(Received for publication, October 13, 1995; and in revised form, January 12, 1996)
From the
The involvement of pancreatic cholesterol esterase (bile
salt-stimulated lipase) in cholesterol absorption through the intestine
has been controversial. We have addressed this issue by using
homologous recombination in embryonic stem cells to produce mice
lacking a functional cholesterol esterase gene. Cholesterol esterase
knockout mice and their wild type counterparts were fed a bolus dose of
[H]cholesterol and a trace amount of
[
-
C]sitosterol by gavage. The ratio of the
two radiolabels excreted in the feces over a 24-h period was found to
be similar in the control and cholesterol esterase-null mice. Similar
results were observed when the radiolabeled sterols were supplied in an
emulsion with phospholipid and triolein or in lipid vesicles with
phosphatidylcholine. Cholesterol absorption results were similar
between the control and cholesterol esterase-null mice regardless of
whether the animals were fed a low fat diet or a high fat/high
cholesterol diet. The rate of [
H]cholesterol
appearance in the serum of the gene-targeted mice paralleled that
observed in control animals. In contrast to these results, when
experiments were performed with [
H]cholesteryl
oleate instead of [
H]cholesterol, a higher amount
of the
H radiolabel was found excreted in feces and
dramatically less of the radiolabel was detected in the serum of the
cholesterol esterase-null mice in comparison with that detected in
control animals. Serum cholesterol levels were not significantly
different between control and cholesterol esterase-null mice fed either
control or an atherogenic diet. These results indicate that cholesterol
esterase is responsible for mediating intestinal absorption of
cholesteryl esters but does not play a primary role in free cholesterol
absorption.
Cholesterol esterase, also called bile salt-stimulated lipase or
carboxyl ester lipase (abbreviated as CEL), ()is a lipolytic
enzyme capable of hydrolyzing triacylglycerol, phospholipid,
lysophospholipid, and cholesteryl esters. The enzyme is synthesized in
the acinar cells of the pancreas and is stored in zymogen granules.
Cholesterol esterase is released into the intestinal lumen upon food
ingestion and constitutes 1-2% of total protein in pancreatic
juice(1) .
While the high concentration of CEL in pancreatic juice suggests that it may play a role in mediating nutrient absorption, the precise physiologic function of the enzyme remains controversial. Early studies with isolated intestinal cells suggested a role for CEL in dietary cholesterol absorption(2) . However, subsequent studies yielded contradictory results. For example, using pancreatic diverted rats, Watt and Simmonds (3) showed normal absorption and esterification of cholesterol. In contrast, using the same experimental system, Gallo et al.(4) showed an 80% reduction in cholesterol absorption, which could be restored by infusion of pancreatic juice containing CEL but not by juice depleted of the enzyme.
Cholesterol absorption has also been studied using a variety of inhibitors. Bennett Clark and Tercyak (5) demonstrated a reduction in cholesterol transmucosal transport in rats with inhibited acyl CoA:cholesterol acyltransferase and normal pancreatic function, which suggested that acyl CoA:cholesterol acyltransferase, and not CEL, was responsible for this process. However, using similar inhibitors, Gallo et al. (6) showed no inhibition of cholesterol absorption, which again suggested the involvement of CEL. In later studies, CEL inhibitors, such as the phenoxyphenyl carbamates WAY-121,751 and WAY-121,898, were shown to be effective inhibitors of cholesterol absorption in normal and cholesterol-fed rats and dogs(7) . Thus, whole animal studies have not consistently shown the importance of CEL in cholesterol absorption.
The possible role of CEL in mediating intestinal absorption of cholesterol has also been investigated in vitro without resolution. Bhat and Brockman (8) showed that incubation of rat intestinal sacs with cholesterol-containing micelles in the presence of CEL resulted in a 3-5-fold enhancement of intracellular cholesterol and cholesteryl ester accumulation compared with intestinal sacs incubated in the absence of the enzyme. More recently, Lange and colleagues, using Caco-2 cells as a model for intestinal epithelium, showed that CEL addition was necessary for the transfer of exogenous cholesterol to a ``physiologically important pool'' that could be esterified and assembled into lipoproteins(9) . In contrast to these results, our laboratory could not demonstrate CEL-mediated uptake of unesterified cholesterol by Caco-2 cells(10) . Our in vitro data were confirmed and extended in a recent publication by Fisher and colleagues(11) . Both laboratories reported that the enzyme was only effective in facilitating cellular uptake of esterified cholesterol.
In an attempt to resolve this controversy, we have used the approach of gene targeting in embryonic stem (ES) cells to produce mice lacking in CEL. The CEL(-/-) mice provide a unique in vivo model to assess the physiological function of the bile salt-stimulated cholesterol esterase.
A 4.7-kb SacI DNA fragment, encoding
sequences from 540 bp upstream of exon 1 to intron 7 of the mouse
cholesterol esterase gene, was subcloned into a similarly-digested
PTZ18U plasmid. A 1.75-kb fragment containing a thymidine kinase
promoter-driven neomycin resistance gene (neo) was
isolated from SspI/HincII-digested pMC1Neo
(Stratagene) and subcloned into the unique BalI site in exon 4
of the 4.7-kb SacI clone (Fig. 1). A plasmid containing neo
inserted in the same orientation as the CEL
gene was selected for the gene-targeting experiment. After CsCl
purification, the targeting vector was digested with SacI, and
the 6.5-kb DNA fragment containing the disrupted CEL gene sequence was
purified by agarose gel electrophoresis.
Figure 1:
Diagram of the mouse cholesterol
esterase gene and targeting construct. Panel A, partial
restriction map and exon/intron arrangement of the mouse CEL gene.
Exons are indicated by boxes. Panel B, construct used
for targeting. neo (shaded box) was
inserted into the BalI site in exon 4 of the 4.7-kb SacI fragment. Panel C, PstI/SacI
5` probe used for screening ES colonies and mice for homologous
recombination. Panel D, BglII/SalI 3` probe
used for screening ES colonies and mice for homologous recombination.
Restriction enzymes indicated are as follows: BalI (B), EcoRI (E), HindIII (H), NcoI (N), SphI (P), SacI (S), and XbaI (X). Arrows indicate the approximate positions of the primers used for
polymerase chain reaction analysis of mice.
The presence of neo was determined using the primers described by
Kim and Smithies(22) , which amplify a 555-bp portion of this
gene. The CEL gene was analyzed with these primers and an additional
set of primers that amplifies exon 4. The upstream CEL primer,
5`-CCCTTTCAGTGTCCCACAACCT-3`, and the downstream CEL primer,
5`-TCACTATTCCCGCTCTTACAGTC-3`, amplify a 244-bp fragment from the wild
type exon 4 but do not amplify the targeted allele because of the
insertion of neo
between their cognate sequences.
Identical conditions were used for both primer sets but in separate
reactions. A positive result with the exon 4 primers only was scored as
wild type. Positive results with both sets indicated a heterozygote,
and a positive result with the neo
primers only
was scored as a homozygous knockout.
For absorption studies, mice were
housed in metabolic cages where they had free access to food and water.
Animals were allowed to adjust to the cages for at least 24 h before
beginning the test. On the day of the experiment, mice were
administered 50 µl of the test meal by gavage approximately
3-4 h before the beginning of their dark cycle. Feces were
collected for the following 24 h. The samples were homogenized in water
and then extracted with an equal volume of chloroform/methanol (2:1,
v/v). The aqueous phase was re-extracted once with chloroform. The
organic phases from each sample were combined, their volumes were
measured, and an aliquot was used for scintillation counting. Counting
efficiency was calculated using the external standard, channel ratio
method. Cholesterol absorption efficiency, determined as percentage of
administered dose absorbed, was calculated based on the formula
described by Grundy et al.(29) as follows: {1 -
((H-dpm/
C-dpm) in
feces/(
H-dpm/
C-dpm) administered)}
100. Total recovery of the
[
-
C]sitosterol over the 24-h period ranged
from 66 to 97%. Differences in cholesterol absorption between groups
were evaluated for statistical significance by Mann-Whitney rank sum
and Student's t tests using SigmaStat software from
Jandel Corporation.
The
electroporation of 5.5 10
mouse ES cells with the
targeting DNA resulted in approximately 4,800 G418-resistant colonies.
One-fourth of the colonies were picked and expanded individually in
24-well dishes. A total of 268 colonies were selected for Southern blot
analysis to screen for homologous recombination between the targeting
DNA and the resident CEL gene. For the initial screening, ES colony DNA
was digested with EcoRI and hybridized with an
1100-bp PstI/SacI DNA fragment corresponding to genomic
sequence 5` from the targeting DNA (Fig. 1C). As shown
in Fig. 2A, the wild-type allele gives rise to a fragment
>30 kb in length, while a correctly targeted gene yields a fragment
6.5 kb in length due to the insertion of two EcoRI sites present
in the thymidine kinase promoter of neo
(Fig. 1B). To confirm that the putative targeting
events had taken place as planned, additional aliquots of the ES colony
DNA were digested with XbaI and hybridized with an
1100-bp BglII/SalI DNA fragment corresponding to
sequences 3` from the targeting DNA (Fig. 1D). Fig. 2B shows that the wild type allele yields a 7.2-kb
fragment, while the correctly targeted allele yields a 9.0-kb fragment
due to the insertion of 1.75 kb of DNA corresponding to the selectable
marker cassette. Of the 268 colonies screened, 11 were positive in both
tests. The overall targeting efficiency was 4.4%.
Figure 2:
Southern blot analysis of wild type and
CEL gene-targeted ES colonies. A, representative colonies
digested with EcoRI or HindIII and hybridized with
the 5` probe. The wild type allele gives rise to a >30-kb fragment
with EcoRI, while the targeted allele yields a fragment of 6.7
kb due to EcoRI sites present in neo.
When digested with HindIII, the wild type allele is 6.7 kb,
while the targeted allele is 8.5 kb due to the presence of neo
. B, representative colonies digested
with SphI and XbaI and hybridized with the 3` probe.
Wild type DNA yields an 18-kb fragment with SphI, while
targeted DNA yields a 7.5-kb fragment due to an SphI site
present in neo
. When digested with XbaI,
the wild type DNA yields a 7.3-kb fragment, and the targeted DNA yields
a 9.1-kb fragment due to the insertion of neo
. W, wild type; T,
targeted.
Site-specific
integration of the targeting DNA at the CEL locus was confirmed by
additional Southern blot analysis with both the 5`- and 3`-flanking
probes. The addition of the 1.75-kb neo cassette
to the endogenous CEL gene resulted in an 8.3-kb HindIII
fragment that hybridized with the 5` probe in addition to the 6.5-kb
band observed for the controls (Fig. 2A). Using the 3`
probe, a 7.5-kb SphI band resulted from the insertion of an SphI site in neo
in targeted clones in
addition to the 18-kb SphI band observed for the wild type
allele (Fig. 2B). These hybridization patterns were
consistent with those predicted for the site-specific insertion of the neo
cassette into exon 4 of the endogenous CEL
gene (Fig. 1). A total of eight enzymes, informative with either
the 5` or 3` probe, were used to confirm that the gene targeting had
occurred as planned. In addition, a neo
-specific
probe was used to confirm that the targeting DNA had inserted in only
one site in the genome (data not shown).
Two of the 11 cell lines
with proper CEL gene targeting were used to generate chimeric mice. One
cell line yielded only one chimeric mouse, which was female and had
only 5% agouti coat color. However, the second cell line produced
22 chimeric mice (from 119 injected and reimplanted blastocysts), all
with extensive agouti coat color. Nineteen of these were male, and 15
of the 19 were able to transmit the modified gene to their offspring.
Progeny from these test matings (chimerics
Black Swiss), which
carried the modified allele, were bred to generate homozygous knockout
animals. Southern blot analysis of the genomic DNA from representative
wild type, heterozygous, and homozygous CEL-targeted mice is shown in Fig. 3. Because of an apparent restriction fragment length
polymorphism between the ES cells and the outbred mice used in the
initial breeding, XbaI was not informative, and NcoI
was used as a diagnostic enzyme for the 3` end of the recombination.
This enzyme yields a 9.5-kb fragment from the wild type allele and a
6.5-kb fragment from the targeted allele due to the insertion of the NcoI site in the neo
gene (see Fig. 1for details).
Figure 3:
Southern blot analysis of wild type and
CEL gene-targeted mice. A, tail DNA digested with EcoRI. B, tail DNA digested with NcoI. Wild
type DNA yields a 9.2-kb fragment, while targeted DNA yields a 6.9-kb
fragment due to an NcoI site in the neo gene. Wild type, +/+; heterozygotes, +/-;
homozygous knockout, -/-. The 4-kb band seen in panel B is due to spurious hybridization and has not been seen in other
experiments.
To verify that the gene targeting abolished expression of CEL protein, pancreatic homogenates from control, heterozygous, and homozygous CEL gene-targeted mice were examined for CEL expression using immunoblotting techniques (Fig. 4). The levels of CEL protein in pancreatic extracts of the heterozygous animals were approximately half those of the wild type mice. No CEL protein was detected in homogenates of the CEL(-/-) mice. Furthermore, no CEL-immunoreactive polypeptides of any size were detected, indicating that no fusion protein or truncated protein was being produced as a result of the modified gene. These extracts were also assayed for cholesteryl ester hydrolytic activity. Table 1shows that cholesteryl oleate hydrolysis is reduced 98% in the pancreatic extracts of CEL(-/-) animals.
Figure 4:
Western blot of pancreatic extract from
wild type and CEL gene-targeted mice. Twenty µg of protein from the
100,000 g supernatant fraction of wild type and
CEL-targeted mouse pancreas was run in duplicate on a 10%
SDS-polyacrylamide gel electrophoresis gel and either stained with
Coomassie Blue (A) or transferred to nitrocellulose and
reacted first with rabbit anti-rat CEL and then with
I-labeled goat anti-rabbit IgG (B).
In contrast to the
cholesteryl ester result, when unesterified
[H]cholesterol was included in the emulsion
instead of the cholesteryl oleate, no significant difference was found
between wild type and CEL-null mice in absorption of the radiolabeled
sterol (Table 3). Interestingly, males were found to absorb
significantly less (59.6 ± 3.05%) cholesterol than females (72.0
± 1.61%), but this difference was independent of their
CEL genotype. For clarity, results from male and female mice are
combined in Table 3.
The presence of cholesteryl ester in the
core of an emulsion particle has been shown to increase the partition
of free cholesterol from the surface to the core(32) . Our
results show that CEL is necessary for digestion of this core
cholesteryl ester. In the absence of CEL, the free cholesterol may
remain sequestered and unabsorbed. To test this possibility, animals
were fed [H]cholesterol in an emulsion that
contained unlabeled cholesteryl ester along with phospholipid and
triglyceride. Table 3also shows that the presence of the
cholesteryl ester in the core had no effect on the ability of
CEL(-/-) mice to absorb the free cholesterol.
Published
literature indicates that dietary cholesterol and biliary cholesterol
may be absorbed from the intestine by different mechanisms (33) . Experiments were undertaken to determine the ability of
wild type and CEL gene-targeted mice to absorb unesterified cholesterol
presented in a vesicular complex with phospholipids, similar to that
present in the biliary tract.
[-
C]Sitosterol was used as a marker of
recovery as described above. The results showed that, regardless of the
ratio of cholesterol to phospholipid used to prepare the lipid
vesicles, there was no significant difference between the ratios of
[
H]cholesterol to
[
-
C]sitosterol recovered in the feces of
wild type versus CEL gene-targeted mice (Table 3).
The report that phenoxyphenyl carbamate inhibitors of CEL result in
delayed absorption of cholesterol (7) prompted additional
experiments to compare the rate at which the radiolabel from
cholesterol and cholesteryl esters appears in the serum of control and
CEL gene-targeted mice. In these experiments, the amount of H in 15 µl of serum was determined at various times
after gastric infusion of emulsified radiolabeled sterol. The infusion
of the unesterified [
H]cholesterol resulted in
the progressive appearance of the radiolabel in the serum of both wild
type and CEL gene-targeted mice with a maximum at
10 h (Fig. 5) and a slow decline thereafter. No significant
difference in the rate of radiolabeled cholesterol appearance in the
serum was observed between the two groups of animals in this case. In
contrast, when the radiolabel was supplied as emulsified
[
H]cholesteryl oleate, the serum level of the
radiolabel after 12 h was
8-fold higher in the CEL(+/+)
mice than in the CEL-null mice. In fact, very little radiolabel was
detected in the serum of the gene-targeted mice (Fig. 5).
Figure 5:
Appearance of dietary cholesterol in the
circulation of wild type and CEL null mice.
[H]cholesterol (FC) or
[
H]cholesteryl oleate (CE) was administered in an
emulsion with PC, TG, and a trace amount of
[
-
C]sitosterol. Thirty µl of whole
blood (FC, first experiment, solid lines) or 15 µl of
plasma (all other points) was counted at various times after
the label was given. For FC, each point represents the average of two
animals. For the cholesteryl oleate experiment, each point represents
the average of six animals.
and
, wild type and FC;
and
, CEL(-/-) and FC;
, wild type and CE;
, CEL(-/-) and CE. Open and closed
symbols represent different experiments with
FC.
To examine the possibility that CEL plays a role in cholesterol absorption when mice are fed a high fat, high cholesterol, atherogenic diet, we studied the absorption of free and esterified cholesterol in wild type and CEL-null mice fed this diet for 6 weeks. As shown in Table 4, cholesteryl oleate absorption was reduced in the CEL-null mice, while free cholesterol was absorbed similarly by the wild type and CEL gene-targeted mice. The percentage of cholesterol absorbed was decreased relative to normal diet in both the free cholesterol and cholesteryl ester experiments due to the high level of cholesterol in the atherogenic diet. In fact the total mass of absorbed cholesterol is increased.
The results of the current study show that disruption of the CEL gene has no significant effect on the ability of mice to absorb unesterified cholesterol from the gastrointestinal tract. Similar results were observed regardless of the physical characteristics of the substrate or the dietary conditions of the animals. Furthermore, similar results were observed when cholesterol absorption was determined based on the amount of nonabsorbed cholesterol present in the feces or on the appearance of the radiolabeled cholesterol in the serum. These observations demonstrate that CEL is not necessary for cholesterol flux across the intestinal epithelium. In contrast to its effect on unesterified cholesterol absorption this study shows that CEL is necessary for intestinal absorption of esterified cholesterol. However, since cholesteryl esters constitute only a small fraction of total cholesterol in the diet and are absent from the bile (34) , these results strongly suggest that CEL does not play a determining role in the absorption of either dietary or biliary cholesterol.
Although CEL does not appear to be essential for intestinal absorption of unesterified cholesterol, results of this study demonstrate unequivocally that CEL plays a primary role in absorption of cholesteryl esters. These results are consistent with in vitro studies from this laboratory and others that showed a role of CEL in facilitating the uptake of esterified cholesterol but not unesterified cholesterol by intestinal cells(10, 11) . Curiously, our fecal sterol experiments indicate a base-line level of cholesteryl ester absorption that is independent of CEL (Table 2). However, pancreatic extracts from CEL(-/-) mice lack significant esterolytic activity (Table 1). Also, the amount of radiolabel appearing in the serum of cholesteryl ester-fed, CEL-null mice was not consistent with this base line (Fig. 5). One explanation for this discrepancy is that a minor pathway for absorption of these nutrients exists and that our serum assay was insufficiently sensitive. Alternatively, some cholesteryl ester may remain associated with cell membranes or lipid vesicles of the intestinal epithelium due to hydrophobic interactions.
The current study, showing normal absorption of unesterified cholesterol in CEL-null mice, resolves the discrepancy in previously published data. Our results are consistent with those of Watt and Simmonds(3) , which showed that unesterified cholesterol absorption was independent of pancreatic proteins. Gallo et al. (4) showed that CEL-depleted pancreatic juice could not restore cholesterol absorption in pancreatic-diverted rats; however, intestinal lymph flow in the CEL-depleted group was severely compromised in those experiments (11) . Our current results are also in agreement with in vitro data that showed no CEL requirement for unesterified cholesterol uptake by Caco-2 cells(10, 11) . The CEL-stimulated uptake in Caco-2 cells reported by Lopez-Candales et al.(9) , while intriguing, did not reflect physiologically relevant levels of cholesterol in the gastrointestinal tract.
Until the availability of an animal model lacking in CEL, such as the gene-targeted mice described here, the most physiologically relevant experiments regarding the role of this enzyme in cholesterol absorption were performed by feeding animals CEL inhibitors. Two classes of such inhibitors, phenoxyphenyl carbamates (7) and the lipstatins(35) , were reported to reduce cholesterol absorption in normal and cholesterol-fed animals. However, the lipstatins were shown to also inhibit pancreatic lipase(35) . Thus, their inhibitory effect on cholesterol absorption may be related to the inhibition of lipid emulsion hydrolysis and the release of unesterified cholesterol for diffusion through the mucosa (36) . In support of this possibility was the observation that tetrahydrolipstatin had no effect on intestinal uptake of unesterified cholesterol from phospholipid-bile salt mixed micelles(35) . Although the carbamate inhibitors were reported to be more specific for CEL and did not inhibit the activity of pancreatic lipase or acyl CoA:cholesterol acyltransferase in vitro, other possible side effects that can modulate cholesterol absorption in vivo may exist in the carbamate-treated animals.
Although our data show that CEL does not
participate in free cholesterol absorption, the wide range of
absorption values (37-87%) as well as additional studies with
inbred strains of mice support the hypothesis that cholesterol
absorption is regulated by at least one gene. ()The recent
report by Kirk et al. (37) on the responsiveness of
different strains of mice to dietary fat and cholesterol with respect
to cholesterol absorption and serum lipid parameters also supports this
hypothesis. Candidate genes potentially involved in this process
include the cholesterol transfer protein(38) , which may be the
same as, or closely related to, sterol carrier
protein-2(39, 40) , acyl CoA:cholesterol
acyltransferase(5, 41) , pancreatic
lipase(36) , and liver fatty acid binding protein(42) .
Additional experiments are necessary to investigate the physiological
role of these and other proteins in cholesterol absorption.
Although CEL does not play a primary role in free cholesterol absorption, its abundance in the intestinal lumen suggests that it may play a different role in the absorption of lipid-based nutrients. This enzyme may complement other lipolytic enzymes to increase the efficiency of dietary fat absorption by the small intestine. For example, CEL has been shown to be more efficient than pancreatic lipase in the hydrolysis of long chain polyenoic fatty acids(43, 44) . CEL is also capable of hydrolyzing phospholipids and lysophospholipids (45) and thus may play a role in the assimilation of dietary phospholipids. More importantly, a pancreas-derived, vitamin-ester hydrolytic activity has been ascribed to CEL(46, 47) , suggesting its importance in the absorption of fat-soluble vitamins, which are primarily esterified in dietary sources. The presence of CEL in the milk of many mammalian species has led to the proposal that the milk CEL is critically important for digestion of milk triglycerides, the major source of energy in infants, before the maturation of the pancreas(48) . Finally, in addition to its presence in the gastrointestinal tract, CEL is also found to be synthesized by the liver and is present in serum(12, 26, 49, 50, 51) , where its level is correlated to that of serum cholesterol and LDL(52) . Thus, CEL may play a role in the modulation of lipoprotein structure and metabolism. The knockout mice described in this report will provide a useful tool to address the role of CEL in various aspects of lipid absorption and metabolism.