From the Laboratoire de Physiologie de la Nutrition,
Ecole Nationale Supérieure de Biologie Appliquée à la
Nutrition et à l'Alimentation, FRE2328
CNRS/Université de Bourgogne 1, Dijon 21000, France,
the § Unité de Recherches sur les
Obésités, INSERM, Institut Louis Bugnard, Centre
Hospitalier Universitaire de Toulouse, Université Paul Sabatier,
Toulouse 31403, France, and the
Department of Cell and Molecular
Biology, Section for Molecular Signaling, Lund University,
Lund 221 84, Sweden
Received for publication, August 20, 2002, and in revised form, November 21, 2002
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ABSTRACT |
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The identity of the enzymes responsible for
lipase and cholesterol esterase activities in the small intestinal
mucosa is not known. Because hormone-sensitive lipase (HSL) catalyzes
the hydrolysis of acylglycerols and cholesteryl esters, we sought to
determine whether HSL could be involved. HSL mRNA and protein were
detected in all segments of the small intestine by Northern and Western blot analyses, respectively. Immunocytochemistry experiments revealed that HSL was expressed in the differentiated enterocytes of the villi
and was absent in the undifferentiated cells of the crypt. Diacylglycerol lipase and cholesterol esterase activities were found in
the different segments. Analysis of gut from HSL-null mice showed that
diacylglycerol lipase activity was unchanged in the duodenum and
reduced in jejunum. Neutral cholesterol esterase activity was totally
abolished in duodenum, jejunum, and ileum of HSL-null mice. Analysis of
HSL mRNA structure showed two types of transcripts expressed in
equal amounts with alternative 5'-ends transcribed from two exons. This
work demonstrates that HSL is expressed in the mucosa of the small
intestine. The results also reveal that the enzyme participates in
acylglycerol hydrolysis in jejunal enterocytes and cholesteryl ester
hydrolysis throughout the small intestine.
Hormone-sensitive lipase
(HSL)1 is a multifunctional
enzyme with broad substrate specificity (1). It hydrolyzes tri-, di-, and monoacylglycerols, cholesteryl esters, and retinyl esters. The
activity against diacylglycerol is higher than the activity toward tri-
and monoacylglycerols. The enzyme also exhibits cholesterol esterase
activity, which is almost twice the activity toward triacylglycerols. Much has been learned in the recent years about the domain structure of
HSL. Sequence comparisons revealed that HSL belongs to a family of
esterases which is mainly represented by prokaryotic enzymes (2, 3).
From a structural point of view, HSL is the most complex protein of the
family. Sequence alignments together with biochemical experiments
suggest that adipocyte HSL is composed of two structural domains (4,
5). The first 315 amino acids make up the N-terminal domain, which
shows very little sequence similarities to other known proteins. The
region responsible for the interaction with adipocyte lipid-binding
protein (ALBP) was mapped to this domain (6, 7). In adipose tissue,
ALBP could increase the hydrolytic activity of HSL through its ability
to bind and sequester fatty acids and through specific protein-protein interactions. The C-terminal domain is divided in two functional parts,
a catalytic core and a regulatory module. The latter is composed of 150 amino acids, including all of the known phosphorylation sites of HSL.
Unlike other known mammalian triacylglycerol lipases, the activity of
HSL is regulated by phosphorylation. The phosphorylation sites of
protein kinase A, extracellular signal-regulated kinase, and
AMP-dependent protein kinase have been mapped (8-10). The catalytic core is the region that shows homology with the other members
of the family. Modeling of the part revealed that it adopts an
Several forms of HSL transcripts and the exon-intron organization of
the HSL gene have been characterized in humans. The 88-kDa adipocyte
HSL is translated from a 2.8-kb mRNA and encoded by 9 exons (12,
13). The transcription start site was mapped in a short noncoding exon
called exon B. In the adenocarcinoma cell line HT29, two mRNA
species are found, the adipocyte HSL mRNA and a mRNA with a
different 5'-end transcribed from exon A. Two testicular forms of HSL
have been characterized. The 3.9-kb mRNA encodes a 120-kDa protein
that contains a unique N-terminal region encoded by exon T1, a region
that presumably forms a third structural domain in this isoform (14).
The 3.3-kb mRNA encodes a protein that is identical to the
adipocyte HSL form (15). However, the mRNA species differ in their
5'-ends. Exon usage is mutually exclusive, exon T2 being only
transcribed in testis and exon B being transcribed in adipose tissue.
The nature and role of lipases and esterases participating in the
digestion of dietary lipids in the lumen of the gastrointestinal tract
are well established. In addition to the enzymes in the lumen, there is
evidence of lipase activity in enterocytes (16). The presence of
cholesterol esterase activity is more elusive. The exact identity of
the enzymes responsible for the hydrolysis of intracellular
acylglycerols and cholesteryl esters is still unclear. Pancreatic
triacylglycerol lipase, microsomal triacylglycerol hydrolase, and
pancreatic cholesterol esterase have all been suggested to be
responsible for the hydrolytic activities (17-19). Pancreatic triacylglycerol lipase may be synthesized by the small intestine and
accounts for the alkaline lipase activity of the enterocytes (19).
Microsomal triacylglycerol hydrolase could also be involved (18).
However, it has been shown that most of the lipase activity is
cytosolic (20). Pancreatic cholesterol esterase, also called bile
salt-stimulated lipase, is able to hydrolyze cholesteryl esters and
triacylglycerols. In the absence of bile salts, the contribution of
this enzyme is presumably minor (21). Because HSL is a cytosolic enzyme
with a wide range of hydrolytic activities, the purpose of the present
paper was to determine whether HSL was expressed in the intestine and
could contribute to the hydrolysis of intracellular lipids.
Preparation of Intestinal Mucosa--
French guidelines for the
use and care of laboratory animals were followed. Male Swiss mice were
fed ad libitum a standard chow (UAR A04, Usine
d'Alimentation Rationnelle). To study the expression of HSL along the
gastro-colic axis of the gut, the small intestine from the pylorus to
the ileocaecal valvula was removed, flushed with 0.9% NaCl at 4 °C,
and divided into five equal segments. The mucosa was scraped off at
4 °C with a spatula. The first segment is considered to be the
duodenum; segments 2-4, the jejunum; and segment 5, the ileum.
Northern Blot Analysis--
Total RNA was extracted from adipose
tissue and intestinal mucosa by the method of Chomczynski and Sacchi
(22). RNA was denatured, subjected to electrophoresis on a 1% (w/v)
agarose gel, and transferred to GeneScreen membranes (PerkinElmer Life Sciences). Rat intestinal fatty acid-binding protein (I-FABP, a gift
from Dr. J. I. Gordon, Washington University, St. Louis, MO),
mouse ALBP (a gift from Dr. P. Grimaldi, INSERM U 470, Université de Nice Sophia-Antipolis, Nice, France), and mouse HSL cDNA were used as probes. They were labeled with [ Analysis of 5'-cDNA Ends and Real Time Quantitative PCR of
Intestinal HSL mRNAs--
Total RNA was isolated using RNASTAT-60
(AMS Biotechnology). Total RNA (1 µg) was treated with DNase I (DNase
I amplification grade, Invitrogen), then retrotranscribed using random
hexamers (Amersham Biosciences) and Thermoscript reverse transcriptase (Invitrogen) according to the manufacturer's recommendations. Combinations of the different primers and amplicon sizes are shown in
Table I. The PCRs were performed on a
Biometra apparatus with 94 °C for 2 min followed by 35 amplification
cycles (94 °C for 20 s, 58 or 60 °C for 30 s, 72 °C
for 30 s). This was followed by an additional elongation step of 7 min at 70 °C. The PCR products were electrophoresed on agarose gels.
Real time quantitative PCR was performed on a GeneAmp 7000 Sequence
Detection System using SYBR green chemistry (Applied Biosystems). 18 S
rRNA was used as control to normalize gene expression using the
Ribosomal RNA Control Taqman Assay kit (Applied Biosystems).
Preparation of Whole Cell and Cytosolic Homogenates from
Intestine and Adipose Tissue--
Intestinal mucosa were homogenized
in 4 volumes of homogenization buffer (0.25 M sucrose, 1 mM EDTA, pH 7.0, 1 mM dithioerythritol, 20 µg/ml leupeptin, 20 µg/ml antipain) using syringe and needle (19 and 25 gauge) to obtain whole cell homogenates. Homogenates were
centrifuged at 110,000 × g at 4 °C for 45 min to
prepare fat-free cytosolic supernatants. Cytosolic homogenates were
prepared from adipose tissue samples as described for the intestine
except that a glass/Teflon potter was used for homogenization. Protein concentrations were determined with a Bio-Rad protein assay using bovine serum albumin as standard.
Western Blot Analysis of HSL--
Samples of 50 µg of proteins
from intestine supernatants and 15 µg of proteins from adipose tissue
supernatants were subjected to 10% SDS-PAGE, transferred onto
nitrocellulose membrane (Hybond ECL, Amersham Biosciences), and probed
with specific polyclonal anti-rat HSL antibody. Immunoreactive protein
was determined by enhanced chemiluminescence reagent (Amersham
Biosciences) and visualized by exposure to Fujifilm.
Immunocytochemistry--
The jejunum and ileum were rapidly
filled in and rinsed with 10% formalin, pH 7.0. Pieces were removed
and immersed in the same fixative for 6 h at 4 °C. After
rinsing overnight with sodium phosphate, pH 7.4, 20% sucrose,
specimens were frozen ( Enzyme Activity Assays--
In vitro
enzymatic activities were performed on whole cell and cytosolic
fractions. Diacylglycerol lipase and cholesteryl ester hydrolase
activities were measured using phospholipid-stabilized emulsions of
1(3)-monooleoyl-2-O-monooleylglycerol or cholesterol oleate, respectively (21). Total esterase activity was determined using
p-nitrophenyl butyrate as substrate. One unit of hydrolase activity is equivalent to 1 µmol of fatty acid released/min at 37 °C.
Generation of HSL-null Mice--
HSL-null mice were generated by
targeted disruption of the HSL gene in SV129-derived embryonic stem
cells by standard procedures (23). In brief, the cDNA encoding the
Aequorea victoria green fluorescent protein was inserted
in-frame into exon 5 of the HSL gene, followed by a neomycin resistance
gene, thereby disrupting the catalytic domain. The herpes simplex
thymidine kinase gene was inserted at the 3'-end of the construct.
Detailed description of the targeting construct will be published
elsewhere.2 After
electroporation of embryonic stem cells, 96 colonies resistant to both
G418 and ganciclovir were isolated, 10 of which showed homologous
recombination as determined by Southern blot analysis. Two of these
colonies were used for generation of two independent HSL-null mouse
lines. In the present work animals from both of these lines were used
with almost identical results, thus results from only one of the
strains are presented. The studies were approved by the Animal Ethics
Committee at Lund University.
Statistical Analysis--
Data are presented as the means ± S.E. Values from HSL-null and wild-type mice were compared using the
Mann-Whitney nonparametric test (Stat View software, Abacus concepts).
Northern Blot Analysis of HSL mRNA--
The distribution of
HSL mRNA along the gastro-colic axis was analyzed by Northern blot
(Fig. 1A). HSL mRNA is
present in the five segments of the small intestine (Fig.
1B). No expression of HSL mRNA was detected in colon
(data not shown). Using quantitative RT-PCR, the HSL mRNA level was
8.8 ± 0.7-fold higher in adipose tissue than in intestinal mucosa
(n = 5).
To determine that the specificity of the detected HSL mRNA signal
did not derive from contaminating visceral adipose tissue, a series of
hybridization was performed with different probes that were tested on
intestinal and adipose tissue samples. As shown in Fig. 1C,
ALBP mRNA that encodes an adipocyte-specific fatty acid-binding
protein was only detected in the adipose tissue sample. There was no
hybridization of the probe in the intestinal mucosa sample. Considering
the quantities of total RNA loaded (15 µg for adipose tissue and 30 µg for intestine) and the intensity of the ALBP mRNA signal
obtained in adipose tissue, a weak contamination of intestinal sample
by visceral adipose tissue would thus have been detected. On the
contrary, I-FABP, which encodes the intestinal fatty acid-binding
protein, was only detected in the intestinal mucosa sample.
Western Blot Analysis of HSL--
Western blot analysis with
supernatants from the five parts of small intestinal mucosa was
performed and developed with specific antibodies against rat HSL. A
protein with similar apparent molecular mass (82 kDa) as the
adipocyte murine HSL was detected in the different intestinal parts
(Fig. 2). A lower molecular band that may
correspond to a proteolytic fragment was present in intestinal samples.
Immunocytochemistry Analysis of HSL--
Immunocytochemistry
experiments were performed on jejunum (Fig.
3, A and B) and
ileum (Fig. 3, C and D) sections. HSL protein was
detected in the differentiated cells of the villi and was absent in the
undifferentiated cells of the crypt.
Enzyme Activity Assays--
Cholesterol esterase activity was
measured with cholesterol oleate. Using a diacylglycerol analog
in which only the first ester bond can be hydrolyzed,
diacylglycerol lipase activity can be determined without simultaneously
measuring monoacylglycerol lipase activity. Enzymatic
activity determination on intestinal musosa cytosolic fractions
indicated the presence of a cholesterol esterase and a lipase along the
small intestine (Fig. 4). Cholesterol esterase and diacylglycerol lipase activities were 9- and 4-fold lower
in jejunum than in white adipose tissue, respectively. Diacylglycerol lipase activity was inhibited using 100 µM
diethyl-p-nitrophenyl phosphate by 75 ± 3%
(range from 68.2 to 82.5) in all intestinal segments and by 94 ± 1% in white adipose tissue.
Analysis of HSL-null Mice Intestine--
In vitro
enzymatic assays were realized in HSL-null mice intestine and compared
with those from wild-type littermates. Cholesterol esterase activity
was totally abolished in the cytosolic fractions of duodenum, jejunum,
and ileum of HSL-null mice (Fig.
5A). Although diacylglycerol
lipase activity was unchanged in the duodenum of HSL-null mice, it was
significantly reduced in jejunum (Fig. 5B). Enzymatic assays were also performed in whole cell homogenates. There
was almost no cholesterol esterase activity in the various parts of
HSL-null mouse small intestine (Fig.
6A). Diacylglycerol lipase
activity was decreased in jejunum (Fig. 6B). Total esterase activity was not modified by the lack of HSL (Fig. 6C). As
shown in Fig. 7, Western blot analysis
performed on HSL-null mouse intestine showed complete disappearance of
the 82-kDa protein. A higher molecular mass band was detected in
wild-type mice. It may correspond to the 89 kDa band observed in some
rat tissues expressing HSL (24).
Analysis of the 5'-Ends of Intestinal HSL mRNA--
Two forms
of HSL transcripts have been characterized in the
adenocarcinoma cell line HT29 (13). The 5'-ends of the two forms are
transcribed either from exon B or from exon A. In an attempt to
characterize intestinal HSL transcripts, different primers were used in
RT-PCR (Table I) with mRNA from intestinal mucosa. As expected, the
use of primers in exon 1 led to an amplification of a 206-bp PCR
product in adipose tissue and intestine (Fig. 8). Using different antisense primers in
exon 1 with sense primers designed either in exon A or in exon B, we
could detect the different PCR products with the expected size in
intestine. These results suggest that two HSL mRNA with mutually
exclusive 5'-ends coexist in enterocytes. The relative abundance of
exon A- and exon B-containing transcripts was determined using
quantitative RT-PCR on adipose tissue and intestinal mucosa total RNA
(n = 4). The ratio of exon B to exon A transcripts was
4.4 ± 0.2 in the adipose tissue and 1.1 ± 0.1 in the
intestine. The data reveal that exons A and B are used equally in the
enterocytes.
Here we have found that HSL contributes to lipase activity and is
the major cholesterol esterase in the intestinal mucosa. HSL mRNA
and protein were readily detected along the small intestine. Immunohistochemistry data revealed that the enzyme is expressed preferentially in differentiated cells of the villi. Data from HSL-null
mice showed that HSL does not account for a significant part of total
esterase activity. However, the enzyme is responsible for all neutral
cholesteryl ester hydrolase activity both in the cytosolic and
particulate fractions. It also contributes between one-third and
one-half of diacylglycerol lipase activity in the jejunum.
The size of intestinal HSL mRNA and protein corresponds to those of
mouse adipocyte HSL, i.e. a 2.6-2.8-kb mRNA and an
82-kDa protein. The 5'-ends of the mRNA species are transcribed
from two exons that correspond to human exons A and B. Exon A is
located in the mouse gene Lipid processing through the intestine is a complex pathway with
multiple control steps. The intestine is unable to transport neutral
lipids into the lymph at the rate with which they are absorbed,
especially at high input rates. Nearly half of the triacylglycerol mass
infused into rat intestine does not appear in the lymph (29). It is
unlikely that the lipids are oxidized because The nature of the enzyme responsible for the hydrolysis of cholesteryl
esters in the intestine has remained unclear. Pancreatic cholesterol
esterase (bile salt-stimulated lipase) is internalized upon binding to
the surface of enterocytes (33). The esterase could hydrolyze
intracellular cholesteryl esters or conversely participate, at acid pH,
in the esterification of cholesterol (17, 34). However, the
intracellular esterase activity in the absence of cofactors such as
bile salts may be very low. Contribution to cholesterol esterification
is also unlikely because studies on knockout mice revealed that the
enzyme is responsible for mediating intestinal absorption of
cholesteryl esters but does not influence free cholesterol absorption
(35). In contrast, acyl-CoA:cholesterol acyltransferase 2-deficient
mice are resistant to diet-induced hypercholesterolemia (36).
Localization of pancreatic cholesterol esterase in intestinal
epithelium may therefore not be related to intracellular metabolism.
There is evidence that the enzyme, via an apical-to-basolateral
transcytotic pathway, is released at the basolateral membrane level and
may contribute to serum pancreatic cholesterol esterase activity (37).
Our data reveal that HSL and not pancreatic cholesterol esterase
accounts for neutral cholesterol esterase activity in the small intestine.
The expression of HSL in the enterocytes may open new paths in our
understanding of cholesterol intestinal absorption and metabolism.
HSL-mediated hydrolysis of the intracellular pool of cholesteryl esters
may contribute together with the esterification process mediated by
acyl-CoA:cholesterol acyltransferase-2 and cholesterol transport
mediated by ATP-binding cassette (ABC) transporters to the control of
cholesterol homeostasis. Several transporters are expressed in the
intestinal epithelium. ABCA1 is expressed in the small intestine and
may modulate cholesterol absorption. However, data from ABCA1-deficient
mice are conflicting (38, 39). Studies in patients with sitosterolemia
(40, 41) and in transgenic mice overexpressing ABCG5 and ABCG8 (42)
suggest that the half-transporters participate in cholesterol efflux. Hydrolysis of cholesteryl esters by HSL may produce free cholesterol for export through ABC transporters into the lumen. Because of the
unique properties of HSL, the present work paves the way for future
studies on lipid metabolism in the enterocyte.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
-hydrolase fold that harbors the catalytic triad constituted by
Ser423, Asp703, and His733 (4,
11).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP
(3,000 Ci/mmol; Amersham Biosciences) using the Megaprime kit (Amersham
Biosciences). A 24-residue oligonucleotide specific for rat 18 S rRNA
was used as probe to ensure that equivalent quantities of RNA were
loaded and transferred. This oligonucleotide was 5'-end labeled with
T4 polynucleotide kinase and [
-32P]ATP
(3,000 Ci/mmol; Amersham Biosciences).
Primers used for analysis of murine intestinal HSL mRNA
40 °C in isopentane). Sections (10 µm
thick) were sliced on a cryostat HM 500 (Microm) at
25 °C and
mounted on glass slides coated with poly(L-lysine). The
sections were hydrated with NaCl/Pi for 10 min, then
incubated with 5% goat serum in 0.2% Triton X-100,
NaCl/Pi for 20 min. After being washed with 0.2% Triton
X-100, NaCl/Pi, sections were incubated with polyclonal
anti-rat HSL antibodies generated in rabbits (1:500) in 5% goat serum,
0.2% Triton X-100, NaCl/Pi, in a humid chamber for 6 h at room temperature. After several washes with 0.2% Triton,
NaCl/Pi, sections were incubated with fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG (Sigma) at a dilution of
1:160 for 60 min. After washing with NaCl/Pi and addition
of mounting media (Sigma) and coverslips, slides were examined with an
ultraviolet visible confocal microscope (Leica TCS 4D).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Northern blot analysis of HSL
mRNA. A, the small intestine was divided into five
segments. The first segment is considered to be the duodenum
(D); segments 2-4, the jejunum (J); and segment
5, the ileum (I). Total RNA from intestinal mucosa (30 µg)
was resolved on a 1% agarose gel containing 2.2 M
formaldehyde, transferred, and fixed to a nylon membrane. B,
the bar graph represents HSL mRNA data normalized to 18 S rRNA for difference in total RNA loading. Values are the means ± S.E. from three independent determinations. C, RNA from
adipose tissue (15 µg) or from jejunal mucosa (30 µg) was resolved
on a 1% agarose gel containing 2.2 M formaldehyde,
transferred, and fixed to a nylon membrane. Hybridizations were
performed with HSL, ALBP, I-FABP, and 18 S rRNA probes.
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Fig. 2.
Western blot analysis of intestinal HSL.
The small intestine was divided into five parts from duodenum to ileum,
and the mucosa was scraped off. Cytosolic fractions were prepared from
the five intestinal segments (numbered 1-5) and visceral
white adipose tissue (WAT). Lanes were loaded with 50 µg
of protein for intestine and 15 µg of protein for adipose tissue,
subjected to SDS-PAGE, and Western blotted with anti-rat HSL antibody.
The arrow shows the murine HSL protein (82 kDa). The lower
molecular mass band in intestinal samples may correspond to a
proteolytic fragment.
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Fig. 3.
Immunocytochemistry analysis of HSL
expression in the small intestine. A, HSL
immunoreactivity in the jejunal section. B, control
micrograph performed in the jejunal section without anti-HSL antibody.
C, HSL immunoreactivity in the ileal section. D,
control micrograph performed in the ileal section without anti-HSL
antibody. Horizontal bar, 300 µm.
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Fig. 4.
Cholesterol esterase and diacylglycerol
lipase activities in small intestine and visceral white adipose tissue
cytosolic homogenates. The small intestine was divided into five
parts from duodenum to ileum, and the mucosa was scraped off.
Supernatants were prepared from the five intestinal segments (numbered
1-5) and visceral white adipose tissue (WAT).
In vitro hydrolytic activities against a cholesteryl ester
(cholesterol oleate, ) and a diacylglycerol analog
(1(3)-monooleoyl-2-O-monooleylglycerol,
)
substrate were determined in cytosolic homogenates. Values are the
means ± S.E. from three independent determinations.
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Fig. 5.
Cholesterol esterase and diacylglycerol
lipase activities in small intestine cytosolic homogenates from
HSL-null mice. In vitro hydrolytic activities against a
cholesterol ester (cholesterol oleate) (A) and a
diacylglycerol analog
(1(3)-monooleoyl-2-O-monooleylglycerol)
(B) were determined in intestinal cytosolic homogenates from
wild-type ( ) and HSL-null (
) mice. Values are the means ± S.E. from six independent determinations. *, p < 0.05 and **, p < 0.01, HSL-null versus wild-type
mice.
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Fig. 6.
Cholesterol esterase, diacylglycerol lipase,
and total esterase activities in small intestine whole cell homogenates
from HSL-null mice. In vitro hydrolytic activities
against a cholesterol ester (cholesterol oleate) (A), a
diacylglycerol analog
(1(3)-monooleoyl-2-O-monooleylglycerol)
(B), and a nonspecific esterase substrate
(p-nitrophenyl butyrate) (C) were determined in
intestinal whole cell homogenates from wild-type ( ) and HSL-null
(
) mice. Values are the means ± S.E. from three independent
determinations. *, p < 0.05, HSL-null
versus wild-type mice.
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Fig. 7.
Western blot of intestinal mucosa extracts
from wild-type and HSL-null mice. Cytosolic fractions were
prepared from jejunal and ileal segments. Lanes were loaded with 50 µg of protein, subjected to SDS-PAGE, and Western blotted with
anti-rat HSL antibody. The arrow shows the murine HSL
protein (82 kDa). The higher molecular mass band in wild-type mice may
correspond to a previously described HSL form in rat tissues
(24).
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Fig. 8.
RT-PCR amplification of HSL mRNA
5'-ends. Total RNA was extracted from intestinal mucosa
(Int.) and white adipose tissue (WAT). RT-PCR was
performed using primers derived from various exons (Ex.)
with (+) and without ( ) reverse transcriptase. Information on the
primers is provided in Table I.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7 kb upstream of exon B (25). Use of
the two exons is mutually exclusive. Interestingly, we have shown previously that HSL is expressed in the human adenocarcinoma cell line
of intestinal origin HT29 (26). The two mRNA 5'-ends were found in
HT29 HSL mRNA (13). In human and mouse adipose tissue, the main
transcription start site is located in exon B. Exon A-containing transcripts are found at very low levels in humans (13) and are much
less abundant than mRNA with exon B in mice ((25) and present
work). To date, there is little information on the mechanisms controlling tissue-specific expression of HSL. We have shown that the
first 95 bp of human exon T1 5'-flanking region conferred expression of
a reporter gene exclusively in testis of transgenic mice (27). Exon B
5'-flanking region contains an active promoter with an E-box and two
GC-boxes as functional cis-acting elements (28). In adipocytes, the
E-box mediates the glucose-mediated induction of HSL gene expression.
However, the sequences responsible for the adipose tissue-specific
expression of HSL are not present in this region because the pattern of
promoter activity up to 2.4 kb was similar in adipocytes and in HeLa
cells that do not express HSL (13). No data are yet available for the
exon A 5'-flanking region. In the enterocytes, exons A and B are
represented in equal amounts, suggesting that two alternative promoters
control HSL gene expression providing the possibility of distinct
transcriptional regulation.
-oxidation of lipid
entering the mucosa from the lumen is limited. There is no evidence
that triacylglycerols are transported via the portal vein (30). These
studies suggest that some triacylglycerols in the enterocyte are
undergoing hydrolysis. In support of this concept, a mucosal
triacylglycerol pool distinct from the chylomicron triacylglycerol
precursor pool has been characterized (31). Lipolysis of the mucosal
pool has been shown both in vitro and in vivo.
Both acidic and alkaline lipase activities have been described in the
mucosa (20, 32). Because most of the lipolytic activity was found at
neutral or basic pH, the physiological importance of the acidic lipase
is unclear. Here, we confirm that significant neutral lipase activity
is found in the enterocyte. This activity was inhibited by
diethyl-p-nitrophenyl phosphate as shown previously for
mucosal lipolysis in triolein-infused rats (31). Lipase activity was
found in the different parts of the small intestine. Data from HSL-null
mice show that HSL contributes to lipase activity in the distal section
but not in the first part of the small intestine. Recently, Mansbach
and colleagues (19) showed that pancreatic lipase was expressed in the
intestine with most of the enzyme detected in the first quarter (19).
Altogether, the data suggest that the hydrolysis of mucosal
triacylglycerols is caused by pancreatic lipase in the proximal part of
the small intestine and HSL in the more distal parts.
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ACKNOWLEDGEMENTS |
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We acknowledge gratefully the contribution of the staff of the Louis Bugnard Institute Animal Care facility. We thank Birgitta Danielsson (Lund University) for technical assistance and Drs. Pascal and Patricia Degrace (Université de Bourgogne) for help with immunocytochemistry experiments.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by doctoral grants from the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, and the Fondation pour la Recherche Médicale.
** Supported by the Swedish Research Council Project 112 84 and the A. Påhlsson Foundation.
To whom correspondence should be addressed: INSERM U317,
Bâtiment L3, CHU Rangueil, 31403 Toulouse Cedex 4, France.
Tel.: 33-562-172-950; Fax: 33-561-331-721; E-mail:
Dominique.Langin@toulouse.inserm.fr.
Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M208513200
2 H. Mulder, M. Sörhede-Winzell, J.-A. Contreras, M. Fex, K. Ström, T. Ploug, H. Galbo, P. Arner, C. Lundberg, F. Sundler, B. Ahrén, and C. Holm, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: HSL, hormone-sensitive lipase; ABC, ATP-binding cassette; ALBP, adipocyte lipid-binding protein; I-FABP, intestinal fatty acid-binding protein; RT, reverse transcription.
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