George Lyman Duff Memorial Lecture
Role of the Liver in Atherosclerosis
Richard J. Havel
dark and frequently cool basement laboratory that
we occupied, they seemed to think that winter was
coming and many of them rapidly gained weight and
had visibly lipemic serum, whether they were eating
low-fat or high-fat vegetable diets. In some of them,
triglyceride levels reached several thousand mg/dl
and they had creamy serum. When the animals were
caused to hibernate by placing them in the cold
room, they lost weight and their serum triglyceride
levels fell rapidly. Some of the lipemic animals had oil
red O-positive extracellular deposits in the aorta,
coronary arteries, and heart valves, and two of them
had masses of foam cells beneath the endothelium,
even though they had quite high high density lipoprotein (HDL) levels, as estimated by analytical ultracentrifugation.
What struck me was Bragdon's observation that
serum triglyceride levels were correlated with the
extent of fatty infiltration of the liver, which in one
case reached a level of 44% of gross liver weight,
and also that both the severity of the fatty liver and
the hypertriglyceridemia were correlated with the
rate of body weight gain.1 Joe and I talked then about
a junket to Alsace-Lorraine to study the fatty liver and
(we assumed) the hyperlipemia of the force-fed
goose.
eorge Lyman Duff was a pioneer in relating the
quality as well as the quantity of plasma lipoproG
teins to experimental atherosclerosis. His investiga-
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tions and those of John Gofman foreshadowed much
contemporary research on the interactions of lipoproteins with cells and other components of the arterial wall. This current research has, I believe, put to
rest past criticisms of the meetings of our Council, at
which it seemed to some that the artery is hardly
involved in the pathogenesis of atherosclerosis. Perhaps I can then be forgiven for taking as the subject
of this lecture the role of the liver in lipoprotein metabolism and atherosclerosis, and for daring to suggest that if the liver would only do correctly its job of
secreting the "right" kinds of lipoproteins and of taking up, catabolizing, and excreting lipoprotein components, it would be unnecessary for us to be so
concerned about the mechanisms by which lipoprotein-cholesterol accumulates in the arterial intima.
It is a truism that laboratory animals fed standard
chow diets have low levels of atherogenic lipoproteins and do not develop atherosclerosis, whereas
lipid accumulates in arteries and proliferative, spaceoccupying lesions develop when these animals are
fed unnatural diets, sometimes aided by auxiliary
measures that perturb hepatic cholesterol metabolism.
My introduction to this field was in Joseph Bragdon's laboratory at the National Heart Institute in
1953. Bragdon, a pathologist, had read a report that
Alaskan ground squirrels have rather high serum lipid levels and he undertook a systematic investigation
of another species of ground squirrel native to the
northern Rocky Mountains, Citellus colombianusS
These animals did not develop hypercholesterolemia with cholesterol-feeding. However, in the rather
Lipoprotein Synthesis and Secretion
Some may not fully appreciate that in 1953 there
was little understanding of the sites or mechanisms
of lipoprotein biosynthesis, and we knew essentially
nothing about apolipoproteins. Much later, when we
did know something about lipoprotein structure and
metabolism, I encountered in fetal guinea pigs a phenomenon reminiscent of Bragdon's early observations in ground squirrels. In these fetal animals,
which rapidly gain weight during the last part of gestation and are born sufficiently mature that they do
not need to suckle to survive, Thomas Bahmer and I
studied the transplacental transfer of free fatty
acids.2 We showed that a large fraction of fatty acids
derived from maternal adipose tissue is transferred
to the fetal liver and esterified to form triglycerides,
causing fatty liver and hyperlipemia. By electron microscopy, the secretory vesicles of the Golgi apparatus of hepatocytes were seen to be filled with giant,
very low density lipoproteins (VLDL) (the size of chy-
Dr. Richard J. Havel Is at the Cardiovascular Research Institute
and the Department of Medicine, School of Medicine, University of
California, San Francisco, California.
This lecture was given on November 12, 1984, at the 57th
Scientific Session of the American Heart Association at Miami,
Florida.
The author's research since 1971 has been supported by U.S.
Public Health Service Grant HL-14237 Arteriosclerosis SCOR.
Address for reprints: Richard J. Havel, M.D., 1327-M, University
of California, San Francisco, California 94143-0130.
(Arteriosclerosis 5:569-580, November/December 1985)
569
ARTERIOSCLEROSIS VOL 5, No 6,
NOVEMBER/DECEMBER 1985
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Figure 1. Transmission electron photomicrographs of fetal guinea pig liver at 68
days of gestation. A. Golgi region of hepatocyte, showing dilated Golgi cistemae
and secretory vesicles containing lipoprotein particles up to 2000 A in diameter,
x 35,000. B. Portions of two hepatocytes, with extracellular regions between the
cells and at the sinusoidal front filled with lipoprotein particles of similar size to those
in the Golgi apparatus. Several large fat droplets are present in the cell on the left.
Arrow indicates possible point of fusion of secretory vesicle with plasma lemma,
x 16,500. (Reprinted from reference 3, with permission of the publisher.)
lomicrons normally produced after a fat-rich meal)
and similar VLDL were seen in the space of Disse
(Figure 1); both were comparable to the animals'
serum VLDL.3 We concluded that the hyperlipemia in
these animals, in which no large chylomicrons could
have been formed, arose from overloading of the
liver with maternal fatty acids, which were subsequently transported, as VLDL-triglycerides, to adipose tissue for use by the animals after birth. I was
by then convinced that the liver has a tremendous
capacity to secrete triglycerides and, like the gut, can
make bigger VLDL when this is necessitated by the
fat load. It seems to make no difference whether the
fat comes directly from the diet, from preformed free
fatty acids, or from hepatic lipogenesis when large
amounts of carbohydrates are ingested.
In the late 1960s, Robert Hamilton, Albert Jones,
and others defined the pathway of synthesis and
secretion of VLDL in hepatocytes (Figure 2). The
nonpolar cores of the VLDL that the liver secretes
usually contain mainly triglycerides. In species that
store large amounts of cholesteryl esters in the liver
during cholesterol feeding, including rabbits, guinea
pigs, some primates, and (with auxiliary measures)
rats, the liver then secretes VLDL with cholesteryl
ester-enriched cores 56 (Figure 3). Evidently, the liver has the capacity to package and secrete in VLDL
whichever nonpolar lipid is available.
ROLE OF THE LIVER IN ATHEROSCLEROSIS
Havel
571
Hepatic Uptake of Lipoprotelns
Bile
Conaliculus
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Figure 2. Pathway of assembly and secretion of VLDL in
hepatocytes. The lipid particle, which is assembled in the
smooth endoplasmic reticulum (SER), is thought to acquire its protein components, including apo B which is
synthesized in the rough endoplasmic reticulum (RER), at
the smooth-surfaced termini of the latter. The nascent
VLDL are transported to the Golgi apparatus, where glycosylation of the proteins is completed, and concentrated in
secretory vesicles. The latter migrate to the cell surface
and fuse with the plasmalemma, releasing the particles
into the hepatic sinusoid. (Reprinted from reference 4, with
permission of the publisher.)
80
% of Total
Lipoprotein
Mass
It has been known since the early 1960s that free
fatty acids derived from chylomicron triglycerides,
like free fatty acids derived from adipose tissue, can
be esterified to form triglycerides in the liver and then
exported in VLDL. Dietary cholesterol, however, enters the liver with chylomicron remnants. The early
work of Paul Nestel, working with me in San Francisco,7 and the later studies of Emmett Bergman in
sheep and dogs8 and those of Trevor Redgrave, who
first isolated chylomicron remnants from functionally
eviscerated rats,9 defined the two-step pathway of
chylomicron metabolism and showed that the liver is
the major and obligatory site of uptake of dietary
cholesterol. Subsequently, it was found that hepatogenous VLDL are also metabolized by an analogous pathway, the outlines of which had become
apparent by 19741011 (Figure 4). At the same time, it
became apparent that lipoproteins can be catabolized by receptor-dependent endocytosis as a result
of the discovery of the LDL receptor by Michael
Brown and Joseph Goldstein.12 We also knew that
not all VLDL remnants enter the liver, but that some
are further processed to form low density lipoproteins (LDL). Finally, we knew that one of the major
differences between VLDL remnants, which Ole
Mjos isolated from functionally eviscerated rats,13
and LDL was that LDL lack the arginine-rich protein
discovered by Virgie and Bernard Shore,14 now
known as apolipoprotein E. Some of the other features of VLDL metabolism, which are analogous to
those of chylomicrons, including the acquisition of
the cofactor protein for lipoprotein lipase (apo C-ll)
and other C apoproteins from HDL, are also shown in
this 1974 model.
VLDL
IDL
40
CE
• •• RER
0
10
28
84
Days Fed 1% Cholesterol Diet
Figure 3. The composition of the core of VLDL and IDL
isolated from blood plasma of guinea pigs is drastically
altered by feeding the animals a diet containing 1 % cholesterol by weight. In animals fed a low-fat chow, these lipoproteins contain very small amounts of cholesteryl esters
(CE). In cholesterol-fed animals, cholesteryl esters replace
much of the normally predominant triglycerides (TG) in the
core of the particles. The size of VLDL and IDL is not
appreciably changed by cholesterol-feeding. (Graph prepared from data in reference 6.)
Figure 4. This 1974 model of VLDL metabolism has
proven to be generally correct. Nascent VLDL contain apo
B (o), apo E (a , here called hi ARG), and C apoproteins
(A). The role of apo C-ll as a cofactor for lipoprotein lipase
was then known, as was that of HDL as a reservoir for C
apoproteins, but the general role of C apoproteins in preventing the premature uptake of nascent VLDL, prior to
formation of remnant particles,11 was discovered later; so
was the function of apo E as a ligand for lipoprotein receptors and the definitive demonstration that VLDL remnants
are taken up into hepatocytes by receptor-mediated endocytosis. (Reprinted from reference 10, with permission of
the publisher.)
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ARTERIOSCLEROSIS VOL 5, No 6,
It is now appreciated that the uptake of chylomicron remnants by the liver is a receptor-mediated,
endocytic event15 and that apo E has a crucial role in
this process. We know that:
1. HDL containing only apo E compete for uptake
of chylomicron remnants in perfused rat livers.16
2. The addition of apo E to chylomicrons from
estradiol-treated rats increases their uptake into perfused rat livers.17
3. Particles resembling chylomicron remnants
(which contain apo B-48) accumulate in the blood
plasma of persons with familial dysbetalipoproteinemia. 1819
4. The removal of apo B-48 from plasma is grossly impaired in persons with familial dysbetalipoproteinemia caused by at least two mutations of apo E.20
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A modern version of chylomicron metabolism is
shown in Figure 5.21 Several years ago in his Duff
lecture, Donald Zilversmit presented evidence that
chylomicron remnants, enriched in cholesterol, are
responsible for the hypercholesterolemia of cholesterol-fed rabbits.22 His subsequent studies with Katherine Thompson23 have shown that the particles that
accumulate probably represent remnants of VLDL,
secreted from the cholesterol-loaded livers of these
INTESTINAL
MUCOSAL
CELL
NOVEMBER/DECEMBER 1985
animals. They found that chylomicron remnants are
removed normally by perfused livers of cholesterolfed rabbits, whereas the cholesterol-rich particles
that accumulate in their plasma are removed slowly.
The nature of the particles that accumulate in cholesterol-fed animals can also be evaluated by determining the species of apo B that they contain. JeanLouis Vigne and I (Vigne JL, Havel RJ, unpublished
data) have found that mesenteric lymph chylomicrons from both chow-fed and cholesterol-fed rabbits
contain apo B-48, the form of apo B secreted from
the intestine of rats and humans.24 By contrast, the
cholesterol-enriched VLDL of nonfasted, cholesterol-fed rabbits, like the VLDL secreted from their perfused livers, contain predominantly apo B-100 at all
times after cholesterol feeding (Figure 6). The accumulation of cholesterol in the liver evidently has two
effects: 1) secretion of cholesterol-enriched VLDL;
and 2) down-regulation of hepatic LDL receptors.25
Both of these contribute to the accumulation of cholesterol in the blood. Only in the alloxan-diabetic,
cholesterol-fed rabbit, a model introduced by Duff
and McMillan26 do triglyceride-rich particles that contain mainly apo B-48 accumulate in appreciable
amounts (Vigne JL, Havel RJ, unpublished data).
These large particles are probably those that Duff
HDL
Blood
Capillary
Llpo protein
Lipau
FFA
Figure 5. Chylomicron pathway. Chylomicrons secreted from absorptive cells of the small
intestine tranport dietary triglycerides (gray region of particle) and cholesterol, as cholesteryl
esters (darker region of particle). Most of the triglycerides are hydrolyzed in extrahepatic
tissues by lipoprotein lipase. The resulting remnant particles, depleted of trigycerides and
most surface lipids and proteins (but retaining the cholesteryl esters, apo B-48, and apo E),
are taken up into hepatocytes after binding to a chylomicron remnant receptor. Lysosomal
catabolism releases the cholesterol, which thereby regulates a number of key processes in
this cell that influence lipoprotein biosynthesis and catabolism, including cholesterol synthesis, cholesterol esterification, bile acid synthesis, and the activity of LDL receptors. (Reprinted from reference 21, with permission of the publisher.)
ROLE OF THE LIVER IN ATHEROSCLEROSIS
B-100 —<
Havel
573
B100
B-48-
B 48
E -
Cholesterol 63
Triglycerides 188
91
no
118
214
259
93
338
385
188
236
703
37
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Figure 6. Potyacrylamide gel electrophoretograms run in
sodium dodecyl sulfate at a low concentration of polyacrylamide (3.5%) permit the B apoproteins that accumulate in
VLDL of cholesterol-fed rabbits to be visualized. The gels,
from left to right, are of VLDL obtained from seven rabbits,
1 to 90 days after they were placed on diet of pelleted
rabbit chow to which 1% by weight of cholesterol was
added by evaporating an ether solution of cholesterol over
the pellets. In all cases, apo B-100 predominated and only
small amounts of apo B-48 were present.
found to be less atherogenic and which Peter
Brecher and his associates27 have shown to have
less capacity to induce foam cell formation when
incubated with macrophages than the remnant-like
VLDL that accumulate in nondiabetic, cholesterolfed rabbits (in whom lipoprotein lipase is normally
active). In the alloxan-treated animals, apo B-100
remains the predominant form of apo B secreted by
the liver (Figure 7).
These data in cholesterol-fed rabbits are consistent with those that my colleagues and I in San Francisco have obtained in Watanabe heritable hyperlipidemic (WHHL) rabbits in collaboration with Brown,
Goldstein, and their colleagues in Dallas. We found
that apo B-100 accumulates in VLDL, intermediate
density lipoproteins (IDL), and LDL of WHHL homozygotes, but little apo B-48 is found in these lipoproteins.23 Although these animals essentially lack functional hepatic LDL receptors, their livers take up
chylomicron remnants normally.29 By contrast, the
Dallas group has shown that the uptake of VLDLprotein by the liver is greatly retarded.30 From these
studies and those of others,31 it is now evident that
chylomicron and VLDL remnants are removed by
distinct hepatic receptors. Uptake of both forms of
remnants seems to depend upon apo E. Anton Stalenhoef, working with our group in San Francisco,20
has studied the metabolism of apo B-48 and apo B100 of large triglyceride-rich lipoproteins (400-800 A
in diameter) in patients with familial dysbetalipopro-
Figure 7. Polyacrylamide gradient gel electrophoretogram (3% to 15% polyacrylamide) of VLDL (d< 1.010 g/ml)
isolated from the perfusate of a liver of a cholesterol-fed,
alloxan-diabetic rabbit. The liver was perfused for 3 hours.
In this overloaded gel, apo B-100 predominates among the
proteins of high molecular weight. A component with an
apparent molecular weight slightly greater than 200,000
could represent apo B-48.
teinemia, who have dysfunctional mutant forms of
the protein. He found the removal of both B apoproteins from the blood to be grossly impaired. The defect in the structure of apo E in this genetic disorder
thus accounts for the accumulation of both types of
remnant in the blood.
As I have noted, the lipoproteins that accumulate
in WHHL homozygotes include not only LDL, but
appreciable amounts of VLDL and IDL as well. The
concentration of apo E, like that of apo B-100, is
increased severalfold. These findings, taken together with the metabolic studies that I have described in
cholesterol-fed and WHHL rabbits, strongly suggest
that the hepatic uptake of VLDL remnants, as well as
LDL, is mediated by the LDL receptor. In WHHL homozygotes, LDL accumulate not only because their
clearance from the blood is impaired, but also because their formation is increased.32 Toru Kita and
David Bilheimer, working with Brown and Goldstein,30 have suggested that LDL formation is increased because retained VLDL remnants are
574
ARTERIOSCLEROSIS VOL 5, No 6,
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gradually converted to LDL. This conclusion is supported by studies of lipoprotein secretion from the
liver of WHHL homozygotes carried out by Conrad
Hornick and Kita et al.33 They found that the rate of
accumulation of apo B-100 in perfusates of livers
from the mutant animals is not increased; furthermore, virtually all the accumulating apo B-100 was in
VLDL and almost none in LDL. It therefore appears
that the number of VLDL particles secreted is normal
but that fewer VLDL remnants are taken up directly
by the liver and more are converted to LDL in the
absence of a functional LDL receptor.
In humans, Stalenhoef et al.34 have found that apo
B-100 of large VLDL is removed from the blood almost as rapidly as apo B-48 of chylomicrons; little or
none of this apo B-100 is converted to LDL. These
observations have led us to conclude that the extent
to which VLDL remnants are converted to LDL is a
function of particle size. The rapid removal of large
VLDL by the liver presumably gives little opportunity
for further metabolism to yield LDL.
The rapid removal of large VLDL may be related to
the presence of more than one molecule of apo E in
each particle (Table 1).35 A number of years ago,
Thomas Innerarity, Robert Pitas, and Robert Mahley36 concluded that HDL particles that contain several apo E molecules bind to multiple LDL receptor
sites on cultured human fibroblasts, leading to higher
binding affinity of these particles for the receptor than
LDL, which presumably bind to the receptor via a
single site on apo B. Later, we observed similar high
affinity binding of rat VLDL particles containing several molecules of apo E to LDL receptors on liver
membranes.37 The high affinity of such particles can
now be understood in terms of the recently described
structure of the receptor protein: it contains seven
repeated segments of about 40 amino acids in its
cysteine-rich N-terminal region. Each of these segments has a highly negatively charged region that
probably constitutes a receptor-binding domain.38
Thus, rat VLDL particles may bind polyvalently to a
single receptor, accounting for the high binding affinity and, presumably, the more efficient removal of
such particles from the blood.39 The rapid removal of
human chylomicron remnants from the blood may
Table 1. Apo E Content of Human Plasma Very Low
Density Llpoprotelns
Fraction
(diameter, A)
Normal
(% of apo
B mass)
Endogenous
hyperlipemia
(% of apo
B mass)
900
680
520
420
320
75
43
23
13
12
171
99
64
34
23
Calculated from data in reference 35.
NOVEMBER/DECEMBER 1985
also be a function of polyvalent binding via multiple
molecules of apo E on these particles (Table 1).
Nobuhiro Yamada, working with David Shames
and me in San Francisco, has used immunoaffinity
chromatography on columns containing anti-apo E
to characterize VLDL, their remnants, and LDL in
normal rabbits and WHHL homozygotes. We reasoned that VLDL and their remnants, which should
contain apo E as well as apo B-100, would be retained on the affinity columns, whereas LDL, which
contain only apo B-100, would not. We found that
more than one-half of the apo B-100 in plasma of
normal rabbits binds to such columns. All fractions
(VLDL, IDL, and LDL) contain some particles that do
not bind to the columns. The unbound fraction increases with density, but even LDL contain an appreciable portion of particles that contain apo E.
Similar findings were obtained with lipoprotein fractions from WHHL homozygotes. We have also used
immunoaffinity chromatography and multicompartmental analysis to study the conversion of radioiodinated apo B-100 of VLDL to remnants and LDL in
normal and mutant rabbits. In normal rabbits, only a
small fraction of VLDL particles (containing apo E)
appears to be converted to LDL particles that lack
apo E; however, all LDL seem to be derived from
VLDL. The fraction of the VLDL converted to LDL is
increased in WHHL homozygotes, as predicted from
the observations of Kita and his associates.30 However, the overall rate of removal of apo B-100 of
VLDL from the blood considerably exceeds that of
IDL or LDL. We have therefore considered the possibility that some VLDL particles in WHHL rabbits,
which contain more than one molecule of apo E, are
removed by their chylomicron remnant receptor, but
we cannot exclude the possibility that a small number of LDL receptors, present on hepatocytes of the
mutant animals, mediate the clearance of such particles.
Our kinetic studies in normal rabbits also show
that IDL particles, which appear to contain only a
single molecule of apo E, are removed slowly from
the blood, perhaps as slowly as LDL that contain no
apo E. Evidently, particles that contain apo E are not
invariably removed from the blood more efficiently
than LDL. In normal rabbits, the concentration of
remnant-like particles, isolated in the density ranges
of IDL and LDL, is almost as great as that of particles
that contain apo B-100 but no apo E. The apparently
large concentration of VLDL remnants in rabbits (relative to "true" LDL) could be related to the very low
activity of the hepatic, heparin-releasable lipase in
this species40 (Frost PH, Havel RJ, unpublished
data). This enzyme, which hydrolyzes triglycerides
and phospholipids in small triglyceride-rich lipoproteins efficiently in vitro, may be involved in the conversion of VLDL remnants to LDL, accompanied by
loss of apo E39 (Figure 8). In this light, the high concentration of putative VLDL remnants in WHHL homozygotes28 is not surprising. However, humans
with homozygous familial hypercholesterolemia also
ROLE OF THE LIVER IN ATHEROSCLEROSIS
575
Havel
_ ; % PERIPHERAL CELLS
Dietary
Cholesterol
LDL
REMNANT
VLDL
\
/
B-10G E Receptor
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Figure 8. The formation of LDL from VLDL may involve
two lipolytic steps. The first extrahepatic step, catalyzed by
lipoprotein lipase, is well recognized. The second, which
dearty involves further loss of triglycerides and phospholipids, as well as proteins other than apo B-100, may be
catalyzed by hepatic lipase. The removal of both VLDL
remnants and LDL from the blood depends mainly upon
the activity of LDL receptors. According to this scheme,
LDL formation will be promoted by increased activity of
hepatic lipase or reduced activity of hepatic LDL receptors.
have increased levels of IDL and remnant-like VLDL,
and elevated concentrations of apo E (up to 30
mg/dl) which are proportional to the concentration of
plasma total cholesterol (Havel RJ, Bilheimer DW,
unpublished data). Moreover, the conversion of IDL
to LDL is slowed in human homozygotes.41 VLDL
remnants may contribute to atherogenesis in receptor-deficient humans and rabbits; therefore, it cannot
be assumed that LDL are the sole atherogenic lipoprotein in this situation.
Besides causing elevated concentrations of atherogenic Iipoproteins (which can be taken up into the
arterial intima), LDL receptor deficiency may contribute to atherogenesis by impeding the process of reverse cholesterol transport. In mammals that have
an active cholesteryl ester transfer protein in blood
plasma, such as rabbits and humans, the LDL receptor may be a key participant in reverse cholesterol
transport. The cholesteryl esters that are carried by
VLDL remnants and LDL in these species are derived largely from the action of lecithin-cholesterol
acyltransferase. The findings of many investigators,
including those of Christopher and Phoebe Fielding,42 are consistent with the process of reverse cholesterol transport shown in Figure 9. Cholesterol, derived from cell surfaces or plasma Iipoproteins, is
esterified on a minor species of HDL, from which it is
transferred to other Iipoproteins, mainly remnants
and LDL, that are taken up primarily into the liver.
After lysosomal catabolism, the cholesterol can be
excreted into the bile. This process seems to be impeded in many hyperlipidemic states, including familial hypercholesterolemia,43 as a result of impairment of the transfer step.44 At least in some cases,
this abnormality is related to altered composition of
the acceptor particles, VLDL and LDL.45
LI.LK CF_L
Figure 9. The primary pathway for reverse cholesterol
transport in those species that have an active mechanism
for transfer of cholesteryl esters among lipoprotein particles is shown here. It involves the movement of cholesteryl
esters synthesized by lecithin-cholesterol acyltransferase
(LCAT) from a fraction of HDL (to which the enzyme is
bound) to acceptor Iipoproteins, which are mainly those
that contain apo B. This produces a gradient that permits
more cholesterol to move to the site of esterification from
the surfaces of cells or other plasma Iipoproteins. The
process of reverse transport is completed when the acceptor Iipoproteins are taken up by the liver via receptor-mediated endocytosis. In many hyperlipoproteinemic states,
the ability of the acceptor Iipoproteins to receive the newly
synthesized esters is impaired. This altered property
seems to result from an alteration of the composition of the
particle surface (see text). (Reprinted from reference 21,
with permission of the publisher.)
Intracellular Processing of Llpoproteins
in Hepatocytes
For several years, my associates and I have been
studying the intrahepatic processing of remnants
and LDL, especially that mediated by the LDL receptor of hepatocytes. Much of this work has been carried out in ethinyl estradiol-treated rats which, as
shown by Petri Kovanen and others in Dallas46 and
by Yu-sheng Chao and several associates in San
Francisco,47 express up to 20 times the normal number of LDL receptors on hepatocytes.
The intracellular pathway, defined originally by autoradiographic studies carried out in collaboration
with Albert Jones et al.,48 seems to be the same as
that found for other ligands taken up into hepatocytes by receptor-mediated endocytosis.49 In the
course of this work, we found that radioiodinated apo
B of LDL accumulates in multivesicular bodies at the
bile canalicular pole of the cell, close to the Golgi
apparatus. We pursued this observation, with a view
to determining the possible significance of this topo-
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ARTERIOSCLEROSIS VOL 5, No 6,
NOVEMBER/DECEMBER 1985
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Figure 10. A negatively stained multivesicular body is shown here at high magnification ( x 180,000). Rupture of its
membrane (black and white arrow) has permitted the phosphotungstate stain to penetrate and outline the contents. These
include collapsed internal vesicles (white arrowheads); numerous electron-lucent particles (250-800 A in diameter), which
represent remnants of triglyceride-rich lipoproteins; a few particles about 200 A in diameter, which may represent LDL, that
were injected into the rat from which the organelles were isolated (black arrowheads); and occasional discoidal structures
that may represent early stages of lipoprotein degradation (white arrows). This multivesicular body, from the liver of an
estradiol-treated rat, is larger than most observed in normal rats (see text). (Reprinted from reference 50, with permission of
the publisher.)
ROLE OF THE LIVER IN ATHEROSCLEROSIS
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graphic localization for recycling of the receptor and
degradation of the lipoproteins. We have now50 isolated multivesicular bodies from hepatocytes of estradiol-treated rats and have determined some of
their properties. In these animals, the organelles are
filled with triglyceride-rich lipoproteins with the properties of remnants (Figure 10). This observation is
consistent with autoradiographic studies in untreated rats, in which we have shown15 that chylomicrons
and VLDL are processed in the liver by the same
pathway found for LDL. The content lipoproteins of
multivesicular bodies are largely undegraded; indeed, this organelle seems to represent the last prelysosomal way-station in which endocytosed lipoproteins accumulate just before acid hydrolases are
delivered from the nearby Golgi apparatus within primary lysosomes, which fuse with the multivesicular
bodies.51
The traffic of chylomicron and VLDL remnants into
multivesicular bodies of rat hepatocytes seems at
first glance to be extraordinary, but our studies of this
organelle are consistent with current knowledge of
lipoprotein catabolism. In the rat, not only virtually all
chylomicron remnants, but more than 90% of VLDL
remnants, are taken up into the liver within minutes
of their secretion.52-M The ability to localize intact
lipoproteins in multivesicular bodies and to isolate
these structures is also clarifying our understanding
of the role of the Golgi apparatus in processing endocytosed macromolecules and of the properties of nascent lipoproteins that are delivered to the Golgi apparatus from the endoplasmic reticulum and are
finally concentrated in secretory vesicles. Although
the role of the Golgi apparatus in receptor-recycling
remains to be defined, ligands destined for lysosomal degradation do not themselves enter the Golgi
compartment of the cell. Lipoprotein-filled structures
in the Golgi region of hepatocytes, described by
many investigators, may represent elements of distinct secretory or endocytic pathways, and we now
recognize that Golgi-rich fractions of livers, as isolated by common methods, may be seriously contaminated by multivesicular bodies.
It is difficult to distinguish secretory vesicles filled
with nascent VLDL from multivesicular bodies filled
with remnant lipoproteins by ordinary electron microscopic methods. Robert Hamilton et al.50 have used
modified staining techniques that aid this distinction.
Multivesicular bodies, as the name implies, contain a
number of internal bilayer vesicles, about 600 A in
diameter. In hepatocytes these vesicles are obscured by the content lipoproteins unless the bilayer
is heavily stained. The distinction is easier to make
in livers of estradiol-treated rats because the multivesicular bodies are larger and the Golgi secretory
vesicles smaller than in untreated animals. By use of
a procedure for isolation of "intact" Golgi apparatus
in which the secretory vesicles remain attached to
the flattened cisternae, this organelle can be separated from multivesicular bodies. It is thus possible to
isolate and characterize nascent and remnant lipo-
Havel
577
protein populations from the same liver. Application
of these methods promises to provide a new dimension to our understanding of the processes of hepatic
lipoprotein secretion and catabolism. A current concept of the latter, based upon our studies and those
of investigators who have studied the processing of
other ligands by hepatocytes, is shown in Figure 11.
Role of VLDL Remnants in Atherogenesls
Until recently, it was generally considered that few
VLDL remnants are taken up into the liver as such in
humans. Rather, it was thought that the remnants
(more or less equivalent to intermediate density lipoproteins) were first processed to LDL, which are taken up slowly by the liver and other tissues that express LDL receptors. Recent kinetic studies,
however, suggest that humans may not differ so
drastically in this respect from all other mammals,
and that perhaps one-half or more of VLDL may normally be taken up into the human liver as remnants.39
The efficiency of this uptake could be a major determinant of the concentration of atherogenic lipoproteins.
Epidemiologic and other clinical observations
have yielded conflicting data concerning the atherogenicity of VLDL (the concentration of which is closely related to plasma triglyceride levels). In Table 2,1
have listed some of the structural and metabolic
properties of triglyceride-rich lipoproteins that could
promote their atherogenieity. The last, but certainly
not the least, is the activity of hepatic LDL receptors,
which to a substantial extent determines whether
VLDL remnants as well as LDL accumulate in the
blood.
Many years ago, Gofman proposed that small
VLDL and IDL may be more atherogenic than LDL.55
This idea was discounted by a cooperative study of
lipoproteins in patients with coronary heart disease.58 I do not believe, however, that subsequent
studies, which in the main have measured as "LDLcholesterol" the fraction that includes IDL, have
clearly demonstrated that "true LDL" (particles that
contain apo B-100 as the sole protein) are the major
atherogenic lipoprotein in human populations. In an
ongoing intervention study, John Kane, Mary Malloy,
Thomas Ports and I have found that the majority of
asymptomatic familial hypercholesterolemia heterozygotes, whose average age is about 40 years, have
little or no coronary atherosclerosis demonstrable by
angiography. The determinants of premature atherosclerosis in these individuals must include factors
other than the concentration of LDL. These could
include additional lipoprotein determinants, such as
the concentration of VLDL remnants.
The role of hepatic LDL receptors in human lipoprotein catabolism has recently been shown dramatically by Bilheimer and his associates57 in a
young girl with homozygous familial hypercholesterolemia, in whom the desperate clinical situation required cardiac transplantation. The child also re-
ARTERIOSCLEROSIS VOL 5, No 6,
NOVEMBER/DECEMBER 1985
Sinusoid
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Figure 11. This diagram, prepared by Robert Hamilton, depicts current concepts of the intracellular
processing of endocytosed lipoproteins in hepatocytes. The initial binding occurs at sites on the sinusoidal surface of the cell, from which the receptor-ligand complex moves to a coated pit. Endocytosis ensues
and the clathrin coat is rapidly lost. The primary endosomes appear to fuse and then migrate to the bile
canalicular pole of the cell, where they are seen as multivesicular bodies (MVB). Primary lysosomes (L1),
derived from the nearby Golgi apparatus, fuse with the multivesicular bodies, converting them to secondary lysosomes (L2), releasing enzymes that hydrolyze the lipid and protein components. The soluble
products can then diffuse into the cytoplasm. The membrane of the endosomes, including multivesicular
bodies, contains an ATP-drive protein pump, which acidifies the interior of these organelles to a pH of
about 5.5. M The reduced pH serves to dissociate lipoproteins from the receptors and provides a suitable
environment for the action of lysosomal enzymes. After dissociation, the receptor is recycled to the cell
surface. The endosomal compartment from which dissociation occurs has been named CURL (compartment of uncoupling of receptor and ligand) and the process of dissociation has been visualized by
immunocytochemical techniques for some nonlipoproteln receptors.49 The precise site of dissociation
and the pathway of recycling are poorly defined. BC = bile canaliculus.
Table 2. Trlglyceride-Rich Lipoproteins: Potential for Atherogenlclty
Property
Determinants
Content of cholesteryl esters
Composition of nascent particles,
residence time in the blood
Potential for receptor-mediated endocytosis
Loss of C apoproteins,
surface lipid composition,
content and reactivity of apo E
Potential to form LDL
Species of apo B,
size/cholesteryl ester content,
hepatic lipase activity
Capacity to accept LCAT-derived cholesteryl esters
Surface cholesterol concentration
Activity of hepatic vs extrahepatic LDL receptors
Cells' need for cholesterol
ROLE OF THE LIVER IN ATHEROSCLEROSIS
ceived a liver homograft, and has since maintained
lipoprotein levels only slightly above normal. Preoperatively, she had grossly elevated levels of IDL as
well as LDL and cholesterol-enriched VLDL (Havel
RJ, Bilheimer DW, unpublished data).
Conclusions
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It is now recognized that the liver has a central role
in regulating both the synthesis and the catabolism
of the plasma lipoproteins. However, we lack some
crucial pieces of information. Although we understand a good deal about the regulation of hepatic
triglyceride synthesis and secretion, we still do not
know what determines the rate of secretion of VLDL
particles (i.e., the rate of synthesis and secretion of
apo B). We are aware that hepatic lipoprotein receptors are subject to important regulatory influences
related to cholesterol metabolism, but we do not
know how the availability of cholesterol to the cell is
coupled to receptor activity. We know that hepatic
cholesterol homeostasis is regulated in part by biliary
secretion of cholesterol and bile acids, but we do not
know how these processes are regulated, nor do we
understand the basis for the large species differences in the capacity to secrete these substances
into the bile in response to the influx of dietary cholesterol in chylomicron remnants. We also know little
about the determinants of variation of LDL receptor
activity within and among species. Finally, we still do
not know whether or how the liver participates in the
formation of LDL. These problems pose interesting
challenges for investigators of lipid and lipoprotein
metabolism. If the accumulation of lipoproteins that
contain apo B, with or without accompanying apo E,
is as important for atherogenesis as many of us believe, the solution to these problems may be not only
interesting, but also quite useful.
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• triglycerides
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•
LDL-receptor
George Lyman Duff memorial lecture. Role of the liver in atherosclerosis.
R J Havel
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Arterioscler Thromb Vasc Biol. 1985;5:569-580
doi: 10.1161/01.ATV.5.6.569
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