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J. Biol. Chem., Vol. 276, Issue 51, 48048-48057, December 21, 2001
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From the Department of Molecular Biology and Biotechnology,
University of Sheffield, Sheffield S10 2TN, United Kingdom
Received for publication, May 10, 2001, and in revised form, October 10, 2001
The aim of this study was to investigate the
types and characteristics of chylomicron precursors in the lumen of the
secretory compartment of rabbit enterocytes. Luminal contents were
separated into density subfractions in two continuous self-generating
gradients of different density profiles. In enterocytes from rabbits
fed a low fat diet, newly synthesized and immunodetectable apoB48 was
only in the subfraction of density similar to high density lipoprotein (dense particles); the luminal triacylglycerol (TAG) content was low and only in the subfraction of density similar to that
of chylomicrons/very low density lipoproteins (light particles). After
feeding fat, newly synthesized, and immunodetectable apoB48 was in both
dense (phospholipid-rich) and light (TAG-rich) particles. Luminal TAG
mass and synthesis increased after fat feeding and was only in light
particles. Pulse-chase experiments showed that the luminal-radiolabeled
apoB48 lost from the dense particles was recovered in the light
particles and the secreted chylomicrons. All of the light particle
lipids (mass and newly synthesized) co-immunoprecipitated with apoB48.
However, in the dense particles, there was a preferential
co-precipitation of the preexisting rather than newly synthesized
phospholipid. Assembly of apoB48-containing TAG-enriched lipoproteins
is therefore a two-step process. The first step produces dense apoB48
phospholipid-rich particles, which accumulate in the smooth endoplasmic
reticulum lumen. In the second step, these dense particles rapidly
acquire the bulk of the TAG and additional phospholipid in a
single and rapid step.
Dietary lipids are digested in the small intestine, and the
products are transferred across the brush border of the enterocytes. Triacylglycerols (TAG)1 are
resynthesized and assembled into chylomicrons, which are released into
the lamina propria and move via the lymph into the blood. The ability
of enterocytes to assemble and secrete chylomicrons is modulated by a
variety of factors including the amount and composition of the dietary
fats (reviewed in Refs. 1-3).
Chylomicrons consist of droplets of TAG with some cholesterol ester,
stabilized by a shell of phospholipids, cholesterol, and protein
(1-3). The major protein of chylomicrons is apolipoprotein-B48 (apoB48), a truncated form of apoB100 produced through
post-transcriptional editing of the mRNA of apoB100, the
characteristic protein of very low density lipoproteins (VLDL), which
transport endogenous lipid from the liver (4-6). The intracellular
events in the assembly of VLDL and chylomicrons revealed by electron
microscopic studies are basically similar (7-9). ApoB is synthesized
by bound ribosomes in the rough endoplasmic reticulum (RER), the lipid
components are synthesized in the smooth endoplasmic reticulum (SER),
and assembly of the lipoprotein particle occurs within the lumen of the
ER/Golgi compartment. There is evidence for a two-step assembly of VLDL
in which small, dense apoB-containing particles are formed initially in
the lumen of the RER and fuse with TAG-rich particles, which form
separately in the SER lumen (10-12). Generally these observations on
liver have been extrapolated to the enterocyte, and it has been assumed
that chylomicron assembly follows a similar pattern (reviewed in Refs.
2 and 3). However, chylomicrons are considerably larger and contain
more TAG than VLDL. Enterocytes require a far larger capacity for
chylomicron assembly (to accommodate the variations in the amount of
dietary fat) than the liver needs for VLDL assembly. Rat hepatocytes,
which synthesize apoB48 as well as apoB100, do not secrete
chylomicrons, while apobec-1 knockout mice do secrete apoB100 in
chylomicrons (13, 14). Thus, assembly of chylomicrons is not determined
by the form of apoB produced but is a characteristic property of
enterocytes. Consistent with this, there is at least one protein that
is specifically essential for chylomicron and not VLDL formation and is
absent/defective in chylomicron retention disease (15).
Although there have been many studies of VLDL assembly in liver,
isolated hepatocytes, and cultured cell lines, there have been few
studies of chylomicron assembly. This is in part because there is no
good cultured cell model that secretes chylomicrons containing only
apoB48. Important data have been obtained using CaCO2 cells (reviewed
in Refs. 2 and 3); however, these cells secrete relatively dense
lipoproteins that contain apoB100 in addition to apoB48. As we are
interested in the effects of diet on fat absorption, under
physiological conditions, we have recently developed a method for the
preparation of viable isolated rabbit enterocytes, which synthesize
apoB48 and lipid (but not apoB100) and assemble and secrete
chylomicrons (16-18). We have also developed methods for the
separation of the components of the secretory compartment of
enterocytes (RER/SER/Golgi) in a single self-generating gradient that
have allowed us to dissect the intracellular events in chylomicron
assembly (18). We have shown that in enterocytes, although apoB48 is
synthesized by bound ribosomes, it is not detectable in either the
membrane or lumen of the RER; membrane-associated and luminal apoB48
and TAG are both concentrated in the SER (16). This is in contrast to
the hepatocyte, in which luminal-dense apoB100-containing particles and
TAG-rich particles form in the RER and SER, respectively. Our
observations raise questions concerning the sequence of events in
chylomicron assembly vis à vis VLDL assembly. Does
this take place in one or more steps? To answer this question, we have
now investigated the nature of the chylomicron precursors in the lumen of the secretory compartment. Our results are consistent with a
two-step assembly of chylomicrons, which exhibits differences from VLDL
assembly that may be important in the regulation of fat absorption.
Materials--
Liposep (iodixanol solution for lipoprotein
separation), Optiprep (iodixanol solution for cell fractionation), and
Maxidens (inert dense displacement medium) were purchased from Lipotek Ltd., UK. Vivaspin centrifugal concentrators (PES membrane,
100,000 molecular weight cut off) were purchased from Sartorius Ltd., Epsom, UK. Hybridoma cells producing anti-rabbit monoclonal antibody Mac 31 were a gift from Drs. Bowyer and Gherardi, Cambridge
University, UK. All other reagents were as described previously
(16-18) or from Sigma.
Animals and Diets--
Dwarf lop rabbits (~6 months old,
2.56 ± 0.12 kg) were bred in the University of Sheffield Field
Laboratories. Rabbits were maintained on a 12-h light/dark cycle
and allowed free access to water and low fat chow (2.5% fat (w/w)
equivalent to 7% of the dietary energy intake) (chow-fed). In some
experiments, the diets were supplemented with sunflower oil to increase
the TAG content to 7.5% (w/v), equivalent to 21% of the calorie
intake for 3 or 6 days, as described previously (fat-fed) (17).
Separation of Rabbit Plasma Lipoproteins on Self-generating
Gradients--
Rabbit plasma lipoproteins were separated by a
modification of the method we developed for separation of human
lipoproteins (19). Blood was collected by cardiac puncture, and
disodium EDTA (anticoagulant) was added (final concentration 2.7 mM). Red cells were pelleted by centrifugation (1000 × g for 20 min), and 4 volumes of plasma were mixed with 1 volume of Liposep (final concentration 12% iodixanol). 1.4 ml of the
mixture was layered beneath 1.4 ml of 9% iodixanol solution (0.75 parts Liposep mixed with 4.25 parts Hepes-buffered saline, pH 7.8) in
an Optiseal tube for the Beckman TLN100 near vertical Ultracentrifuge
rotor. The tube was topped up with ~0.12 ml of 10 mM
Hepes-buffered saline (pH 7.8), sealed, and centrifuged in the Beckman
Optima MaxE bench top Ultracentrifuge at 100,000 rpm (350,000 × gav) for 2 h and 30 min at 16 °C. The
gradient formed was collected in 10 fractions from the bottom using a
Beckman gradient unloader. Cholesterol and TAG content of the fractions
was determined using kits from Roche Molecular Biochemicals and
Alpha Laboratories, respectively (19). The lipoprotein in each fraction
was identified by agarose gel electrophoresis using Sebia hydragels
(Lipo+Lp(a)) (19). Plasma lipoproteins were also separated in a
gradient with a different profile designed to move the HDL toward the
middle of the centrifuge tube. In this case, the protocol was exactly
the same as above except that 1 part Liposep was added to 4 parts
plasma (15% iodixanol); this was layered beneath 15% iodixanol, and
the tubes were centrifuged for 90 min.
Isolation and Incubation of Enterocytes and Preparation of
Subcellular Fractions--
Villous enterocytes were isolated from the
small intestine of rabbits and incubated for 30 min with lipid and bile
salt micelles containing [14C]oleate (4 µCi) or
[35S]methionine (1 mCi) to radiolabel newly synthesized
TAG and phospholipid and apoB48, respectively (16-18). The enterocytes
were pelleted by centrifugation and homogenized, and total microsomes
were prepared and separated into RER, SER, and Golgi fractions in
self-generating gradients of iodixanol (18). In some experiments,
enterocytes were incubated with labeled micelles for 30 min, isolated
by centrifugation, and reincubated with unlabeled micelles for a range
of times. The cells were reisolated by centrifugation. The media
were transferred to centrifuge tubes for the Beckman MLA rotor, 0.5 ml
of HEPES-buffered saline was carefully pipetted on top of the media,
and the tubes were centrifuged at 50,000 rpm for 1 h. After
centrifugation, the interface between the HEPES-buffered saline layer
and the media was distinct, and the top 0.5 ml, which was milky, was
carefully removed, the apoB48-containing lipoproteins were
immunoprecipitated, and the apoB48 and lipids were determined (18). In
preliminary experiments, it was demonstrated that the infranatant did
not contain detectable radiolabeled or immunologically detectable apoB48.
Preparation and Subfractionation of Secretory Compartment Luminal
Contents--
Microsomal membrane and luminal content fractions were
prepared by treatment with sodium carbonate (18). To remove the sodium carbonate and change the buffer, the luminal contents were concentrated to 0.2 ml by centrifugation at 4000 × g for 2 h
in Vivaspin ultrafiltration devices followed by resuspension to 4 ml
with TBS (10 mM Tris-HCl and 150 mM NaCl, pH
7.4) and reconcentrated as above. The final concentrates were
resuspended to 1.12 ml with TBS and mixed with 0.28 ml of Liposep
(final concentration, 12% iodixanol), and the chylomicron precursors
were separated in self-generated gradients exactly as described above
for plasma lipoproteins.
Imunoprecipitation of ApoB48 from Luminal Content
Subfractions--
ApoB48 was immunoprecipitated from luminal content
subfractions using an anti-rabbit monoclonal antibody (MAC31), which
recognizes the N terminus of apoB100 (20). Samples were
concentrated to 0.2 ml in Vivaspin centrifugal concentrators and mixed
with 0.05 ml of MAC 31 for 4 h at 4 °C, followed by the
addition of 0.2 ml of anti-rat IgG agarose, and then mixed for 16 h at 4 °C. Immunoprecipitated apoB48-containing particles attached
to the agarose beads were pelleted by centrifugation at 1700 × g for 30 min at 4 °C. Immunoprecipitates were washed by
resuspension in TBS and recentrifugation. Controls were carried out
using the same protocol without primary antibody or with an anti-actin
monoclonal antibody (gift of Dr Lynda Partridge in this department). No
immunoprecipitation of apoB48 or lipids occurred in the absence of MAC31.
Analysis of Lipids--
Lipids were extracted from luminal
content subfractions, separated by high performance thin layer
chromatography (HPTLC), and stained, and the mass of TAG,
phosphatidylcholine (PC), and phosphatidylethanolamine (PE) was
determined by laser densitometry (16-18, 21, 22). Incorporation of
[14C]oleate into newly synthesized TAG, PC, and PE was
determined on the same HPTLC plates using a Packard InstantImager
two-dimensional counter as described previously (21, 22).
Analysis of ApoB48--
ApoB48 was detected by immunoblotting
after SDS-PAGE (16, 18). The incorporation of
[35S]methionine into the apoB48 band was determined using
the Packard InstantImager as described previously (23-25).
Rabbit Plasma Lipoprotein Classes Separate on the Basis of Size
and/or Density in Self-generating Gradients of
Iodixanol--
Self-generating gradients of iodixanol have been used
for the separation of human plasma lipoproteins (19) and therefore potentially provide a simple procedure for the determination of the
density distribution of luminal lipoprotein precursors in the secretory
compartment of enterocytes. In initial experiments, rabbit plasma was
used to establish the protocol. After centrifugation in two-step
iodixanol gradients, rabbit plasma VLDL floated to the top of the
gradient (Fig. 1, fraction 1),
LDL peaked in the upper half of the gradient (Fig. 1, fractions
3-5), and HDL peaked in the lower half of the gradient (Fig. 1,
fractions 7-10). Because of their low density (<1.000
g/ml), chylomicrons move to the top of the gradient with the VLDL;
however, chylomicrons were not detectable in the rabbit plasma. The
densities of the fractions in which the lipoproteins were collected
were 1.032-1.21, 1.015-1.032, and 1.002-1.009 g/ml for HDL, LDL, and
VLDL, respectively. It must be emphasized that this is the density of
the iodixanol fractions and that the lipoproteins have slightly
different densities in salt gradients because the salt removes water
from the particles (19).
Lipoprotein Precursors Are in Particles of High (~HDL) and Low
Density (~Chylomicron/VLDL) in the Lumen of the Secretory
Compartment--
We have previously shown that in enterocytes from
rabbits fed low fat chow, most of the intracellular TAG is associated
with the SER membrane, and apoB48 is both membrane-bound and in the luminal contents (18). After feeding fat, the membrane-bound TAG
remains relatively unchanged, but there is a large increase in the ER
luminal TAG; however, there was little change in the apoB48 (18). To
determine the nature of the chylomicron precursors under these
different dietary conditions, we analyzed the ER luminal contents from
rabbit enterocytes fed a low fat chow diet and a high fat diet for 3 or
6 days. Rabbit enterocytes were incubated with micelles containing
[35S]methionine (to radiolabel newly synthesized apoB48)
and [14C]oleate (to radiolabel newly synthesized lipid).
The microsomal luminal contents were prepared and separated in the
gradient developed for the separation of plasma lipoproteins. In
luminal gradient fractions from chow-fed rabbit enterocytes,
immunodetectable apoB48 and newly synthesized radiolabeled apoB48 were
located only in the bottom fractions 8-10, which have a density of
1.067-1.21 g/ml, similar to that of plasma HDL (Fig.
2). In fractions from fat-fed rabbit
enterocytes, both newly synthesized radiolabeled and immunodetectable
apoB48 were found in the bottom fractions 7-10 and also in the top
fraction 1, which has the density, <1.0086 and corresponds to
chylomicrons/VLDL (Fig. 2). Although there was an increased synthesis
of apoB48 after feeding fat, there was no apparent increase in the
apoB48 detected by immunoblotting carried out under identical
conditions (Fig. 2B).
The luminal TAG content was low in gradient fractions from enterocytes
of chow-fed rabbits and only detectable in the top fraction 1 of the
gradient (Fig. 3A). After
feeding sunflower oil, there was an increase in both the newly
synthesized TAG and the mass of TAG in this light gradient fraction
(Fig. 3A). The increase was related to the length of time
the high fat diet was fed from 0 to 3 and from 3 to 6 days. Almost
100% of the TAG mass and the radiolabeled TAG in the luminal light
fractions 1-2 was co-immunoprecipitated with apoB48 (Fig.
3B), indicating that under all dietary conditions
investigated, luminal TAG is only in apoB48-containing particles of the
density of VLDL/chylomicrons.
Phosphatidylcholine (~80% of the phospholipid mass) and
phosphatidylethanolamine were the only detected phospholipids in the luminal contents, and they showed similar density distributions in the
gradient fractions (Figs. 4 and 5). Newly
synthesized phospholipids were at a low
concentration in the fractions from
chow-fed rabbit enterocytes, and the amount of phospholipid in each
fraction was only significant in the densest gradient fraction 10, which also contained the highest concentration of apoB48 (Figs.
4A and 5A). After fat feeding, the mass of
phospholipid increased in the denser fractions 7-10, coincident with
the immunodetectable apoB48. Newly synthesized phospholipids showed a
different distribution from phospholipid mass after feeding fat with a
greater incorporation of radiolabel into fraction 1.
As the amounts of material in gradient fractions are small, especially
in the samples from chow-fed rabbit enterocytes, gradient fractions
were pooled into light (fractions 1-3), intermediate (fractions 4-7),
and dense (fractions 8-10) to investigate the association between
luminal phospholipids and apoB48. In the fat-fed enterocytes,
all of the mass of the phospholipids and the newly synthesized
phospholipids were co-immunoprecipitated with apoB48 in the luminal
light and intermediate fractions (Figs. 4B and 5B). This was also the case in fractions from chow-fed
enterocytes, although the amounts of phospholipid were very small, and
accurate measurement was not possible. In contrast, although a large
fraction of the mass of the phospholipids (70-80%) in the dense
fractions co-immunoprecipitated with apoB48, only ~30% of the newly
synthesized radiolabeled phospholipids were co-precipitated (Figs.
4B and 5B). The specific activity of
phospholipids that co-immunoprecipitated with apoB from the dense
fractions was ~8-fold lower than that of phospholipids in the
supernatant. Therefore, preformed phospholipids are preferentially
incorporated into the dense apoB48-containing particle, whereas newly
synthesized phospholipids are incorporated into the light particles.
The lipid composition of luminal gradient fractions was not altered by
feeding fat. The light fraction contained 68.6 ± 0.3%, 23.7 ± 0.5%, and 7.8 ± 1.1% (n = 3) TAG,
phosphatidylcholine, and phosphatidylethanolamine, respectively. The
dense fraction contained 13.3 ± 1.1%, 67.3 ± 0.4%, and
19.0 ± 0.2% TAG, PC, and PE, respectively. The TAG content of
the light particles compared with the phospholipid content is ~20%
less than that usually reported for chylomicrons. However, to measure
the very small amounts of lipids in the luminal content fractions, it
was necessary to use a very sensitive technique, HPTLC to separate the
lipids followed by staining with cupric acetate, which reacts with
double bonds in the acyl moieties, followed by quantification by laser
densitometry (21, 22). Triolein was used as the standard TAG. As the
hepatic TAG contains a mixture of fatty acids of different degrees of saturation, this would stain to a smaller extent per molecule than
triolein, thus underestimating the amount of TAG.
The Dense ApoB48-containing Particles Float at a Similar Density to
HDL on Denser Iodixanol Gradients--
In iodixanol solutions,
proteins have a density of ~1.26 g/ml (26) and would be expected to
form a pellet in the 9-12% gradients used to separate luminal
contents. However, using this gradient, we cannot exclude the
possibility that some apoB48 in the dense fractions is free or
denatured protein. Therefore, in order to check that the
apoB48-containing dense fractions behave as lipoprotein particles, the
luminal contents from chow-fed enterocytes were also separated in 15%
iodixanol gradients in addition to 9-12% gradients. Under these
conditions, plasma HDL move to the middle of the tube, peaking in
fractions 5-7, which have densities of 1.055-1.069 g/ml, while LDL
move to the top of the tube in fractions of densities 1.024-1.039 g/ml
(Fig. 6). VLDL are lost because they are
considerably lighter than the density of the top fraction, which they
move through, and adhere to the shoulders of the Optiseal tubes. In
these gradients, the apoB48-containing fractions, which are at the
bottom of the 9-12% gradient, moved to the middle fractions of
densities 1.039-1.061 g/ml, overlapping the densities of HDL in the
same gradients (Fig. 7). The small amount
of TAG in the dense particles, both the mass and the radiolabeled,
comigrated with the apoB48 in the gradient, as did most of the
phospholipid. However, some of the phospholipid also remained in the
bottom dense fractions, suggesting that there may be
phospholipid-protein complexes denser than those containing
apoB48 in the enterocyte secretory compartment. As apoB48 was not
detected in the densest fractions of this gradient, we conclude that
there is no lipid-free apoB48 in the luminal contents.
Chylomicron Precursors, ApoB48 and TAG, Are Both Located in the SER
Lumen--
To determine whether the luminal and dense and light
apoB48-containing particle precursors are in separate intracellular
compartments, the distributions of luminal apoB and TAG in subfractions
of ER separated in self-generating gradients were determined. In
gradient fractions from chow-fed enterocytes, both radiolabeled apoB48 and immunodetectable apoB48 were mainly in the SER lumen (fractions 4-8) and not detected in fraction 10, which contains most of the RER
(18) (Fig. 8). Therefore, the dense
particles that predominate under these dietary conditions are in the
SER lumen. After feeding a high fat diet for 6 days, distribution of
apoB48, detected by immunoblotting under the same conditions, was not
altered (not illustrated); however, there was a large increase in the
newly synthesized TAG and TAG mass in the SER lumen (Fig. 8). The
TAG-rich light particles are therefore also in the SER lumen.
Luminal ApoB48-containing Dense Particles Are Precursors of
Light Particles and Secreted
Chylomicrons--
To determine
the relationship between the luminal-dense apoB48 particles and the
light particles and whether these are precursors of secreted
lipoproteins, isolated enterocytes were incubated (pulse)
with radiolabeled [35S]methionine for 30 min followed by
reincubation (chase) with unlabeled micelles for 45 min
(Fig. 9). The time selected for the chase was determined in preliminary
experiments in which we showed that the secretion of radiolabeled
apoB48 and loss of luminal apoB48 were linear for 60 min (23). At the
end of the pulse, the luminal-radiolabeled apoB48 was in the dense
particles. At the end of the chase, radiolabeled apoB48 in the dense
particles decreased, and radiolabeled apoB48 in the light particles and in the lipoproteins secreted into the incubation medium increased. 92%
of the radiolabeled apoB48 initially in the dense particles was
recovered with 23% of this in the light particles and the remainder in
the medium. The secreted lipoproteins were collected from the
incubation medium by flotation into a buffer of density >1.006
using a relatively short centrifugation time. These conditions are
generally used for the flotation of chylomicrons from plasma. We
therefore conclude that the secreted apoB48 is predominantly in
chylomicrons.
The aim of this investigation was to determine the nature of the
chylomicron precursors in the ER lumen of enterocytes to formulate a
model for assembly. Our previous observation, that both TAG and apoB48
enter the lumen of the secretory compartment in the SER, had raised the
possibility that chylomicrons form in a single step (18). However, the
present results show that there are apoB48-containing particles of
density similar to that of HDL in the ER lumen and that these are
precursors of secreted lipoproteins. After feeding fat, luminal apoB48
also appears in particles of the density of chylomicrons/VLDL,
consistent with a two-step model, and these light particles accumulate
in the SER lumen. This is consistent with the observations of Mansbach and co-workers (27-29), who showed that in chylomicron secretion, there is a rate-limiting step between the ER and Golgi. This step is
apparently saturated under conditions of fat feeding.
The small intestine secretes particles of the density of VLDL in the
fasted state and of the density of chylomicrons in the fed state (1).
The methods we have used previously and in the present investigation to
isolate secreted lipoproteins from the incubation medium or from
plasma are those normally used to prepare chylomicrons (16-19). A
short centrifugation step is not sufficient to float most VLDL, which
are usually isolated by adjusting the density of plasma to 1.019 g/ml
followed by 18-24 h centrifugation at >100,000 g. However,
we cannot exclude the possibility that isolated enterocytes secrete a
range of particles from the density of light VLDL to that of
chylomicrons. However, all of the particles contain apoB48. Similarly
the luminal light particles are in the fraction of density
1.0019-1.0086 g/ml. However, as plasma VLDL are also recovered in this
fraction, it is possible that the luminal contents contain particles of
the density of VLDL as well as of chylomicrons.
There are several possible sequences of events by which chylomicrons
may be formed in the second step (reviewed in Ref. 3). The dense
particles may fuse with a preformed TAG-rich light particle, or there
may be core expansion by sequential or continuous addition of lipid to
the dense particles. In the latter case, it would be expected that a
range of TAG-containing particles of intermediate density would be
produced. However, our results reveal only dense and light
apoB48-containing particles and light TAG-containing particles. There
is some phospholipid in the intermediate density fractions from fat-fed
enterocytes. However, TAG in these fractions is almost undetectable,
and apoB48 is at a low concentration. Thus, when the chylomicron
production rate is increased, there may be some intermediate density
particles relatively enriched in phospholipid, but these contain only a
small fraction of the luminal apoB48. The results indicate that the
bulk of the TAG must be acquired by the dense particles in one step.
All of the lipid in the light particles co-immunoprecipitates with
apoB48, indicating that transfer of TAG into the SER lumen must be
either co-incident with or followed rapidly by fusion with dense
particles. A large proportion of the apoB48 in the SER is
membrane-bound (17). This may include dense particles in the process of
fusion with TAG droplets in the membrane. Indeed, Rustaeus et
al. (30) have shown that in McA7777 cells, part of the
membrane-associated apoB is not integrated and is removed by
mild detergent treatment. There is evidence from studies of genetically
manipulated mice (deficient in intestinal apoB) that chylomicron-sized
particles lacking apoB48 can form in the ER lumen of the enterocytes in addition to the large accumulation of cytosolic TAG droplets (31). These ER droplets are not observed in the suckling or adult mice in
which TAG only accumulates in the cytosol (32). The reason for these
conflicting observations is not clear; however, the enterocytes of
fetal mice are essentially fasted and are not involved in the active
absorption of dietary fat. In the normal adult enterocytes, TAG
transfer into the lumen may be facilitated by fusion with apoB-containing dense particles but occur to a low extent in the absence of apoB.
We have previously calculated that in chow-fed enterocytes, ~97% of
the cellular TAG is recovered in the microsomal membrane (17, 18).
During a 90-min incubation, secreted TAG is equivalent to ~40% of
the initial cellular TAG (17, 18). The microsomal membrane therefore
contains sufficient TAG to account for that secreted. In the fat-fed
enterocytes, ~60% of the cellular TAG is recovered in the microsomal
membranes, and ~40% is recovered in the cytosol (17, 18, 33). During
a 90-min incubation, secretion of TAG accounts for about 60% of the
cellular TAG. It is possible in this case that cytosol TAG contributes
to secreted TAG in addition to that in the membranes. The TAG content
of the SER membrane is ~33 µg/mg of phospholipid in chow-fed
enterocytes and ~66 µg/mg of phospholipid in the fat-fed
enterocytes (18). This is sufficient to dissolve in the membrane
bilayer (34). However, TAG may be sequestered in regions of the
membrane and separate by phase partition for incorporation into luminal
lipoproteins. We have previously suggested (18) that the transfer of
TAG from the SER membrane into the lumen for incorporation into light
particles may be rate-limiting in the chow-fed enterocytes so that
light particles are immediately secreted. The present results are
consistent with this as only dense particles are present in the lumen
of the chow-fed enterocytes. In the enterocytes from fat-fed rabbits, light particles are detected in the luminal fractions. As we have suggested before (18), the movement of chylomicron precursors from the
SER to the Golgi may also be saturatable under conditions of increased
chylomicron assembly and secretion. The mechanisms involved in the
transfer of TAG remain to be elucidated.
Diacylglycerol acyltransferase, the enzyme involved in the final step
of TAG synthesis, has recently been shown to be active at both the
cytosolic and luminal side of the ER membrane (35). This raises the
possibility that triglycerides destined for secretion and for the
cytosol are synthesized at different sites. However, transfer of TAG
into the ER lumen is saturatable, and under conditions of high fat
load, TAG is also transferred to the cytosol, so the two enzyme
activities must be coordinately regulated (17). Luminal transfer does
not appear to be limited by the availability of "acceptor" dense
particles, which are present under all dietary conditions investigated.
Transfer of the membrane-bound TAG to the apoB48-containing particle
may involve a number of factors including microsomal
triglyceride transfer protein (MTP), which has been implicated in
intestinal fat absorption (36). We have previously shown that
inhibition of MTP reduces the transfer of SER membrane-bound TAG into
the lumen and its subsequent secretion (18). Although in
vitro MTP transfers lipid by a shuttle mechanism, it does form a
complex with apoB48 (37-39) and thus may facilitate the association of
the apoB48 in the dense particles with TAG-rich droplets.
ApoB is an extremely hydrophobic protein and must be stabilized during
its translocation into the ER lumen and in the dense particles. Our
observations suggest that phospholipids, particularly PC, may fulfill
this role. This is consistent with the observation that the initial
steps in lipoprotein formation involve binding of phospholipid by the N
terminus of apoB (40). We did not detect other lipid classes in the
dense particle fractions, although it is possible that small amounts
are present below the limits of detection by HPTLC. In our short term
experiments, stabilization of apoB48 in the dense particles uses
preformed rather than newly synthesized phospholipids. However, newly
synthesized TAG and phospholipids appear in the light particles.
Luchoomun and Hussain (41) have also shown that chylomicrons
secreted by CaCO2 cells tend to contain preexisting phospholipids,
whereas newly synthesized TAG is preferentially secreted. Our results
suggest that this is due to compartmentalization of the lipids
during the assembly process.
In summary, this study shows directly that chylomicron assembly in
adult enterocytes involves formation of dense apoB48-containing particles, which are stabilized by phospholipids. In contrast to VLDL
assembly in hepatocytes, these particles accumulate in the SER lumen.
The bulk of the chylomicron TAG is acquired in a single step, which
also occurs in the SER lumen. However, we did not detect TAG-rich
apoB48-deficient particles, suggesting that in enterocytes, the second
assembly step may involve either the budding of TAG-containing
particles into the lumen followed by rapid fusion with dense particles
or that assembly may take place at the SER membrane surface. The
regulation of the second step is important in the determination of the
size, composition, and rate of production of chylomicrons.
*
This research was supported by grants from the
Biotechnology and Biological Sciences Research Council.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, October 23, 2001, DOI 10.1074/jbc.M104229200
The abbreviations used are:
TAG, triacylglycerol;
HDL, high density lipoprotein(s);
LDL, low
density lipoprotein(s);
VLDL, very low density lipoprotein(s);
ER, endoplasmic reticulum;
SER, smooth endoplasmic reticulum;
RER, rough
endoplasmic reticulum;
HPTLC, high performance thin layer
chromatography;
PC, phosphatidylcholine;
PE, phosphatidylethanolamine.
Direct Evidence for a Two-step Assembly of ApoB48-containing
Lipoproteins in the Lumen of the Smooth Endoplasmic Reticulum of Rabbit
Enterocytes*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (33K):
[in a new window]
Fig. 1.
Separation of rabbit plasma lipoproteins in
9-12% iodixanol gradients. Rabbit plasma (total of 1.25 ml) was
separated in self-generating gradients of iodixanol and collected in 10 fractions (fraction 1 is the light end of the gradient) as described
under "Experimental Procedures," using 9-12% two layers. Aliquots
of the fractions were separated on agarose gels, and the cholesterol
and TAG content was determined in other aliquots. The top figure
shows the distribution profile of cholesterol (µmoles/fraction)
(left-hand axis) and the distribution profile of TAG
(µmoles/fraction) (right-hand axis). The middle
figure illustrates an agarose gel; 1-10 are gradient
fractions, and plasma is an aliquot of total plasma
separated in a separate gel. The position of the lipoproteins, HDL,
LDL, and VLDL, and the origin, where chylomicrons remain, are
indicated. The bottom figure shows the density profile of
the gradient determined from the refractive indices of the fractions
collected (19). The results plotted illustrate a separation of one
plasma sample. However, similar patterns were observed in many repeated
analyses.
View larger version (44K):
[in a new window]
Fig. 2.
Density distribution of apoB48 in luminal
gradient fractions. Isolated enterocytes from rats fed chow or fat
for 3 and 6 days were incubated with micelles containing
[35S]methionine for 30 min. Microsomes were isolated, the
luminal contents were prepared and separated in self-generated
gradients of iodixanol, and the gradient fractions were analyzed as
described under "Experimental Procedures." A, the
density distribution of radiolabeled apoB48 is plotted for the three
dietary states ±SD (n = 3). B,
immunodetectable apoB48 in the gradient fractions from chow-fed and
fat-fed for 6 days is illustrated.
View larger version (30K):
[in a new window]
Fig. 3.
Density distribution of lipids in luminal
gradient fractions. Isolated enterocytes from rats fed chow or fat
for 3 and 6 days were incubated with micelles containing
[14C]oleate for 30 min. Microsomes were isolated, the
luminal contents were prepared and separated in self-generated
gradients of iodixanol, and the lipids of the gradient fractions were
extracted and analyzed as described under "Experimental
Procedures." A, the density distribution of radiolabeled
TAG is plotted for the three dietary states ±SD (n = 3); fractions 1-2, 3-5, and 7-9 contain particles of the density of
chylomicrons/VLDL, LDL, and HDL, respectively. B, fractions
1 and 2 from microsomes from chow-fed and fat-fed for 6 days were
pooled, and the apoB48 was immunoprecipitated. The percentage of
radiolabeled TAG and TAG mass co-immunoprecipitating with apoB
(%co-ppt) is plotted.
View larger version (35K):
[in a new window]
Fig. 4.
Density distribution of PC in luminal
gradient fractions. The experiment was conducted as in the legend
for Fig. 3. A, the density distributions of radiolabeled PC
respectively, are plotted for the three dietary states ±SD
(n = 3). B, fractions 1-3, 4-7, and 8-10
from microsomes from chow-fed and fat-fed for 6 days were pooled, and
the apoB48 was immunoprecipitated. The percentage of radiolabeled PC
and PE mass co-immunoprecipitating with apoB (%co-ppt) is
plotted.
View larger version (36K):
[in a new window]
Fig. 5.
Density distribution of PE
in luminal gradient fractions. The experiment was conducted as in
the legend for Fig. 3. A, the density distributions of
radiolabeled PE, respectively, are plotted for the three dietary states
±SD (n = 3). B, fractions 1-3, 4-7, and
8-10 from microsomes from chow-fed and fat-fed for 6 days were pooled,
and the apoB48 was immunoprecipitated. The percentage of radiolabeled
PE mass co-immunoprecipitating with apoB (%co-ppt) is
plotted.
View larger version (45K):
[in a new window]
Fig. 6.
Separation of rabbit plasma lipoproteins in
15% iodixanol gradients. Rabbit plasma (total of 1.25 ml) was
separated on self-generating gradients of iodixanol and collected in 10 fractions (fraction 1 is the light end of the gradient) as described
under "Experimental Procedures," using 15% layers. Aliquots of the
fractions were separated on agarose gels, and the cholesterol was
determined in other aliquots. The top figure illustrates an
agarose gel; 1-10 are gradient fractions. The positions of
HDL and LDL are indicated. The bottom graph shows the
density profile of the gradient fractions determined from the
refractive index (19).
View larger version (19K):
[in a new window]
Fig. 7.
Density distribution of apoB48 in luminal
gradient fractions separated in 15% iodixanol gradients. Isolated
enterocytes from chow-fed rabbits were incubated with micelles
containing [35S]methionine and [3H]oleate
for 30 min. Microsomes were isolated, the luminal contents were
prepared and separated in self-generated gradients of 15% iodixanol,
and the gradient fractions were analyzed as described under
"Experimental Procedures." A, the density
distribution of radiolabeled apoB48 and of radiolabeled and
masses of PC and TAG. The distribution of PE was similar to that of PC
and is not plotted. B, immunodetectable apoB48 in the
gradient fractions.
View larger version (18K):
[in a new window]
Fig. 8.
Density distribution of TAG and apoB48 in
subfractions of the ER. Isolated enterocytes from rats fed chow or
fat for 6 days were incubated with micelles containing
[14C]oleate and [35S]methionine for 30 min.
Golgi, SER, and RER fractions were prepared, and the luminal contents
and membranes of each gradient sub-fraction were separated as described
previously (18). Newly synthesized TAG (counts/fraction), the mass of
TAG (µg/fraction), and newly synthesized apoB48 (counts/fraction)
were determined as described under "Experimental Procedures."
A, the distribution of each luminal component in the
gradient. Open triangles, fat-fed; closed
triangles, chow-fed. B, immunodetectable apoB in the
gradient fractions from chow-fed enterocytes. A similar pattern was
observed in gradient fractions from fat-fed enterocytes. The position
of the Golgi, SER, and RER fractions (18) is shown in panel
B.
View larger version (26K):
[in a new window]
Fig. 9.
Fate of newly synthesized luminal
apoB48 in dense particles in isolated enterocytes. Isolated
enterocytes from fat-fed rabbits were incubated with
[35S]methionine for 30 min (pulse). The cells
were isolated by centrifugation and reincubated with unlabeled micelles
and an excess of unlabeled methionine (18) for 45 min
(chase). At the end of the pulse and the chase periods, the
cells were pelleted by centrifugation, and secreted lipoproteins were
isolated from the incubation medium. Luminal content fractions were
prepared and separated in 9-12% density gradients, and radiolabeled
apoB48 in the fractions was determined as described under
"Experimental Procedures." The radiolabeled apoB48 in the luminal
fractions and the medium was normalized to 1 g of
enterocytes. The data plotted are from a typical experiment.
A, distribution of radiolabeled apoB48 at the end of the
pulse and the end of the chase. B, sum of the radiolabeled
apoB48 in the luminal fractions at the end of the pulse and the end of
the chase and recovered from the media as described under
"Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
To whom correspondence should be addressed: Dept. of Molecular
Biology and Biotechnology, University of Sheffield, Sheffield, S10 2TN,
UK; Tel.: 44-114-2224235; Fax: 44-114-2222793; E-mail: J.Higgins@sheffield.ac.uk.
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