in total tissue to that of isolated adipocytes from the same tissue. With this method it was
concluded that the specific LPL activity (activity/mass) was reduced only in the extracellular
11
fraction of rat adipose tissue upon fasting [74]. It is noteworthy that the extracellular
fraction of LPL in adipose tissue represented approximately 70% of the total LPL pool. In
following studies of the perfused rat heart it was shown that only 10-20% of the total LPL
pool was released upon heparin addition [75]. It remains to be determined if the residual
pool of LPL resides within the interstitial spaces or inside cardiomyocytes. The specific
activity of LPL in homogenates of heart is not affected by nutrient status. Instead there was a
shift in the amount of heparin-releasable LPL that was increased during fasting. Conversely,
heparin-releasable LPL mass was low in the fed state but could be reverted by the
administration of actinomycin D [75]. These observations suggest a regulatory mechanism
involving a protein responsible for transporting LPL to its endothelial site of action, rather
than an effect on the catalytic function of LPL as seen in adipose tissue. A strong candidate
for this regulation is GPIHBP1 that has been found to transport LPL across the capillary
endothelium [76]. More studies are needed to understand how GPIHBP1 transports LPL to
the capillary lumen and if additional factors are involved in this process. The mechanisms for
regulation of LPL activity in skeletal muscle is less understood than in heart and adipose
tissue. Changes in LPL activity were reported to correlate with LPL mRNA and enzymatic
activity has been found to increase during fasting. Several studies correlate acute exercise
with increased expression of LPL and with increased LPL activity in skeletal muscle [71].
Hindlimb unloading (inactivity) demonstrated a dramatic reduction in LPL activity in the
soleus muscle of rats compared with that in the muscles of ambulatory control rats. The
observed loss in LPL activity could not be explained by a decrease in tissue LPL mass.
However, the amount of heparin-releasable LPL mass was markedly reduced. The reduction
in LPL activity associated with physical inactivity was prevented by administration of
actinomycin D, but also reverted by treadmill walking [77]. Taken together, LPL regulation in
skeletal muscle show several similarities to that of the heart and point toward GPIHBP1 as
part of the regulatory process in times when there is a local energy demand.
Lipoprotein lipase in atherosclerosis
The role of LPL in atherosclerosis is dual-faceted. It was early postulated that the action of
LPL may be pro-atherogenic because remnant lipoproteins, depleted of their triglyceride
core, should be capable of entering the arterial wall while unprocessed large TRLs remain in
the circulation [78,79]. In support of this presumption, individuals with homozygous loss-of-
function mutations in the LPL gene, or in genes responsible for proper enzymatic
functionality, are not associated with severely increased CVD risk [31]. However, the
involvement of LPL in atherosclerosis is far more complex than originally anticipated.
Individuals with heterozygous loss-of-function mutations in the LPL gene have been reported
to be predisposed to premature atherosclerosis [80]. Although unprocessed circulating TRLs
are not able to cross the endothelial barrier, evidence point toward involvement of remnant
lipoproteins with not fully depleted triglyceride cores in the development of atherosclerosis.
To further add complexity, it was shown that severely hypertriglyceridemic mice, deficient of
12
either LPL or GPIHBP1, spontaneously develop atherosclerosis on a normal diet [81,82].
Whether these observations are species-specific effects warrant further studies
It was suggested that lipolysis products derived from LPL may promote atherosclerosis by
causing vascular injury and increased endothelial permeability. This was based on in vitro
studies using aortic endothelial cells [83]. Previous studies had shown that injection of
labeled human VLDLs followed by injections of LPL increased the amount of VLDL-label in the
arterial wall of perfused rat carotid arteries compared to when no LPL was injected.
Injections of HDL prior to injections of the labeled VLDLs and LPL prevented the VLDL-label to
enter the arterial wall [84]. Assuming that LPL is not present at the luminal endothelium of
arteries, due to the lack of GPIHBP1 in these vessels [7], the pro-atherogenic effects of
lipolysis products should be due to either effects of lipolysis spillover in adjacent tissues
[reviewed [85]] – or to that catalytically active LPL within the arterial wall (e.g. macrophage
LPL) may account for such effects by hydrolysis of triglycerides in retained remnant
lipoproteins. Irrespectively, the observation regarding the protective effects of HDL is highly
interesting. Possibly a similar mechanism could account for the anti-atherogenic effects seen
by overexpression of LPL in atherosclerotic mice models that lacks the LDL-receptor (LDLR)
or its ligand APOE. When fed an atherogenic diet, the combined Ldlr-knockout and LPL-
transgenic mice displayed an 18-fold reduction in atherosclerotic mean lesion area
compared to Ldlr-knockout mice. As expected, overexpression of LPL increased HDL-
cholesterol levels and erased the relatively mild hypertriglyceridemia associated with the
LDLR deficiency. Interestingly, LPL overexpression reduced plasma LDL-cholesterol levels by
approximately 50% without effects on circulating APOB levels [86]. Similar experiments on
Apoe-knockout mice showed a more modest two-fold reduction in mean lesion area upon
LPL overexpression. The lesion area size was in the same order of magnitude as that
observed for Ldlr-knockout mice in the previous study. Analogous to the previous study the
combined Apoe-knockout and LPL-transgenic mice showed lower plasma triglycerides levels
compared to Apoe-knockout mice while there were no differences in circulating APOB levels.
However, in contrast to the Ldlr-knockout mice, total cholesterol levels remained unaffected
[87]. The detailed mechanism behind why LPL overexpression protects against
atherosclerosis is intriguing but remains unknown. However, it is striking that cholesterol
levels were only affected in Ldlr-knockout mice who also benefitted dramatically by LPL
overexpression in terms of reduced atherosclerotic lesion area. Studies on transgenic rabbits
overexpressing LPL show conflicting results. One study showed that overexpression of LPL
protected against diet-induced atherosclerosis [88]. The same group later showed that
overexpression of LPL promoted atherosclerosis [89]. Their suggestion for these
discrepancies were that in the first study reduced lesion formation was a result of reduced
plasma cholesterol in the transgenic animals, while in the second study cholesterol levels
were similar between transgenic and wild-type rabbits. The authors proposed that the anti-
atherogenic effects of LPL are due to increased remnant removal while the pro-atherogenic
effects of LPL are caused by an increased amount of atherogenic LDL particles due to a more
rapid and extensive depletion of VLDL-triglycerides. The latter suggestion is in contrast to the
13
observations seen in mice deficient of either LDLR or APOE, as discussed above. Possibly
there are confounding effects which differ among species.
In contrast to the anti-atherogenic properties of endothelial bound LPL in mice, LPL in
macrophages is presumed to be pro-atherogenic. By ablating endogenous macrophages in
Ldlr-knockout mice using lethal irradiation, Babaev et al. showed that transplantation of
macrophages from an LPL-deficient donor reduced the mean lesion area by approximately
30% when compared with irradiated littermates transplanted with macrophages which had
normal LPL expression levels [90]. In this study the lipid profiles were not different between
the groups of mice, suggesting that macrophage transplantation had no impact on
circulating TRLs. Reductions in mean lesion areas were seen upon irradiation followed by
macrophage transplantations in wild-type C57BL/6 mice [91,92]. It should be mentioned that
these mice only developed minor lesions even when LPL-expressing macrophages were
transplanted. Gustafsson et al. were successful in reproducing the observations previously
made by Babaev et al. with macrophage transplantation from LPL-deficient donors into
irradiated Ldlr-knockout mice [93]. They also showed that LDL retention in the aortae was
reduced in mice with LPL-deficient macrophage transplants compared to those transplanted
with LPL-expressing macrophages. They overexpressed both catalytically active and inactive
LPL in macrophages on an APOE-deficient background. Modest overexpression of
macrophage LPL in Apoe-knockout mice increased lesion size area by approximately 30%
independently of catalytic ability, suggesting that the main effects are due to the non-
catalytic properties of LPL [93]. Wu et al. showed that local transient overexpression of
either catalytically active or inactive LPL in the carotid artery of rabbits receiving a normal
diet resulted in rapid lipid deposition in the arterial wall [94]. It is evident that LPL exerts pro-
atherogenic functions in the arterial wall, mainly by non-enzymatic effects. LPL can mediate
binding and subsequent uptake of atherogenic lipoproteins in macrophages independent of
LDLR [95]. It was also shown that LPL can bridge lipoproteins to the extracellular matrix,
suggesting that LPL could promote retention of atherogenic lipoproteins in the artery wall
[96].
Angiopoietin-like proteins
Several lines of evidence implicate that members of the angiopoietin-like protein family
(ANGPTL3, -4, -8) are important LPL modulators. Transgenic mouse models overexpressing
either ANGPTL3, -4 or -8 all display increased circulating triglyceride levels, while
corresponding knockout models all show reduced triglyceride levels compared to wild-type
mice [71]. Importantly, ANGPTLs differ in their expression, regulation and tissue distribution
suggesting that they have distinct roles in lipid metabolism. In support of this is that Angptl3-
knockout mice show increased LPL activity only in the fed state while the increase in LPL
activity for Angptl4-knockout mice is more pronounced in the fasted state [97]. This suggests
that the ANGPTLs may act as control proteins for LPL activity but under different metabolic
conditions.
14
ANGPTL4 is under the transcriptional control of peroxisome proliferator-activated receptors
(PPARs) [98,99]. Fatty acids are PPAR agonists, suggesting a feed-back mechanism involving
ANGPTL4 with the aim to control lipid homeostasis [100,101,102]. Mice display a complex
regulation of ANGPTL4 partly due to overlapping functions between PPAR isoforms.
ANGPTL4 is synthesized by numerous tissues in mice, including white and brown adipose
tissue, skeletal muscle, heart, liver and macrophages, supporting that it has a role as a
paracrine factor to control LPL activity [71]. Hepatocytes do not normally produce LPL, while
ANGPTL4 is mainly produced by the liver in humans. ANGPTL4 is found in blood, and
intravenous injection of recombinant ANGPTL4 to mice, or overexpression of ANGPTL4 in
mouse livers, have effects on LPL activity in post-heparin plasma, suggesting effects on the
total tissue pool of LPL [97,103]. Thus, an endocrine role of ANGPTL4 circulating in blood
cannot be excluded [71]. There are conflicting results on whether ANGPTL4 may affect
hepatic lipase activity. Transgenic overexpression of ANGPTL4 in mice showed no effects on
post-heparin hepatic lipase activity in one study, while a 50% reduction was found in another
study [97,104]. There are no reports whether ANGPTL4 have effects on endothelial lipase.
ANGPTL4 consists of two domains, an N-terminal domain containing a coil-coiled region,
followed by a C-terminal fibrinogen-like domain. Both the full-length protein and the N-
terminal domain can inhibit LPL activity, while the C-terminal domain alone has no
inactivating effect on LPL [99,103]. The mechanism for LPL inhibition is presumed to be due
to conversion of active LPL dimers to inactive monomers [69]. Recently, another study
suggested that ANGPTL4 inhibits LPL via a non-competitive, reversible mechanism and
thereby prevents LPL to reach its site of action in vivo [105]. It is noteworthy that in this
study deoxycholic acid was used to prevent the spontaneous inactivation of LPL. In the
presence of other stabilizing factors like substrate lipoproteins, LPL is less susceptible to
become inactivated by ANGPTL4 [106].
Generation of Angptl4-knockout mice, ANGPTL4-transgenic mice as well as injection of
recombinant ANGPTL4 or injection of an neutralizing antibody against ANGPTL4 to different
mouse models infer effects on LPL in vivo [97,103,107]. Interestingly, intercrossing Angptl4-
knockout mice with atherosclerotic-inducible Apoe-knockout mice suppressed foam cell
formation and protected against atherosclerosis compared with APOE-deficient control mice
[108]. In contrast, Angptl4-knockout mice given a diet high in saturated fat developed a
lethal phenotype with lipid-laden (foamy) macrophages in the mesenteric lymph nodes
[101]. The authors proposed that ANGPTL4 prevents macrophage activation by reducing
fatty acid uptake from triglycerides in chyle mediated by LPL. Another study showed that
ANGPTL4 mimics the effects of tetrahydrolipstatin, an active-site inhibitor of LPL [109],
regarding reduced uptake of oxidized LDL in macrophages [110]. In the same study,
transgenic mice overexpressing ANGPTL4 were intercrossed with atherosclerosis-prone
apolipoprotein E*3-Leiden mice. Combined ANGPTL4-transgenic and E*3-Leiden mice
showed no difference in atherosclerosis susceptibility but displayed a 34% reduction in total
lesion area size compared to E*3-Leiden mice. Previous studies have demonstrated that LPL
15
activity is under control of ANGPTL4 in macrophages [111]. Together these studies raise the
possibility that the catalytic activity of LPL in macrophages may have pro-atherogenic effects
in terms of atherosclerotic lesion progression.
In population studies it was found that a missense mutation in the ANGPTL4 gene, E40K, was
present in approximately 3% of European Americans and resulted in reduced levels of
plasma triglycerides and increased levels of HDL-cholesterol [112]. Later studies showed that
a recombinant N-terminal fragment of ANGPTL4 containing the E40K mutation was unable
to bind to LPL explaining the lack of function [113]. Other rare mutations within the
ANGPTL4 gene have also been associated with reduced levels of plasma triglycerides
[114,115]. Although loss-of-function mutations in the ANGPTL4 gene are associated with an
anti-atherogenic lipid profile, there are conflicting results regarding the beneficial effect of
these mutations on cardiovascular outcomes. One study showed that E40K-ANGPTL4 carriers
were protected from coronary heart disease (CHD) compared to non-carriers [116]. In
contrast, another study showed an association between E40K-ANGPTL4 and increased risk
for CHD [115]. Further studies are needed to validate the role of loss-of-function mutations
in the gene of ANGPTL4 and their implications for CVD. In addition to effects on lipid
metabolism, ANGPTL4 has other non-metabolic functions, presumably linked to the C-
terminal fibrinogen-like domain [reviewed [117]].
ANGPTL3 was the first member of this gene family to be recognized for its involvement in
lipid metabolism. A mutant strain of KK obese mice (KK/San) showed marked reductions in
plasma free fatty acids, and levels of total cholesterol (including HDL-cholesterol) and
triglyceride compared to KK mice. It was later found that the hypotriglyceridemic mice were
homozygous for a mutation in the Angptl3 gene and that mutations in the human ortholog
gene also gave rise to hypolipidemia [118,119]. Fasted Angptl3-knockout mice displayed
approximately 50% increased post-heparin LPL and hepatic lipase activities compared to
wild-type mice [120]. Another study showed opposite effects, where Angptl3-knockout mice
had slightly increased LPL activity in the fasted state while activity was dramatically
increased in the fed state compared to wild-type mice [97]. ANGPTL3 was also reported to
inhibit endothelial lipase activity and suggested to account for the low levels of HDL-
cholesterol seen in ANGPTL3-deficient mice [121].
The biochemical mechanism for how ANGPTL3 inhibits LPL activity is unclear. Compared to
ANGPTL4, several-fold more ANGPTL3 was required to cause LPL inactivation [122]. Based on
kinetic studies it was suggested that ANGPTL3 causes inhibition of LPL rather than
irreversible inactivation of the enzyme [123]. It was proposed that ANGPTL3 increases the
susceptibility of LPL to become cleaved by proprotein convertases [124]. Similar effects were
reported for ANGPTL4 [125]. One possible explanation for this could be that LPL is more
prone to cleavage by proteinases in its monomeric conformational state [126]. LPL
monomers are formed on inactivation by ANGPTL4 and these data suggests that also
ANGPTL3 may inactivate LPL by a dimer to monomer conversion.
16
ANGPTL3 is exclusively expressed in the liver both in mice and humans. Like ANGPTL4,
ANGPTL3 is found in blood. This suggests the effects of ANGPTL3 on the LPL system is
accomplished by the circulating protein. Due to the modest effects of ANGPTL3 on LPL in the
presence of TRLs [106], it is hard to understand the mode of action of ANGPTL3 in vivo. It
was recently suggested that ANGPTL3 acts in concert with ANGPTL8 [127]. ANGPTL8 is
expressed in liver, brain and adipose tissue in mice and humans. Interestingly, the expression
of ANGPTL8 is increased by feeding and decreased during fasting (in mice), suggesting a role
of ANGPTL8 as a LPL controller in the fed state. More studies are needed to corroborate
these observations, and to unravel the biochemical mechanisms for the effects of the
ANGPTLs on the LPL system, both in mice and humans.
GPI-anchored HDL-binding protein 1
Previous dogma held that LPL interacted with heparane sulphate proteoglycans at its site of
action on the luminal capillary endothelium. This view has been changed with the discovery
of GPIHBP1, a GPI-anchored protein of capillary endothelial cells, and the evidence for the
importance of GPIHBP1 for plasma triglyceride metabolism [76]. It was clearly demonstrated
by Stephen Young and coworkers that GPIHBP1 binds to LPL in the subendothelial spaces
and shuttles the enzyme to the capillary lumen where it can interact with TRLs. When
GPIHBP1 is absent, LPL remains in the interstitial spaces. This mislocalization of LPL results in
severe hypertriglyceridemia [128]. Recently it was shown that the complex of LPL and
GPIHBP1 on endothelial cells is crucial for the so called margination of TRLs, referring to the
attachment of the TRLs to the luminal side of the endothelium allowing triglyceride
hydrolysis by LPL to proceed [129]. After the discovery of GPIHBP1, several human loss-of-
function mutations in the gene of GPIHBP1 have been reported [reviewed [130]]. Common
for these mutations are that homozygous carriers have extremely low post-heparin LPL
activity in plasma, and that they display a similar phenotype as those with LPL-deficiency.
Heterozygous carriers have approximately 50% of the LPL mass and corresponding activity in
post-heparin plasma compared to non-carriers. The plasma triglyceride levels are however
usually within the normal range [131]. This is an interesting observation suggesting that
GPIHBP1 is not rate-limiting in terms of plasma triglyceride removal, but it does indicate that
the subendothelial pool of LPL is in excess compared to GPIHBP1. These facts opens up for
that the transport of LPL to the capillary lumen might very well serve as a regulatory element
for controlling fatty acid delivery to cells in striated muscle, as discussed previously.
Human GPIHBP1 is made up of two domains followed by a GPI-anchor which attaches the
mature protein to the endothelial cell surface layer. The N-terminal domain consists of a
stretch of 25 amino acid residues highly enriched in negatively charged aspartic and glutamic
acid residues. Deletion of this domain or alanine-replacements of all negatively charged
amino acid residues in the N-terminal domain, abolished interaction with LPL. In the C-
terminal domain 10 cysteine residues are highly conserved across species. These residues
are presumed to form intramolecular disulphide bonds, similar to those observed in
UPAR/ly6 proteins. By cysteine to alanine replacement studies it was found that each
17
individual cysteine residue was essential for the interaction with LPL but not for secretion of
GPIHBP1 to the cell surface. In addition, it was found that an epitope adjacent to the N-
terminal acidic domain was still exposed in loss-of-function mutations in the C-terminal ly6-
domain [132,133]. These observations suggest that the C-terminal ly6-domain binds directly
to LPL, alternatively that it positions the N-terminal acidic domain in such a way that it can
interact with LPL.
Gpihbp1-knockout mice are severely hypertriglyceridemic due to the mislocalization of LPL
within interstitial spaces [128]. When mice that were genetically manipulated to express LPL
in endothelial cells were intercrossed with Gpihbp1-knockout mice, the margination of TRLs
was not stimulated, while plasma triglycerides were markedly reduced. The authors
suggested that this was due to lipolysis by LPL secreted to the blood stream, rather than to
lipolysis ocurring on the plasma membranes of the endothelial cells. Interestingly,
intercrossing Gpihbp1-knockout mice with Angptl4-knockout mice resulted in offspring with
mild hypertriglyceridemia. It was shown that GPIHBP1 protects LPL against both ANGPTL3-
and ANGPTL4-mediated inactivation under in vitro conditions. In addition, administration of
an antibody against ANGPTL4 in fasted Gpihbp1-knockout mice almost normalized their
triglyceride levels while the effects were much less pronounced using a corresponding
antibody against ANGPTL3 in fed Gpihbp1-knockout mice [122]. What is peculiar about these
findings is that in the absence of GPIHBP1 no LPL should be transported to the capillary
lumen. Another mystery is that in contrast to humans, LPL can be slowly released to blood
by heparin in Gpihbp1-knockout mice. The release of LPL is markedly delayed in these mice.
Yet, LPL mass and specific activity is similar to wild-type mice after 10 minutes, suggesting
that the LPL pool is not inactivated [134]. With this in mind it is hard to understand how
ANGPTL4-deficiency could ameliorate the hypertriglyceridemia associated with GPIHBP1-
deficiency. Another possibility for such an observation could be that ANGPTL4 enables LPL to
passively travel across the endothelial barrier to the capillary lumen.
Apolipoproteins
Apolipoproteins constitute the protein content of lipoproteins (see Table 1) and are located
on the surface of the lipoprotein particles. The APOBs initiate lipoprotein assembly and are
present in one copy per lipoprotein particle and remain attached during the entire metabolic
cycle of the lipoprotein. In contrast, the other major apolipoproteins (APOAs, APOCs and
APOE) are not permanently attached to one lipoprotein particle. Instead, these
apolipoproteins are frequently exchanged among the lipoproteins and function as receptor
ligands and enzyme cofactors/inhibitors. The exchangeable apolipoproteins share a
secondary structural motif referred to as an amphipathic α-helix [135]. The amphipathic α-
helix has a non-polar face which is presumed to associate with the proximal parts of the fatty
acyl chains of the surface phospholipids on the lipoproteins. On the opposing face of the
helix there are charged amino acid residues disposed such that they can interact with
solvent molecules and the polar headgroups of the phospholipids [136,137]. The
18
amphipathic properties of the α-helix prevent apolipoproteins to bury into the hydrophobic
environment of lipoproteins. As a result, the apolipoproteins remain at the lipid/water
interface which facilitates transfer between lipoproteins and enables interaction with
enzymes and cell-surface receptors [138].
In humans the genes for the main exchangeable apolipoproteins are primarily clustered at
two distinct chromosomal regions. It was suggested that apolipoproteins have diverged
through evolution from a common ancestral protein [139]. The genes (APOA1/C3/A4/A5)
and (APOE/C1/C4/C2) are located on chromosome 11 and 19 respectively. The gene for
APOA2 is on chromosome 1 and the gene for the main non-exchangeable apolipoprotein
APOB is on chromosome 2 [138]. Below follows a brief description of the most studied
apolipoproteins with focus on the APOCs and their relationship to LPL. In addition, another
apolipoprotein, APOA5, is discussed for its particular, and yet not fully understood,
importance for plasma triglyceride metabolism.
Apolipoprotein A1
Individuals with a premature stop mutation in the gene of APOA1 have markedly decreased
levels of HDL-cholesterol and develop premature CHD [140]. APOA1 is mainly synthesized in
the liver where it interacts with ABCA1 which aids in the formation of nascent HDL by the
addition of phospholipids and cholesterol. APOA1 is also synthesized by enterocytes and
becomes associated with chylomicrons. During intravascular lipolysis HDL is matured by the
release of chylomicron surface material along with APOA1 in a process that depends on both
LPL and PTLP [141,142]. The role of LPL for HDL metabolism is complex [as discussed [143]].
HDL has major functions in reverse cholesterol transport as acceptors for cholesterol,
mediated from cells by ABC-transporters. APOA1 activates LCAT which enables incorporation
of cholesterol into the lipid core of HDL through conversion of free cholesterol to cholesteryl
esters [144]. In addition, APOA1 acts as a ligand for SR-B1 and thus facilitates cholesterol
uptake in the liver [145].
Apolipoprotein B
APOB is synthesized in both liver and intestine where it initiates intracellular lipoprotein
assembly with the aid of MTP. Liver-specific expression of APOB (APOB100) translates into a
polypeptide chain with 4536 amino acid residues, while the intestine expresses an truncated
form corresponding to approximately 48% of the amino acid sequence of the full-length
protein. Hence, it is referred to as APOB48 [146]. Consequently, in humans chylomicrons are
assembled with APOB48 while VLDLs are assembled with APOB100 [146]. In contrast, mice
synthesize APOB48 lipoproteins also in the liver [147]. The main difference between the two
isoforms is that APOB48 lacks the ability to interact with members of the LDL-receptor family,
and therefore cannot be internalized into cells via this pathway. In order for chylomicron
remnants to interact with members of the LDL-receptor family these lipoproteins must carry
APOE. The importance of APOB100 is highlighted in patients suffering from a condition
19
referred to as familial defective apolipoprotein B100. These individuals display
hypercholesterolemia and have increased risk for developing premature atherosclerosis due
nonsense mutations in the gene of APOB resulting in a truncated protein of 3500 amino acid
residues [146].
Apolipoprotein C1
There are no known monogenic loss-of-function mutations in the human APOC1 gene.
APOC1 is a 57 amino acid residue polypeptide primary synthesized in the liver. APOC1 is a
constituent of chylomicrons, VLDL and HDL in the circulation [148]. APOC1 inhibits LPL
activity in vitro, but the mechanism for this effect has not been previously described [149].
Structurally, APOC1 is composed of two long amphipathic α-helices both with polar faces
composed of negative residues (Figure 4) [150]. Point mutations in either of the two helices
strongly influence the ability of APOC1 to bind to, to modify and to be retained on
lipoprotein-like structures [151]. There are currently no reported mutagenesis studies on
APOC1 that examine effects on LPL inhibition. It was shown that APOC1 can interfere with
APOE-dependent, but not APOB100-dependent, uptake of remnant lipoproteins. These
observations were suggested to be the result of either conformational changes of APOE or
displacement of this protein from the lipoprotein by the presence of APOC1 [152,153].
Surprisingly, Apoc1-knockout mice display slightly elevated plasma triglyceride levels and are
more prone to develop hypercholesterolemia compared to wild-type mice when challenged
with a severe atherogenic diet, suggesting impaired lipoprotein remnant clearance [154].
Early studies on APOC1-transgenic mice showed increased plasma cholesterol and
triglyceride levels compared to wild-type mice. The effect of APOC1 was presumed to be due
to impaired clearance of VLDL [155]. APOC1-transgenic mice that were intercrossed with
Apoe-knockout mice displayed increased plasma cholesterol and triglyceride levels
compared to Apoe-knockout mice. However, triglyceride levels were several-fold more
increased than the corresponding cholesterol levels. The authors suggested that the main
effect of overexpression of APOC1 was due to inhibition of LPL [156]. Animal studies also
suggest that physiological levels of APOC1 have effects on plasma lipid levels. By comparing
combined Apoe/Apoc1-knockout mice with Apoe-knockout mice it was proposed that
endogenous levels of APOC1 increase VLDL production and inhibit LPL activity [157]. APOC1
was also suggested to be involved in reverse cholesterol transport by acting as an activator
for LCAT [144] and as an inhibitor of CETP in humans [158].
20
Figure 4. High-resolution structure of human APOC1 in complex with sodium dodecyl sulfate micelles [150].
Apolipoprotein C2
Homozygous loss-of-function mutations in the human gene of APOC2 are rare and manifests
in chylomicronemia and reduced levels of HDL-cholesterol, almost as severe as seen on LPL
deficiency [159,160]. Several mutations that result in low or absent plasma levels of APOC2
have been reported and include premature stop mutations, donor splice-site mutations,
exon missense mutations or null mutations within the promotor region [reviewed [161]].
Individuals heterozygous for loss-of-function mutations in the APOC2 gene display normal
plasma triglyceride levels, unless genetic or environmental confounding factors are present
[162]. Interestingly, one case of drug-resistant hypertriglyceridemia was reported and
suggested to be due to high levels of plasma APOC2 [163].
APOC2 is a 79 amino acid residue polypeptide primarily synthesized in the liver. APOC2 is
present on chylomicrons, VLDL and HDL [6,164]. High concentration of APOC2 inhibits LPL
activity in vitro, but the explanation for these observations has not been studied in detail
[149]. At physiological concentrations, APOC2 increases the activity of LPL on TRLs and
emulsified long-chain triglyceride substrates [56,165]. However, there are important
differences in the dependency of LPL for APOC2 with different types of lipid emulsions. For
instance, synthetic lipid emulsions of triglycerides made up from long-chain fatty acids are
hydrolyzed to some extent by LPL even without APOC2, referred to as the basal activity of
LPL. Addition of APOC2 may increase the basal activity about 5-fold [166]. However, with
APOC2-deficient chylomicrons as substrate, the basal activity of LPL was extremely low and it
therefore increased >100-fold upon addition of exogenous APOC2 [167]. Structurally APOC2
is made up of three amphipathic α-helices all with negative polar faces spanning between
residues 16–38, 45–57, and 65–74 respectively (Figure 5) [168,169]. Helix 1 and 2 are
presumed to constitute the main lipid binding properties of APOC2 [170,171]. It was
21
concluded that the N-terminal APOC2 fragment (residues 1-50) could not activate LPL while
the C-terminal fragment (residues 51-79) was responsible for activation of LPL [172]. It was
shown that an APOC2 fragment spanning residues 50-79 could increase LPL activity against
synthetic lipid emulsion particles, while full-length APOC2 was required when APOC2-
deficient chylomicrons were used as substrate. The same authors suggested that the surface
pressure for the individual lipid particles could account for these differences [167]. It was
proposed by others that an APOC2 fragment (residues 39-62) could activate LPL [173], but
these results could not be reproduced [174]. More recent studies confirmed the importance
of helix 3 in LPL activation and indicate a direct interaction with the enzyme. In this study the
nature of the fully conserved residues among animal species within the polar face of helix 3
were shown to be more critical for LPL activation than the hydrophobic residues on the
opposite side of the helix [174].
There are no reports on successful generation of Apoc2-knockout mice. APOC2-transgenic
mice develop hypertriglyceridemia attributed to delayed clearance of VLDL. Plasma APOC2
levels correlated positively with plasma triglycerides [175]. APOC2 was reported to impair
the cellular uptake of remnant lipoproteins by either displacement of, or conformational
modifications of, APOE [153]. However, it is also possible that APOC2-transgenic mice exhibit
impaired intravascular lipolysis. Intercrossing APOC2-transgenic mice with muscle-specific
LPL-transgenic mice reduced the hypertriglyceridemia associated with APOC2
overexpression [176]. This support the notion that excessive amounts of APOC2 inhibit LPL
activity in vivo and suggest that LPL can compete with excessive APOC2 residing on TRLs.
Dogma holds that APOC2 is the LPL cofactor. The exact mechanism for how these proteins
interact has not been resolved. For this, studies of the lipid-bound states for both APOC2 and
LPL are necessary and steady state kinetics cannot be obtained for the required time due to
lipid hydrolysis by LPL followed by remodeling of the lipid. Thus innovative approaches are
required to explain how LPL is activated by APOC2.
22
Figure 5. High-resolution structure of human APOC2 in complex with dodecylphosphocholine micelles [177].
Apolipoprotein C3
Human subjects that are heterozygous for a premature stop mutation within the signal
peptide R19X* in the APOC3 gene have reduced levels of plasma APOC3 compared to non-
carriers. As a result, carriers display lower levels of plasma triglycerides and increased levels
of HDL-cholesterol. In addition, carriers were shown to be less prone to develop coronary
arterial calcification [178,179]. Other rare mutations in the APOC3 gene were reported to be
associated with reduced levels of plasma APOC3, and with concomitant reductions in plasma
triglyceride levels compared with controls; these include two splice site mutations [180], the
A23T* and E58K* variants [181,182]. For the A23T and the splice site mutations, plasma
APOB levels were not affected, suggesting more efficient intravascular lipolysis (for the E58K
mutation no APOB measurements were reported). Recently it was shown that loss-of-
function mutations in APOC3 are associated with protection against CVD [183,184].
* R19X refer to the position 19 with position one being the first amino acid residue of the signal peptide. A23T and E58K refer to the positions 23 and 58 respectively where position one being the first amino acid residue of the secreted protein.
23
APOC3 is a 79 amino acid residue glycoprotein. It is mainly synthesized by the liver, but is
also synthesized in the intestine [164]. APOC3 is a constituent of chylomicrons, VLDL and
HDL [6]. APOC3 inhibits LPL activity in vitro [185,186]. Alaupovic and coworkers concluded
from studies on hypertriglyceridemic patients that plasma from these subjects had inhibitory
effects on LPL activity and that this effect was due to APOC3. Using kinetic analysis the
authors suggested that APOC3 inhibits LPL activity by a direct interaction with the enzyme
[187]. A reduced degree of glycosylation of APOC3 was recently proposed to account for
improved postprandial triglyceride clearance due to lowering the inhibition of APOC3 on LPL
activity [188]. The authors also suggested that an altered distribution of APOC3 on plasma
lipoproteins could account for their observation. From our unpublished observations using
synthetic emulsions we could not see any difference between APOC3 glycosylation variants
regarding inhibition of LPL activity. Thus, other explanations are needed. APOC3 is composed
of 6 amphipathic helices with positively charged residues accessing the solvent for helices 1
and 2 while negatively charged residues comprise the polar face of the C-terminal helices 4
and 5 (Figure 6) [189]. Thrombin cleavage of APOC3 generates two fragments (residues 1–40
and 41–78), where the C-terminal fragment is mainly responsible for lipid binding and LPL
inhibition [190,191]. For the rare natural mutation A23T in the APOC3 gene, the associated
low levels of plasma triglycerides could not be explained by reduced inhibition of LPL
compared to the wild-type protein when a synthetic lipid emulsion was used as substrate
[182]. Moreover, the E58K mutation has not been shown to have impaired ability to inhibit
LPL activity. Recently, it was proposed that APOC3 plays a crucial role in VLDL assembly,
independently of MTP, and that both A23T and E58K were unable to incorporate bulk
triglycerides into precursor lipoproteins [192,193]. APOC3 impairs APOE-mediated, but not
APOB100-mediated, uptake of remnant lipoproteins [153]. From studies of VLDL from
hypertriglyceridemic subjects, Breyer et al. demonstrated that APOE was redistributed from
VLDL to HDL at increasing APOC3 concentrations [194]. Interestingly, the redistribution of
APOE was dependent on VLDL size, with large particles being less capable of ejecting APOE
from the lipid/water interface.
Unlike Apoc1-knockout mice, Apoc3-knockout mice have decreased plasma cholesterol and
triglyceride levels and do not display hypercholesterolemia when put on an atherogenic diet
[195,196]. Combined Apoe/Apoc3-knockout mice display moderate reductions in plasma
cholesterol and significant reductions in circulating triglyceride levels compared with Apoe-
knockout mice. The postprandial response to an oral lipid load was blunted in both Apoc3-
knockout mice and the combined Apoc3/Apoe-knockout mice. Particle clearance was not
affected, suggesting profound effects on intravascular lipolysis. Chylomicron production
rates were lower in combined knockout mice compared to Apoe-knockout mice, but the
reductions in chylomicron secretion could not account for the dramatic effects seen on the
postprandial lipid response [196].
Several species have been genetically modified to overexpress human APOC3 and in all cases
the animals develop severe hypertriglyceridemia [197,198,199]. APOC3-transgenic mice
24
intercrossed with Apoe-knockout mice displayed increased plasma cholesterol and
triglyceride levels without effects on lipoprotein production rates. Triglyceride levels were
several-fold more increased than corresponding cholesterol levels suggesting that the main
effect of APOC3 overexpression was due to inhibition of LPL activity [200].
Overexpression of APOC3 in mice causes a phenotype with high resemblance to that seen on
overexpression of APOC1. Possibly all these observations can be explained by a
redistribution of APOE from TRLs to other lipoproteins, causing impaired ability for the
APOC3-containing TRLs to interact with LPL, and prolonged residency for APOB48 remnant
lipoproteins in blood. The redistribution of APOE might be a consequence of the excess of
APOC3 that competes out APOE from TRLs. In support of this is that combined APOE-
transgenic and APOC3-transgenic mice display normal levels of plasma triglycerides [201].
Additionally, APOC3 can displace APOC2 from lipid emulsion droplets [202]. Thus, it is
conceivable that APOC3 (and APOC1) can displace APOC2 from TRLs and thereby impair
lipolysis. In contrast, there are differences when Apoc1- and Apoc3-knockout mice are
compared, either on a wild-type or an APOE-deficient, background suggesting distinct roles
for these APOCs in lipoprotein metabolism.
Figure 6. High-resolution structure of human APOC3 in complex with sodium dodecyl sulfate micelles [189].
Apolipoprotein E
APOE deficiency in humans is associated with premature CVD and manifests in moderately
increased levels of plasma triglycerides and massive accumulation of cholesterol in remnant
25
lipoproteins due to impaired hepatic uptake via receptor-mediated endocytosis [203]. The
human APOE gene is represented by three common alleles; APOE2, APOE3 and APOE4,
where APOE3 is the normal allele with regard to all known functions. The APOE isoforms
differ by having different combinations of arginine and/or cysteine residues at position 112
and 158, respectively. Allele-specific effects have been extensively studied in the fields of
cardiovascular disease and alzheimers disease [reviewed [204]].
APOE is a ligand for members of the LDL-receptor family which internalize lipoproteins into
endosomes and lysosomes of cells. The main function of APOE is to mediate uptake of
remnant lipoproteins (chylomicron remnants and IDL) in the liver, mainly via LDL receptor-
related protein 1 (LRP1) and LDLR. APOE is especially important for clearance of
chylomicrons remnants because APOB48 cannot interact with members of the LDL-receptor
family [12]. APOE is much more potent than APOB100 as ligand for LDLR, and may therefore
regulate the residence time of lipoproteins in the circulation [204]. Unlike most other
apolipoproteins, APOE is synthesized by numerous tissues. Large amounts are produced in
the brain where APOE functions as a lipid transport vehicle in cerebrospinal fluid. APOE plays
a central role in the neuronal response to injury by providing lipids which promote neuronal
repair [204]. Macrophages secrete significant amounts of APOE. It was shown that APOE
stimulate efflux of cholesterol from cholesterol-enriched macrophages to HDL in vitro
[205,206].
Apoe-knockout mice show a similar phenotype as humans with dysfunctional APOE due to
impaired removal of APOB48 remnant lipoproteins. In contrast to humans wild-type mice do
not spontaneously develop atherosclerosis, while Apoe-knockout mice develop
atherosclerotic plaques when put on a diet enriched in cholesterol [207]. Mice deficient of
APOE, except for in macrophages, were protected against atherosclerosis when compared
with Apoe-knockout mice [208]. Direct evidence for the importance of APOE for cholesterol-
efflux from macrophages, and for prevention of atherosclerosis, was obtained by studies
with bone-marrow transplantations. Transplantation of bone marrow from APOE deficient
donor mice to wild-type mice showed that the recipient animals developed 10-fold more
atherosclerosis than their littermates reconstituted with bone marrow from wild-type
donors [209].
Apoe-knockout mice intercrossed with LPL-transgenic mice were protected against diet-
induced atherosclerosis [87]. In this study, overexpression of LPL reduced plasma triglyceride
levels while total cholesterol and APOB levels remained unchanged. These observations
demonstrated that increased triglyceride hydrolysis of APOB48 remnant lipoproteins did not
improve lipoprotein remnant particle removal from the circulation, nor did an excess of LPL
under these conditions improve remnant uptake via cell surface heparan sulphate as
previously proposed [210].
In in vitro experiments APOE inhibits LPL activity dose-dependently, both with lipid emulsion
particles and APOE-deficient VLDL as substrate. Injection of lipid emulsion particles
26
containing recombinant APOE showed impaired lipolysis in hepatectomized rats dependent
on the dose of APOE [211,212]. APOE-transgenic mice show moderately increased plasma
triglyceride levels where plasma APOE levels correlate positively with plasma triglyceride
levels and negatively with APOC2 on VLDL. This suggests that APOE may impair lipolysis, at
least in part, by displacement of APOC2 from the TRLs. In addition, VLDL secretion was
higher in APOE-transgenic mice. This could in part account for the observed
hypertriglyceridemia [213]. As discussed above, APOE-transgenic mice ameliorate the
hypertriglyceridemia associated with APOC3 overexpression. Thus, the inhibition of LPL
activity by APOE in vivo warrants further studies.
Apolipoprotein A5
Homozygous nonsense mutations in the human gene of APOA5 are associated with severe
hypertriglyceridemia and low levels of HDL-cholesterol [214,215]. Typically the
hypertriglyceridemia is not as severe as that observed in homozygous loss-of-function
mutations in any of the genes for LPL, GPIHBP1, APOC2 or LMF1.
APOA5 is mainly produced by the liver and associates with chylomicrons, VLDL and HDL
[216,217]. Peculiar for this apolipoprotein are the low circulating levels in plasma ranging
from 1 to 10 nmol/l [217]. In comparison, plasma levels of APOB100 and APOCs in fasted
subjects were reported to be in the micromolar range [161,218]. The low plasma
concentrations of APOA5 suggest intracellular functions for this protein. Ryan and coworkers
have postulated that APOA5 promotes lipid droplet formation in hepatocytes and thereby
modulate hepatic triglyceride stores by retarding VLDL assembly and subsequent secretion
[219,220]. However, certain human mutations in the gene of APOA5 cause chylomicronemia
suggesting effects also on circulating TRLs [214].
Transgenic APOA5-mice displayed reductions in plasma triglycerides without affecting
cholesterol levels. Moreover, Apoa5-knockout mice displayed approximately 4-fold
increased levels of plasma triglycerides, while cholesterol levels remained unaffected
compared to wild-type mice [216]. This is a relatively mild phenotype compared to knockout
mice models for other human mutations that cause severe hypertriglyceridemia (e.g. LPL,
GPIHBP1, LMF1, APOC2). Possibly this is a species-dependent effect, but it could also
indicate that other confounding factors are present in humans. Due to their opposing
phenotypes; Apoc3-knockout mice were intercrossed with Apoa5-knockout mice. Similarly
APOC3-transgenic mice were intercrossed with APOA5-transgenic mice. In both cases the
resulting double knockout and double transgenic progeny displayed lipid levels within the
normal range, compared to wild-type mice. Based on this the authors suggested that APOA5
and APOC3 affect plasma triglyceride levels independently but in an opposing manner [221].
27
Aims of the thesis
To investigate the molecular mechanisms by which APOC3 inhibits LPL activity
To compare the molecular mechanisms for inhibition of LPL by APOC1 and APOC3
To investigate if fatty acids bind to ANGPTL4 and whether fatty acids affect the
inactivation of LPL by ANGPTL4
To conduct a small molecule screen designed to identify compounds that would block
the ability of ANGPTL4 to inactivate LPL
To perform structure-activity relationship studies on promising “hit” compounds to
find lead compounds with improved potency to preserve LPL activity
28
Results and discussion
Paper I
In this study we present evidence for that APOC1 or APOC3, when bound to TRLs, prevents
binding of LPL to the lipid/water interface of the substrate lipid droplets. This results in
decreased lipolysis by the enzyme. In the presence of APOC2 more APOC1 or APOC3 was
needed to prevent LPL from binding to lipid emulsion particles, and consequently to inhibit
lipase activity. We found that prevention of LPL from binding to the lipid/water interface of
TRLs and lipid emulsion particles rendered LPL more susceptible to inactivation by ANGPTL4.
Mutagenesis of APOC3 revealed that alanine replacements within helix 3 and 4 of (W42A
and F47A) were the most important changes that caused decreased lipid binding of APOC3
and consequently decreased inhibition of LPL activity.
Method development and considerations
To be able to assess binding of LPL to lipid emulsion particles, a filtration method was
developed. By filtering the incubation mixtures through a syringe filter, LPL that was bound
to lipid particles larger than the pore size of the filter would be retained. Previous methods
had utilized the buoyant properties of triglyceride-rich particles to investigate LPL binding.
The large triglyceride-rich lipid particles can be recovered from the top layer after
centrifugation of the incubation mixtures and analyzed for their content of LPL [56,167].
However, lipolysis will go on during the time required for the centrifugation and this may
impair the buoyant properties of the lipid particles. Another problem is that pressure effects
will occur during the centrifugation [222]. Our technique avoids lipolysis by allowing for a
very rapid separation of the incubation mixture. A disadvantage shared with the flotation
techniques is that LPL bound to lipid particles smaller than the filter pore size are not
distinguished from unbound LPL. This problem could probably be solved by using filters with
an even smaller pore size. For centrifugation, the density of the mixtures could be increased
to flotate even the smaller particles. However, the filtration method is quick and allows
control regarding separation based on particles size. A potential disadvantage by using the
filter approach is that unbound proteins may get stuck in the filter. Because of this we used
filters composed of membranes with low protein-binding properties. We also used albumin
in the incubation mixtures to block unspecific protein binding. In control experiments we
observed >90% recovery of LPL from the separations, even in the absence of lipid particles,
suggesting that binding of LPL to the filter was minimal.
Discussion
Previous studies, based on conventional analysis of enzyme kinetics, had proposed that
APOC3 inhibits LPL by direct interaction with the enzyme at a site other than the active site
[187]. However, such an interaction has not been possible to demonstrate. In contrast, our
results suggest that APOC1 or APOC3 affects the properties of the substrate so that binding
29
of LPL to the lipid lipid/water interface is reduced. It is known that analyses of lipase kinetics
are complicated due to that the lipid substrate is aggregated in emulsion droplets [223]. This
makes the normal tests for enzyme inhibition non-applicable. A mechanism similar to ours
was reported for an inhibitor of trypsin and thrombin, by which the inhibitor affected the
substrates rather than interacted with the enzymes directly [224].
The exchangeable apolipoproteins differ in their ability to bind to and be retained on
lipoproteins. It was demonstrated that when apolipoproteins penetrates a phospholipid
monolayer the surface pressure increases as the interface becomes more crowded [225]. It
was predicted that APOC1 has a higher affinity for lipoproteins than APOC2. Therefore it was
speculated by others that binding of APOC1 to a TRL will result in a local increase of the
surface pressure, leading to the displacement of APOC2 [151]. By using lipid emulsion
particles carrying APOC2 it was shown that APOC3 inhibited LPL activity with a concomitant
loss of APOC2 from the lipid/water interface [202]. We did not investigate if APOC1 or
APOC3 displaced APOC2 in our systems, but in light of the previous report it is likely that this
was the case also in our systems. In the absence of APOC2 the ability of LPL to hydrolyze
triglycerides in TRLs is severely impaired [167], while with lipid emulsions containing long-
chain triglycerides (e.g. Intralipid) LPL shows some triglyceride hydrolysis even in the
absence of APOC2, suggesting that interaction between LPL and the surface lipids occur [56].
Our results suggest that APOC3 prevents this interaction, while APOC2 may promote such
interactions. Based on our observations it should be expected that LPL is unable to bind to
APOC2-deficient TRLs. This was, however, reported not to be the case with APOC2-deficient
human TRLs that were isolated by flotation [167]. Further studies are needed to investigate
these ambiguities that are likely due to that binding of both LPL and the apolipoproteins is
highly dependent on the lipid composition and physical properties of the surface layer of the
substrate emulsion particles, and that these properties may differ between synthetic
emulsions and plasma lipoproteins.
Several studies have addressed the effects of APOCs on receptor-mediated endocytosis of
lipoproteins. It was reported that addition of APOCs to VLDLs could displace APOE and that
that this caused a reduction in cellular lipoprotein uptake. There are, however, conflicting
opinions about whether the degree of APOE-displacement (APOE was reduced but still
present) could account for the effects on lipoprotein uptake via the LRP1 [152,153,226]. This
phenomenon could be due to displacement of LPL by APOCs, because it has been shown that
LPL strongly enhance binding of APOE-containing lipoproteins to LRP1 [227]. Supporting that
LPL could act as a ligand in vivo is that LPL is present in plasma at concentrations of about
100 ng/ml [228]. Interestingly, transgenic mice overexpressing LPL intercrossed with Apoe-
knockout mice display low levels of plasma triglycerides, while circulating APOB levels are
not affected compared to those in Apoe-knockout mice [87]. Possibly the combined
presence of APOE and LPL on the remnant lipoproteins is necessary for receptor-mediated
uptake.
30
A mysterious component in triglyceride metabolism is APOA5. It was suggested that APOA5
and APOC3 affect plasma triglyceride levels independently, but in an opposing manner [221].
In this study the combined APOC3/APOA5-trangenic mice overexpressed both proteins to
the same magnitude, approximately 500-fold higher than their respective founder line.
Given the observations that APOC3 can displace APOC2 and APOE, it is not unlikely that
APOA5 could be displaced as well [194,202]. If APOC3 and APOA5 bind competitively to the
lipid/water interface of TRLs, it is conceivable that the amount bound of each protein will
depend on their respective concentrations and binding affinities. Thus, the observed effect
that the combined transgenic mice displayed normal triglyceride levels was most likely
dependent on the relative concentrations of the apolipoproteins on the surface of the TRLs.
This cannot be considered as representing independent mechanisms for control of TRL
metabolism. LPL-transgenic mice were able to normalize plasma triglycerides in Apoa5-
knockout mice. It was suggested that APOA5 enhance intravascular lipolysis by guiding TRLs
to proteoglycan-bound LPL [229]. This is an interesting hypothesis, especially in light of
recent advances in triglyceride metabolism. As discussed above, several lines of evidence
suggest that LPL is bound to its transporter protein GPIHBP1 also at the luminal side of the
capillary endothelial cells. TRL margination in capillaries depends on LPL, but only when the
enzyme is in complex with GPIHBP1 [129]. Thus, the hypothesis originally proposed by
Merkel et al. might hold true, [229], with the updated modification that APOA5 via its affinity
to GPIHBP1 could enhance LPL-dependent lipolysis by guiding TRLs to LPL anchored to the
endothelial cells by GPIHBP1. APOA5 was shown to bind to GPIHBP1 in vitro. Later studies
showed that recombinant APOA5 fails to reduce plasma triglyceride levels in Gpihbp1-
knockout mice, but ameliorates much of the hypertriglyceridemia in Apoa5-knockout mice
[76,230]. It should be noted, however, that Gpihbp1-knockout mice have extremely low
amounts of LPL in capillaries. Taken together, these are interesting data that implicate an
important role for APOA5 to enhance LPL-dependent lipolysis through interaction with
GPIHBP1 and thereby tethering the TRLs to the endothelium.
We propose another consequence of the competition by APOC1 and APOC3 with LPL for
binding to the surface of lipid particles, namely an increased susceptibility for LPL to become
inactivated by ANGPTL4. Given that ANGPTL4 can readily inactivate LPL when the enzyme is
free in solution, and that previous studies had shown that TRLs or lipid emulsion prevent this
inactivation, we expected that APOC1 or APOC3 should promote inactivation of LPL by
ANGPTL4 in the presence of lipid droplets. Our data in paper I show that this is in fact the
case. Whether these effects occur also in vivo remain to be demonstrated. Transgenic mice
that overexpressed APOC1 displayed increased levels of LPL activity in post-heparin plasma
compared to wild-type mice [156]. With our proposed mechanism in mind this observation
may be due to the inability of TRLs to remove LPL from the endothelium. Transgenic
miniature pigs that overexpressed APOC3 did not show reduced post-heparin LPL activity
after the effects of APOC3 were corrected for by serial dilution, while other studies
demonstrated reduced LPL activity but had not taken this effect into account [199]. Thus,
transgenic animal models that overexpress APOC1 or APOC3 do not indicate that LPL is more
31
susceptible to inactivation by ANGPTL4, but recombinant injection or overexpression of
ANGPTL4 in mice reduces post-heparin LPL activity [97]. Later studies showed that this effect
was reproducible in fasted mice, but interestingly not in littermates that had been given an
olive oil bolus [106]. The same study suggested that the prime action of ANGPTL4 on LPL
occurs by paracrine effects within interstitial spaces rather than at the capillary lumen. This
was based on the knowledge about the low plasma concentrations of ANGPTL4 in humans
and the protective effects on LPL activity seen by TRLs in blood. Our present observations
could predict that under certain conditions, when plasma concentrations of APOC1 and/or
APOC3 and ANGPTL4 are elevated, LPL activity could also be modulated at the capillary
lumen.
Recently two independent studies have shown that rare heterozygous loss-of-function
mutations in the gene of APOC3 are associated with reduced CVD risk [183,184]. Carriers
show reductions in plasma triglyceride levels (-39%, -44%) and increased levels of HDL-
cholesterol (+22%, +24%) compared to non-carriers. Plasma triglyceride levels were similar
for all mutations which included two splice-site mutations, the nonsense mutation R19X and
the missense mutation A23T. Interestingly, the A23T mutation is comparable to wild-type
APOC3 for inhibition of LPL activity in incubation systems in vitro [182]. Therefore other
mechanisms for the effect of APOC3 on plasma triglyceride levels must be considered. A
recently proposed mechanism for the function of APOC3 was to assist in VLDL assembly and
secretion. The A23T variant abolished the ability to stimulate VLDL secretion in vitro [192].
This is an interesting observation and challenges the preexisting view regarding the function
of APOC3 for inhibition of intravascular lipolysis when this apolipoprotein is expressed at
normal levels. It is possible to inhibit LPL activity on TRLs in blood by i.v. injections of tri-
block copolymers (e.g. triton wr-1339 or poloxamer 407) and thereby assess lipoprotein
production rates [231]. In such experiments there were no differences in VLDL production
rates between Apoc3-knockout mice compared to wild-type mice, suggesting that the main
effect by APOC3 on plasma triglyceride levels is due to inhibition of LPL activity and/or
impaired receptor mediated uptake [196]. Thus, more studies are needed to understand the
mechanisms of the A23T mutation in vivo. The in vitro systems are not representative for the
situation in blood which differs in two fundamental ways. Firstly, apolipoproteins can
redistribute among the lipoprotein classes in blood. This is not possible in systems using lipid
emulsions. It was shown that, compared to wild-type APOC3, the A23T mutation caused
reduced affinity of LPL for phospholipid liposomes, while the inhibition of this mutant on LPL
activity against emulsified triglycerides was not affected [182]. Mutants like the A23T may
cause small changes of the properties of lipoproteins that reduce the affinity for LPL. These
effects may not be detected in in vitro systems using lipid emulsions, whereas in blood they
may cause an altered distribution of apolipoprotiens among lipoprotein classes, resulting in
an impaired ability to inhibit LPL activity. Secondly, most mechanistic studies on
apolipoproteins in relation to LPL activity have been conducted in test tube experiments
with LPL free in solution. The recent findings that LPL is bound to GPIHBP1, also at its site of
action in capillaries, give rise to a fundamental question; are TRLs more dependent on
32
apolipoproteins for efficient triglyceride hydrolysis when LPL is bound to GPIHBP1 compared
to when LPL is free in solution? If this is the case, some known apolipoproteins may serve
other functions than those we know of today. As discussed above, APOA5 might guide TRLs
to LPL, and APOC2 may support binding of TRLs to the LPL-GPIHBP1 complex in addition to
its direct enzyme activating properties.
In summary, I suggest that APOC1 and APOC3 retard TRL catabolism by preventing LPL from
interaction with the lipid particles and by competition with other apolipoproteins for binding
to the lipid/water interface. APOCs retard receptor-mediated uptake by displacement of
APOE and my studies suggest that also LPL could be displaced. Further studies are needed to
understand the details of the function of APOC1 and APOC3 in vivo by using in vitro systems
that mimic the situation in capillaries where LPL is bound to GPIHBP1 and all lipoprotein
classes are present.
Paper II
In this study we used surface plasmon resonance, isothermal titration calorimetry, and
fluorescent quenching to investigate binding of fatty acids to ANGPTL4. It was shown that
fatty acids bind with very high affinity to ANGPTL4 and that ANGPTL4, in the presence of
fatty acids, has limited capacity to inactivate LPL. Addition of bovine serum albumin to
incubation mixtures containing fatty acids, ANGPTL4 and LPL removed the protective effect
of fatty acids on LPL activity, and dose-dependently promoted ANGPTL4-mediated
inactivation of LPL.
Discussion
It was previously shown that LPL is inhibited by its products when incubated with long-chain
triglyceride substrates, and that the inhibition is relieved by addition of a fatty acid acceptor
like albumin [58]. In this study the authors proposed that the inhibition was mainly due to
binding of fatty acids to LPL, and that the function might be to serve as a feed-back
regulation to prevent excessive fatty acid delivery to cells. In the present study we found
that fatty acids can bind even stronger to ANGPTL4 than to LPL. We also found that LPL is
less prone to become inactivated by ANGPTL4 in the presence of fatty acids. Previous studies
had demonstrated that TRLs protect LPL against ANGPTL4-mediated inactivation [106].
Possibly the fatty acids released during lipolysis of TRLs prevent ANGPTL4 from interacting
with LPL. This may explain part of the observed protective effect. Because albumin was
present during those incubations in vitro, and should sequester the fatty acids, it is also likely
that binding of LPL to TRLs had protective effects on LPL activity.
In an in vivo situation it is difficult to reconcile the effects of fatty acids on LPL activity in
adipose tissue in the fasted state when the expression of ANGPTL4 is up-regulated by
activated PPARs, while the LPL system is tuned down and fatty acids are mobilized from
intracellular lipid stores, presumably with the aid of ANGPTL4 [68,69]. The reduced ability of
ANGPTL4 to inactivate LPL in the presence of fatty acids is not in accordance with the rapid
33
down-regulation of LPL activity during fasting [68]. Intriguingly, LPL activity in adipose tissue
of obese individuals was shown to be unresponsive to a meal, while lean subjects displayed
decreased specific LPL activity during fasting indicating inactivation of the enzyme [232].
Administration of the transcription inhibitor actinomycin D to old obese rats in order to
block protein synthesis did not increase LPL activity in the fasted state as would have been
expected due to decreased synthesis of ANGPTL4. This suggested impaired ability of
ANGPTL4 to inactivate LPL in the obese animals [68]. In obesity, plasma free fatty acids are
usually elevated due to insulin resistance, and hypoxia may occur in white adipose tissue
[233,234]. Both factors may promote upregulation of ANGPTL4 expression. In human
adipocytes hypoxia causes dramatic increases in ANGPTL4 expression [235]. Later studies
have shown that in addition to hypoxia, fatty acids may elevate ANGPTL4 expression and
secretion in human adipocytes [236]. Thus, it would be expected that adipose tissue LPL
should be strongly suppressed in obesity. Evidently this is not the case. A possibility could be
that the increased concentration of fatty acid that accompanies obesity neutralizes the
ability of ANGPTL4 to inactivate LPL. More studies are obviously needed to understand the
physiological relevance of the high affinity of ANGPTL4 for fatty acids.
Paper III and IV
In Paper III we have employed a small molecule screening approach to identify compounds
that could protect LPL against inactivation by ANGPTL4. Paper IV is a follow-up structure-
activity relationship (SAR) study aimed to develop lead compounds for drug development
efforts for prevention of CVD. Small molecules that prevent inactivation of lipoprotein lipase
(LPL) were identified. One hit compound (Figure 7) prevented both ANGPTL4-dependent and
heat-dependent inactivation of LPL. This occurred by stabilization of the active LPL dimer.
The hit compound reduced postprandial triglyceride levels in Apoa5-knockout mice. SAR
studies resulted in a series of lead compounds, some of which showed approximately 2-3
fold improved potency compared to the original hit compound. SAR analysis identified the
carboxyl-group (Figure 7, marked in orange) as highly important for retaining biological
activity of the compound. Increasing the steric hindrance by substituent scrambling of the
central phenyl ring (blue) decreased the compound potency and solubility. Modifications of
the amide group (green) seemed tolerable. Hydrophilic substituents of either the piperidinyl
group (yellow) or the para-tolyl group (red) reduced biological activity of the compound
while more lipophilic substituents increased compound potency but decreased solubility.
34
Figure 7. Chemical structure of the hit compound used as starting point for our SAR investigation. The encapsulations denote chemical groups that were investigated by modification and/or substitution.
Although administration of our original hit compound showed promising lipid-lowering
effects in Apoa5-knockout mice [Paper III], the phthalimide moiety (white) was shown to be
metabolically unstable in the presence of human liver microsomes [Paper IV]. Primary
metabolites were less potent than the original hit compound in vitro. Secondary metabolites
did not show in vitro activity at 25 µM or below. For this reason, analogous heterocyclic
compounds were synthesized to investigate if improved metabolic stability could increase
compound efficacy in vivo. Although certain heterocyclic analogues showed improved
potency in vitro the effects were moderate compared to the dramatic effects observed in
vivo, suggesting that the original hit compound had a shorter half-life than the modified
compounds in vivo (Table 2).
Time
(min)
Buffer
(µg TG/ml)
Original hit
compound
(µg TG/ml)
Hetero-
cycle-A
(µg TG/ml)
Hetero-
cycle-B
(µg TG/ml)
Hetero-
cycle-C
(µg TG/ml)
Hetero-
cycle-D
(µg TG/ml)
Hetero-
cycle-E
(µg TG/ml)
0 349±112 325±104 263±97 271±76 217±73 196±75* 222±73
60 759±249 531±256 446±167** 297±114*** 379±161** 323±121*** 413±124**
120 921±241 802±377 507±110** 324±110*** 359±162*** 485±123** 521±283*
180 1043±656 1079±301 600±122 456±109** 421±96** 440±90** 436±159**
Table 2: In vivo effects of heterocyclic phthalimide substituents. Female C57B6J mice (n=56, n=8/group) aged 8-9 weeks with an average weight of 18 g were used for the in vivo experiments. The mice were housed at room temperature with free access to tap water and standard chow. The mice were treated in the morning for 3 days with 0.5 ml intraperitoneal injections (buffer composition as that described in Paper III containing 1 mM compound). On the 3
rd day the mice had been fasted 8 h from midnight and injected a final 4
th time. One hour
after the final injection the mice were given an oral olive oil gavage of 0.2 ml. Blood samples were collected to EDTA coated capillaries from the tail vein at baseline and for every 60 min until 180 min. They were analyzed for their triglyceride contents. Data for blood triglyceride (TG) levels are represented as mean values with standard deviations. *p < 0.05, **p < 0.01, ***p<0.001.
35
Method development and considerations
In the pharmaceutical industry high-throughput screening (HTS) is part of the early drug-
discovery process. Once the decision has been made to try to affect the function of a target
(e.g. protein of interest) the next step is to test if drug-like compounds possess biological
activity towards the target. Typically more than 100000 compounds are screened in fully
automated facilities. Therefore it is necessary to develop robust assays that are amenable to
automation. Usually traditional benchtop assays for a given target are transformed to a
microtiter plate format to enable robotic pipetting and facilitate endpoint readouts. Assay
reliability is easily assessed by applying various statistics based on controls (e.g. no
compound) or the total signal distribution [237]. Reliable assays are able to identify
biologically active compounds, usually referred to as “hits”, while discarding inactive ones. In
the screening process it is most common to use one concentration at one time and then
cherry-pick hits for re-screening by using the same assay. Hits are further validated by
secondary assays and by dose-response studies to better assess if a hit compound interacts
with the target, or if the effects are due to off-target effects. Using a compound library of
approximately 17000 compounds these steps were carried out in the facilities of
Laboratories for Chemical Biology Umeå and form the basis for Paper III. Compounds that
remain after validation studies may be used as starting points for lead optimization studies.
In the lead optimization step SAR studies are used to study the relationship between a
compounds molecular structure and its biological activity. By the identification and
substitution of functional groups and structurally relevant properties, these studies aim to
increase the potency of the original hit compounds. A lead optimization was carried out for
our hit compound and is described in Paper IV.
Traditional benchtop assays for measuring LPL activity include triglyceride-based substrates
(e.g. TRLs or lipid emulsions) and water-soluble ester substrates (e.g. para-nitrophenyl
butyrate). When triglyceride substrates are used, a subsequent method for quantification of
fatty acids is required. Quantification of radiolabelled fatty acids is most commonly used in
in vitro assays for LPL. This procedure is not easily transformed to an automated format. An
alternative quantification method for fatty acid is titration using a pH-stat, but this is too
laborious in a screening environment. An attractive but expensive option was to use
quantification of non-labelled fatty acids by an enzymatic calorimetric assay. To obtain an
affordable, yet reliable, assay we used a water-soluble substrate for LPL in our primary
screen. LPL has catalytic activity to both emulsified triglyceride substrates and water-soluble
ester substrates. However, previous studies had shown that on inactivation of LPL the
enzymatic activity disappears more quickly towards long-chain triglyceride emulsions than
towards water-soluble substrates [238]. This is presumably because the water-soluble
substrates do not require binding of LPL to the lipid/water interface of emulsion particles
[55]. Thus, by using water-soluble esters as substrates we expected that we should not miss
out on potential hits. Rather, we took the risk of identifying compounds that showed effects
on LPL with water-soluble ester substrates, but not with emulsified triglyceride substrates.
36
However, we reasoned that such compounds could be identified and later discarded by using
a long-chain triglyceride-based substrate in a secondary screen. We first used the water-
soluble para-nitrophenyl butyrate as substrate [239]. However, numerous compounds
appeared to catalyze hydrolysis of this substrate themselves, leading to large numbers of
false positives. We therefore turned to an umbelliferone-ester (pivaloyl-umbelliferone)
which had shown better resistance to non-specific hydrolysis [240]. There were yet no
reports regarding whether this ester could function as a substrate for LPL. Interestingly, in
comparison with para-nitrophenyl butyrate, the hydrolysis of the umbelliferone-ester was
linear over a very long time suggesting that LPL was stabilized by the substrate and/or by the
product formed. Addition of ANGPTL4 caused marked reductions in product formation. We
therefore decided to use this substrate in our primary screen. Our second concern was to
keep LPL stable during the automated process. One problem in this regard was that
traditional stabilizers of LPL activity (e.g. bile salts or fatty acids) did not enable ANGPTL4 to
inactivate LPL. Other known stabilizers, such as heparin and similar polyanions, are less
efficient and bind LPL by ionic interactions that could prevent interactions with compounds.
For these reasons we decided to use conditions for preserving LPL activity without the
addition of stabilizers. It appeared that LPL was relatively stable in phosphate buffer for
activity against pivaloyl-umbelliferone for sessions up to three hours. Our screening
campaign had a throughput of approximately 600 compounds per hour. Given the relatively
small compound library, we did try to increase throughput further. Validation and follow-up
studies are described in detail in Paper III.
Discussion
The idea of finding a drug that prevents inactivation of LPL by ANGPTL4 in order to reduce
the risk for CVD is based on the knowledge that increased levels of LPL activity is associated
with an anti-atherogenic lipid profile and that carriers of the LPL-S447X gain-of-function
mutation have been associated with reduced CVD risk [31,41,42]. Also, the E40K mutation of
ANGPTL4, that has impaired ability to inactivate LPL in vitro, is associated with an anti-
atherogenic lipid profile [112,113]. However, there are conflicting results regarding the
effects on CVD risk for this ANGPTL4 mutation [115,116]. Transgenic mice overexpressing
LPL at locations other than in macrophages are protected against atherosclerosis, while
overexpression of LPL in macrophages is pro-atherogenic and was found to mainly be due to
the non-catalytic functions of LPL [93]. An alarming fact is that when Angptl4-knockout mice
are fed a diet enriched in saturated fat they develop lipid-laden macrophages in the
mesenteric lymph nodes [101]. It was also shown that ANGPTL4 suppresses LPL activity in
macrophages and that this may reduce atherosclerotic lesion progression [110,111]. Given
these contradictory findings for the role of LPL with regard to presumed CVD risk it is
impossible to speculate on the outcomes for a drug that would increase LPL activity in vivo.
Our goal was, however, to find a drug that can reduce the levels of plasma triglycerides. The
obvious target for this effort is LPL. Irrespective of outcome, a compound that directly
affects LPL activity is an interesting tool for future studies.
37
Recently, another group reported on a small molecule as a novel LPL agonist [241]. They
used para-nitrophenyl butyrate as substrate for LPL and ANGPTL4 for inactivation, an
approach almost identical to that of our first screening effort. Similar to several compounds
in our primary screen, the published LPL agonist failed to show activity in our secondary
screen using triglyceride-based substrates [Paper III]. The reason for this discrepancy is
currently unknown, but as discussed in Paper III it might be due to lack of stabilization of the
necessary lipid-binding properties of LPL. Other drugs that may affect LPL activity are those
which prevent the hypertriglyceridemia induced by triblock co-polymers (e.g. triton wr-
1339). It should be noted that the mechanism by which triblock co-polymers inhibit LPL is
not understood. One compound class which prevented triton wr-1339 induced
hypertriglyceridemia showed structural similarities to our compounds [Paper IV,[242]].
However, this compound class did not show activity in our assays. Furthermore
administration of triton wr-1339 to mice receiving our original hit compound showed a
similar rise in plasma triglyceride levels as mice receiving vehicle only. The drug Ibrolipim
(NO-1886) was reported to increase post-heparin LPL activity, and to decrease plasma
triglycerides, increase HDL-cholesterol and protect against atherosclerosis in rats and rabbits
[243,244]. The exact mechanism by which Ibrolipim increases LPL activity has not been
elucidated. Both LPL mRNA levels and LPL mass is increased upon Ibrolipim administration,
suggesting transcriptional effects [245]. Ibrolipim protect against diet-induced
atherosclerosis in miniature pigs [246]. The same animals displayed increased post-heparin
LPL activity, but they also had increased levels of ABCA1 mRNA and mass, presumably due to
increased levels and activation of the liver X receptor alpha (LXRα) [246]. Activation of LXR
has many effects and protects against atherosclerosis [reviewed [247]]. This makes it hard to
discriminate the exact mode of action by which Ibrolipim may protect against
atherosclerosis.
Small molecules with biological activity in our assays show a common property; they are all
anionic amphiphilic compounds. This property is shared with fatty acids and is also a
property in common with deoxycholic acid that is known to efficiently stabilize LPL in vitro
[Paper II][248]. We have observed that deoxycholic acid prevents inactivation of LPL by
ANGPTL4 (unpublished data). Both fatty acids and deoxycholic acid function as ligands for
nuclear receptors involved in lipid metabolism where fatty acids primarily activate PPARs,
while deoxycholic acid activates the farnesoid X receptor [249,250,251]. Thus, it is highly
important to investigate if our compounds show off-target effects on nuclear receptors and
if these effects may account for our observations regarding plasma lipids in vivo. Both LPL
and ANGPTL4 bind with high affinity to fatty acids as well as to our original hit compound
[Paper II, Paper III]. It is therefore likely that our lead compounds bind to both proteins as
well.
With regard to the mode of action of ANGPTL4 on LPL an appealing idea has emerged during
our studies. It is evident that LPL and ANGPTL4 show similar physical properties with regard
to hydrophobic and charged sites. It was previously shown that rapid subunit exchange
38
occurs in the active LPL dimer [238]. Thus, it is possible that ANGPTL4 mimics the structure
of an LPL monomer, and once a transiently free LPL monomer encounters ANGPTL4 they
may bind to each other. The suboptimal partner ANGPTL4 may not be able to stabilize the
conformation of the LPL subunit as well as another LPL subunit. Therefore the interaction
with ANGPTL4 results in irreversible inactivation of LPL. The presence of anionic amphiphilic
compounds may shift the equilibrium in favour of the dimeric state of LPL and thereby
prevent the enzyme from inactivation by ANGPTL4 (Figure 8). Another possibility is that the
anionic amphiphiles simply prevent interaction between ANGPTL4 and LPL by blocking the
binding site.
Figure 8. Model for stabilization of the active LPL dimer by compounds and ANGPTL4-mediated inactivation involving binding of ANGPTL4 to dissociated, but still natively folded LPL monomers.
In terms of the lipid-lowering effects of our compounds, preliminary in vivo data are highly
encouraging. Administration of our stable heterocyclic lead compounds to wild-type mice
caused dramatic reductions in plasma triglycerides. Studies are ongoing to validate these
compounds regarding off-target effects and to assess if they are effective in the prevention
of atherosclerosis in animal models.
39
Conclusions
APOC1 and APOC3 inhibit LPL activity by similar mechanisms. Both proteins prevent
LPL from binding to the lipid-water interface of TRLs and lipid emulsion particles.
Hydrophobic amino acid residues centrally located in APOC3 are the most important
residues for lipid-binding, and consequently for inhibition of LPL activity.
ANGPTL4 and LPL both have high affinity for fatty acids. More studies are needed to
understand the physiological relevance of this interaction.
Anionic amphiphilic small molecules stabilize the LPL homodimer and prevent
inactivation of LPL by ANGPTL4.
Heterocyclic substitution of our original hit compound caused dramatic
improvements in plasma lipid parameters in vivo, presumably by improved metabolic
stability of the compound.
40
Acknowledgements
Gunilla tack för att du alltid tagit dig tid att lyssna på mina tankar kring forskning. Du har gett
mig den frihet och vägledning jag behövde för att få utvecklas och komma till insikt med vad
jag vill göra i min fortsatta yrkeskarriär. För detta är jag mycket tacksam.
Nästa tack går till alla medarbetare på fysiologisk kemi. Jag uppskattar verkligen all hjälp, ert
sällskap och trevliga stunder. Aivar tack, för all hjälp och trevliga diskussioner. Jag hoppas
fortfarande komma till Tallinn och hälsa på. Solveig, en riddare utan rustning. Såväl röd som
svart är hon den vänligaste av oss alla. Elena, ”I will kill you” har fått en helt ny innebörd.
Numera associerar jag det med omtänksamma människor och nektariner. Lasse har ett
problem – han glömmer inte – vare sig allmäna kunskaper eller var han inte har lagt sitt
kaffekort. Stort tack för tillfällen då ett lyssnande öra behövts. Thomas is still going strong,
en sann forskare. Stefan, hade vänligheten att bjuda in mig till tjockisfältet, olyckligtvis blev
jag tunnare. ”Krabban” a.k.a byggar-Rakel, Mållgan hälsar att han inte behöver fler
imaginära buffertar. Evelina har ingenting emot LPL aktivitet, vilket antyder att hon måste ha
det starkaste psyket i vintergatan. Madde, vi ses på six flags! Fredrik och Niklas det känns
tryggt att lämna över till er, kom ihåg att låten ”Ai Se Eu Te Pego” kan ge nektariner. Oleg, en
blivande sann forskare. Slava, Jessica och Massi thank you for many laughs at the end.
Ett stort tack till tjejerna i tarmen på våning två. Öppna dörr nummer ett och du tas emot
och blir servad på det bästa av vis av en alltid så glad Åsa. Innanför dörr nummer två huserar
”aldrig ett problem”-Carina, där man tyvärr aldrig behöver vistas så länge. Bakom dörr
nummer tre sliter vår hjälte Terry såväl sena kvällar som helger. Längst in, finner vi en annan
slitvarg och även den bästa av bordsdamer Karin.
Ett alldeles eget tack tillägnas Clara som även hon har sett till att mitt arbete har förflutit
sömlöst.
En eloge till tjejerna på pediatrik som stått ut med surgubben i labbet intill. Yvonne, jag
håller på dig! Sussi stay frosty. Anna fortsätt att le. Carina du skrattar högst, fortsätt med
det. Catarina slår du hammaren såsom ELISA-plattan tycker jag synd om plankan. Lotta till
skillnad från solen har du inga fläckar.
Stort tack till mina vänner på organisk kemi. Remi your diligence and professionalism
inspires me. Mikael för att du ställer upp i tid och otid, uppskattas! P-A, en vänlig själ.
Tack även till alla er som gjort 6M till en bra och trivsam arbetsplats. Maria B dig skulle man
klona! Tack för all hjälp. Ingegärd tack för trevliga stunder och ugnsbak. Maria L som ställer
upp och sekvenserar fastän du inte behöver. Monica och Maria H blir det något lopp? Lelle
för att du muntrar upp. Malin för att du är så okonstlat bra. Angelica för att du är kul. Nina
för att du fått mig att försöka vara en pistvakt och Urban för att du bistått i mina försök. Åsa
kom och ät. Lisbeth, Mikael, Heidi för att ni alltid är glada. Emma för bästa humorn. Linda
41
för att jag får försöka programmera. Sofia för att du är snäll. Per som delar min passion för
litterära mästerverk. Dan, Elin och Martin, jag hoppas vi ses igen.
Cecilia Elofsson, en räddare i nöden. Tack!
Till sist, tack till mina föräldrar och min bror. En annan tid, ett annat liv–jag önskar vi får vara
tillsammans även då.
42
References
[1] G.L. Zubay, Biochemistry, 4th ed., Wm.C. Brown Publishers, Dubuque, IA, 1998. [2] M.E. Lowe, Pancreatic triglyceride lipase and colipase: insights into dietary fat digestion,
Gastroenterology 107 (1994) 1524-1536. [3] M.M. Hussain, Intestinal lipid absorption and lipoprotein formation, Curr Opin Lipidol 25 (2014)
200-206. [4] D.I. Abramson, P.B. Dobrin, Blood vessels and lymphatics in organ systems, Academic Press,
Orlando, 1984. [5] I. Ramasamy, Recent advances in physiological lipoprotein metabolism, Clin Chem Lab Med (2013)
1-33. [6] R.J. Havel, J.P. Kane, M.L. Kashyap, Interchange of apolipoproteins between chylomicrons and
high density lipoproteins during alimentary lipemia in man, J Clin Invest 52 (1973) 32-38. [7] S.G. Young, R. Zechner, Biochemistry and pathophysiology of intravascular and intracellular
lipolysis, Genes Dev 27 (2013) 459-484. [8] P.J. Nestel, High-density lipoprotein turnover, Am Heart J 113 (1987) 518-521. [9] A.D. Cooper, Hepatic uptake of chylomicron remnants, J Lipid Res 38 (1997) 2173-2192. [10] S. Tiwari, S.A. Siddiqi, Intracellular trafficking and secretion of VLDL, Arterioscler Thromb Vasc
Biol 32 (2012) 1079-1086. [11] S.J. Murdoch, W.C. Breckenridge, Effect of lipid transfer proteins on lipoprotein lipase induced
transformation of VLDL and HDL, Biochim Biophys Acta 1303 (1996) 222-232. [12] R.W. Mahley, Z.S. Ji, Remnant lipoprotein metabolism: key pathways involving cell-surface
heparan sulfate proteoglycans and apolipoprotein E, J Lipid Res 40 (1999) 1-16. [13] A. Zambon, S. Bertocco, N. Vitturi, V. Polentarutti, D. Vianello, G. Crepaldi, Relevance of hepatic
lipase to the metabolism of triacylglycerol-rich lipoproteins, Biochem Soc Trans 31 (2003) 1070-1074.
[14] R.W. Mahley, Apolipoprotein E: cholesterol transport protein with expanding role in cell biology, Science 240 (1988) 622-630.
[15] M.S. Brown, J.L. Goldstein, A receptor-mediated pathway for cholesterol homeostasis, Science 232 (1986) 34-47.
[16] J.L. Goldstein, M.S. Brown, The LDL receptor, Arterioscler Thromb Vasc Biol 29 (2009) 431-438. [17] M. Cofan Pujol, [Basic mechanisms: absorption and excretion of cholesterol and other sterols],
Clin Investig Arterioscler 26 (2014) 41-47. [18] R.S. Kiss, D.C. McManus, V. Franklin, W.L. Tan, A. McKenzie, G. Chimini, Y.L. Marcel, The
lipidation by hepatocytes of human apolipoprotein A-I occurs by both ABCA1-dependent and -independent pathways, J Biol Chem 278 (2003) 10119-10127.
[19] J. Huuskonen, V.M. Olkkonen, M. Jauhiainen, C. Ehnholm, The impact of phospholipid transfer protein (PLTP) on HDL metabolism, Atherosclerosis 155 (2001) 269-281.
[20] G. Li, H.M. Gu, D.W. Zhang, ATP-binding cassette transporters and cholesterol translocation, IUBMB Life 65 (2013) 505-512.
[21] J.A. Glomset, E.T. Janssen, R. Kennedy, J. Dobbins, Role of plasma lecithin:cholesterol acyltransferase in the metabolism of high density lipoproteins, J Lipid Res 7 (1966) 638-648.
[22] A. Leiva, H. Verdejo, M.L. Benitez, A. Martinez, D. Busso, A. Rigotti, Mechanisms regulating hepatic SR-BI expression and their impact on HDL metabolism, Atherosclerosis 217 (2011) 299-307.
[23] T. Chajek, C.J. Fielding, Isolation and characterization of a human serum cholesteryl ester transfer protein, Proc Natl Acad Sci U S A 75 (1978) 3445-3449.
[24] C.J. O'Donnell, E.G. Nabel, Genomics of cardiovascular disease, N Engl J Med 365 (2011) 2098-2109.
[25] G.K. Hansson, Inflammation, atherosclerosis, and coronary artery disease, N Engl J Med 352 (2005) 1685-1695.
43
[26] I. Tabas, K.J. Williams, J. Boren, Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications, Circulation 116 (2007) 1832-1844.
[27] C. Baigent, L. Blackwell, J. Emberson, L.E. Holland, C. Reith, N. Bhala, R. Peto, E.H. Barnes, A. Keech, J. Simes, R. Collins, Efficacy and safety of more intensive lowering of LDL cholesterol: a meta-analysis of data from 170,000 participants in 26 randomised trials, Lancet 376 (2010) 1670-1681.
[28] M. Miller, N.J. Stone, C. Ballantyne, V. Bittner, M.H. Criqui, H.N. Ginsberg, A.C. Goldberg, W.J. Howard, M.S. Jacobson, P.M. Kris-Etherton, T.A. Lennie, M. Levi, T. Mazzone, S. Pennathur, Triglycerides and cardiovascular disease: a scientific statement from the American Heart Association, Circulation 123 (2011) 2292-2333.
[29] A.M. Gotto, Jr., J.E. Moon, Pharmacotherapies for lipid modification: beyond the statins, Nat Rev Cardiol 10 (2013) 560-570.
[30] J.P. Despres, HDL cholesterol studies--more of the same?, Nat Rev Cardiol 10 (2013) 70-72. [31] C.T. Johansen, R.A. Hegele, The complex genetic basis of plasma triglycerides, Curr Atheroscler
Rep 14 (2012) 227-234. [32] T. Gotoda, K. Shirai, T. Ohta, J. Kobayashi, S. Yokoyama, S. Oikawa, H. Bujo, S. Ishibashi, H. Arai,
S. Yamashita, M. Harada-Shiba, M. Eto, T. Hayashi, H. Sone, H. Suzuki, N. Yamada, Diagnosis and management of type I and type V hyperlipoproteinemia, J Atheroscler Thromb 19 (2012) 1-12.
[33] C.T. Johansen, S. Kathiresan, R.A. Hegele, Genetic determinants of plasma triglycerides, J Lipid Res 52 (2011) 189-206.
[34] G.G. Schwartz, A.G. Olsson, M. Abt, C.M. Ballantyne, P.J. Barter, J. Brumm, B.R. Chaitman, I.M. Holme, D. Kallend, L.A. Leiter, E. Leitersdorf, J.J. McMurray, H. Mundl, S.J. Nicholls, P.K. Shah, J.C. Tardif, R.S. Wright, Effects of dalcetrapib in patients with a recent acute coronary syndrome, N Engl J Med 367 (2012) 2089-2099.
[35] W.E. Boden, J.L. Probstfield, T. Anderson, B.R. Chaitman, P. Desvignes-Nickens, K. Koprowicz, R. McBride, K. Teo, W. Weintraub, Niacin in patients with low HDL cholesterol levels receiving intensive statin therapy, N Engl J Med 365 (2011) 2255-2267.
[36] M. Jun, C. Foote, J. Lv, B. Neal, A. Patel, S.J. Nicholls, D.E. Grobbee, A. Cass, J. Chalmers, V. Perkovic, Effects of fibrates on cardiovascular outcomes: a systematic review and meta-analysis, Lancet 375 (2010) 1875-1884.
[37] P.F. Hahn, Abolishment of Alimentary Lipemia Following Injection of Heparin, Science 98 (1943) 19-20.
[38] E.D. Korn, Clearing factor, a heparin-activated lipoprotein lipase. I. Isolation and characterization of the enzyme from normal rat heart, J Biol Chem 215 (1955) 1-14.
[39] V. Murthy, P. Julien, C. Gagne, Molecular pathobiology of the human lipoprotein lipase gene, Pharmacol Ther 70 (1996) 101-135.
[40] M. Merkel, R.H. Eckel, I.J. Goldberg, Lipoprotein lipase: genetics, lipid uptake, and regulation, J Lipid Res 43 (2002) 1997-2006.
[41] M.K. Jensen, E.B. Rimm, D. Rader, E.B. Schmidt, T.I. Sorensen, U. Vogel, K. Overvad, K.J. Mukamal, S447X variant of the lipoprotein lipase gene, lipids, and risk of coronary heart disease in 3 prospective cohort studies, Am Heart J 157 (2009) 384-390.
[42] J. Rip, M.C. Nierman, C.J. Ross, J.W. Jukema, M.R. Hayden, J.J. Kastelein, E.S. Stroes, J.A. Kuivenhoven, Lipoprotein lipase S447X: a naturally occurring gain-of-function mutation, Arterioscler Thromb Vasc Biol 26 (2006) 1236-1245.
[43] H. Wong, M.C. Schotz, The lipase gene family, J Lipid Res 43 (2002) 993-999. [44] H. van Tilbeurgh, A. Roussel, J.M. Lalouel, C. Cambillau, Lipoprotein lipase. Molecular model
based on the pancreatic lipase x-ray structure: consequences for heparin binding and catalysis, J Biol Chem 269 (1994) 4626-4633.
[45] H. Wong, R.C. Davis, T. Thuren, J.W. Goers, J. Nikazy, M. Waite, M.C. Schotz, Lipoprotein lipase domain function, J Biol Chem 269 (1994) 10319-10323.
44
[46] J.C. Osborne, Jr., G. Bengtsson-Olivecrona, N.S. Lee, T. Olivecrona, Studies on inactivation of lipoprotein lipase: role of the dimer to monomer dissociation, Biochemistry 24 (1985) 5606-5611.
[47] H. Wong, R.C. Davis, J.S. Hill, D. Yang, M.C. Schotz, Lipase engineering: a window into structure-function relationships, Methods Enzymol 284 (1997) 171-184.
[48] Y. Kobayashi, T. Nakajima, I. Inoue, Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl-terminal domain, Eur J Biochem 269 (2002) 4701-4710.
[49] G. Bengtsson-Olivecrona, T. Olivecrona, Phospholipase activity of milk lipoprotein lipase, Methods Enzymol 197 (1991) 345-356.
[50] G. Bengtsson-Olivecrona, T. Olivecrona, Binding of active and inactive forms of lipoprotein lipase to heparin. Effects of pH, Biochem J 226 (1985) 409-413.
[51] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling, Bioinformatics 22 (2006) 195-201.
[52] L. Bordoli, F. Kiefer, K. Arnold, P. Benkert, J. Battey, T. Schwede, Protein structure homology modeling using SWISS-MODEL workspace, Nat Protoc 4 (2009) 1-13.
[53] H. van Tilbeurgh, L. Sarda, R. Verger, C. Cambillau, Structure of the pancreatic lipase-procolipase complex, Nature 359 (1992) 159-162.
[54] T.O. Olivecrona, G., Cellular Lipid Metabolism, Springer Berlin Heidelberg, 2009. [55] A. Lookene, G. Bengtsson-Olivecrona, Chymotryptic cleavage of lipoprotein lipase. Identification
of cleavage sites and functional studies of the truncated molecule, Eur J Biochem 213 (1993) 185-194.
[56] G. Bengtsson, T. Olivecrona, Lipoprotein lipase: some effects of activator proteins, Eur J Biochem 106 (1980) 549-555.
[57] J.S. Hill, D. Yang, J. Nikazy, L.K. Curtiss, J.T. Sparrow, H. Wong, Subdomain chimeras of hepatic lipase and lipoprotein lipase. Localization of heparin and cofactor binding, J Biol Chem 273 (1998) 30979-30984.
[58] G. Bengtsson, T. Olivecrona, Lipoprotein lipase. Mechanism of product inhibition, Eur J Biochem 106 (1980) 557-562.
[59] C.F. Semenkovich, C.C. Luo, M.K. Nakanishi, S.H. Chen, L.C. Smith, L. Chan, In vitro expression and site-specific mutagenesis of the cloned human lipoprotein lipase gene. Potential N-linked glycosylation site asparagine 43 is important for both enzyme activity and secretion, J Biol Chem 265 (1990) 5429-5433.
[60] R. Busca, M.A. Pujana, P. Pognonec, J. Auwerx, S.S. Deeb, M. Reina, S. Vilaro, Absence of N-glycosylation at asparagine 43 in human lipoprotein lipase induces its accumulation in the rough endoplasmic reticulum and alters this cellular compartment, J Lipid Res 36 (1995) 939-951.
[61] M. Peterfy, O. Ben-Zeev, H.Z. Mao, D. Weissglas-Volkov, B.E. Aouizerat, C.R. Pullinger, P.H. Frost, J.P. Kane, M.J. Malloy, K. Reue, P. Pajukanta, M.H. Doolittle, Mutations in LMF1 cause combined lipase deficiency and severe hypertriglyceridemia, Nat Genet 39 (2007) 1483-1487.
[62] O. Ben-Zeev, M. Hosseini, C.M. Lai, N. Ehrhardt, H. Wong, A.B. Cefalu, D. Noto, M.R. Averna, M.H. Doolittle, M. Peterfy, Lipase maturation factor 1 is required for endothelial lipase activity, J Lipid Res 52 (2011) 1162-1169.
[63] P.H. Weinstock, C.L. Bisgaier, K. Aalto-Setala, H. Radner, R. Ramakrishnan, S. Levak-Frank, A.D. Essenburg, R. Zechner, J.L. Breslow, Severe hypertriglyceridemia, reduced high density lipoprotein, and neonatal death in lipoprotein lipase knockout mice. Mild hypertriglyceridemia with impaired very low density lipoprotein clearance in heterozygotes, J Clin Invest 96 (1995) 2555-2568.
[64] K.J. Excoffon, G. Liu, L. Miao, J.E. Wilson, B.M. McManus, C.F. Semenkovich, T. Coleman, P. Benoit, N. Duverger, D. Branellec, P. Denefle, M.R. Hayden, M.E. Lewis, Correction of hypertriglyceridemia and impaired fat tolerance in lipoprotein lipase-deficient mice by
45
adenovirus-mediated expression of human lipoprotein lipase, Arterioscler Thromb Vasc Biol 17 (1997) 2532-2539.
[65] M. Shimada, H. Shimano, T. Gotoda, K. Yamamoto, M. Kawamura, T. Inaba, Y. Yazaki, N. Yamada, Overexpression of human lipoprotein lipase in transgenic mice. Resistance to diet-induced hypertriglyceridemia and hypercholesterolemia, J Biol Chem 268 (1993) 17924-17929.
[66] P. Benlian, Genetics of dyslipidemia, Kluwer Academic Publishers, Boston, 2001. [67] M. Bergo, G. Olivecrona, T. Olivecrona, Forms of lipoprotein lipase in rat tissues: in adipose
tissue the proportion of inactive lipase increases on fasting, Biochem J 313 ( Pt 3) (1996) 893-898.
[68] M. Bergo, G. Wu, T. Ruge, T. Olivecrona, Down-regulation of adipose tissue lipoprotein lipase during fasting requires that a gene, separate from the lipase gene, is switched on, J Biol Chem 277 (2002) 11927-11932.
[69] V. Sukonina, A. Lookene, T. Olivecrona, G. Olivecrona, Angiopoietin-like protein 4 converts lipoprotein lipase to inactive monomers and modulates lipase activity in adipose tissue, Proc Natl Acad Sci U S A 103 (2006) 17450-17455.
[70] W. Dijk, S. Kersten, Regulation of lipoprotein lipase by Angptl4, Trends Endocrinol Metab 25 (2014) 146-155.
[71] S. Kersten, Physiological regulation of lipoprotein lipase, Biochim Biophys Acta 1841 (2014) 919-933.
[72] T. Ruge, M. Bergo, M. Hultin, G. Olivecrona, T. Olivecrona, Nutritional regulation of binding sites for lipoprotein lipase in rat heart, Am J Physiol Endocrinol Metab 278 (2000) E211-218.
[73] G.Q. Liu, T. Olivecrona, Pulse-chase study on lipoprotein lipase in perfused guinea pig heart, Am J Physiol 261 (1991) H2044-2050.
[74] G. Wu, G. Olivecrona, T. Olivecrona, The distribution of lipoprotein lipase in rat adipose tissue. Changes with nutritional state engage the extracellular enzyme, J Biol Chem 278 (2003) 11925-11930.
[75] G. Wu, L. Zhang, J. Gupta, G. Olivecrona, T. Olivecrona, A transcription-dependent mechanism, akin to that in adipose tissue, modulates lipoprotein lipase activity in rat heart, Am J Physiol Endocrinol Metab 293 (2007) E908-915.
[76] A.P. Beigneux, B.S. Davies, P. Gin, M.M. Weinstein, E. Farber, X. Qiao, F. Peale, S. Bunting, R.L. Walzem, J.S. Wong, W.S. Blaner, Z.M. Ding, K. Melford, N. Wongsiriroj, X. Shu, F. de Sauvage, R.O. Ryan, L.G. Fong, A. Bensadoun, S.G. Young, Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 plays a critical role in the lipolytic processing of chylomicrons, Cell Metab 5 (2007) 279-291.
[77] L. Bey, M.T. Hamilton, Suppression of skeletal muscle lipoprotein lipase activity during physical inactivity: a molecular reason to maintain daily low-intensity activity, J Physiol 551 (2003) 673-682.
[78] D.B. Zilversmit, A proposal linking atherogenesis to the interaction of endothelial lipoprotein lipase with triglyceride-rich lipoproteins, Circ Res 33 (1973) 633-638.
[79] B.G. Nordestgaard, D.B. Zilversmit, Large lipoproteins are excluded from the arterial wall in diabetic cholesterol-fed rabbits, J Lipid Res 29 (1988) 1491-1500.
[80] J.R. Mead, A. Cryer, D.P. Ramji, Lipoprotein lipase, a key role in atherosclerosis?, FEBS Lett 462 (1999) 1-6.
[81] M.M. Weinstein, L. Yin, Y. Tu, X. Wang, X. Wu, L.W. Castellani, R.L. Walzem, A.J. Lusis, L.G. Fong, A.P. Beigneux, S.G. Young, Chylomicronemia elicits atherosclerosis in mice--brief report, Arterioscler Thromb Vasc Biol 30 (2010) 20-23.
[82] X. Zhang, R. Qi, X. Xian, F. Yang, M. Blackstein, X. Deng, J. Fan, C. Ross, J. Karasinska, M.R. Hayden, G. Liu, Spontaneous atherosclerosis in aged lipoprotein lipase-deficient mice with severe hypertriglyceridemia on a normal chow diet, Circ Res 102 (2008) 250-256.
[83] L. Eiselein, D.W. Wilson, M.W. Lame, J.C. Rutledge, Lipolysis products from triglyceride-rich lipoproteins increase endothelial permeability, perturb zonula occludens-1 and F-actin, and induce apoptosis, Am J Physiol Heart Circ Physiol 292 (2007) H2745-2753.
46
[84] J.C. Rutledge, A.E. Mullick, G. Gardner, I.J. Goldberg, Direct visualization of lipid deposition and reverse lipid transport in a perfused artery : roles of VLDL and HDL, Circ Res 86 (2000) 768-773.
[85] F. Karpe, J.R. Dickmann, K.N. Frayn, Fatty acids, obesity, and insulin resistance: time for a reevaluation, Diabetes 60 (2011) 2441-2449.
[86] M. Shimada, S. Ishibashi, T. Inaba, H. Yagyu, K. Harada, J.I. Osuga, K. Ohashi, Y. Yazaki, N. Yamada, Suppression of diet-induced atherosclerosis in low density lipoprotein receptor knockout mice overexpressing lipoprotein lipase, Proc Natl Acad Sci U S A 93 (1996) 7242-7246.
[87] H. Yagyu, S. Ishibashi, Z. Chen, J. Osuga, M. Okazaki, S. Perrey, T. Kitamine, M. Shimada, K. Ohashi, K. Harada, F. Shionoiri, N. Yahagi, T. Gotoda, Y. Yazaki, N. Yamada, Overexpressed lipoprotein lipase protects against atherosclerosis in apolipoprotein E knockout mice, J Lipid Res 40 (1999) 1677-1685.
[88] J. Fan, H. Unoki, N. Kojima, H. Sun, H. Shimoyamada, H. Deng, M. Okazaki, H. Shikama, N. Yamada, T. Watanabe, Overexpression of lipoprotein lipase in transgenic rabbits inhibits diet-induced hypercholesterolemia and atherosclerosis, J Biol Chem 276 (2001) 40071-40079.
[89] T. Ichikawa, S. Kitajima, J. Liang, T. Koike, X. Wang, H. Sun, M. Okazaki, M. Morimoto, H. Shikama, T. Watanabe, N. Yamada, J. Fan, Overexpression of lipoprotein lipase in transgenic rabbits leads to increased small dense LDL in plasma and promotes atherosclerosis, Lab Invest 84 (2004) 715-726.
[90] V.R. Babaev, M.B. Patel, C.F. Semenkovich, S. Fazio, M.F. Linton, Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in low density lipoprotein receptor-deficient mice, J Biol Chem 275 (2000) 26293-26299.
[91] V.R. Babaev, S. Fazio, L.A. Gleaves, K.J. Carter, C.F. Semenkovich, M.F. Linton, Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in vivo, J Clin Invest 103 (1999) 1697-1705.
[92] M. Van Eck, R. Zimmermann, P.H. Groot, R. Zechner, T.J. Van Berkel, Role of macrophage-derived lipoprotein lipase in lipoprotein metabolism and atherosclerosis, Arterioscler Thromb Vasc Biol 20 (2000) E53-62.
[93] M. Gustafsson, M. Levin, K. Skalen, J. Perman, V. Friden, P. Jirholt, S.O. Olofsson, S. Fazio, M.F. Linton, C.F. Semenkovich, G. Olivecrona, J. Boren, Retention of low-density lipoprotein in atherosclerotic lesions of the mouse: evidence for a role of lipoprotein lipase, Circ Res 101 (2007) 777-783.
[94] X. Wu, J. Wang, J. Fan, M. Chen, L. Chen, W. Huang, G. Liu, Localized vessel expression of lipoprotein lipase in rabbits leads to rapid lipid deposition in the balloon-injured arterial wall, Atherosclerosis 187 (2006) 65-73.
[95] S.C. Rumsey, J.C. Obunike, Y. Arad, R.J. Deckelbaum, I.J. Goldberg, Lipoprotein lipase-mediated uptake and degradation of low density lipoproteins by fibroblasts and macrophages, J Clin Invest 90 (1992) 1504-1512.
[96] I. Tabas, Y. Li, R.W. Brocia, S.W. Xu, T.L. Swenson, K.J. Williams, Lipoprotein lipase and sphingomyelinase synergistically enhance the association of atherogenic lipoproteins with smooth muscle cells and extracellular matrix. A possible mechanism for low density lipoprotein and lipoprotein(a) retention and macrophage foam cell formation, J Biol Chem 268 (1993) 20419-20432.
[97] A. Koster, Y.B. Chao, M. Mosior, A. Ford, P.A. Gonzalez-DeWhitt, J.E. Hale, D. Li, Y. Qiu, C.C. Fraser, D.D. Yang, J.G. Heuer, S.R. Jaskunas, P. Eacho, Transgenic angiopoietin-like (angptl)4 overexpression and targeted disruption of angptl4 and angptl3: regulation of triglyceride metabolism, Endocrinology 146 (2005) 4943-4950.
[98] S. Kersten, S. Mandard, N.S. Tan, P. Escher, D. Metzger, P. Chambon, F.J. Gonzalez, B. Desvergne, W. Wahli, Characterization of the fasting-induced adipose factor FIAF, a novel peroxisome proliferator-activated receptor target gene, J Biol Chem 275 (2000) 28488-28493.
47
[99] H. Ge, J.Y. Cha, H. Gopal, C. Harp, X. Yu, J.J. Repa, C. Li, Differential regulation and properties of angiopoietin-like proteins 3 and 4, J Lipid Res 46 (2005) 1484-1490.
[100] F. Mattijssen, S. Alex, H.J. Swarts, A.K. Groen, E.M. van Schothorst, S. Kersten, Angptl4 serves as an endogenous inhibitor of intestinal lipid digestion, Mol Metab 3 (2014) 135-144.
[101] L. Lichtenstein, F. Mattijssen, N.J. de Wit, A. Georgiadi, G.J. Hooiveld, R. van der Meer, Y. He, L. Qi, A. Koster, J.T. Tamsma, N.S. Tan, M. Muller, S. Kersten, Angptl4 protects against severe proinflammatory effects of saturated fat by inhibiting fatty acid uptake into mesenteric lymph node macrophages, Cell Metab 12 (2010) 580-592.
[102] A. Georgiadi, L. Lichtenstein, T. Degenhardt, M.V. Boekschoten, M. van Bilsen, B. Desvergne, M. Muller, S. Kersten, Induction of cardiac Angptl4 by dietary fatty acids is mediated by peroxisome proliferator-activated receptor beta/delta and protects against fatty acid-induced oxidative stress, Circ Res 106 (2010) 1712-1721.
[103] K. Yoshida, T. Shimizugawa, M. Ono, H. Furukawa, Angiopoietin-like protein 4 is a potent hyperlipidemia-inducing factor in mice and inhibitor of lipoprotein lipase, J Lipid Res 43 (2002) 1770-1772.
[104] L. Lichtenstein, J.F. Berbee, S.J. van Dijk, K.W. van Dijk, A. Bensadoun, I.P. Kema, P.J. Voshol, M. Muller, P.C. Rensen, S. Kersten, Angptl4 upregulates cholesterol synthesis in liver via inhibition of LPL- and HL-dependent hepatic cholesterol uptake, Arterioscler Thromb Vasc Biol 27 (2007) 2420-2427.
[105] M.J. Lafferty, K.C. Bradford, D.A. Erie, S.B. Neher, Angiopoietin-like protein 4 inhibition of lipoprotein lipase: evidence for reversible complex formation, J Biol Chem 288 (2013) 28524-28534.
[106] S.K. Nilsson, F. Anderson, M. Ericsson, M. Larsson, E. Makoveichuk, A. Lookene, J. Heeren, G. Olivecrona, Triacylglycerol-rich lipoproteins protect lipoprotein lipase from inactivation by ANGPTL3 and ANGPTL4, Biochim Biophys Acta 1821 (2012) 1370-1378.
[107] U. Desai, E.C. Lee, K. Chung, C. Gao, J. Gay, B. Key, G. Hansen, D. Machajewski, K.A. Platt, A.T. Sands, M. Schneider, I. Van Sligtenhorst, A. Suwanichkul, P. Vogel, N. Wilganowski, J. Wingert, B.P. Zambrowicz, G. Landes, D.R. Powell, Lipid-lowering effects of anti-angiopoietin-like 4 antibody recapitulate the lipid phenotype found in angiopoietin-like 4 knockout mice, Proc Natl Acad Sci U S A 104 (2007) 11766-11771.
[108] H. Adachi, Y. Fujiwara, T. Kondo, T. Nishikawa, R. Ogawa, T. Matsumura, N. Ishii, R. Nagai, K. Miyata, M. Tabata, H. Motoshima, N. Furukawa, K. Tsuruzoe, J. Kawashima, M. Takeya, S. Yamashita, G.Y. Koh, A. Nagy, T. Suda, Y. Oike, E. Araki, Angptl 4 deficiency improves lipid metabolism, suppresses foam cell formation and protects against atherosclerosis, Biochem Biophys Res Commun 379 (2009) 806-811.
[109] A. Lookene, N. Skottova, G. Olivecrona, Interactions of lipoprotein lipase with the active-site inhibitor tetrahydrolipstatin (Orlistat), Eur J Biochem 222 (1994) 395-403.
[110] A. Georgiadi, Y. Wang, R. Stienstra, N. Tjeerdema, A. Janssen, A. Stalenhoef, J.A. van der Vliet, A. de Roos, J.T. Tamsma, J.W. Smit, N.S. Tan, M. Muller, P.C. Rensen, S. Kersten, Overexpression of angiopoietin-like protein 4 protects against atherosclerosis development, Arterioscler Thromb Vasc Biol 33 (2013) 1529-1537.
[111] E. Makoveichuk, V. Sukonina, O. Kroupa, P. Thulin, E. Ehrenborg, T. Olivecrona, G. Olivecrona, Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed, Biochem Biophys Res Commun 425 (2012) 138-143.
[112] S. Romeo, L.A. Pennacchio, Y. Fu, E. Boerwinkle, A. Tybjaerg-Hansen, H.H. Hobbs, J.C. Cohen, Population-based resequencing of ANGPTL4 uncovers variations that reduce triglycerides and increase HDL, Nat Genet 39 (2007) 513-516.
[113] V. Sukonina, Angiopoietin-like protein 4: an unfolding chaperone regulating lipoprotein lipase activity, Diss., Medical Biosciences, Umeå University, Umeå, 2007, pp. 50.
48
[114] S. Romeo, W. Yin, J. Kozlitina, L.A. Pennacchio, E. Boerwinkle, H.H. Hobbs, J.C. Cohen, Rare loss-of-function mutations in ANGPTL family members contribute to plasma triglyceride levels in humans, J Clin Invest 119 (2009) 70-79.
[115] P.J. Talmud, M. Smart, E. Presswood, J.A. Cooper, V. Nicaud, F. Drenos, J. Palmen, M.G. Marmot, S.M. Boekholdt, N.J. Wareham, K.T. Khaw, M. Kumari, S.E. Humphries, ANGPTL4 E40K and T266M: effects on plasma triglyceride and HDL levels, postprandial responses, and CHD risk, Arterioscler Thromb Vasc Biol 28 (2008) 2319-2325.
[116] A.R. Folsom, J.M. Peacock, E. Demerath, E. Boerwinkle, Variation in ANGPTL4 and risk of coronary heart disease: the Atherosclerosis Risk in Communities Study, Metabolism 57 (2008) 1591-1596.
[117] P. Zhu, Y.Y. Goh, H.F. Chin, S. Kersten, N.S. Tan, Angiopoietin-like 4: a decade of research, Biosci Rep 32 (2012) 211-219.
[118] R. Koishi, Y. Ando, M. Ono, M. Shimamura, H. Yasumo, T. Fujiwara, H. Horikoshi, H. Furukawa, Angptl3 regulates lipid metabolism in mice, Nat Genet 30 (2002) 151-157.
[119] I. Minicocci, A. Montali, M.R. Robciuc, F. Quagliarini, V. Censi, G. Labbadia, C. Gabiati, G. Pigna, M.L. Sepe, F. Pannozzo, D. Lutjohann, S. Fazio, M. Jauhiainen, C. Ehnholm, M. Arca, Mutations in the ANGPTL3 gene and familial combined hypolipidemia: a clinical and biochemical characterization, J Clin Endocrinol Metab 97 (2012) E1266-1275.
[120] K. Fujimoto, R. Koishi, T. Shimizugawa, Y. Ando, Angptl3-null mice show low plasma lipid concentrations by enhanced lipoprotein lipase activity, Exp Anim 55 (2006) 27-34.
[121] M. Shimamura, M. Matsuda, H. Yasumo, M. Okazaki, K. Fujimoto, K. Kono, T. Shimizugawa, Y. Ando, R. Koishi, T. Kohama, N. Sakai, K. Kotani, R. Komuro, T. Ishida, K. Hirata, S. Yamashita, H. Furukawa, I. Shimomura, Angiopoietin-like protein3 regulates plasma HDL cholesterol through suppression of endothelial lipase, Arterioscler Thromb Vasc Biol 27 (2007) 366-372.
[122] W.K. Sonnenburg, D. Yu, E.C. Lee, W. Xiong, G. Gololobov, B. Key, J. Gay, N. Wilganowski, Y. Hu, S. Zhao, M. Schneider, Z.M. Ding, B.P. Zambrowicz, G. Landes, D.R. Powell, U. Desai, GPIHBP1 stabilizes lipoprotein lipase and prevents its inhibition by angiopoietin-like 3 and angiopoietin-like 4, J Lipid Res 50 (2009) 2421-2429.
[123] L. Shan, X.C. Yu, Z. Liu, Y. Hu, L.T. Sturgis, M.L. Miranda, Q. Liu, The angiopoietin-like proteins ANGPTL3 and ANGPTL4 inhibit lipoprotein lipase activity through distinct mechanisms, J Biol Chem 284 (2009) 1419-1424.
[124] J. Liu, H. Afroza, D.J. Rader, W. Jin, Angiopoietin-like protein 3 inhibits lipoprotein lipase activity through enhancing its cleavage by proprotein convertases, J Biol Chem 285 (2010) 27561-27570.
[125] X. Lei, F. Shi, D. Basu, A. Huq, S. Routhier, R. Day, W. Jin, Proteolytic processing of angiopoietin-like protein 4 by proprotein convertases modulates its inhibitory effects on lipoprotein lipase activity, J Biol Chem 286 (2011) 15747-15756.
[126] L. Zhang, A. Lookene, G. Wu, G. Olivecrona, Calcium triggers folding of lipoprotein lipase into active dimers, J Biol Chem 280 (2005) 42580-42591.
[127] F. Quagliarini, Y. Wang, J. Kozlitina, N.V. Grishin, R. Hyde, E. Boerwinkle, D.M. Valenzuela, A.J. Murphy, J.C. Cohen, H.H. Hobbs, Atypical angiopoietin-like protein that regulates ANGPTL3, Proc Natl Acad Sci U S A 109 (2012) 19751-19756.
[128] B.S. Davies, A.P. Beigneux, R.H. Barnes, 2nd, Y. Tu, P. Gin, M.M. Weinstein, C. Nobumori, R. Nyren, I. Goldberg, G. Olivecrona, A. Bensadoun, S.G. Young, L.G. Fong, GPIHBP1 is responsible for the entry of lipoprotein lipase into capillaries, Cell Metab 12 (2010) 42-52.
[129] C.N. Goulbourne, P. Gin, A. Tatar, C. Nobumori, A. Hoenger, H.B. Jiang, C.R.M. Grovenor, O. Adeyo, J.D. Esko, I.J. Goldberg, K. Reue, P. Tontonoz, A. Bensadoun, A.P. Beigneux, S.G. Young, L.G. Fong, The GPIHBP1-LPL Complex Is Responsible for the Margination of Triglyceride-Rich Lipoproteins in Capillaries, Cell Metab 19 (2014) 849-860.
[130] O. Adeyo, C.N. Goulbourne, A. Bensadoun, A.P. Beigneux, L.G. Fong, S.G. Young, Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 and the intravascular processing of triglyceride-rich lipoproteins, J Intern Med 272 (2012) 528-540.
49
[131] R. Franssen, S.G. Young, F. Peelman, J. Hertecant, J.A. Sierts, A.W. Schimmel, A. Bensadoun, J.J. Kastelein, L.G. Fong, G.M. Dallinga-Thie, A.P. Beigneux, Chylomicronemia with low postheparin lipoprotein lipase levels in the setting of GPIHBP1 defects, Circ Cardiovasc Genet 3 (2010) 169-178.
[132] A.P. Beigneux, GPIHBP1 and the processing of triglyceride-rich lipoproteins, Clin Lipidol 5 (2010) 575-582.
[133] A.P. Beigneux, P. Gin, B.S. Davies, M.M. Weinstein, A. Bensadoun, L.G. Fong, S.G. Young, Highly conserved cysteines within the Ly6 domain of GPIHBP1 are crucial for the binding of lipoprotein lipase, J Biol Chem 284 (2009) 30240-30247.
[134] M.M. Weinstein, L. Yin, A.P. Beigneux, B.S. Davies, P. Gin, K. Estrada, K. Melford, J.R. Bishop, J.D. Esko, G.M. Dallinga-Thie, L.G. Fong, A. Bensadoun, S.G. Young, Abnormal patterns of lipoprotein lipase release into the plasma in GPIHBP1-deficient mice, J Biol Chem 283 (2008) 34511-34518.
[135] J.P. Segrest, M.K. Jones, H. De Loof, C.G. Brouillette, Y.V. Venkatachalapathi, G.M. Anantharamaiah, The amphipathic helix in the exchangeable apolipoproteins: a review of secondary structure and function, J Lipid Res 33 (1992) 141-166.
[136] J.P. Segrest, R.L. Jackson, J.D. Morrisett, A.M. Gotto, Jr., A molecular theory of lipid-protein interactions in the plasma lipoproteins, FEBS Lett 38 (1974) 247-258.
[137] J.P. Segrest, H. De Loof, J.G. Dohlman, C.G. Brouillette, G.M. Anantharamaiah, Amphipathic helix motif: classes and properties, Proteins 8 (1990) 103-117.
[138] W.H. Li, M. Tanimura, C.C. Luo, S. Datta, L. Chan, The apolipoprotein multigene family: biosynthesis, structure, structure-function relationships, and evolution, J Lipid Res 29 (1988) 245-271.
[139] C.C. Luo, W.H. Li, M.N. Moore, L. Chan, Structure and evolution of the apolipoprotein multigene family, J Mol Biol 187 (1986) 325-340.
[140] E.J. Schaefer, R.D. Santos, B.F. Asztalos, Marked HDL deficiency and premature coronary heart disease, Curr Opin Lipidol 21 (2010) 289-297.
[141] M.C. Cheung, S.D. Sibley, J.P. Palmer, J.F. Oram, J.D. Brunzell, Lipoprotein lipase and hepatic lipase: their relationship with HDL subspecies Lp(A-I) and Lp(A-I,A-II), J Lipid Res 44 (2003) 1552-1558.
[142] A.R. Tall, P.H. Green, R.M. Glickman, J.W. Riley, Metabolic fate of chylomicron phospholipids and apoproteins in the rat, J Clin Invest 64 (1979) 977-989.
[143] S. Kaser, A. Sandhofer, B. Holzl, R. Gander, C.F. Ebenbichler, B. Paulweber, J.R. Patsch, Phospholipid and cholesteryl ester transfer are increased in lipoprotein lipase deficiency, J Intern Med 253 (2003) 208-216.
[144] A.K. Soutar, C.W. Garner, H.N. Baker, J.T. Sparrow, R.L. Jackson, A.M. Gotto, L.C. Smith, Effect of the human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of lecithin: cholesterol acyltransferase, Biochemistry 14 (1975) 3057-3064.
[145] S. Acton, A. Rigotti, K.T. Landschulz, S. Xu, H.H. Hobbs, M. Krieger, Identification of scavenger receptor SR-BI as a high density lipoprotein receptor, Science 271 (1996) 518-520.
[146] S.G. Young, Recent progress in understanding apolipoprotein B, Circulation 82 (1990) 1574-1594.
[147] J. Greeve, I. Altkemper, J.H. Dieterich, H. Greten, E. Windler, Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoB-containing plasma lipoproteins, J Lipid Res 34 (1993) 1367-1383.
[148] S.J. Lauer, D. Walker, N.A. Elshourbagy, C.A. Reardon, B. Levy-Wilson, J.M. Taylor, Two copies of the human apolipoprotein C-I gene are linked closely to the apolipoprotein E gene, J Biol Chem 263 (1988) 7277-7286.
[149] R.J. Havel, C.J. Fielding, T. Olivecrona, V.G. Shore, P.E. Fielding, T. Egelrud, Cofactor activity of protein components of human very low density lipoproteins in the hydrolysis of triglycerides by lipoproteins lipase from different sources, Biochemistry 12 (1973) 1828-1833.
50
[150] A. Rozek, J.T. Sparrow, K.H. Weisgraber, R.J. Cushley, Conformation of human apolipoprotein C-I in a lipid-mimetic environment determined by CD and NMR spectroscopy, Biochemistry 38 (1999) 14475-14484.
[151] N.L. Meyers, L. Wang, O. Gursky, D.M. Small, Changes in helical content or net charge of apolipoprotein C-I alter its affinity for lipid/water interfaces, J Lipid Res 54 (2013) 1927-1938.
[152] K.H. Weisgraber, R.W. Mahley, R.C. Kowal, J. Herz, J.L. Goldstein, M.S. Brown, Apolipoprotein C-I modulates the interaction of apolipoprotein E with beta-migrating very low density lipoproteins (beta-VLDL) and inhibits binding of beta-VLDL to low density lipoprotein receptor-related protein, J Biol Chem 265 (1990) 22453-22459.
[153] E. Sehayek, S. Eisenberg, Mechanisms of inhibition by apolipoprotein C of apolipoprotein E-dependent cellular metabolism of human triglyceride-rich lipoproteins through the low density lipoprotein receptor pathway, J Biol Chem 266 (1991) 18259-18267.
[154] J.H. van Ree, M.H. Hofker, W.J. van den Broek, J.M. van Deursen, H. van der Boom, R.R. Frants, B. Wieringa, L.M. Havekes, Increased response to cholesterol feeding in apolipoprotein C1-deficient mice, Biochem J 305 ( Pt 3) (1995) 905-911.
[155] N.S. Shachter, T. Ebara, R. Ramakrishnan, G. Steiner, J.L. Breslow, H.N. Ginsberg, J.D. Smith, Combined hyperlipidemia in transgenic mice overexpressing human apolipoprotein Cl, J Clin Invest 98 (1996) 846-855.
[156] J.F. Berbee, C.C. van der Hoogt, D. Sundararaman, L.M. Havekes, P.C. Rensen, Severe hypertriglyceridemia in human APOC1 transgenic mice is caused by apoC-I-induced inhibition of LPL, J Lipid Res 46 (2005) 297-306.
[157] M. Westerterp, W. de Haan, J.F. Berbee, L.M. Havekes, P.C. Rensen, Endogenous apoC-I increases hyperlipidemia in apoE-knockout mice by stimulating VLDL production and inhibiting LPL, J Lipid Res 47 (2006) 1203-1211.
[158] T. Gautier, D. Masson, J.P. de Barros, A. Athias, P. Gambert, D. Aunis, M.H. Metz-Boutigue, L. Lagrost, Human apolipoprotein C-I accounts for the ability of plasma high density lipoproteins to inhibit the cholesteryl ester transfer protein activity, J Biol Chem 275 (2000) 37504-37509.
[159] W.C. Breckenridge, J.A. Little, G. Steiner, A. Chow, M. Poapst, Hypertriglyceridemia associated with deficiency of apolipoprotein C-II, N Engl J Med 298 (1978) 1265-1273.
[160] R. Fellin, G. Baggio, A. Poli, J. Augustin, M.R. Baiocchi, G. Baldo, M. Sinigaglia, H. Greten, G. Crepaldi, Familial lipoprotein lipase and apolipoprotein C-II deficiency. Lipoprotein and apoprotein analysis, adipose tissue and hepatic lipoprotein lipase levels in seven patients and their first degree relatives, Atherosclerosis 49 (1983) 55-68.
[161] M.C. Jong, M.H. Hofker, L.M. Havekes, Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2, and ApoC3, Arterioscler Thromb Vasc Biol 19 (1999) 472-484.
[162] A.A. Kei, T.D. Filippatos, V. Tsimihodimos, M.S. Elisaf, A review of the role of apolipoprotein C-II in lipoprotein metabolism and cardiovascular disease, Metabolism 61 (2012) 906-921.
[163] P. Fornengo, A. Bruno, R. Gambino, M. Cassader, G. Pagano, Resistant hypertriglyceridemia in a patient with high plasma levels of apolipoprotein CII, Arterioscler Thromb Vasc Biol 20 (2000) 2329-2339.
[164] V.I. Zannis, F.S. Cole, C.L. Jackson, D.M. Kurnit, S.K. Karathanasis, Distribution of apolipoprotein A-I, C-II, C-III, and E mRNA in fetal human tissues. Time-dependent induction of apolipoprotein E mRNA by cultures of human monocyte-macrophages, Biochemistry 24 (1985) 4450-4455.
[165] J.C. LaRosa, R.I. Levy, P. Herbert, S.E. Lux, D.S. Fredrickson, A specific apoprotein activator for lipoprotein lipase, Biochem Biophys Res Commun 41 (1970) 57-62.
[166] Y. Andersson, A. Lookene, Y. Shen, S. Nilsson, L. Thelander, G. Olivecrona, Guinea pig apolipoprotein C-II: expression in E. coli, functional studies of recombinant wild-type and mutated variants, and distribution on plasma lipoproteins, J Lipid Res 38 (1997) 2111-2124.
51
[167] G. Olivecrona, U. Beisiegel, Lipid binding of apolipoprotein CII is required for stimulation of lipoprotein lipase activity against apolipoprotein CII-deficient chylomicrons, Arterioscler Thromb Vasc Biol 17 (1997) 1545-1549.
[168] C.A. MacRaild, D.M. Hatters, G.J. Howlett, P.R. Gooley, NMR structure of human apolipoprotein C-II in the presence of sodium dodecyl sulfate, Biochemistry 40 (2001) 5414-5421.
[169] J. Zdunek, G.V. Martinez, J. Schleucher, P.O. Lycksell, Y. Yin, S. Nilsson, Y. Shen, G. Olivecrona, S. Wijmenga, Global structure and dynamics of human apolipoprotein CII in complex with micelles: evidence for increased mobility of the helix involved in the activation of lipoprotein lipase, Biochemistry 42 (2003) 1872-1889.
[170] C.E. MacPhee, G.J. Howlett, W.H. Sawyer, Mass spectrometry to characterize the binding of a peptide to a lipid surface, Anal Biochem 275 (1999) 22-29.
[171] M. Dahim, W.E. Momsen, M.M. Momsen, H.L. Brockman, Specificity of the lipid-binding domain of apoC-II for the substrates and products of lipolysis, J Lipid Res 42 (2001) 553-562.
[172] T.A. Musliner, E.C. Church, P.N. Herbert, M.J. Kingston, R.S. Shulman, Lipoprotein lipase cofactor activity of a carboxyl-terminal peptide of apolipoprotein C-II, Proc Natl Acad Sci U S A 74 (1977) 5358-5362.
[173] C.E. MacPhee, D.M. Hatters, W.H. Sawyer, G.J. Howlett, Apolipoprotein C-II39-62 activates lipoprotein lipase by direct lipid-independent binding, Biochemistry 39 (2000) 3433-3440.
[174] Y. Shen, A. Lookene, L. Zhang, G. Olivecrona, Site-directed mutagenesis of apolipoprotein CII to probe the role of its secondary structure for activation of lipoprotein lipase, J Biol Chem 285 (2010) 7484-7492.
[175] N.S. Shachter, T. Hayek, T. Leff, J.D. Smith, D.W. Rosenberg, A. Walsh, R. Ramakrishnan, I.J. Goldberg, H.N. Ginsberg, J.L. Breslow, Overexpression of apolipoprotein CII causes hypertriglyceridemia in transgenic mice, J Clin Invest 93 (1994) 1683-1690.
[176] L.K. Pulawa, D.R. Jensen, A. Coates, R.H. Eckel, Reduction of plasma triglycerides in apolipoprotein C-II transgenic mice overexpressing lipoprotein lipase in muscle, J Lipid Res 48 (2007) 145-151.
[177] C.A. MacRaild, G.J. Howlett, P.R. Gooley, The structure and interactions of human apolipoprotein C-II in dodecyl phosphocholine, Biochemistry 43 (2004) 8084-8093.
[178] T.I. Pollin, C.M. Damcott, H. Shen, S.H. Ott, J. Shelton, R.B. Horenstein, W. Post, J.C. McLenithan, L.F. Bielak, P.A. Peyser, B.D. Mitchell, M. Miller, J.R. O'Connell, A.R. Shuldiner, A null mutation in human APOC3 confers a favorable plasma lipid profile and apparent cardioprotection, Science 322 (2008) 1702-1705.
[179] I. Tachmazidou, G. Dedoussis, L. Southam, A.E. Farmaki, G.R. Ritchie, D.K. Xifara, A. Matchan, K. Hatzikotoulas, N.W. Rayner, Y. Chen, T.I. Pollin, J.R. O'Connell, L.M. Yerges-Armstrong, C. Kiagiadaki, K. Panoutsopoulou, J. Schwartzentruber, L. Moutsianas, E. Tsafantakis, C. Tyler-Smith, G. McVean, Y. Xue, E. Zeggini, A rare functional cardioprotective APOC3 variant has risen in frequency in distinct population isolates, Nat Commun 4 (2013) 2872.
[180] A.E. Bochem, J.C. van Capelleveen, G.M. Dallinga-Thie, A.W. Schimmel, M.M. Motazacker, I. Tietjen, R.R. Singaraja, M.R. Hayden, J.J. Kastelein, E.S. Stroes, G.K. Hovingh, Two novel mutations in apolipoprotein C3 underlie atheroprotective lipid profiles in families, Clin Genet 85 (2014) 433-440.
[181] A. von Eckardstein, H. Holz, M. Sandkamp, W. Weng, H. Funke, G. Assmann, Apolipoprotein C-III(Lys58----Glu). Identification of an apolipoprotein C-III variant in a family with hyperalphalipoproteinemia, J Clin Invest 87 (1991) 1724-1731.
[182] H. Liu, C. Labeur, C.F. Xu, R. Ferrell, L. Lins, R. Brasseur, M. Rosseneu, K.M. Weiss, S.E. Humphries, P.J. Talmud, Characterization of the lipid-binding properties and lipoprotein lipase inhibition of a novel apolipoprotein C-III variant Ala23Thr, J Lipid Res 41 (2000) 1760-1771.
[183] J. Crosby, G.M. Peloso, P.L. Auer, D.R. Crosslin, N.O. Stitziel, L.A. Lange, Y. Lu, Z.Z. Tang, H. Zhang, G. Hindy, N. Masca, K. Stirrups, S. Kanoni, R. Do, G. Jun, Y. Hu, H.M. Kang, C. Xue, A. Goel, M. Farrall, S. Duga, P.A. Merlini, R. Asselta, D. Girelli, O. Olivieri, N. Martinelli, W. Yin, D.
52
Reilly, E. Speliotes, C.S. Fox, K. Hveem, O.L. Holmen, M. Nikpay, D.N. Farlow, T.L. Assimes, N. Franceschini, J. Robinson, K.E. North, L.W. Martin, M. DePristo, N. Gupta, S.A. Escher, J.H. Jansson, N. Van Zuydam, C.N. Palmer, N. Wareham, W. Koch, T. Meitinger, A. Peters, W. Lieb, R. Erbel, I.R. Konig, J. Kruppa, F. Degenhardt, O. Gottesman, E.P. Bottinger, C.J. O'Donnell, B.M. Psaty, C.M. Ballantyne, G. Abecasis, J.M. Ordovas, O. Melander, H. Watkins, M. Orho-Melander, D. Ardissino, R.J. Loos, R. McPherson, C.J. Willer, J. Erdmann, A.S. Hall, N.J. Samani, P. Deloukas, H. Schunkert, J.G. Wilson, C. Kooperberg, S.S. Rich, R.P. Tracy, D.Y. Lin, D. Altshuler, S. Gabriel, D.A. Nickerson, G.P. Jarvik, L.A. Cupples, A.P. Reiner, E. Boerwinkle, S. Kathiresan, Loss-of-function mutations in APOC3, triglycerides, and coronary disease, N Engl J Med 371 (2014) 22-31.
[184] A.B. Jorgensen, R. Frikke-Schmidt, B.G. Nordestgaard, A. Tybjaerg-Hansen, Loss-of-function mutations in APOC3 and risk of ischemic vascular disease, N Engl J Med 371 (2014) 32-41.
[185] W.V. Brown, M.L. Baginsky, Inhibition of lipoprotein lipase by an apoprotein of human very low density lipoprotein, Biochem Biophys Res Commun 46 (1972) 375-382.
[186] R.J. Havel, V.G. Shore, B. Shore, D.M. Bier, Role of specific glycopeptides of human serum lipoproteins in the activation of lipoprotein lipase, Circ Res 27 (1970) 595-600.
[187] C.S. Wang, W.J. McConathy, H.U. Kloer, P. Alaupovic, Modulation of lipoprotein lipase activity by apolipoproteins. Effect of apolipoprotein C-III, J Clin Invest 75 (1985) 384-390.
[188] A.G. Holleboom, H. Karlsson, R.S. Lin, T.M. Beres, J.A. Sierts, D.S. Herman, E.S. Stroes, J.M. Aerts, J.J. Kastelein, M.M. Motazacker, G.M. Dallinga-Thie, J.H. Levels, A.H. Zwinderman, J.G. Seidman, C.E. Seidman, S. Ljunggren, D.J. Lefeber, E. Morava, R.A. Wevers, T.A. Fritz, L.A. Tabak, M. Lindahl, G.K. Hovingh, J.A. Kuivenhoven, Heterozygosity for a loss-of-function mutation in GALNT2 improves plasma triglyceride clearance in man, Cell Metab 14 (2011) 811-818.
[189] C.S. Gangabadage, J. Zdunek, M. Tessari, S. Nilsson, G. Olivecrona, S.S. Wijmenga, Structure and dynamics of human apolipoprotein CIII, J Biol Chem 283 (2008) 17416-17427.
[190] J.T. Sparrow, H.J. Pownall, F.J. Hsu, L.D. Blumenthal, A.R. Culwell, A.M. Gotto, Lipid binding by fragments of apolipoprotein C-III-1 obtained by thrombin cleavage, Biochemistry 16 (1977) 5427-5431.
[191] D.A. Lambert, A.L. Catapano, L.C. Smith, J.T. Sparrow, A.M. Gotto, Jr., Effect of the apolipoprotein C-II/C-III1 ratio on the capacity of purified milk lipoprotein lipase to hydrolyse triglycerides in monolayer vesicles, Atherosclerosis 127 (1996) 205-212.
[192] M. Sundaram, S. Zhong, M. Bou Khalil, P.H. Links, Y. Zhao, J. Iqbal, M.M. Hussain, R.J. Parks, Y. Wang, Z. Yao, Expression of apolipoprotein C-III in McA-RH7777 cells enhances VLDL assembly and secretion under lipid-rich conditions, J Lipid Res 51 (2010) 150-161.
[193] W. Qin, M. Sundaram, Y. Wang, H. Zhou, S. Zhong, C.C. Chang, S. Manhas, E.F. Yao, R.J. Parks, P.J. McFie, S.J. Stone, Z.G. Jiang, C. Wang, D. Figeys, W. Jia, Z. Yao, Missense mutation in APOC3 within the C-terminal lipid binding domain of human ApoC-III results in impaired assembly and secretion of triacylglycerol-rich very low density lipoproteins: evidence that ApoC-III plays a major role in the formation of lipid precursors within the microsomal lumen, J Biol Chem 286 (2011) 27769-27780.
[194] E.D. Breyer, N.A. Le, X. Li, D. Martinson, W.V. Brown, Apolipoprotein C-III displacement of apolipoprotein E from VLDL: effect of particle size, J Lipid Res 40 (1999) 1875-1882.
[195] N. Maeda, H. Li, D. Lee, P. Oliver, S.H. Quarfordt, J. Osada, Targeted disruption of the apolipoprotein C-III gene in mice results in hypotriglyceridemia and protection from postprandial hypertriglyceridemia, J Biol Chem 269 (1994) 23610-23616.
[196] M.C. Jong, P.C. Rensen, V.E. Dahlmans, H. van der Boom, T.J. van Berkel, L.M. Havekes, Apolipoprotein C-III deficiency accelerates triglyceride hydrolysis by lipoprotein lipase in wild-type and apoE knockout mice, J Lipid Res 42 (2001) 1578-1585.
[197] Y. Ito, N. Azrolan, A. O'Connell, A. Walsh, J.L. Breslow, Hypertriglyceridemia as a result of human apo CIII gene expression in transgenic mice, Science 249 (1990) 790-793.
53
[198] Y. Ding, Y. Wang, H. Zhu, J. Fan, L. Yu, G. Liu, E. Liu, Hypertriglyceridemia and delayed clearance of fat load in transgenic rabbits expressing human apolipoprotein CIII, Transgenic Res 20 (2011) 867-875.
[199] J. Wei, H. Ouyang, Y. Wang, D. Pang, N.X. Cong, T. Wang, B. Leng, D. Li, X. Li, R. Wu, Y. Ding, F. Gao, Y. Deng, B. Liu, Z. Li, L. Lai, H. Feng, G. Liu, X. Deng, Characterization of a hypertriglyceridemic transgenic miniature pig model expressing human apolipoprotein CIII, FEBS J 279 (2012) 91-99.
[200] T. Ebara, R. Ramakrishnan, G. Steiner, N.S. Shachter, Chylomicronemia due to apolipoprotein CIII overexpression in apolipoprotein E-null mice. Apolipoprotein CIII-induced hypertriglyceridemia is not mediated by effects on apolipoprotein E, J Clin Invest 99 (1997) 2672-2681.
[201] H.V. de Silva, S.J. Lauer, J. Wang, W.S. Simonet, K.H. Weisgraber, R.W. Mahley, J.M. Taylor, Overexpression of human apolipoprotein C-III in transgenic mice results in an accumulation of apolipoprotein B48 remnants that is corrected by excess apolipoprotein E, J Biol Chem 269 (1994) 2324-2335.
[202] R.L. Jackson, S. Tajima, T. Yamamura, S. Yokoyama, A. Yamamoto, Comparison of apolipoprotein C-II-deficient triacylglycerol-rich lipoproteins and trioleoylglycerol/phosphatidylcholine-stabilized particles as substrates for lipoprotein lipase, Biochim Biophys Acta 875 (1986) 211-219.
[203] E.J. Schaefer, R.E. Gregg, G. Ghiselli, T.M. Forte, J.M. Ordovas, L.A. Zech, H.B. Brewer, Familial Apolipoprotein-E Deficiency, Journal of Clinical Investigation 78 (1986) 1206-1219.
[204] R.W. Mahley, S.C. Rall, Jr., Apolipoprotein E: far more than a lipid transport protein, Annu Rev Genomics Hum Genet 1 (2000) 507-537.
[205] T. Mazzone, C. Reardon, Expression of heterologous human apolipoprotein E by J774 macrophages enhances cholesterol efflux to HDL3, J Lipid Res 35 (1994) 1345-1353.
[206] P.G. Yancey, H. Yu, M.F. Linton, S. Fazio, A pathway-dependent on apoE, ApoAI, and ABCA1 determines formation of buoyant high-density lipoprotein by macrophage foam cells, Arterioscler Thromb Vasc Biol 27 (2007) 1123-1131.
[207] S.H. Zhang, R.L. Reddick, J.A. Piedrahita, N. Maeda, Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E, Science 258 (1992) 468-471.
[208] S. Bellosta, R.W. Mahley, D.A. Sanan, J. Murata, D.L. Newland, J.M. Taylor, R.E. Pitas, Macrophage-specific expression of human apolipoprotein E reduces atherosclerosis in hypercholesterolemic apolipoprotein E-null mice, J Clin Invest 96 (1995) 2170-2179.
[209] S. Fazio, V.R. Babaev, A.B. Murray, A.H. Hasty, K.J. Carter, L.A. Gleaves, J.B. Atkinson, M.F. Linton, Increased atherosclerosis in mice reconstituted with apolipoprotein E null macrophages, Proc Natl Acad Sci U S A 94 (1997) 4647-4652.
[210] S. Eisenberg, E. Sehayek, T. Olivecrona, I. Vlodavsky, Lipoprotein lipase enhances binding of lipoproteins to heparan sulfate on cell surfaces and extracellular matrix, J Clin Invest 90 (1992) 2013-2021.
[211] P.C. Rensen, T.J. van Berkel, Apolipoprotein E effectively inhibits lipoprotein lipase-mediated lipolysis of chylomicron-like triglyceride-rich lipid emulsions in vitro and in vivo, J Biol Chem 271 (1996) 14791-14799.
[212] M.C. Jong, V.E. Dahlmans, M.H. Hofker, L.M. Havekes, Nascent very-low-density lipoprotein triacylglycerol hydrolysis by lipoprotein lipase is inhibited by apolipoprotein E in a dose-dependent manner, Biochem J 328 ( Pt 3) (1997) 745-750.
[213] Y.D. Huang, X.Q. Liu, S.C. Rall, J.M. Taylor, A. von Eckardstein, G. Assmann, R.W. Mahley, Overexpression and accumulation of apolipoprotein E as a cause of hypertriglyceridemia, Journal of Biological Chemistry 273 (1998) 26388-26393.
[214] S. Calandra, C. Priore Oliva, P. Tarugi, S. Bertolini, APOA5 and triglyceride metabolism, lesson from human APOA5 deficiency, Curr Opin Lipidol 17 (2006) 122-127.
54
[215] C. Priore Oliva, F. Carubbi, F.G. Schaap, S. Bertolini, S. Calandra, Hypertriglyceridaemia and low plasma HDL in a patient with apolipoprotein A-V deficiency due to a novel mutation in the APOA5 gene, J Intern Med 263 (2008) 450-458.
[216] L.A. Pennacchio, M. Olivier, J.A. Hubacek, J.C. Cohen, D.R. Cox, J.C. Fruchart, R.M. Krauss, E.M. Rubin, An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing, Science 294 (2001) 169-173.
[217] P.J. O'Brien, W.E. Alborn, J.H. Sloan, M. Ulmer, A. Boodhoo, M.D. Knierman, A.E. Schultze, R.J. Konrad, The novel apolipoprotein A5 is present in human serum, is associated with VLDL, HDL, and chylomicrons, and circulates at very low concentrations compared with other apolipoproteins, Clin Chem 51 (2005) 351-359.
[218] D.S. Cho, S. Woo, S. Kim, C.D. Byrne, J.H. Kong, K.C. Sung, Estimation of plasma apolipoprotein B concentration using routinely measured lipid biochemical tests in apparently healthy Asian adults, Cardiovasc Diabetol 11 (2012) 55.
[219] X. Gao, T.M. Forte, R.O. Ryan, Influence of apolipoprotein A-V on hepatocyte lipid droplet formation, Biochem Biophys Res Commun 427 (2012) 361-365.
[220] R.B. Weinberg, V.R. Cook, J.A. Beckstead, D.D. Martin, J.W. Gallagher, G.S. Shelness, R.O. Ryan, Structure and interfacial properties of human apolipoprotein A-V, J Biol Chem 278 (2003) 34438-34444.
[221] N. Baroukh, E. Bauge, J. Akiyama, J. Chang, V. Afzal, J.C. Fruchart, E.M. Rubin, J. Fruchart-Najib, L.A. Pennacchio, Analysis of apolipoprotein A5, c3, and plasma triglyceride concentrations in genetically engineered mice, Arterioscler Thromb Vasc Biol 24 (2004) 1297-1302.
[222] S. Formisano, H.B. Brewer, Jr., J.C. Osborne, Jr., Effect of pressure and ionic strength on the self-association of Apo-A-I from the human high density lipoprotein complex, J Biol Chem 253 (1978) 354-359.
[223] F. Ferrato, F. Carriere, L. Sarda, R. Verger, A critical reevaluation of the phenomenon of interfacial activation, Methods Enzymol 286 (1997) 327-347.
[224] F.S. Steven, M.M. Griffin, T.P. Hulley, P. Brooman, Interaction of alpha, beta-diethyl stilbestrol 4,4'-bisphosphate with arginyl substrates resulting in apparent inhibition of trypsin and thrombin, Eur J Biochem 125 (1982) 305-309.
[225] L. Wang, D. Atkinson, D.M. Small, Interfacial properties of an amphipathic alpha-helix consensus peptide of exchangeable apolipoproteins at air/water and oil/water interfaces, J Biol Chem 278 (2003) 37480-37491.
[226] R.C. Kowal, J. Herz, K.H. Weisgraber, R.W. Mahley, M.S. Brown, J.L. Goldstein, Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein, J Biol Chem 265 (1990) 10771-10779.
[227] U. Beisiegel, W. Weber, G. Bengtsson-Olivecrona, Lipoprotein lipase enhances the binding of chylomicrons to low density lipoprotein receptor-related protein, Proc Natl Acad Sci U S A 88 (1991) 8342-8346.
[228] P. Tornvall, G. Olivecrona, F. Karpe, A. Hamsten, T. Olivecrona, Lipoprotein lipase mass and activity in plasma and their increase after heparin are separate parameters with different relations to plasma lipoproteins, Arterioscler Thromb Vasc Biol 15 (1995) 1086-1093.
[229] M. Merkel, B. Loeffler, M. Kluger, N. Fabig, G. Geppert, L.A. Pennacchio, A. Laatsch, J. Heeren, Apolipoprotein AV accelerates plasma hydrolysis of triglyceride-rich lipoproteins by interaction with proteoglycan-bound lipoprotein lipase, J Biol Chem 280 (2005) 21553-21560.
[230] X. Shu, L. Nelbach, M.M. Weinstein, B.L. Burgess, J.A. Beckstead, S.G. Young, R.O. Ryan, T.M. Forte, Intravenous injection of apolipoprotein A-V reconstituted high-density lipoprotein decreases hypertriglyceridemia in apoav-/- mice and requires glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1, Arterioscler Thromb Vasc Biol 30 (2010) 2504-2509.
[231] J.S. Millar, D.A. Cromley, M.G. McCoy, D.J. Rader, J.T. Billheimer, Determining hepatic triglyceride production in mice: comparison of poloxamer 407 with Triton WR-1339, J Lipid Res 46 (2005) 2023-2028.
55
[232] J.M. Ong, P.A. Kern, Effect of feeding and obesity on lipoprotein lipase activity, immunoreactive protein, and messenger RNA levels in human adipose tissue, J Clin Invest 84 (1989) 305-311.
[233] G. Boden, Obesity, insulin resistance and free fatty acids, Curr Opin Endocrinol Diabetes Obes 18 (2011) 139-143.
[234] N. Hosogai, A. Fukuhara, K. Oshima, Y. Miyata, S. Tanaka, K. Segawa, S. Furukawa, Y. Tochino, R. Komuro, M. Matsuda, I. Shimomura, Adipose tissue hypoxia in obesity and its impact on adipocytokine dysregulation, Diabetes 56 (2007) 901-911.
[235] B. Wang, I.S. Wood, P. Trayhurn, Dysregulation of the expression and secretion of inflammation-related adipokines by hypoxia in human adipocytes, Pflugers Arch 455 (2007) 479-492.
[236] P. Gonzalez-Muniesa, C. de Oliveira, F. Perez de Heredia, M.P. Thompson, P. Trayhurn, Fatty acids and hypoxia stimulate the expression and secretion of the adipokine ANGPTL4 (angiopoietin-like protein 4/ fasting-induced adipose factor) by human adipocytes, J Nutrigenet Nutrigenomics 4 (2011) 146-153.
[237] N. Malo, J.A. Hanley, S. Cerquozzi, J. Pelletier, R. Nadon, Statistical practice in high-throughput screening data analysis, Nat Biotechnol 24 (2006) 167-175.
[238] A. Lookene, L. Zhang, M. Hultin, G. Olivecrona, Rapid subunit exchange in dimeric lipoprotein lipase and properties of the inactive monomer, J Biol Chem 279 (2004) 49964-49972.
[239] K. Shirai, R.L. Jackson, Lipoprotein lipase-catalyzed hydrolysis of p-nitrophenyl butyrate. Interfacial activation by phospholipid vesicles, J Biol Chem 257 (1982) 1253-1258.
[240] E. Leroy, N. Bensel, J.L. Reymond, A low background high-throughput screening (HTS) fluorescence assay for lipases and esterases using acyloxymethylethers of umbelliferone, Bioorg Med Chem Lett 13 (2003) 2105-2108.
[241] W.J. Geldenhuys, D. Aring, P. Sadana, A novel Lipoprotein lipase (LPL) agonist rescues the enzyme from inhibition by angiopoietin-like 4 (ANGPTL4), Bioorg Med Chem Lett 24 (2014) 2163-2167.
[242] G. Shattat, R. Al-Qirim, Y. Al-Hiari, G.A. Sheikha, T. Al-Qirim, W. El-Huneidi, M. Shahwan, Synthesis and anti-hyperlipidemic evaluation of N(benzoylphenyl)-5-fluoro-1H-indole-2-carboxamide derivatives in Triton WR-1339-induced hyperlipidemic rats, Molecules 15 (2010) 5840-5849.
[243] K. Tsutsumi, Y. Inoue, A. Shima, K. Iwasaki, M. Kawamura, T. Murase, The novel compound NO-1886 increases lipoprotein lipase activity with resulting elevation of high density lipoprotein cholesterol, and long-term administration inhibits atherogenesis in the coronary arteries of rats with experimental atherosclerosis, J Clin Invest 92 (1993) 411-417.
[244] W. Yin, K. Tsutsumi, Z. Yuan, B. Yang, Effects of the lipoprotein lipase activator NO-1886 as a suppressor agent of atherosclerosis in aorta of mild diabetic rabbits, Arzneimittelforschung 52 (2002) 610-614.
[245] W. Yin, K. Tsutsumi, Lipoprotein lipase activator NO-1886, Cardiovasc Drug Rev 21 (2003) 133-142.
[246] C. Zhang, W. Yin, D. Liao, L. Huang, C. Tang, K. Tsutsumi, Z. Wang, Y. Liu, Q. Li, H. Hou, M. Cai, J. Xiao, NO-1886 upregulates ATP binding cassette transporter A1 and inhibits diet-induced atherosclerosis in Chinese Bama minipigs, J Lipid Res 47 (2006) 2055-2063.
[247] C. Fievet, B. Staels, Liver X receptor modulators: effects on lipid metabolism and potential use in the treatment of atherosclerosis, Biochem Pharmacol 77 (2009) 1316-1327.
[248] G. Bengtsson, T. Olivecrona, Binding of deoxycholate to lipoprotein lipase, Biochim Biophys Acta 575 (1979) 471-474.
[249] M. Makishima, A.Y. Okamoto, J.J. Repa, H. Tu, R.M. Learned, A. Luk, M.V. Hull, K.D. Lustig, D.J. Mangelsdorf, B. Shan, Identification of a nuclear receptor for bile acids, Science 284 (1999) 1362-1365.
[250] D.J. Parks, S.G. Blanchard, R.K. Bledsoe, G. Chandra, T.G. Consler, S.A. Kliewer, J.B. Stimmel, T.M. Willson, A.M. Zavacki, D.D. Moore, J.M. Lehmann, Bile acids: natural ligands for an orphan nuclear receptor, Science 284 (1999) 1365-1368.
56
[251] I.J. A, N.S. Tan, L. Gelman, S. Kersten, J. Seydoux, J. Xu, D. Metzger, L. Canaple, P. Chambon, W. Wahli, B. Desvergne, In vivo activation of PPAR target genes by RXR homodimers, EMBO J 23 (2004) 2083-2091.