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INTRODUCTION |
The cholesterol content of all cells is tightly controlled. When
cholesterol is in excess, cells decrease both lipoprotein cholesterol
uptake and cholesterol synthesis to prevent accumulation of this
potentially toxic sterol. In contrast, when cells have insufficient
cholesterol to meet their sterol requirements, lipoprotein cholesterol
uptake is increased as are rates of cholesterol biosynthesis. This end
product feedback mechanism of regulating the amount of cellular
cholesterol has been extensively studied and characterized (for a
review, see Ref. 1). Understanding of how a cell senses and regulates
the amount of cholesterol has been advanced by the characterization of
two transcription factors called sterol regulatory element-binding
proteins (SREBPs),1 SREBP-1
and -2 (for a review, see Ref. 2). SREBPs are bound to membranes of the
endoplasmic reticulum and nuclear envelope. Under conditions of
cholesterol deficiency, a two-step proteolytic process releases from
the membrane "precursor" protein a "mature" form of the protein
(3). This active form of the protein enters the nucleus and binds to a
10-base pair sterol regulatory element that enhances the transcription
of target genes that encode enzymes regulating cholesterol and fatty
acid synthesis (4-8). SREBP-1 and -2 are derived from two separate
genes. Two isoforms of SREBP-1 exist, 1a and 1c (9). SREBP-1a appears
to be the more potent transcription factor and regulates genes of both
fatty acid and cholesterol pathways (10). SREBP-1c, in contrast, tends
to be more active in regulating genes of the fatty acid biosynthetic pathway (10). SREBP-2 preferentially enhances several genes of the
cholesterol biosynthetic pathway (11-13).
We have recently characterized gene expression for SREBP-1 and -2 in
hamster small intestine and have implicated SREBP-2 as responsible for
regulating cholesterol synthesis and SREBP-1c as responsible for
regulating fatty acid synthesis in intestine (14). We have also
demonstrated that changes in cholesterol flux across the intestinal
cell regulate gene expression of SREBP-1c and several sterol-responsive
genes, including SREBP-2. In the present study, we have extended our
investigations into the regulation of intestinal SREBPs by studying the
regulation of SREBP gene and protein expression in hamster small
intestine by changes in cholesterol flux. The results demonstrate that
a diet enriched in cholesterol increases gene expression of SREBP-1c,
whereas a cholesterol-depletion diet markedly decreases its expression. In contrast, a cholesterol-depletion diet results in a substantial increase in the expression of SREBP-2 and other sterol-responsive genes, HMG-CoA reductase, HMG-CoA synthase, and the LDL receptor, while
dietary cholesterol had little, if any effect on the expression of
these genes. In the intestines of hamsters on control or cholesterol diets, the mature form of SREBP-2 is in very low abundance. In intestines of animals on the cholesterol-depletion diet, the precursor and mature forms of SREBP-2 are increased.
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MATERIALS AND METHODS |
Tritiated water and [32P]UTP were purchased from
PerkinElmer Life Sciences. Protease inhibitors, TRI REAGENT, INFINITY
cholesterol reagent, and cholesterol were purchased from Sigma.
MAXIscript T7 and RPA III kits were supplied by Ambion (Austin, TX).
Medium M199 was from Life Technologies, Inc. Lovastatin was obtained from Merck. Cholestyramine was obtained from Bristol-Myers Squibb Co.
Animals--
Male Golden Syrian hamsters weighing 90-120 g were
purchased from Harlan Sprague-Dawley, Inc. They were maintained for a
week on NIH-31 modified mouse/rat diet number 7013 (Harlan/Teklad, Madison, WI) before starting the test diets. The animals were fed the
test diets ad libitum, and the light cycle was
0600-1800 h. The control chow diet was prepared by adding 1% olive
oil to Teklad 4% mouse/rat meal, number 7001. The cholesterol-enriched diet was prepared by dissolving 1% cholesterol in the olive oil and
thoroughly mixing with the control chow diet. To prepare the cholesterol-depletion diet, the control chow diet was supplemented with
4% (w/w) cholestyramine and 0.15% (w/w) lovastatin (dissolved in
olive oil). The start of the dietary period was staggered in the
hamsters so that all animals were fed the respective diets for exactly
14 days.
Isolation of Enterocytes--
Hamsters were killed between 0800 and 0900 h by inhalation of CO2. The small intestine
was removed in its entirety and placed in a beaker containing cold
0.9% saline. Isolated intestinal cells were prepared by a procedure
described by Cartwright and Higgins that was modified for hamster
intestine (14, 15). The lumen of the intestine was flushed with
solution A (117 mM NaCl, 5.4 mM KCl, 0.96 mM NaH2PO4, 26 mM
NaHCO3, 5 mM HEPES, 5.5 mM glucose) to remove fecal material and debris. The intestine was then filled with
solution B (67.5 mM NaCl, 1.5 mM KCl, 0.96 mM NaH2PO4, 26 mM
NaHCO3, 5 mM HEPES, 5.5 mM glucose,
27 mM sodium citrate) and placed in a beaker containing
0.9% saline at 37 °C. The beaker was gently agitated for 15 min.
Solution B was removed, and the intestine was divided into three equal
segments representing duodenum, jejunum, and ileum. Each segment was
filled with solution C (115 mM NaCl, 5.4 mM
KCl, 0.96 mM NaH2PO4, 26 mM NaHCO3, 1.5 mM EDTA, 0.5 mM dithiothreitol, 5 mM HEPES, 5.5 mM glucose) and placed in a beaker containing 0.9% saline
at 37 °C. The beakers were gently agitated in a shaking water bath
to isolate cells along the villus. Solution C was emptied into plastic
conical centrifuge tubes every 5 min and replaced. The tubes containing
the cells were kept on ice until 12 fractions were collected. Two
sequential fractions were then pooled from the first eight fractions,
and the last four fractions were combined to obtain a total of five fractions (16). Cells were collected by centrifugation at 1000 × g for 10 min at 4 °C.
Cholesterol and Fatty Acid Synthesis--
Cholesterol and fatty
acid synthesis were estimated by incorporation of tritiated water into
these lipids 4 h after intraperitoneal injection of 100 mCi of
tritiated water (1 Ci/g; PerkinElmer Life Sciences) (17). Intestinal
cell fractions were collected from duodenum, jejunum, and ileum as
described above. The cells were then suspended in 2 ml of 90% methanol
containing 0.5 M NaOH and kept in boiling water for 1 h to saponify the lipids. After adding an equal volume of acidic water,
the lipids were extracted twice at pH 3.0 with 4 ml of hexanes. To
remove residual labeled water, the hexane extract was washed once with
1 ml of acidic water. The hexanes were evaporated under a stream of
nitrogen, and the lipids were dissolved in 0.1 ml of chloroform. Fatty
acids and cholesterol were separated by thin layer chromatography on
silica gel plates using hexanes/diethyl ether/acetic acid/methanol
(70:30:1:1, v/v/v/v) as solvent. The lipids were visualized by exposure
to iodine, and the radioactivity in fatty acids and cholesterol
fractions was determined by scraping the bands and counting in a
Packard Tricarb 2100-TR liquid scintillation counter.
Estimation of Cholesterol Mass--
Cellular lipids were
extracted with hexane/isopropyl alcohol/water (3:2:0.1, v/v/v).
Cholesteryl esters were separated from unesterified cholesterol by TLC
on silica gel plates using a solvent system of hexanes/diethyl
ether/acetic acid (80:20:1, v/v/v). The cholesteryl ester band was
eluted from the silica gel by extraction with acidic
water/chloroform/methanol (0.9:1:1, v/v/v). To each sample, 10 µg of
cholesterol-free egg phosphatidylcholine was added, and the chloroform
was evaporated under a stream of nitrogen. The sample containing
cholesteryl esters or total cholesterol was dissolved by vortexing in
10 µl of isopropyl alcohol followed by 90 µl of water. The color
was developed by adding 100 µl of a 2× solution of INFINITY
cholesterol reagent. The samples were read at 500 nm with known plasma
standards. Protein was estimated with BCA protein reagent purchased
from Pierce.
RNase Protection Assays--
RNA was extracted from
isolated cells with TRI REAGENT. The RNA probes to estimate mRNA
for hamster SREBP-1a, SREBP-1c, SREBP-2, HMG-CoA reductase, HMG-CoA
synthase, and LDL receptor were prepared as described by Shimomura
et al. (12, 14). The RNA probe for SREBP-1c, however,
contained 0.207 instead of 0.114 kilobases as described. pTRI RNA 18 S
probe was obtained from Ambion (Austin, TX). The probes were labeled
with [32P]UTP using MAXIscript T7 kit from Ambion. 20 µg of total RNA was used per assay. The assay was performed per a
protocol for RPA III described by Ambion. The protected fragments were
resolved on a 6% polyacrylamide gel. Radioactivity was measured by
autoradiography using a Storm phosphor screen and quantitated using
ImageQuant software (Molecular Dynamics, Sunnyvale, CA). To compare
mRNA levels among hamsters on the three diets, RNA from a hamster
on each of the three diets was analyzed by RNase protection assays simultaneously under identical conditions.
Immunoblot Analysis--
Immunoblot analysis was performed as we
have described with the following modifications (18). The cell
fractions from a hamster on one of the three diets were prepared
each day. All buffers contained 50 µg/ml
N-acetyl-leucyl-leucyl-norleucinal, 0.5 mM
phenylmethylsulfonyl fluoride, 5 µg/ml pepstatin A, 10 µg/ml
leupeptin, 1 mM pefabloc, 10 mM dithiothreitol,
2 µg/ml aprotinin, 0.1 mM zinc sulfate, and 0.1 mM copper sulfate. The cells were suspended in 20 mM Tris/HCl, pH 8, 150 mM NaCl, and 1 mM CaCl2 and kept on ice for 30 min with
occasional mixing. The cells were homogenized by passing five times
through a 25-gauge needle. Total membranes were prepared by
centrifuging the homogenate for 45 min at 100,000 × g
at 4 °C. The total membrane pellet was suspended in 0.1-0.4 ml of
125 mM Tris/HCl (pH 6), 2 mM CaCl2, 160 mM NaCl, 1% Triton X-100 and sonicated briefly. The
membrane extract was subsequently centrifuged for 30 min at 19,000 × g at 4 °C. The supernatant was used for
immunoblotting. Proteins were resolved by SDS-PAGE on the same day of
preparation and blotted onto a polyvinylidene difluoride membrane. The
blot was prepared, dried, stored, and probed using a nonblock technique
described by Sadra et al. (19). Briefly, the blot was
incubated with 1× TBS for 30 min followed by a rinse with water. It
was air-dried for 15 min followed by three rinses with 1:1
methanol/water, kept in methanol for 2 min, and air-dried. It was
stored at room temperature between two sheets of filter paper. All
blots were dried under vacuum for 10 min just before probing with the
antibodies. To compare the bands representing precursor and mature
forms of SREBPs among hamsters on the three diets, one blot from a
hamster on each of the three diets was probed simultaneously under
identical conditions. The blots were incubated with the primary
antibody for 1 h in Tris-buffered saline containing 1% goat or
donkey serum, 2% nonfat milk, 0.1% Triton X-100. They were rinsed
with Tris-buffered saline plus 0.1% Triton X-100 followed by secondary
horseradish peroxidase antibody for 1 h in Tris-buffered saline
containing 1% goat or donkey serum, 2% nonfat milk, 0.1% Triton
X-100. Rabbit polyclonal antibody against hamster SREBP-2 (kindly
provided by Dr. Jay Horton, Department of Molecular Genetics,
Univ. of Texas Southwestern Medical Center, Dallas, TX.) and mouse
monoclonal antibody against the amino-terminal end of human SREBP-1a,
IgG-2A4, were used. Goat anti-rabbit horseradish peroxidase (A-6154;
Sigma) or donkey anti-mouse horseradish peroxidase (code 715-035-150; Jackson Immunoresearch Laboratory Inc., West Grove, PA) were used as
secondary antibodies. In preliminary experiments, nonspecific bands in
the 80-150-kDa range were observed in the absence of primary
monoclonal antibody when anti-mouse horseradish peroxidase antibodies
were used that were not adsorbed for hamster proteins. The donkey
anti-mouse horseradish peroxidase antibody did not react with hamster
protein in the absence of the monoclonal antibody. The horseradish
peroxidase signal was detected on the blots with SuperSignal West Femto
maximum sensitivity substrate (Pierce).
Hepatic and intestinal mucosa nuclear and membrane fractions were
prepared exactly as described by Sheng et al. (11).
Statistical Analysis--
To determine if the differences among
the animals fed the three diets were significant, the data were
analyzed at p < 0.05 by one-way analysis of variance
and Student-Newman-Keuls method using SIGMASTAT software.
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RESULTS |
In hamsters ingesting a 1% cholesterol diet for 2 weeks, plasma
cholesterol levels were increased 2.6-fold compared with levels observed in animals ingesting the chow diet, 100 ± 8.1 versus 262 ± 18 mg/dl, n = 8. Plasma
cholesterol levels were markedly decreased in animals ingesting a
cholesterol-depleted diet of cholestyramine and lovastatin, 9 ± 2.5 mg/dl, n = 8.
Gene Expression--
Following 2 weeks on the respective diets,
the intestines were divided into three equal segments representing
duodenum, jejunum, and ileum. Isolated cells were prepared from the
three segments representing upper (fractions 1 and 2), middle
(fractions 3 and 4), and lower (fraction 5) villus cells (14). RNA was
extracted from the different cell populations, and mRNA levels were
estimated by RNase protection assays for SREBP-1a, SREBP-1c, SREBP-2,
HMG-CoA synthase, LDL receptor, and HMG-CoA reductase.
Fig. 1a shows a representative
RNase protection assay from a single animal in each of the dietary
groups. Fig. 1b shows the mRNA levels in the different
cell populations of the villus in the three segments, and Fig.
1c shows the cumulative average of the mRNA levels in
duodenum, jejunum, and ileum. From the data shown in the representative
RNase protection assay, it is clear that, compared with SREBP-1a,
SREBP-1c was the predominant transcript in the intestine of
hamsters fed the control chow diet. Moreover, the expression of the
transcript for SREBP-1c was highest in cells of the villus tip and
decreased as cells of the lower villus were reached (Fig. 1,
a and b). In contrast, expression of SREBP-2 and
the other sterol-responsive genes was lowest in cells of the upper
villus and increased as cells of the lower villus were reached. Compared with duodenum, expression of these genes was modestly higher
in jejunum and ileum.

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Fig. 1.
Effect of diets on gene expressions of
SREBP-1a, SREBP-1c, SREBP-2, HMG-CoA synthase, LDL receptor, and
HMG-CoA reductase in hamster intestine. Isolated intestinal cells
were prepared from duodenum, jejunum, and ileum of hamsters fed for 2 weeks a control chow diet or a cholesterol-rich (cholesterol) or
cholesterol-depletion (cholestyramine plus lovastatin) diet as
described under "Materials and Methods." RNA was isolated from each
of the five fractions, and mRNA levels were estimated by RNase
protection assay. 18 S rRNA was used as an internal control to confirm
that equal amounts of RNA were used for the assays. Fraction 1 represents mRNA from cells of the uppermost villus, and fraction 5 represents mRNA from cells of the crypts. a, a
representative RNase protection assay from a single hamster on each
diet; b, mRNA levels for the genes in the different cell
populations of the villus from duodenum, jejunum, and ileum. The data
represent the means ± S.E. from three animals fed the respective
diets. c, cumulative mRNA levels in duodenum, jejunum,
and ileum. Open bar, control chow;
diagonal bar, cholesterol-rich; cross-hatch
bar, cholesterol-depletion. *, cholesterol-rich values are
significantly different from control chow values at p < 0.05. **, cholesterol-depletion values are significantly different
from control chow values at p < 0.05. ***,
cholesterol-depletion values are significantly different from
cholesterol-rich values at p < 0.05.
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Compared with the expression of SREBP-1a in intestines of animals on
the control chow diet, neither the cholesterol diet nor the
cholesterol-depletion diet altered SREBP-1a gene expression (Fig. 1,
b and c). In contrast, in hamsters ingesting the
cholesterol diet compared with animals on the control chow diet,
SREBP-1c expression was increased predominantly in cells of the upper
villus. Moreover, there was a profound decrease in gene expression of SREBP-1c in all cells along the villus of the entire small intestine of
hamsters fed the cholesterol-depleted diet.
In animals fed the cholesterol-depletion diet, SREBP-2, HMG-CoA
synthase, HMG-CoA reductase, and LDL receptor gene expressions were
dramatically increased in all cell populations of the villus and in all
three segments of the small intestine (Fig. 1, b and c). In contrast, cholesterol feeding had minimal if any
effect on mRNA levels of these genes.
Protein Expression--
In preliminary experiments, we found the
mature form of both intestinal SREBPs to be in very low abundance and
detection difficult. To help in identifying the bands representing
precursor and mature peptides of SREBPs and to support our results in
intestine, protein expression of SREBP-1 and -2 from livers and
intestines of hamsters ingesting the three different diets was estimated.
Because of the relative paucity of recoverable protein in the five
fractions of enterocytes prepared from the villus, it was not possible
to isolate a nuclear and membrane fraction to estimate SREBP
expression. Thus, to determine if a total membrane preparation could be
used to estimate the amount of SREBPs in liver and intestine, we first
prepared total membrane fractions from livers of hamsters on the
respective diets. Fig. 2a
shows an immunoblot for SREBP-1 and -2 using increasing amounts of a
total membrane preparation isolated from the liver of one animal from
each of the diets. From this blot, it is clear that compared with the
hamster ingesting cholesterol, there was more precursor and mature form
of SREBP-2 in hepatic membranes prepared from the hamster on the
cholesterol-depleted diet. The results for SREBP-1 differed. Compared
with the amount of the precursor form of SREBP-1 in the animal
ingesting the cholesterol-depleted diet, there was more precursor of
SREBP-1 in the liver of the hamster ingesting cholesterol. Two bands
flanking the molecular marker of 66 kDa were observed for the
mature peptide of SREBP-1. Both bands were reduced in the liver of the
hamster ingesting the cholesterol-depletion diet. No clear changes were
observed, however, in the mature peptide of the animal fed
cholesterol. Total membranes were then prepared from the livers of
three individual animals on the different diets, and the precursor and
mature forms of SREBP-2 were analyzed (Fig. 2b). Compared
with hamsters ingesting the cholesterol diet, both the precursor and
mature forms of SREBP-2 were increased in livers of hamsters ingesting
the cholesterol-depletion diet. Compared with animals fed the control
chow, the precursor form of SREBP-2 was decreased in livers of animals
fed cholesterol. The mature form was in low abundance in both control
chow-fed and cholesterol-fed animals. No clear difference was observed in the mature form between these two dietary groups. These results lend
support to the notion that total membranes prepared from cell fractions
of intestines of hamsters on the respective diets could be used to
estimate SREBP mass.

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Fig. 2.
Effect of diets on hepatic SREBP-1 and -2 mass. Total membranes were prepared from livers of hamsters fed
control chow, cholesterol-rich or cholesterol-depletion diets. The
membranes were analyzed for SREBP-1 and SREBP-2 mass by immunoblot as
described under "Materials and Methods." a, an
immunoblot from a single animal on each of the diets. 6-100 µg of
protein was applied per lane. P, precursor form;
M, mature forms. b, an immunoblot of total
membranes prepared from livers of three individual animals fed the
respective diets. P, precursor; M, mature. 50 µg of protein was applied per lane.
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Isolated villus cell fractions were prepared from three intestinal
segments of five animals on each of the diets. Total membranes were
prepared, and the amount of SREBP expression was estimated. Fig.
3a shows a representative
immunoblot of SREBP-1 and SREBP-2 from a single animal in each of the
dietary groups. Because of the low abundance of the mature form of both
SREBPs and to accurately measure changes in the amount of the precursor
form, the blots were exposed to film for different times. The exposure
time for estimating the amount of the precursor form was 3-4 times
less than the exposure time used to detect and estimate the amount of
the mature form. Fig. 3b shows the amount of the precursor form of SREBP-1 and -2 in the different cell populations of the villus
in the three segments. Fig. 3c shows the cumulative average of the precursor mass in duodenum, jejunum, and ileum. The results depicted in the representative blot demonstrate that in membranes prepared from intestines of hamsters on all three diets, the precursor forms of both SREBP-1 and -2 were readily detectable. In general, in
animals fed control chow and the cholesterol-enriched diets, the amount
of the precursor form of SREBP-2 tended to be lower in membranes
prepared from cells of the upper villus and higher in cells of the
lower to middle villus. The reverse tended to be true for the precursor
form of SREBP-1, particularly in the duodenum (Fig. 3, a and
b). It should be appreciated here that the antibody for
SREBP-1 detects the mass of both SREBP-1a and SREBP-1c. From the
cumulative data shown in Figs. 3, b and c, it is
apparent that the amount of precursor form of SREBP-2 in intestinal
cells of animals fed the cholesterol-enriched diet was similar to the
amount present in intestines of animals on the control chow diet. There
was, however, significantly more SREBP-2 precursor in duodenum and
jejunum of hamsters fed the cholesterol-depleted diet than in hamsters
fed either the control chow or cholesterol diet. In ileum, significant
differences in SREBP-2 precursor forms were only observed between
hamsters ingesting the cholesterol-depleted diet and animals on control
chow. The mature form of SREBP-2 was difficult to detect in intestines
of animals fed the control chow or the cholesterol diet. In all five animals, a distinct band above background noise was never fully appreciated (Fig. 3a). However, in intestines of animals on
the cholesterol-depleted diet, particularly in the proximal intestine, the mature form of SREBP-2 was readily detectable and was consistently increased. In contrast, although there was a tendency for SREBP-1 precursor to be present in higher amounts in jejunum of hamsters fed
the cholesterol-enriched diet, because of variability among animals,
this did not reach statistical significance (Fig. 3, b and
c). Like the mature form of SREBP-2, the mature form of SREBP-1 was also in low abundance and difficult to detect. Although in
Fig. 3a, it appears that the mature form of SREBP1 was
increased in intestines of animals fed the cholesterol diet, the mature protein did not consistently appear as a distinct band. Thus, we were
unable to make any conclusions regarding the regulation of mature
SREBP-1 by dietary cholesterol.

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Fig. 3.
Effect of diets on intestinal SREBP-1
and -2 mass. Total membranes were prepared from cells isolated
from the villus of duodenum, jejunum, and ileum of five hamsters fed
the respective diets. The membranes were analyzed for SREBP-1 and
SREBP-2 mass by immunoblot. a, a representative immunoblot
from a single hamster on each diet. To detect and estimate the amount
of precursor and mature forms, the blots were exposed for different
times. Precursor forms of SREBP-1 and SREBP-2 were estimated after 30-s
and 5-min exposure to film, respectively. Mature forms of SREBP-1 and
SREBP-2 were estimated after 5- and 15-min exposure, respectively.
P, precursor; M, mature. 125 µg of protein was
applied per lane. Fraction 1 represents membranes prepared from cells
of the upper villus (tips), and fraction 5 represents membranes
prepared from cells of the crypts. b, the amount of
precursor form of SREBP-1 and SREBP-2 in the different cell populations
of the villus from duodenum, jejunum, and ileum. The data represent the
means ± S.E. from five animals fed the respective diets.
c, cumulative amounts of precursor forms of SREBP-1 and
SREBP-2 in duodenum, jejunum, and ileum. The data represent the
means ± S.E. of five animals fed the respective diets.
Open bar, control chow; diagonal
bar, cholesterol-rich; cross-hatched bar,
cholesterol-depletion. **, cholesterol-depletion values are
significantly different from control chow values at p < 0.05; ***, cholesterol-depletion values are significantly different
from cholesterol-rich values at p < 0.05.
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Cholesterol Mass--
To determine whether the diets altered the
cholesterol content of cells along the intestine, cholesterol mass was
estimated in the five villus cell populations of duodenum, jejunum, and ileum (Fig. 4). In all three segments of
intestines of hamsters fed the control chow diet, there was a trend
indicating more cholesterol mass in cells of the upper rather than the
lower villus. Cholesterol feeding did not alter this trend but caused
significant accumulation of cholesterol in all cell populations of the
villus. More cholesterol accumulated in cells of the jejunum and ileum
than duodenum. The cholesterol-depleted diet decreased the amount of
cholesterol in intestines of these animals, except perhaps, in lower
villus cells of the ileum. The cholesteryl ester content mirrored the results of total cholesterol, accounting for ~3%, 0.9%, and 9% of
total cholesterol in intestinal cell fractions of animals on chow,
cholesterol-depletion, and cholesterol-enriched diets,
respectively.

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Fig. 4.
Effect of diets on intestinal cholesterol
mass. Cholesterol mass was estimated in cell populations along the
villus of duodenum, jejunum, and ileum of hamsters fed the respective
diets. The data represent the means ± S.E. of nine animals on
each diet. , control chow; , cholesterol-rich; ,
cholesterol-depletion. Fraction 1 represents cells of the upper villus,
and fraction 5 represents cells of the crypts. Cumulative values for
each segment are shown in the bar graph on the
right. Open bar, control chow;
diagonal bar, cholesterol-rich;
cross-hatched bar, cholesterol-depletion. *,
cholesterol-rich values are significantly different from control chow
values at p < 0.05; **, cholesterol-depletion values
are significantly different from control chow values at
p < 0.05; ***, cholesterol-depletion values are
significantly different from cholesterol-rich values at
p < 0.05.
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Cholesterol and Fatty Acid Synthesis--
Rates of cholesterol and
fatty acid synthesis in intestinal cell fractions were estimated by
tritiated water incorporation into cholesterol and fatty acids
following the dietary periods (Fig. 5).
In animals on all three diets, rates of cholesterol synthesis were
lowest in cells of the villus tips and increased as cells of the lower
villus were reached. Rates of cholesterol synthesis were similar in
intestines of animals fed the chow or cholesterol-enriched diets. In
contrast, rates of cholesterol synthesis were markedly increased in
intestines of hamsters on the cholesterol-depletion diet.

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Fig. 5.
Effect of diets on intestinal cholesterol and
fatty acid synthesis. Synthesis of cholesterol and fatty acids was
estimated by incorporation of [3H]water into the lipids
in cell populations along the villus of duodenum, jejunum, and ileum of
hamsters fed control chow ( ), cholesterol-rich ( ), or
cholesterol-depletion ( ) diets. The values represent the mean ± S.E. of three hamsters on each diet. Fraction 1 represents cells of
the upper villus, and fraction 5 represents cells of the crypts.
Cumulative values for each segment are shown in the bar
graph on the right. Open
bar, control chow; diagonal bar,
cholesterol-rich; cross-hatched bar, cholesterol-depletion;
**, cholesterol-depletion values are significantly different from
control chow values at p < 0.05; ***,
cholesterol-depletion values are significantly different from
cholesterol-rich values at p < 0.05.
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Rates of fatty acid synthesis were also similar in intestines of
animals on control chow or cholesterol diets. In contrast, in all cells
along the villus and in all three segments of small intestine, rates of
fatty acid synthesis were increased in animals on the
cholesterol-depletion diet.
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DISCUSSION |
The results of this study clearly show that SREBP-1a is a minor
transcript in intestinal cells. Moreover, gene expression of SREBP-1a
in intestine was not altered by rather dramatic changes in cholesterol
flux. These findings, taken together with earlier results in isolated
intestinal segments, which showed that gene expression of SREBP-1a was
not regulated by cholesterol flux, suggest that SREBP-1a does not
regulate cholesterol synthesis in intestine (14). In a previous study,
gene expression of SREBP-1a in livers of hamsters ingesting a
cholesterol-enriched or a cholesterol-depletion diet was also not
altered (conditions that would have had profound effects on hepatic
cholesterol synthesis) (12). Because the liver and intestine account
for most cholesterol synthesis within the body, the results from both
studies suggest that SREBP-1a has only a small role in
regulating total body cholesterol synthesis. This is not to imply that
SREBP-1a does not regulate genes of the cholesterol biosynthetic
pathway. This is clearly not the case. In livers of transgenic mice
that overexpress a truncated form of SREBP-1a, massive accumulation of
cholesterol occurs and rates of cholesterol synthesis and expressions
of sterol-responsive genes are markedly increased (20). Furthermore,
transfection of cultured cells with SREBP-1a increases rates of
transcription of sterol-responsive genes (10). Despite these findings
that support the view that SREBP-1a is a rather potent transcription factor for genes that regulate cholesterol synthesis, SREBP-1a does not
appear to be an important regulator of cholesterol synthesis in hamster
intestine or liver.
In contrast to SREBP-1a, changes in cholesterol flux do regulate gene
expression of intestinal SREBP-1c and SREBP-2. The regulation of these
genes by cholesterol flux, however, is not a simple matter. In cell
culture experiments, changes in cholesterol flux cause coordinate
regulation of expression of both SREBP-1 and -2 (21-23). It is quite
obvious from the results presented here in intact animals that these
transcripts are not coordinately regulated by cholesterol flux; in
fact, quite the opposite is true. SREBP-1c is thought to be a weaker
transcription factor than SREBP-1a and more specific for enhancing the
transcription of genes in the fatty acid synthetic pathway (10).
Transgenic animals overexpressing SREBP-1c in liver display a moderate
increase in fatty acid synthesis without altering cholesterol synthesis
(10). In our previous study, we postulated that SREBP-1c was
responsible for regulating fatty acid synthesis but not cholesterol
synthesis in intestine (14). The present results also support the
notion that SREBP-1c plays little or no role in regulating cholesterol
synthesis in intestine. In response to changes in cholesterol flux,
changes in gene expression of SREBP-1c were reciprocal to the changes observed in expressions of the three sterol-responsive genes and to
cholesterol synthesis. Whereas the previously observed correlation between SREBP-1c gene expression and fatty acid synthesis holds true in
intestines of chow-fed animals (14), the present results might argue
against a regulatory role for SREBP-1c in fatty acid synthesis in
intestine following changes in cholesterol flux. Despite a profound
decrease in SREBP-1c gene expression in intestines of hamsters
ingesting the cholesterol-depletion diet, fatty acid synthesis in the
intestines of these animals was enhanced severalfold. Moreover,
cholesterol feeding caused a dramatic increase in gene expression of
intestinal SREBP-1c, yet fatty acid synthesis in intestines of animals
on the cholesterol-enriched diet was similar to animals on the chow
diet. Thus, it would appear that, at least at a transcriptional level,
SREBP-1c is responsible for maintaining basal levels of fatty acid
synthesis in intestine. In response to changes in cholesterol flux,
however, mechanisms other than those involving transcription of
SREBP-1c need to be entertained.
A mechanism has now been provided that explains the regulation of
SREBP-1c gene expression by changes in cholesterol flux. In a recent
study performed in mice, Repa et al. (24) have shown that
the SREBP-1c gene is a target of the nuclear receptor RXR/LXR transcription factor. By feeding mice either a high cholesterol diet or
RXR/LXR agonists, both hepatic and intestinal mRNA levels and
protein expression of SREBP-1c were increased. An RXR/LXR DNA-binding
site in the promoter of SREBP-1c was identified and found to be
required for this regulation. Despite an increase in SREBP-1c gene
expression by cholesterol feeding, hepatic levels of the lipogenic
target genes, fatty acid synthase and acetyl-CoA carboxylase, were not
increased. Only stearoyl-CoA desaturase gene expression was increased
in livers of mice ingesting cholesterol. Perhaps this lack of effect of
cholesterol feeding on these lipogenic target genes explains why in the
present study, fatty acid synthesis was not increased in intestines of
animals having increased gene expression of SREBP-1c.
The present results would suggest that with changes in cholesterol
flux, particularly cholesterol depletion, SREBP-2 assumes the role for
regulating cholesterol and fatty acid synthesis in intestine. In
intestines of hamsters fed the cholesterol-depletion diet, rates of
cholesterol and fatty acid synthesis were increased, as were mRNA
levels for SREBP-2 and the sterol-responsive genes. Moreover, the
amount of precursor and mature forms of SREBP-2 was also increased. In
transgenic animals overproducing the mature form of SREBP-2, hepatic
fatty acid synthesis and triglyceride content were increased 4-fold
(13). Thus, although SREBP-2 appears to be more specific for increasing
the expression of genes of the cholesterol biosynthetic pathway,
SREBP-2 can enhance fatty acid synthesis as well. Thus, under
conditions of cholesterol depletion, we would postulate that SREBP-2
supplants the function of SREBP-1 in intestine and augments cholesterol
and fatty acid synthesis.
In intestines of hamsters ingesting a cholesterol-enriched diet, a diet
that increased plasma cholesterol levels 2-fold, mRNA levels of the
sterol-responsive genes and SREBP-2 were minimally decreased if at all.
Similarly, rates of cholesterol and fatty acid synthesis were not
altered. This was somewhat unexpected. With a large influx of
cholesterol, a more dramatic suppression of genes that regulate
cholesterol metabolism and rates of cholesterol synthesis was expected.
Perhaps this did not occur because cholesterol taken up from the
lumen does not enter a critical "regulatory pool." The absorbed
sterol escapes putative "cholesterol sensors" in endoplasmic
reticulum or plasma membrane that would, in turn, down-regulate the
SREBP pathway. A similar possible mechanism was postulated in
experiments using CaCo-2 cells (25). Cholesterol taken up from micelles
was shown to displace cholesterol of the plasma membrane, directing it
to the ACAT pool for esterification and transport. Since plasma
membrane serves as a large "sink" for absorbed cholesterol, it is
likely that the SREBP pathway would not be activated or, at least, not
activated until a certain threshold amount of cholesterol in plasma
membrane (or endoplasmic reticulum) is reached. Another possible
explanation for the modest changes observed in sterol-responsive gene
expression by dietary cholesterol comes from results in chow-fed
animals. In intestinal cells of these animals, there were abundant
amounts of the precursor form of SREBP-2. The mature form, however, was
not detectable. This implies that there is an adequate supply of
cholesterol in these cells that prevents cleavage of the precursor
protein. Thus, the expression of sterol-responsive genes, including
SREBP-2 itself, and rates of cholesterol synthesis would be chronically
suppressed. To demonstrate further suppression by dietary cholesterol
might not be possible. This is not the first study that has failed to observe a significant decrease in intestinal cholesterol synthesis by
dietary cholesterol in intact animals (for a review, see Ref. 26).
It has been postulated that in an intestinal absorptive cell there
exists a regulatory pool of cholesterol whose size determines rates of
cholesterol synthesis. Newly synthesized cholesterol, plasma membrane
cholesterol, absorbed cholesterol, and cholesterol derived from
lipoproteins contribute, albeit in different proportions, to this pool
(27). The present data support this notion. In intestines of hamsters
fed a chow diet, there was a clear trend for the rates of cholesterol
synthesis and the expressions of sterol-responsive genes, SREBP-2,
HMG-CoA reductase, HMG-CoA synthase, and the LDL receptor, to be lower
in cells of the upper villus (most differentiated) and higher in cells
of the distal villus (least differentiated). This suggests that
compared with differentiated cells, the regulatory pool of cholesterol
is smaller in undifferentiated cells, which in turn, enhances
cholesterol synthesis by increasing the expression of genes in the
cholesterol synthetic pathway via activation of the SREBP pathway.
Indeed, assuming that cholesterol mass reflect differences in this
regulatory cholesterol pool, cells of the upper villus did contain more
cholesterol than cells of the lower villus. Moreover, feeding
cholesterol markedly increased the amount of cellular cholesterol,
particularly in cells of jejunum and ileum, without altering this
gradient in cells along the villus. Thus, with cholesterol feeding,
rates of cholesterol synthesis and expressions of sterol-responsive
genes maintained a gradient along the villus axis that was similar to
that observed in control chow-fed animals. In contrast, by feeding a
cholesterol-depletion diet, this regulatory cholesterol pool would
decrease, stimulating the SREBP pathway and enhancing
transcription of genes that regulate cholesterol synthesis. Marked
stimulation of the SREBP pathway obscured the gradient of cholesterol
synthesis and gene expression that was appreciated along the villus
axis in intestines of animals on chow and cholesterol diets. It seems
clear in intestine, at least, that activation of the SREBP pathway by
decreasing the regulatory cholesterol pool is a much stronger stimulus
than suppressing the SREBP pathway by expanding the regulatory
cholesterol pool by dietary cholesterol.
In the classic SREBP pathway described in cultured cells, cholesterol
and fatty acid synthesis are enhanced by the cleavage of a
membrane-bound precursor form of SREBP (regulated intramembrane proteolysis), releasing a nuclear or active form (28). This "mature" protein then enters the nucleus, binds to a sterol
regulatory element in promoters of genes in these two pathways, and
enhances their transcription (4-8). The results of this study,
however, suggest another possible mechanism for the regulation of
cholesterol and fatty acid synthesis by SREBPs in intestine. The
correlation that was observed between gene expression and the amount of
precursor mass of SREBP-1 and -2 in individual cell populations of the
intestine, although not exact, was fairly striking. Because our
antibody does not distinguish between SREBP-1a and -1c and will detect both, this correlation is best illustrated for SREBP-2. Both mRNA levels and precursor mass of SREBP-2 were lower in cells of the upper
villus and higher in cells of the lower to middle villus. Moreover, in
intestines of animals ingesting the cholesterol-depletion diet, there
was a marked increase in both mRNA abundance and precursor mass of
SREBP-2. It was only in intestines of this group of animals that the
mature form of SREBP-2 could be detected. If regulated intramembrane
proteolysis were the only mechanism for formation of the mature form
following cholesterol depletion, it would have been expected that the
amount of precursor mass would decrease as the amount of mature form
increased. This did not occur. In fact, the amount of precursor mass
markedly increased with cholesterol depletion. These findings are at
least suggestive that in intestine, the mass of SREBP-2, both precursor
and mature, is regulated at the level of transcription. Other
investigators have also observed a dissociation between the amount of
mature form of SREBP-1 and -2 and gene expression for enzymes involved
in cholesterol and fatty acid synthesis, suggesting that the amount of
the mature form can be regulated by changes in synthesis of its
precursor (29, 30).