Am J Physiol Gastrointest Liver Physiol 294: G344–G352, 2008.

First published November 15, 2007; doi:10.1152/ajpgi.00123.2007.

Why does the gut choose apolipoprotein B48 but not B100
for chylomicron formation?
Chun-Min Lo,1 Brian K. Nordskog,1 Andromeda M. Nauli,1 Shuqin Zheng,1 Sarah B. vonLehmden,1
Qing Yang,1 Dana Lee,1 Larry L. Swift,3 Nicholas O. Davidson,2 and Patrick Tso1
1
Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio; 2Department of Internal
Medicine, Washington University School of Med icine, St. Louis, Missouri; and 3Department of Pathology, Vanderbilt
University, Nashville, Tennessee
Submitted 9 March 2007; accepted in final form 15 November 2007

Lo CM, Nordskog BK, Nauli AM, Zheng S, vonLehmden SB,
Yang Q, Lee D, Swift LL, Davidson NO, Tso P. Why does the gut
choose apolipoprotein B48 but not B100 for chylomicron formation?
Am J Physiol Gastrointest Liver Physiol 294: G344–G352, 2008. First
published November 15, 2007; doi:10.1152/ajpgi.00123.2007.—Chy-
lomicrons produced by the human gut contain apolipoprotein (apo)
B48, whereas very-low-density lipoproteins made by the liver contain
apo B100. To study how these molecules function during lipid
absorption, we examined the process as it occurs in apobec-1 knock-
out mice (able to produce only apo B100; KO) and in wild-type mice
(of which the normally functioning intestine makes apo B48, WT).
Using the lymph fistula model, we studied the process of lipid
absorption when animals were intraduodenally infused with a lipid
emulsion (4 or 6 ␮mol/h of triolein). KO mice transported triacyl-
glycerol (TG) as efficiently as WT mice when infused with the lower
lipid dose; when infused with 6 ␮mol/h of triolein, however, KO mice
transported significantly less TG to lymph than WT mice, leading to
the accumulation of mucosal TG. Interestingly, the size of lipoprotein
particles from both KO and WT mice were enlarged to chylomicron-
size particles during absorption of the higher dose. These increased-
size particles produced by KO mice were not associated with in-
creased apo AIV secretion. However, we found that the gut of the KO
mice secreted fewer apo B molecules to lymph (compared with WT),
during both fasting and lipid infusion, leading us to conclude that the
KO gut produced fewer numbers of TG-rich lipoproteins (including
chylomicron) than the wild-type animals. The reduced apo B secretion
in KO mice was not related to reduced microsomal triglyceride
transfer protein lipid transfer activity. We propose that apo B48 is the
preferred protein for the gut to coat chylomicrons to ensure efficient
chylomicron formation and lipid absorption.
very low-density lipoprotein; lymph
TRIACYLGLYCEROL (TG) is the major component of human di-
etary fat. After consumption, TG molecules are hydrolyzed by
pancreatic lipase yielding the products 2-monoglycerol (2-MG)
and free fatty acids (FA), which are then able to be absorbed by
the intestinal enterocytes either by passive diffusion or through
carrier-mediated processes (5, 20, 27, 35). Following uptake by
the enterocytes, the 2-MG and FA are resynthesized back to
form TG through the MG pathway (17, 26). TG is then
packaged with phosphatidylcholine, cholesteryl ester, free cho-
lesterol, and apolipoproteins (apo) (B48, AI, and AIV) forming
either chylomicrons or very-low-density lipoprotein-sized
(VLDL) lipoproteins (36). The secretion of these respective
intestinal lipoprotein particles is regulated by the physiological
state of the small intestine; specifically, during the fasting state
or during active lipid absorption. In periods of fasting, VLDL-
sized lipoproteins are the predominant particles secreted by the
small intestine (23, 31). After ingestion of a meal rich in TG,
the small intestine continues to form VLDL; however, the
predominant TG-rich lipoprotein particles secreted by the gut
are the larger chylomicron particles (13, 32). An interesting
question dealing with the mechanism of chylomicron forma-
tion is whether the intestine produces a larger number of
chylomicrons, or an equivalent number of chylomicron parti-
cles that are larger, during active lipid absorption. The answer
is that the number of chylomicron particles produced by the
small intestine remains unchanged during active lipid absorp-
tion; to accommodate the large amount of lipid to be trans-
ported by the gut, the small intestine produced larger chylo-
microns during this period (6, 13).
TG synthesized by the liver is packaged with apo B100 to
form VLDL particles, which are subsequently secreted to
plasma circulation. Using isolated hepatocytes in vitro, it has
been demonstrated that incubation with oleic acid results in an
increased production and secretion of TG into the culture
media (9, 24). Additionally, a positive correlation was ob-
served among hepatic TG secretion and apo B100 secretion (3,
10). Thus oleate supplementation increased the number of apo
B particles secreted by the liver, but not the size, since the
products secreted by the liver were consistently the smaller
VLDL particles (8, 15, 38). Clearly, there are differences in the
ways the gut and liver respond physiologically to increased
lipid flux. The question at hand is whether apo B48 and apo
B100 themselves play a role in mediating these differing
physiological responses. The purpose of this study is to answer
the question of whether or not there is a possible functional
advantage of exclusive apo B48 expression in the small intes-
tine for the purpose of chylomicron formation leading to the
most efficient handling of dietary lipid loads.
Tissue-specific production of apo B48 is mediated by post-
transcriptional cytidine deamination of the nuclear apo B
transcript by an RNA-specific cytidine deaminase, apobec-1
(29). Targeted deletion of the apobec-1 gene yields mice in
which only apo B100 is produced in both the liver and the
small intestine; thus circulating lipoprotein particles contain
exclusively apo B100 (14, 21–22). Earlier studies have shown
no significant difference in total plasma TG concentrations
between ad libitum fed or 4-h fasted knockout (KO) mice and

Address for reprint requests and other correspondence: P. Tso, Dept. of The costs of publication of this article were defrayed in part by the payment
Pathology and Laboratory Medicine, Univ. of Cincinnati, 2120 E. Galbraith of page charges. The article must therefore be hereby marked “advertisement”
Rd., Cincinnati, OH 45237 (e-mail: tsopp@email.uc.edu.). in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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 APOLIPOPROTEIN B AND CHYLOMICRON FORMATION
wild-type (WT) mice (fed a standard chow diet), and that the
KO mice appeared to be totally capable of absorbing both lipid
and fat-soluble vitamins normally (14, 22). Thus it was origi-
nally concluded that apo B48 is not required for the assembly
of intestinal lipoproteins and that apo B100 can serve as a
substitute for apo B48 in the intestine, still resulting in normal
lipid transport (12, 38). In contrast to these conclusions, how-
ever, was the observation that KO mice had higher TG con-
centrations in plasma chylomicron fractions and that KO mice
accumulated lipid droplets within enterocytes after being fed a
high-fat chow diet (16). These data seem to hint that lipids
ingested by a KO animal are not absorbed and transported as
efficiently as they are in WT mice and that the apo B100
chylomicrons are not as efficiently catabolized in circulation as
the chylomicrons assembled with apo B48. More recent studies
of apobec-1 KO mice bred into pure C57 genetic background
substantiated these subtle differences in intestinal chylomicron
assembly and secretion from isolated enterocytes. Thus it is
necessary to reevaluate whether or not apo B48 and B100 are
indeed truly interchangeable, with respect to the formation of
chylomicrons and the delivery of the transported TG to lym-
phatic circulation (37).
With the establishment of the lymph fistula mouse model,
we are in a uniquely apt position to address this question.
Accordingly, this present study was conducted to determine
whether the apo B48-producing intestine (WT) absorbs and
transports lipid into lymph more efficiently than the apo B100-
producing intestine and whether the differences previously
observed are reflective of the quantity of lipid presented for
absorption.
MATERIAL AND METHODS
Animal Surgery and Postoperative Care
Male apobec-1 KO mice, backcrossed as described (14, 37) to yield
animals that are almost of pure C57BL/6 genetic background. Six
animals of each genotype were used in the modest-dose triolein study
and seven mice of each genotype were used for the high-dose triolein
study. The main mesenteric lymph duct of each animal was cannu-
lated with a polyvinylchloride tube, which was then secured with a
drop of Krazy Glue (4). A second polyvinylchloride tube was intro-
duced into the stomach through the fundus and threaded into the
duodenum. The fundal incision was closed by purse-string suture.
After surgery, the animals were infused via duodenal catheter with a
saline solution containing 5% glucose at a rate of 0.3 ml/h to
compensate for fluid and electrolyte loss due to lymphatic drainage.
The animals were allowed to recover for at least 24 h in restraining
cages situated in a warm chamber (⬃30°C) prior to the start of the
experiments. All procedures were approved by the University of
Cincinnati Internal Animal Care and Use Committee and complied
with the NIH Guide for the Care and Use of Laboratory Animals.
TG Infusion
Lipid was infused into KO mice (experimental group) and WT
mice (control group) at two different doses. The lower dose lipid
emulsion contained 4 ␮mol/h triolein (labeled with [9,10-3H (N)]oleic
acid, 1 ␮Ci/0.3 ml, Perkin Elmer, Boston, MA), 0.78 ␮mol/0.3 ml of
cholesterol and phospholipid (PL; 0.78 ␮mol/0.3 ml) in a 19 mM
sodium taurocholate solution. The lipid emulsion was infused in-
traduodenally at a constant rate of 0.3 ml/h for 6 h. Studies involving
the higher dose of triolein (6 ␮mol/0.3 ml/h triolein, labeled with
[9,10-3H(N)]triolein, 1 ␮Ci/0.3 ml) utilized the same lipid emulsion
composition (0.78 ␮mol/0.3 ml of cholesterol, PL 0.78 ␮mol/0.3 ml
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G345
in a 19 mM sodium taurocholate solution). This lipid emulsion was
infused intraduodenally to the KO and WT mice at a rate of 0.3 ml/h
for 6 h.
Lymphatic, Mucosal, and Luminal Lipid Content Determined
by Radioactive Assay
Fasting lymph was collected for 1 h prior to lipid infusion, and
hourly lymph samples were collected for 6 h after the start of lipid
infusion. At the end of the infusion period, the stomach, small
intestine, and colon were taken from each animal and luminal lipids
were collected by washing the lumen of the organs three times with a
0.5% sodium taurodeoxycholate solution. The small intestine (from
duodenum to ileum) was divided into four equal-length segments and
each of the four mucosal samples was used in the determination of
radioactive lipid content. Radioactive luminal lipid content was de-
termined by liquid scintillation counting.
Particle Size of Chylomicron and VLDL
To study the size of the lipoprotein particles, fresh lymph samples
from three animals each of both WT and KO groups were stained with
2% phosphotungstic acid (pH 6.0) for 5 min and examined with a
transmission electron microscope (TEM, JEOL, Peabody, MA) and
images were documented with an AMT Advantage Plus CD camera
(31). Eight hundred lipoprotein particles per group were measured by
manual counting using a magnifying glass for further magnification of
printed images of the respective fields of view. All images were
evaluated blindly, without the examiner knowing the animals’ geno-
type, to avoid any bias in the sizing of the lipoprotein particles.
Lipoproteins in the diameter range between 200 and 800 Å were
representative of VLDL, and particles ⬎ 800 Å were classified as
chylomicrons (31).
Lymph Lipoprotein and Apolipoproteins Determination
Samples of mouse lymph were collected for 1 h and aliquoted (i.e.,
1/60 of the volume collected over 60 min) to prepare the equivalent of
a 1-min lymph collection. The rationale for using the 1-min sample is
to overcome the variations in lymph flow between different time
points, both within the same animal and also between animals of the
same group. An equal volume of 2⫻ SDS sample buffer was then
added to each sample. Samples were boiled for 5 min and then loaded
into a 4 –20% Tris 䡠 HCl gradient gel (Bio-Rad Laboratories, Hercules,
CA). Gels were run at a constant voltage (60 V) until the protein
standards were well separated. Proteins were then transferred to a
polyvinylidene difluoride membrane (Bio-Rad Laboratories) for 2 h at
a constant current of 350 mA. After blocking nonspecific binding sites
on the membranes for 1 h with a 5% solution of nonfat milk in
Tris-buffer saline with 0.1% Tween (TBS-T), membranes were then
incubated with polyclonal rabbit anti-rat apo B antibody (1:7,500
dilution in 5% nonfat milk in TBS-T) or with polyclonal goat anti-rat
apo AIV antibody (1:7,500 dilution in 5% nonfat milk in TBS-T).
After incubation with the primary antibody, the blots were washed
with 1.25% nonfat milk in TBS-T and then incubated either with
horseradish peroxidase-conjugated goat anti-rabbit antibodies or with
horseradish peroxidase-conjugated rabbit anti-goat antibodies (Dako,
Cytomation) diluted 1:5,000 with 5% nonfat milk in TBS-T for 2 h.
Detection of apo B was achieved by using the enhanced chemilumi-
nescence system (ECL Western Blotting Detection Reagents, Amer-
sham Biosciences, Buckinghamshire, UK), and X-OMAT AR films
(Kodak) were used for development and visualization of the mem-
branes. Net apo B secretion during each subsequent hour of infusion
was quantified by subtracting the 0 h (fasting) lymph apo B content
from relative apo B levels at each hourly time point.
MTP Lipid Transfer Activity
After 4 h of triolein infusion (6 ␮mol triolein 䡠 0.3 ml⫺1 䡠 h⫺1, 0.78
␮mol/0.3 ml of cholesterol, PL 0.78 ␮mol/0.3 ml in a 19 mM sodium
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G346 APOLIPOPROTEIN B AND CHYLOMICRON FORMATION
taurocholate solution), the first two segments of mucosa (M1 and M2,
M1 ⫽ duodenum and M2 ⫽ upper jejunum) in WT and KO mice were
homogenized in 10 mM Tris-buffer (FisherBiotech, Fair Lawn, NJ)
plus protease inhibitors (Roche, Penzberg, Germany). Ten microliters
of either homogenate (40 ␮g), purified liver MTP (0.5 ␮g/␮l), or
water (control group) were incubated with 4 ␮l of donor and 4 ␮l of
acceptor particles at 37°C according to the protocol provided by the
manufacturer (Roar Biomedical, New York, NY). After 5-h incuba-
tion, MTP lipid transfer activity was measured at an excitation
wavelength of 465 nm and emission wavelength of 538 nm in a
fluorescence spectrophotometer (1, 18). Data from duplicate samples
were measured. The MTP activity is expressed as percent TG transfer
by MTP and is the fraction of the total fluorescent lipid transferred
from donor to acceptor vesicles in a 5-h assay period with a known
amount of mucosal protein.
Statistical Analysis
All values are means ⫾ SE. Student’s t-test and two-way repeated
ANOVA were performed for comparison of all groups throughout the
6-h lipid infusion. Statistical analyses were performed via GraphPad
Prism (version 3.0, San Diego, CA), and differences were considered
significant if P values were ⬍0.05.
RESULTS
Modest Dose (4 ␮mol/h) Triolein Study
Lymph flow rate. The fasting lymph flow rate was 0.22 ml/h
in the WT animals and 0.26 ml/h in the apobec-1 KO animals,
a difference that is not statistically significant (Fig. 1). Unlike
what we and others have observed in rats, lymph flow rate did not
change during fat absorption in either the WT or KO mice (33).
Lymphatic [3H]TG output. KO animals had lower lymphatic
transport of the [3H]TG than WT mice in the first hour;
however, this difference is not considered significant (P ⬍
0.064) (Fig. 2). From the second to sixth hour of lipid infusion,
a similar increase in output was observed in both groups and
the output became steady at the third hour after the beginning
of lipid infusion (⬃58% of hourly infused lipid) (Fig. 2).
Lymphatic transport of the [3H]TG in KO mice was not
different from that of the WT animals during the subsequent
6 h infusion period (P ⫽ 0.93; not significant). Total recovery
of lymphatic radioactive TG in the KO mice (45.56 ⫾ 4.42%)
Fig. 1. Hourly lymphatic flow rate during continuous intraduodenal infusion
of 4 ␮mol/h triolein. Six mice of both [knockout (KO) and wild-type (WT)]
genotypes were used in each study (n ⫽ 6). Lymphatic flow was monitored
during the 6-h lipid infusion period. Values are means ⫾ SE. *P ⬍ 0.05.
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Fig. 2. Lymphatic [3H]triglyceride (TG) transport into lymph during contin-
uous intraduodenal infusion of 4 ␮mol/h of triolein. Mice were implanted with
intraduodenal and lymph cannulas and were intraduodenally infused with a
lipid emulsion containing labeled [3H]TG continuously for 6 h (n ⫽ 6). Lymph
was collected hourly following the start of lipid infusion and analyzed. Values
are means ⫾ SE. *P ⬍ 0.05.
was slightly lower than the WT animals (52.28 ⫾ 3.12%), but
again, this difference is not statistically significant (Fig. 3).
Luminal and mucosal recovery of [3H]TG. There was no
statistical difference in the luminal recovery of [3H]TG be-
tween the WT and the KO animals at the end of the 6-h
infusion of the modest dose of lipid infusate (Fig. 3). However,
the accumulation of TG in the mucosa of KO mice (19.28 ⫾
2.50%) was significantly higher than that of WT mice (11.90 ⫾
1.02%, P ⬍ 0.02). These findings suggest that whereas WT and
KO mice absorb the digestion products of ingested TG with
comparable efficiency, the KO mice are less efficient in deliv-
ering the absorbed lipids to lymph as chylomicrons, resulting
in greater accumulation of lipids in the mucosa of KO animals.
When the recovery of radioactive mucosal lipids from the four
equal-length intestinal segments was analyzed, most of the
radiolabeled TG was detected in the proximal jejunal segment
of the small intestine and some remained in the second seg-
ment; only trace amounts of the [3H]TG remained in the third
and fourth intestinal segments in both KO and WT animals
(Fig. 4). The KO mice (14.06 ⫾ 1.43%) accumulated signifi-
Fig. 3. [3H]TG content in the WT and KO mice at the end of 6-h continuous
infusion of 4 ␮mol/h triolein. Luminal contents, small intestinal mucosa, and
lymph were harvested at the end of the experiment and radioactive lipids were
determined by scintillation counting (n ⫽ 6). Values are means ⫾ SE. *P ⬍ 0.05.
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 APOLIPOPROTEIN B AND CHYLOMICRON FORMATION G347

Fig. 4. Distribution of dietary TG in the intestinal segments. After continuous

infusion of 4 ␮mol/h triolein for 6 h, 4 equal-length segments of the small
intestine of each animal from proximal to distal (M1, M2, M3, M4) were
collected in the WT and KO mice (n ⫽ 6). The amount of [3H]lipids in these
segments was determined by scintillation counting. Values are expressed
mean ⫾ SE. *P ⬍ 0.05.

cantly more radioactive TG than the WT mice (9.20 ⫾ 0.85%)

in the first segment of the small intestine. Thus the duodenum
and the proximal jejunum are the major sites for lipid absorp-
tion in both KO and WT mice, but the KO mice are less
efficient in exporting absorbed lipid to lymph.
Negative staining and sizing of the lymph lipoproteins. With
slightly less lymphatic TG transport observed in KO mice Fig. 5. Size distribution of lymph lipoproteins in apobec-1 KO and WT mice.
compared with WT animals, we wondered whether this differ- Lymph lipoproteins collected during fasting (A) and during active lipid
absorption of a modest lipid dose (B). Particles of 3 mice from each group were
ence could be attributed to a difference in the size of the examined. Lymph was collected during glucose/saline infusion (fasting) and
chylomicron particles produced by the enterocytes of the KO during the 3rd to 4th hour of intraduodenal lipid infusion (4 ␮mol/h); lymph
vs. the WT animals. In this study, fresh lipoproteins from samples were subsequently stained with 2% phosphotungstic acid for obser-
lymph of both WT and KO mice were negatively stained and vation with transmission electron microscopy. The size distribution histogram
represents 800 particles per mice. Bar ⫽ 2,500 Å.
sized (31). The KO and WT animals secreted predominantly
VLDL particles during fasting (99.0% and 99.3%, respec-
tively) (Fig. 5A). In response to lipid feeding, the pattern of ence was observed in the rate of lymph flow between the WT
lipoprotein secretion in WT animals shifted from mostly and the KO animals (P ⬍ 0.016).
VLDL-sized particles to particles of chylomicron size (lymph Lymphatic [3H]TG output. As shown in Fig. 7, the KO mice
samples from the third and fourth hour were analyzed; data are transported lipids less efficiently than WT mice at the second
shown in Fig. 5B). Of the total numbers of lymph lipoproteins hour (P ⫽ 0.017) and third hour (P ⫽ 0.003) of lipid infusion.
counted, ⬃39% were in the chylomicron size range in the WT The largest difference was observed at the third hour, when
animals (the rest in the VLDL range), whereas the KO mice both groups reached their maximum TG output; the WT group
only had 12% of their total lipoprotein particles as chylomi-
crons during the third to fourth hour of lipid infusion. Thus
there was more of a shift in size in the lymphatic lipoproteins
secreted by WT mice during active fat absorption, whereas, in
contrast, lymph lipoproteins secreted by KO mice mostly
remained as VLDL-sized particles. This observed decrease in
the production of chylomicron-sized particles is possibly re-
flective of the observation that KO mice seem to be slightly
less efficient than WT mice in transporting absorbed TG as
chylomicrons during active fat absorption.
High-Dose (6 ␮mol/h) Triolein Study
Lymph flow rate. As shown in Fig. 6, the fasting lymph flow
was 0.23 ml/h in the WT and 0.28 ml/h in the KO animals, but
this difference is not statistically significant. Following lipid
infusion, the rate of lymph flow in both groups of mice fell; Fig. 6. Hourly lymphatic flow rate during continuous intraduodenal infusion of 6
with the effect more marked in the KO animals. At the third ␮mol/h triolein. Data were collected from 7 mice of both genotypes (n ⫽ 7).
hour following the start of lipid infusion, a significant differ- Lymphatic flow rate is expressed in ml/h. Values are means ⫾ SE. *P ⬍ 0.05.

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G348 APOLIPOPROTEIN B AND CHYLOMICRON FORMATION
Fig. 7. Lymphatic [3H]TG transport to lymph during continuous intraduodenal
infusion of 6 ␮mol/h triolein (n ⫽ 7). Mice were implanted with intraduodenal
and lymphatic cannulas and were intraduodenally infused with the lipid
emulsion containing labeled [3H]TG at a constant rate for 6 h. Lymph was
collected hourly and analyzed. Values are means ⫾ SE. *P ⬍ 0.05.
measured 48.16 ⫾ 3.91% of initial dose, whereas the KO mice
only reached 26.75 ⫾ 4.33% (P ⬍ 0.003) (Fig. 7). The total
output of lymphatic TG of the KO mice (25.04 ⫾ 3.86%) was
significantly lower (P ⬍ 0.012) than that of the WT mice
(38.65 ⫾ 2.51%) (Fig. 8). These data showed that KO mice did
not transport TG into lymph as efficiently as WT animals when
the dose of the lipid emulsion infused increased by 50%.
Lymph, luminal, and mucosal recovery of [3H]TG. By the
end of the 6 h lipid infusion period, there was a greater level of
radioactive TG accumulation in the mucosa of KO mice than in
WT mice (39.57 ⫾ 3.86 vs. 21.66 ⫾ 5.94% respectively, P ⬍
0.026). This increase in overall mucosal accumulation of
[3H]TG can be attributed to the vast difference in radioactivity
observed in segment 1 (duodenum ⫹ proximal jejunum) of the
small intestine in the KO mice (28.58 ⫾ 3.73%) relative to the
WT animals (16.05 ⫾ 4.19%) (P ⬍ 0.045) (Fig. 9). These
observations demonstrate that KO mice are clearly less effi-
cient than WT mice in transporting absorbed lipid into lymph
as chylomicron particles, resulting in a marked accumulation
Fig. 8. Luminal, mucosal, and lymphatic recovery of [3H]TG in WT and KO
mice (n ⫽ 7) at the end of 6 h of continuous infusion of 6 ␮mol/h triolein.
Luminal contents, small intestinal mucosa, and lymph were harvested at the
end of the experiment and radioactive lipid content was determined by
scintillation counting. Values are means ⫾ SE. *P ⬍ 0.05.
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Fig. 9. Distribution of mucosal [3H]TG in the small intestinal segments. At the
end of 6 h of continuous infusion of 6 ␮mol/h triolein, 4 equal parts of the
small intestine, from proximal to distal (M1, M2, M3, M4), of the WT and KO
mice (n ⫽ 7) were collected. The amount of [3H]lipid in these segments was
determined by scintillation counting. Values are means ⫾ SE. *P ⬍ 0.05.
of the radioactive TG in the intestine, especially in the first
mucosal segment.
Negative staining and sizing of the lymph lipoproteins. As
shown in Fig. 10, A and B, respectively, both the KO and the
WT mice secreted large chylomicron-sized lipoprotein par-
ticles to the lymphatic circulation during active lipid ab-
sorption after the beginning of infusion of the 6 ␮mol/h TG
emulsion. The size of lipoproteins secreted by the KO mice
was not significantly different from that secreted by WT
mice (P ⬍ 0.68). Overall, during the third to fourth hour,
37% of the total number of secreted particles was of chy-
lomicron size in the KO animals, compared with 32% for
WT animals during the same period (Fig. 10C). We can
draw two conclusions from these data: 1) apo B100-produc-
ing enterocytes of apobec-1 KO animals are indeed capable
of secreting chylomicron-size particles; and 2) despite the
ability to secrete chylomicrons, the apobec-1 KO animals
are still significantly less capable of delivering the absorbed
TG into lymph than the WT animals.
Lymphatic apo B and apo AIV secretion. We next examined
whether decreased apo B secretion might account for the
differences in chylomicron-mediated triglyceride transport
observed between WT and KO mice. To compare the output
from various animals, we examined the apo B content of
timed lymph collections (a sample equivalent to a 1-min
lymph sample was taken from the total lymph collected over
60 min). The hourly lymphatic apo B mass output of the WT
group was higher than that of KO mice during fasting as
well as throughout the 6-h lipid-infusion period (Fig. 11A).
The mass of apo B secreted by the KO mice was 20% less
than that secreted by WT animals for the 6 h of lipid
infusion (Fig. 11B). Collectively, one can infer from the
data that the reason KO mice secrete less TG than WT mice
during the infusion period is probably due to the lower
number of apo B100 chylomicron particles secreted by the
KO group; this is certainly a plausible explanation for the
decrease in efficacy of KO animals in their transportation of
TG to lymph. Furthermore, we examined whether KO rats
produced larger particles with more TG due to increased apo
AIV mass. The hourly output of apo AIV mass by the KO
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 APOLIPOPROTEIN B AND CHYLOMICRON FORMATION G349

Fig. 10. Size distribution of lymph lipoproteins

produced by apobec-1 KO and WT mice during
intraduodenal infusion of 6 ␮mol/h TG. Lymph
from 3 mice from both groups was examined.
Lymph was collected during the 3rd to 4th hour
of lipid infusion and was then stained with 2%
phosphotungstic acid for observation. A and B
show the electron micrographs of the negatively
stained lipoprotein images of lymph collected
during the 3rd to 4th hour of lipid infusion for
the KO mice (A) and the WT mice (B) respec-
tively. The size distribution after measurement
of the diameter of 800 lipoprotein particles is
depicted in C. Bar ⫽ 2,500 Å.

mice did not differ significantly from that secreted by WT
mice during fasting and during lipid infusion (Fig. 11A).
These observations suggest that KO mice secrete fewer but
larger chylomicrons without changing the apo AIV mass
output following lipid infusion.
MTP lipid transfer activity. Next, we determined whether
the reduced apo B secretion in the KO animals relative to the
WT animals was related to less activity of MTP, an enzyme
intimately involved in lipidation of prechylomicrons in
endoplasmic reticulum (ER). Figure 12 showed that KO

Fig. 11. Lymphatic apolipoprotein (apo) B secretion in apobec-1 KO and WT mice. Lymph was collected hourly during the 6 h of continuous intraduodenal
infusion of 6 ␮mol/h triolein (4 mice per group); 1 min volumes (based on flow rate) of lymph were analyzed by a 4 –20% polyacrylamide/SDS gel
electrophoresis followed by Western blot detection of apo B and apo AIV using a polyclonal antibody in A. With the baseline apo B content subtracted, the net
apo B secretion over 6 h of lipid infusion for the KO and the WT animals is depicted in B. *P ⬍ 0.05.

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G350 APOLIPOPROTEIN B AND CHYLOMICRON FORMATION
Fig. 12. MTP transfer activity in apobec-1 KO and WT mice. After 4 h of
triolein infusion (6 ␮mol/h triolein), 10 ␮l of mucosal homogenate (40 ␮g of
proteins) (from duodenum to jejunum) were incubated with 4 ␮l of donor and
4 ␮l of acceptor particles at 37°C for 5-h incubation. Fluorescence units were
measured at an excitation wavelength of 465 nm and emission wavelength of
538 nm.
mice had 29.4 ⫾ 4.0% MTP lipid transfer activity in the
duodenum plus jejunum whereas WT groups displayed
28.6 ⫾ 1.6% after 4 h of 6 ␮mol/h infusion of TG (the
difference is not statistically significant from the KO ani-
mals). These data imply that MTP lipid transfer activity is
not responsible for the less TG lipidation in ER and thus
results in reduced apo B secretion in KO mice.
DISCUSSION
Apo B48 found in chylomicrons produced by the wild-type
intestine clearly plays a critical role in the transportation of
lipids from intestinal enterocytes to the lymphatic circulation.
To examine the different physiological roles of apo B48 and
apo B100, we used the lymph fistula model to compare lipid
transport in the apobec-1 KO and the WT animals. The study
using a modest dose of infused TG (4 ␮mol/h) demonstrated
that the lymphatic flow rate, uptake, and lymphatic output of
TG in the KO mice were not significantly different from those
observed in WT mice, except for the slightly lower TG output
of the KO group, which was observed during the first hour.
After the second hour of lipid infusion, KO animals seemed to
transport lipids as well as the WT mice, and these observations
are consistent with earlier conclusions that KO mice absorbed
fat just as well as WT mice (14, 22), which was further
supported by the fact that plasma lipid concentrations between
the two groups was not significantly different (14, 22). Inter-
estingly, the present study revealed that KO mice accumulated
significantly more mucosal TG than the WT animals; these
new data imply that the KO mice are actually less efficient in
TG transport. Secondly, we also observed that the majority of
the mucosal radioactive TG is found in the first quarter of the
small intestine in both KO and WT mice. Therefore, the
duodenum and the jejunum are the predominant sites of lipid
uptake (33) for both groups of animals, leading us to conclude
that there is no shift in the major location of lipid absorption to
the distal small intestine in KO animals relative to the WT
controls.
To further investigate a possible deficit in the capacity to
transport lipid as chylomicrons in KO mice, a larger dose of
radioactive TG (6 ␮mol/h) was infused in KO and WT groups
of mice. The KO mice transported lipids to the lymphatic
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circulation at a significantly lower rate than the WT mice
(26.75 ⫾ 4.33 vs. 48.16 ⫾ 3.90%, respectively) at 3 h into the
infusion period. Associated with the reduced lymphatic trans-
port of the absorbed TG, there was a significant increase in the
amount of radioactive TG retained in the mucosa of the KO
animals relative to the WT animals (P ⬍ 0.026). This finding
coincides with the observation that the KO mice had slightly
higher mucosal TG content compared with WT mice following
exposure to a 2-wk ad libitum feeding of a high-fat chow diet
(16). Therefore, the gut producing exclusively apo B100 in the
KO mice appears to be generally less capable of transporting
absorbed TG into lymph than the apo B48-producing gut of
WT animals. There are three possibilities for this reduced
capacity to transport absorbed lipids to the lymph: 1) a de-
creased ability to synthesize and secrete chylomicrons, which
may be associated with the apo B100-producing gut; 2) an
inability to increase the number of secreted chylomicrons to
adequately handle a lipid load when they are assembled with
apo B100; or 3) a combination of both possibilities.
In the present study, the WT mice produced larger chylo-
microns to facilitate lipid transport after infusion of a modest or
a high dose of TG; these findings are in agreement with our
previous observations in rats (32). In contrast, the KO animals
produced and transported mostly VLDL-sized particles to
lymph during infusion of a modest dose of TG. However, when
we increased the dose of infused TG fed by 50%, we found that
both KO and WT animals secreted larger chylomicrons. Fur-
thermore, when we examined the size distribution of the
lipoprotein particles found in the lymph of both groups by
electron microscopy, no difference in the size distribution of
TG-rich lipoproteins in lymph was observed. This finding is in
agreement with the earlier observations of Morrison et al. (21).
The findings are also consistent with our own data obtained
from isolated enterocytes in vitro, harvested from WT and KO
mice, in which large chylomicron particles were observed in
the media of cells of both genotypes (37). From the morphol-
ogy data, we can conclude that when the KO and WT mice
were infused with a modest dose of lipid, there was an apparent
lack in the ability of KO animals to secret larger TG-rich
chylomicron particles. However, this apparent deficiency was
diminished when a larger dose of the TG emulsion was
infused. Therefore, it can be concluded that the reduced lym-
phatic transport of absorbed lipids seen in KO animals is not
due to a lack in the ability to make chylomicrons, but rather
due to less of an affinity for chylomicron formation when
handling lipid loads, clearly highlighting a physiological dif-
ference between apo B100 and apo B48.
Further evaluation was undertaken to answer the question of
whether the impaired lipid transport seen in KO mice is a result
of a reduced number of secreted TG-rich lipoproteins follow-
ing administration of the high-dose lipid infusate. Previous
studies have demonstrated that one VLDL-sized particle con-
tains one apo B100 molecule and that one molecule of apo B48
is associated with one chylomicron particle (11, 25). Accord-
ingly, the mass of lymphatic apo B secretion was used as a
surrogate measure to evaluate the relative production of chy-
lomicron and VLDL particles by the gut (13). This approach
revealed that the gut that produces exclusively apo B100
secretes less total apo B mass (therefore a significant less
number of chylomicron particles) than the gut that produces
apo B48 in response to administration of the higher TG dose.
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 APOLIPOPROTEIN B AND CHYLOMICRON FORMATION
Previous reports showed that increased apo AIV expression in
IPEC-1 cells produced larger lipoprotein particles (19). We
speculated that increased apo AIV mass may be associated
with larger chylomicrons secreted by KO mice following
high-dose TG infusion. However, our present study showed
that apobec-1 KO mice produced a comparable amount of apo
AIV mass but reduced apo B mass in the lymph after high-dose
TG treatment. This observation is consistent with normal apo
AIV synthesis and secretion in enterocytes of KO mice (29).
The data suggest that apo AIV secretion is not influenced by
apo B isoform composition of intestinal lipoproteins.
Dietary fatty acids are absorbed and resynthesized in the
small intestine (30). Apo B and MTP are essential for the
assembly and secretion of apo B-containing lipoproteins (2, 7).
MTP transfers lipid to the apo B containing prechylomicrons in
the lumen of the ER (2, 7). MTP expression is highest in
duodenum and jejunum and its expression decreases in the
ileum (28). MTP lipid transfer activity in duodenum and
jejunum of KO mice did not differ from the WT mice. The
observation implies that reduced MTP lipid transfer activity is
not responsible for the reduced number of chylomicron parti-
cles produced by the gut of the KO mice after the high-dose TG
infusion. The mechanism resulting in reduced apo B secretion
of KO mice is not yet clear and will require further evaluation.
We propose, however, that reduced apo B mass secretion
(which in turn reduces the number of TG-rich lipoproteins
produced by KO enterocytes) is responsible for the diminished
efficacy of lipid transport in the KO mice.
In conclusion, apobec-1 KO mice can absorb and transport a
modest dose of dietary TG as well as WT mice, but the ability
of KO mice to transport lipid becomes limited upon adminis-
tration of higher doses of TG. The reduction in lymphatic
transport of absorbed TG is associated with an observed
enterocyte TG accumulation. The deficit in lymphatic transport
of absorbed lipid by the KO animals relative to the WT
controls is likely a result of the reduction of lymphatic apo B
secretion by the gut, in turn leading to a reduction in the
number of TG rich lipoprotein particles produced. The reduced
production of apo B is not associated with a change in the MTP
lipid transfer activity in ER. We are able to conclude from this
study that an important functional adaptation associated with
the exclusive production of apo B48 by the gut is the facilita-
tion of the formation and secretion of chylomicrons. The
unique ability of apo B48 to direct chylomicron formation and
secretion ensures that the naturally occurring gut is optimally
suited for the transport of large amounts of absorbed dietary
TG to the lymphatic system.
GRANTS
This work was supported in part by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-38180, DK-56910, DK-56863,
DK-76928, and DK-59630 and by a postdoctoral fellowship from the Amer-
ican Heart Association, Ohio Valley Affiliate. N. O. Davidson was supported
by National Institutes of Health Grants HL-38180, DK-56260, and DK-52574.
REFERENCES
1. Athar H, Iqbal J, Jiang XC, Hussain MM. A simple, rapid, and sensitive
fluorescence assay for mivrosomal triglyceride transfer protein. J Lipid
Res 45: 764 –772, 2004.
2. Atzel A, Wetterau JR. Identification of two classes of lipid molecule
binding sites on the microsomal triglyceride transfer protein. Biochemistry
33: 15382–15388, 1994.
AJP-Gastrointest Liver Physiol • VOL
Downloaded from journals.physiology.org/journal/ajpgi (001.038.044.185) on June 8, 2020.
G351
3. Arbeeny CM, Meyers DS, Bergquist KE, Gregg RE. Inhibition of fatty
acid synthesis decreases very low density lipoprotein secretion in hamster.
J Lipid Res 33: 843– 851, 1992.
4. Bollman JL, Cain JC, Grindlay JH. Techniques for the collection of
lymph from the liver, small intestine or thoracic duct of the rat. J Lab Clin
Med 33: 1349 –1352, 1948.
5. Chow SL, Hollander D. A dual, concentration dependent absorption
mechanism of linoleic acid by rat jejunum in vitro. J Lipid Res 20:
349 –356, 1979.
6. Davidson NO, Drewek MJ, Gordon JI, Elovson J. Rat intestinal
apolipoprotein B gene expression. J Clin Invest 82: 300 –308, 1988.
7. Davidson NO, Shelness GS. APOLIPOPROTEIN B: mRNA editing,
lipoprotein assembly, and presecretory degradation. Annu Rev Nutr 20:
169 –193, 2000.
8. Davis RA. Cell and molecular biology of the assembly and secretion of
apolipoprotein B-containing lipoproteins by the liver. Biochim Biophys
Acta 1440: 1–31, 1999.
9. Davis RA, Boogaerts JR. Interhepatic assembly of very low density
lipoproteins. Effects of fatty acids on triacylglycerol and lipoprotein
synthesis. J Biol Chem 257: 10908 –10913, 1982.
10. Dixon JL, Furukawa S, Ginsberg H. Oleate stimulates secretion of
apolipoprotein B-containing lipoproteins from HepG2 cells by inhibiting
early intracellular degradation of apolipoproteins B. J Biol Chem 268:
5080 –5086, 1991.
11. Elovson J, Chatterton JE, Bell GT, Schumaker VN, Reuben MA,
Puppione DL, Reeve JR Jr, Young NL. Plasma very low density
lipoproteins contain a single molecular of apo B lipoprotein. J Lipid Res
29: 1461–1473, 1988.
12. Farese RV Jr, Veniant MM, Cham CM, Flynn LM, Pierotti V, Loring
JF, Traber M, Ruland S, Stokowsi RS, Huszar D, Young SG. Pheno-
typic analysis of mice expressing exclusively apolipoprotein B48 or
apolipoprotein B100. Proc Natl Acad Sci USA 93: 6393– 6398, 1996.
13. Hayashi H, Fujimoto K, Cardelli JA, Nutting DF, Bergstedt S, Tso P.
Fat feeding increases size, but not number, of chylomicrons produced by
small intestine. Am J Physiol Gastrointest Liver Physiol 259: G709 –G719,
1990.
14. Hirano K, Young SG, Farese RV, Ng J, Sande E, Warburton C,
Powell-Braxton LM, Davidson NO. Targeted disruption of the mouse
apobec-1 gene abolishes apolipoprotein B mRNA editing and eliminates
apolipoprotein B48. J Biol Chem 271: 9887–9890, 1996.
15. Hoffman CJ, Miller RH, Hultin MB. Correlation of factor VII activity
and antigen with cholesterol and triglycerides in healthy young adults.
Arterioscler Thromb 12: 271–277, 1992.
16. Kendrick JS, Chan L, Higgins JA. Superior role of apolipoprotein B48
over apolipoprotein B100 in chylomicron assembly and fat absorption: an
investigation of apobec-1 knock-out and wild-type mice. Biochem J 356:
821– 827, 2001.
17. Levy E, Mehran M, Seidman E. Caco-2 cells as a model for intestinal
lipoprotein synthesis and secretion. FASEB J 9: 626 – 635, 1995.
18. Lewis GF, Uffelman K, Naples M, Szeto L, Haidari M, Adeli K.
Intestinal lipoprotein overproduction, a newly recognized component of
insulin resistance, is ameliorated by the insulin sensitizer rosiglitazone:
studies in the fructose-fed Syrian golden hamster. Endocrinology 146:
247–255, 2005.
19. Lu S, Yao Y, Cheng X, Mitchell S, Leng S, Meng S, Gallagher JW,
Shelness GS, Morris G, Mahan J, Frase S, Mansbach CM, Weinberg
RB, Black DD. Overexpression of apolipoprotein AIV enhanced lipid
secretion in IPEC-1 cells by increasing chylomicron size. J Biol Chem
281: 3473–3483, 2006.
20. Mattson FH, Volpenhein RA. The digestion and absorption of triglyc-
erides. J Biol Chem 239: 2772–2777, 1964.
21. Morrison JR, Pászity C, Stevens ME, Hughes SD, Forte T, Scott J,
Rubin EM. Apolipoproteins B RNA editing enzyme-deficient mice are
viable despite alterations in lipoprotein metabolism. Proc Natl Acad Sci
USA 93: 7154 –7159, 1996.
22. Nakamuta M, Chang BH, Zsigmond E, Kobayashi K, Lei H, Ishida
BY, Oka K, Li E, Chan L. Complete phenotypic characterization of
apobec-1 knockout mice with a wild-type genetic background and a
human apolipoprotein B transgenic background and restoration of apoli-
poprotein B mRNA editing by somatic gene transfer of apobec-1. J Biol
Chem 271: 25981–25988, 1996.
23. Ockner RK, Hughes FB, Isselbacher KJ K. Very low density lipopro-
teins in intestinal lymph: role in triglyceride and cholesterol transport
during fat absorption. J Clin Invest 48: 2367–2373, 1969.
294 • JANUARY 2008 • www.ajpgi.org
G352 APOLIPOPROTEIN B AND CHYLOMICRON FORMATION
24. Patsch W, Tamai T, Schonfeld G. Effect of fatty acids on lipid and
apoprotein secretion and association in hepatocyte cultures. J Clin Invest
72: 371–378, 1983.15.
25. Phillips ML, Pullinger C, Kroes I, Kroes J, Hardman DA, Chen G,
Curtiss LK, Gutierrez MM, Kane JP, Schumaker VN. A single copy of
apolipoprotein B-48 is present on the human chylomicron remnant. J Lipid
Res 38: 1170 –1177, 1997.
26. Small DM. The effects of glyceride structure on absorption and metabo-
lism. Annu Rev Nutr 11: 413– 434, 1991.
27. Stremmel W. Uptake of fatty acids by jejunal mucosal cells is mediated
by fatty acid binding membrane protein. J Clin Invest 82: 2001–2010,
1988.
28. Swift LL, Jovanovska A, Kakkad B, Ong DE. Microsomal triglyceride
transfer protein expression in mouse intestine. Histochem Cell Biol 123:
475– 482, 2005.
29. Teng B, Burant CF, Davidson NO. Molecular cloning of an apolipopro-
tein B messenger RNA editing protein. Science 260: 1816 –1819, 1993.
30. Tso P, Balint JA. Formation and transport of chylomicrons by enterocytes
to the lymphatics. Am J Physiol Gastrointest Liver Physiol 250: G715–
G726, 1986.
31. Tso P, Drake DS, Black DD, Sabesin SM. Evidence for separate
pathways of chylomicron and very low-density lipoprotein assembly and
AJP-Gastrointest Liver Physiol • VOL
Downloaded from journals.physiology.org/journal/ajpgi (001.038.044.185) on June 8, 2020.
transport by rat small intestine. Am J Physiol Gastrointest Liver Physiol
247: G599 –G610, 1984.
32. Tso P, Lindström MB, Borgström B. Factors regulating the formation of
chylomicrons and very-low-density lipoproteins by the rat small intestine.
Biochim Biophys Acta 922: 304 –313, 1987.11.
33. Tso P, Pitts V, Granger DN. Role of lymph flow in intestinal chylomi-
cron transport. Am J Physiol Gastrointest Liver Physiol 249: G21–G28,
1985.
34. Tso P, Ragland JB, Sabesin SM. Isolation and characterization of
lipoprotein of density⬍1.006 g/ml from rat hepatic lymph. J Lipid Res 24:
810 – 820, 1983.
35. Thomson ABR, Grag ML, Clandinin MT. Intestinal aspects of lipid
absorption. Can J Physiol Pharmacol 67: 179 –191, 1989.
36. Van Greevenbroek MMJ, de Bruin TWA. Chylomicron synthesis by
intestinal cells in vitro and in vivo. Atherosclerosis 141: S9 –S16, 1998.
37. Xie Y, Nassir F, Luo J, Buhman K, Davidson NO. Intestinal lipoprotein
assembly in apobec-1⫺/⫺ mice reveals subtle alterations in triglyceride
secretion coupled with a shift to larger lipoprotein. Am J Physiol Gastroin-
test Liver Physiol 285: G735–G746, 2003.
38. Yao Z, McLeod RS. Synthesis and secretion of hepatic apolipoproteins
B-containing lipoproteins. Biochim Biophys Acta 1212: 152–166, 1994.
39. Yao Z, Tran K, McLeod RS. Intracellular degradation of newly synthe-
sized apolipoprotein B. J Lipid Res 38: 1937–1953, 1997.
294 • JANUARY 2008 • www.ajpgi.org