|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 273, Issue 47, 31215-31221, November 20, 1998
From the Departments of The pancreas secretes several different lipases.
The most abundant is pancreatic triglyceride lipase (PTL). The pancreas
also synthesizes two homologues of PTL, the pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2). Cytotoxic T-lymphocytes also express PLRP2 under certain conditions. We sought to determine the role
of PLRP2 in fat absorption and in T-cell cytotoxicity by creating a
PLRP2-deficient mouse. Adult PLRP2-deficient mice had normal fat
absorption. In contrast, suckling PLRP2-deficient mice had fat
malabsorption evidenced by increased fecal weight, increased fecal
fats, and the presence of undigested and partially digested dietary
triglycerides in the feces. As a result, the PLRP2-deficient pups had a
decreased rate of weight gain. To assess T cell cytotoxicity, we
immunized PLRP2-deficient mice with a mastocytoma cell line, P815, and
determined the ability of splenocytes from the immunized mice to kill
P815 cells in a 51Cr release assay. PLRP2-deficient
cells had deficient killing activity in this assay, and PLRP2-deficient
splenocytes released fewer fatty acid from the target cells than did
control cells. Our results provide the first evidence of a
physiological function for PLRP2. PLRP2 participates in T cell
cytotoxicity, and PLRP2 performs a crucial role in the digestion of
dietary fats in suckling animals.
During the last 10 years, data from cDNA and genomic cloning
have provided evidence for a lipase gene family. The first speculation about a lipase gene family began after the cloning of cDNAs for hepatic lipase, lipoprotein lipase, and pancreatic triglyceride lipase
(PTL)1 showed similarities
among the predicted amino acid sequences of these lipases (1).
Subsequently, the cloning of other cDNAs encoding lipases from
various species and the realization that many lipases and other
hydrolases share a common protein folding pattern expanded the family
greatly (2). Two of the recently described lipases have higher sequence
homology to PTL than any other members of the gene family. Because of
their similarities to PTL, Giller et al. (3) named these two
lipases pancreatic lipase-related proteins 1 and 2 (PLRP1 and PLRP2)
(3).
One of these related proteins, PLRP2, was also independently cloned for
reasons other than its similarity to PTL. Mouse PLRP2 was cloned as an
interleukin-4-inducible gene in CD8+ T cells (4). Rat PLRP2 was cloned
as GP3, a zymogen granule membrane-associated protein (5). These
studies suggested potential physiological functions for PLRP2 in T
cell-mediated cytotoxicity, in zymogen granule fusion with the acinar
cell plasma membrane, and in the digestion of dietary fats.
Additional investigations of PLRP2 have supported some of these
functions. Each of these postulated functions requires that PLRP2 have
lipase activity. Studies of recombinant PLRP2 have demonstrated a high
level of triglyceride lipase activity similar to PTL (6). Unlike PTL,
PLRP2 also hydrolyzes other lipid substrates and possesses
phospholipase and galactolipase activities (6, 7). PLRP2 is also
secreted by the pancreas and by isolated pancreatic acinar cells in
culture (8, 9). This secretion places PLRP2 in the duodenum, where
dietary fat digestion occurs. Secretion of PLRP2 by T cells has not
been reported. Finally, the pancreas expresses mRNA encoding PLRP2
at birth but does not express mRNA encoding PTL until near the
suckling-weanling transition (10). This temporal pattern of expression
for mRNA encoding PLRP2 and PTL led to the speculation that PLRP2
mediates dietary fat digestion in suckling animals.
We sought to define the function of PLRP2 in dietary fat metabolism and
in T cell cytotoxicity. To accomplish this aim, we created a line of
PLRP2-deficient mice by homologous recombination. We characterized T
cell function in adult animals and dietary fat absorption in animals at
various ages.
Targeting Construct and Generation of Mice with Null Allele for
PLRP2--
The targeting construct was made by cloning a
SmaI/ClaI fragment of the mouse PLRP2 gene into a
vector containing a thymidine kinase cassette (Fig. 1) (11). The gene
fragment contained exons I-V out of the 12 total PLRP2 exons (12). A
LacZ/neomycin cassette was cloned into a unique BamHI site
in exon IV (13). The LacZ fusion was created so that cells expressing
the gene encoding PLRP2 could be detected in situ.
D3 embryonic stem cells were transfected with the targeting construct.
We screened for targeted stem cells by Southern blot of genomic DNA
isolated from neomycin-resistant clones and digested with
EcoRI. The blot was hybridized to a 5'-probe, which detected a 5-kilobase pair wild-type band and a 7-kilobase pair fragment from
the targeted allele. The targeting efficiency was 3.6% (7/190). Embryonic stem cells from a targeted clone were injected into BALB/c
mouse blastocysts to generate chimeric animals whose offspring from
BALB/c matings were subsequently screened for the targeted allele by a
polymerase chain reaction with three primers (lipase upstream,
5'-GTAACTGTATTTGCGTTGA-3'; lipase downstream,
5'-ACCGACAACACTTGCACCAA-3'; and LacZ, 5'-ATTCAGGCTGCGCAACTGTT-3') (Fig.
1). These primers amplified a 113-base pair wild-type fragment and a
190-base pair fragment from the targeted allele (Fig.
1). Mice with the targeted allele were
backcrossed six generations to BALB/c mice before intercrossing.
RNA Analysis--
We isolated total RNA from the pancreas of
animals at birth, 10 days, 21 days, and 4 months of age as described
previously (10). The integrity of the RNA was assessed by denaturing
agarose gel electrophoresis (14). The mRNA encoding PTL, PLRP1, and PLRP2 was quantitated by an RNase protection assay utilizing 300-base pair probes complementary to each mRNA species. The PTL and PLRP1 probes went from the middle of exon 3 to the middle of exon 5, and the
PLRP2 probe extended from the middle of exon 2 to the middle of exon 4 and was 5' of the LacZ-neomycin cassette. Standard curves were prepared
by hybridizing the probe to known amounts of in vitro
transcribed mRNA encoding each individual lipase. Values were
normalized to levels of cyclophyllin. The cyclophyllin standard
cDNA was obtained from Ambion, Inc. mRNA transcription was done
with reagents from Ambion, Inc. (Austin TX) according to the
manufacturer's directions. The Ambion RPA II kit was used for the
RNase protection assay. The products were separated by acrylamide gel
electrophoresis according to the instructions with the kit and were
transferred to Ambion's BrightStar Plus membrane in a semidry
electroblot apparatus (Bio-Rad). Bands were detected with the
BrightStar BioDetect (Ambion) kit according to the instructions. The
signal from the standard curves and the unknowns was quantitated using
SigmaGel software (SSPS, Inc., Chicago, IL), and the amount of mRNA
was determined from the standard curve.
Immunohistochemistry and
Animal Diets--
Diets were from PicoLabTM. The
standard chow was 5053 and contained 11.9% of energy as fat, 23.6% as
protein, and 64.5% as carbohydrate. The medium fat diet was 5058, and
the high fat diet was a special test diet. The former contained 21.6%
of energy as fat, 21.9% as protein, and 56.5% as carbohydrate. The
latter had 59.7% of energy as fat, 15% as protein, and 25.3% as
carbohydrate. The fat was derived from lard and corn oil in the same
proportions for all diets. Ad libitum access to food and
water was allowed. The mice were adapted to the diet for at least 2 weeks before samples were collected. Nursing mothers were fed standard
5053 chow.
Fecal Fat Analysis--
Adult mice were placed in a cage with a
metabolic screen. They were given water but no food during the 4-h
collection period. The stool was dried to a constant weight, and fats
were extracted as described (16). To obtain stool from suckling
animals, 10-day-old animals were sacrificed, and the entire colon was
removed. The feces was removed by extrusion and processed as for adult
animals. The percentage of recovery was 90 ± 2.0% as determined
by the recovery of radiolabeled triolein.
Analysis of Lipid Classes--
Extracted fecal fats from 100 mg
of dried feces were dissolved in 1 ml of chloroform, and 10 µl of
each sample was spotted onto a silica G TLC plate along with a standard
mixture containing 10 µg each of monoolein, 1,2-diolein, 1,3-diolein,
and triolein. The plate was developed by a two-stage, one-dimensional
TLC and developed in cupric acetate/phosphoric acid as described (17, 18).
T Cell Cytotoxicity Assays--
Three wild-type and
PLRP2-deficient mice were injected intraperitoneally with 3 × 107 P815 cells. After 10 days, the mice were sacrificed,
and the spleens were removed. Pooled single cell suspensions were
diluted for use in the cytotoxicity assay or for in vitro
stimulation. Spleen cells were cultured at 5 × 106
cells/ml and stimulated with 3 × 104 mitomycin
C-treated P815 cells/ml. After 24 h, 20 units/ml interleukin-2 was
added to each culture. The cells were cultured for 6 days before use as
effectors in the secondary cytotoxicity assay.
To test cytotoxicity, 2 × 107 P815 cells were labeled
with 200 µCi of sodium chromate-51 (NEN Life Science Products) in 10 ml at 37 °C for 1 h or with 3H-labeled oleic acid
for 18 h. Labeled target cells were washed extensively and plated
in a round bottom 96-well microtiter plate at a concentration of 2 × 104/well. Effector cells were added to the wells in
triplicate at the indicated effector:target ratios in a total volume of
200 µl. Plates were spun down, and supernatants were collected after 3 h for fatty acid release or 4 h for chromium release.
Samples were counted in a Statistical Analysis--
The data were analyzed by Student's
t test and by two-way ANOVA followed by the Tukey test for
pairwise multiple comparison procedures. The SigmaStat statistical
package was used for all calculations.
PLRP2-deficient Mice--
To investigate the role of PLRP2 in
dietary fat digestion and T cell-mediated cytotoxicity, we generated
PLRP2-deficient mice by targeted homologous recombination. Adult and
suckling PLRP2-deficient mice were grossly indistinguishable from
wild-type and heterozygote littermates. Adult mice had normal numbers
and populations of cells in peripheral lymphoid organs. The frequency
of PLRP2-deficient mice in heterozygote by heterozygote matings was
diminished. The ratio of wild-type:heterozygous:homozygous deficient
pups was 1.3:1.9:0.8 in over 300 live births.
Analysis of PLRP2 Expression--
Expression of PLRP2 was
determined by immunohistochemistry and by
The temporal pattern of expression for mRNA encoding PTL, PLRP1,
and PLRP2 was quantitated by an RNase protection assay. Total RNA was
prepared from wild-type and PLRP2-deficient pancreas taken from mice at
different ages. Fig. 3 shows the temporal
pattern of expression for the mRNAs encoding the three lipase
species. The expression pattern of mRNA encoding PLRP1 and PLRP2 in
wild-type mice mirrored the pattern previously reported in the rat
(Fig. 3, A and B). Both were present at birth,
and expression persisted throughout adulthood. mRNA encoding PTL
was not detected at birth or 10 days of age but was present in both
wild-type and PLRP2-deficient mice at 21 days of age and 4 months of
age (adults). A faint signal with the PLRP2 probe was found in the
PLRP2-deficient animals. The signal was about 5% of the signal in
wild-type mice and, presumably, represented a chimeric mRNA between
the 5'-end of the PLRP2 mRNA and the Absorption of Dietary Fats--
We next addressed the role of
PLRP2 in dietary fat digestion. The secretion of PLRP2, a lipase with
high triglyceride lipase activity, by the pancreas suggests that it may
play a role in dietary fat digestion. Because earlier studies showed
that newborn and suckling animals express PLRP2 but not PTL, we
examined both suckling and adult animals. We determined if PLRP2
contributes significantly to fat digestion by measuring fecal fat. The
feces from the PLRP2-deficient suckling mice were loose, yellow, and of
larger volume than the formed brown feces of wild-type and heterozygous
pups. This observation was supported by the increased dry weight of the
colonic contents from the PLRP2-deficient pups (Table
I). These findings suggested that the
PLRP2 pups had steatorrhea.
We confirmed that the pups had fatty stools by measuring the fecal fat
content in the stools (Fig. 4). Fats
comprised 55% of the fecal dry weight in the PLRP2-deficient pups
compared with 10.6% in heterozygous and 7.5% in wild-type pups. There
were no differences in fecal fat between wild-type and PLRP2-deficient mice by 23 days of age. Challenging the adult mice with higher fat
diets did not cause the PLRP2-deficient mice to malabsorb fat (Fig. 4).
These findings demonstrated that PLRP2 makes a major contribution to
the digestion of dietary fats in suckling animals but not in adult
animals. The observation that the defect disappears as the pups age can
be explained by the induction of pancreatic triglyceride lipase, which
compensates for the lack of PLRP2 activity in the older pups.
The malabsorbed lipid classes were identified by thin layer
chromatography. The extracted lipids were separated in a two-solvent, one-dimensional system and quantitatively stained with cupric acetate/phosphoric acid. Fatty acids were the predominant species in
the feces from wild-type and heterozygous suckling mice (Fig. 5, lanes 1 and
2) and in adult wild-type and PLRP2-deficient mice (Fig. 5,
lanes 5 and 6). In contrast,
diglycerides and triglycerides comprised a much greater proportion of
the lipids in the feces of PLRP2-deficient animals (Fig. 5,
lanes 3 and 4). These results confirm
that suckling animals require PLRP2 for efficient dietary fat digestion
and show that diglycerides and triglycerides are physiological
substrates for PLRP2.
Growth of PLRP2-deficient Mice--
If the PLRP2-deficient mice
malabsorb fat, their rate of weight gain might be affected. To test
this possibility, we weighed all of the pups in multiple litters of
heterozygous intercrosses from day 4 to weaning at day 23 (Fig.
6). The curves for the wild-type and
heterozygote pups overlap and were not significantly different by
two-way ANOVA analysis. The rate of weight gain for the PLRP2-deficient pups differed from the rates for the wild-type and heterozygote pups
(0.33 g/day versus 0.43 and 0.44 g/day). Two-way ANOVA
revealed a significant difference between the PLRP2-deficient pups and the wild-type pups (F (1, 19) = 78.9, p < 0.0001) and the heterozygote pups (F (1, 19) = 87.9, p < 0.0001). The decreased weight gain in
PLRP2-deficient pups confirmed the importance of PLRP2 in dietary fat
digestion by suckling animals.
Cytotoxic Activity in PLRP2-deficient Mice--
Since murine PLRP2
was originally identified as an interleukin-4-inducible gene in CD8+ T
cells, we also examined the PLRP2-deficient mice for alterations in
cytotoxic activity (4). To determine whether PLRP2 plays an important
role in T cell-mediated cytotoxicity, we immunized wild-type and
PLRP2-deficient mice with the P815 mastocytoma cell line. P815 was
derived from DBA/2 mice and is syngeneic at the major
histocompatibility complex locus (H-2d) but has other
histocompatibility differences from the BALB/c background of the mice.
Ten days following injection of tumor cells, mice were sacrificed and
spleen cells were tested as effectors in a 51Cr release
assay. Control splenocytes displayed a dose-dependent cytotoxicity against 51Cr-labeled P815 targets (Fig.
7A). However, PLRP2-deficient
splenocytes killed targets at a significantly lower level.
In parallel, splenocytes were expanded in vitro by
stimulation with mitomycin C-treated P815 cells. After 6 days in
culture, cells were used as effectors in a cytotoxic assay with
51Cr-labeled P815 targets. Cytotoxicity as measured by
51Cr release was similar to the results of the primary
assay. CTL from wild-type mice killed targets in a
dose-dependent manner, but killing was less efficient by
PLRP2-deficient cells (Fig. 7B). This deficiency in killing
activity was not due to differences in the percentages of CD8+ cells in
the cultures (data not shown). Furthermore, levels of granzyme A
production by wild-type and PLRP2-deficient mice were
indistinguishable. A similar defect in killing was seen in assays using
RENCA cells, another syngeneic tumor target (data not shown).
CTL-mediated lysis has been shown to result in the liberation of free
fatty acids from target cells through an undefined mechanism (19). To
determine whether free fatty acid release was affected by PLRP2
deficiency, we used [3H]oleic acid-labeled P815 cells as
targets in the CTL assay described above. Fig. 7C
demonstrates that free fatty acid release from syngeneic targets is
decreased when PLRP2-deficient CTLs are used as effectors compared with
wild-type cells. This correlates with the decreased cytotoxicity seen
in the 51Cr release assays.
Lipases are ubiquitous enzymes present in many tissues and
organisms. One recently described lipase, PLRP2, is expressed in pancreas and cytotoxic T cells (4, 5, 10). Although the function of
PLRP2 in these tissues is not known, one hypothesis is that pancreatic
PLRP2 participates in dietary fat digestion and that cytotoxic T cell
PLRP2 mediates cell lysis by hydrolyzing membrane lipids. To address
the physiological function of PLRP2, we created and characterized mice
deficient in PLRP2. Our results show that PLRP2 contributes to
cytotoxic T cell cytotoxicity and to fat digestion in the suckling animal.
PLRP2 in Cytotoxic T Cells--
PLRP2-deficient mice had a clear
decrease in cytotoxicity as measured by both 51Cr and
3H-fatty acid release when compared with normal cytotoxic T
cells. The decrease was greater than the decrease in cytotoxicity
observed in granzyme B-deficient mice, but the defect was not as great as when pathways dependent on perforin or Fas were compromised (20-22). Each of these pathways has been implicated in cytotoxic T
cell killing activity. These findings suggested that PLRP2 may play an
auxiliary role in some types of cytotoxic T cell-mediated lysis. The
molecular details of PLRP2-mediated cytotoxicity remain unclear, and
further studies are necessary to elucidate these mechanisms.
Expression in Pancreas--
Our evaluation of PLRP2 expression by
immunohistochemistry and PLRP2 in Dietary Fat Digestion--
Although the role of
pancreatic lipases in dietary fat absorption is widely accepted, the
participation of PLRP2 in fat digestion has not been established. The
PLRP2-deficient mice provided the opportunity to directly test the
function of PLRP2 in dietary fat digestion. Our results demonstrate
that adult mice do not require PLRP2 for efficient dietary fat
digestion. Although PLRP2 did not make a significant contribution to
overall fat absorption in the adult, we did not eliminate the
possibility that PLRP2 hydrolyzes a minor species of dietary fat. Also,
the potential participation of PLRP2 in zymogen granule release was not
tested in these studies. If PLRP2 has an important function in the
adult animals, additional investigations are required to define that role.
In contrast, PLRP2 was essential for efficient fat digestion in
suckling mice. We found that the PLRP2-deficient suckling mice had
10-15 times more fat in their feces than wild-type or heterozygote
animals, suggesting a marked decrease in fat absorption. The fecal fat
from PLRP2-deficient mice contained undigested and partially digested
acylglycerides, confirming the importance of PLRP2 in dietary
triglyceride digestion. Additionally, the weight gain of the suckling
animals was decreased compared with wild type and heterozygotes, which
is consistent with significant loss of calories through fat
malabsorption. Clearly, PLRP2 contributes significantly to dietary fat
digestion in suckling animals.
Our findings provide an explanation for the efficient fat digestion in
suckling animals. Newborns have a relative PTL deficiency (25). Yet,
newborn infants do not have significant steatorrhea despite consuming
3-5-fold more fat per kg, body weight, than adults. Previous models of
fat absorption in newborns centered on the ready absorption of
undigested monoacylglycerols, which made the complete digestion of
dietary fats unnecessary for efficient absorption, or on the presence
of bile salt-stimulated lipase in breast milk, which could compensate
for the lack of PTL (26-28).
Against these models are in vitro experiments demonstrating
that a combination of gastric lipase, bile salt-stimulated lipase, and
PTL are required for efficient digestion of breast milk triglycerides (28-30). Digestion was incomplete unless all three lipases were present. In addition, there is ample evidence that the low intraluminal concentrations of bile salts found in premature and full-term infants
not only decrease bile salt-stimulated lipase activity but favor the
absorption of fatty acids over monoacylglycerols, making the complete
digestion of triglycerides beneficial for the newborn (31-33).
Given these data, the low levels of PTL in newborns should hamper the
digestion of dietary fats, but most newborns efficiently absorb their
large dietary fat intakes, suggesting that other lipases may be active.
PLRP2 is clearly one of those lipases.
*
This work was supported by National Institutes of Health
Grants CA-56462 and DK 53100 and by the Mathers Foundation.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.
¶
To whom correspondence should be addressed: Dept. of
Pediatrics, Washington University School of Medicine, Box 8116, St.
Louis, MO 63110. Tel.: 314-454-2576; Fax: 314-454-4218; E-mail:
Lowe{at}Kids.wustl.edu.
**
Special Fellow of the Leukemia Society of America.
¶¶
Scholar of the Leukemia Society of America.
The abbreviations used are:
PTL, pancreatic
triglyceride lipase; PLRP1, pancreatic lipase related protein 1; PLRP2, pancreatic lipase related protein 2; PBS, phosphate-buffered saline; ANOVA, analysis of variance; CTL, cytotoxic T lympocytes.
2
M. Lowe, unpublished observations.
Decreased Neonatal Dietary Fat Absorption and T Cell Cytotoxicity
in Pancreatic Lipase-related Protein 2-Deficient Mice*
§¶,
**,,, and§§¶¶
Pediatrics and
§ Molecular Biology and Pharmacology, Washington University
School of Medicine, St. Louis, Missouri 63110, the Department of
Immunology and Infectious Diseases, Harvard School of Public Health,
Boston, Massachusetts 02115, the §§ Department
of Medicine, Harvard Medical School, Boston, Massachusetts 02115, and
The Whitehead Institute for Biomedical
Research, Cambridge, Massachusetts 02142
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (39K):
[in a new window]
Fig. 1.
Targeting construct for creating
PLRP2-deficient mice and polymerase chain reaction (PCR)
screening of tail DNA from matings of mice heterozygous for the PLRP2
allele. The top half of the
figure shows a schematic map of the PLRP2 wild-type allele
(top), targeting construct (middle), and the
targeted allele (bottom). Exons are numbered with roman
numerals. The positions of the polymerase chain reaction primers and
fragments used for Southern analysis are indicated. tk,
thymidine kinase. The bottom half of the
figure shows a representative polymerase chain reaction
screening of tail DNA. Samples were separated by agarose gel
electrophoresis and stained with ethidium bromide. Markers are in the
first lane. The next nine
lanes show the results from screening. The upper, 190-base
pair band is the targeted allele, and the lower, 113-base pair band is
the wild-type allele.
-Galactosidase Staining--
For
Immunoperoxidase staining, tissues were fixed in 10% formalin and
embedded in paraffin, and thin sections were made. Paraffin was removed
with xylene and isopropyl alcohol washes followed by three 5-min rinses
in PBS-T (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4·H2O, 1.4 mM KH2PO4, 0.2% Tween 20).
Endogenous peroxidase activity was quenched with 0.3% hydrogen
peroxide in H2O. After washing three times for 5 min in
PBS-T, the slides were incubated in blocking serum, 1.5% preimmune
goat serum in PBS-T for 30 min. They were then incubated with the
primary antibody diluted 1:2000 in PBS-T overnight at 4 °C. The
primary antibody was a rabbit polyclonal antibody prepared against a
unique peptide from mouse PLRP2
(422TVQRGKDGKEFNFC435) coupled to keyhole
lympet hemocyanin. The slides were washed with PBS-T three times for 5 min each wash and processed following the directions in the
Vectastain Elite ABC Kit (Vector Laboratories, Burlingame
CA). The slides were viewed under a standard light microscope.
-Galactosidase staining was done on fresh frozen sections embedded
in O.C.T. (Sakura Finetek, Inc., Torrance CA). The sections were
air-dried and postfixed in 0.5% glutaraldehyde in PBS (PBS-T without
the Tween 20) containing 2 mM MgCl2. The slides
were then processed as described (15).
-counter. The percentage of cytotoxicity
was calculated as (experimental release 
spontaneous
release)/(maximum release spontaneous release). Spontaneous
release was less than 10% of the maximum in lipid release assays and
less than 20% of the maximum in chromium release assays.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-galactosidase
histochemistry. A polyclonal rabbit antibody against a
carboxyl-terminal peptide unique to PLRP2 was generated. This antibody
recognized purified rat PLRP2 but not purified rat PTL or PLRP1 on a
protein immunoblot.2 Sections
of pancreas from wild-type and PLRP2-deficient mice were
immunoperoxidase-stained using the anti-PLRP2 antibody as the primary
reagent. No staining was detected in the acinar cells of the
PLRP2-deficient pancreas (Fig.
2A), whereas acinar cells in
the wild-type pancreas stained with the antibody (Fig. 2B). The staining pattern was heterogeneous with some acinar cells staining
darkly and others not staining at all. The absence of PLRP2
immunoreactivity confirms that the mice containing the targeted allele
did not synthesize PLRP2 protein.
View larger version (89K):
[in a new window]
Fig. 2.
PLRP2 expression in the pancreas determined
by immunohistochemistry and -galactosidase histochemistry.
Sections of pancreas from wild-type and PLRP2-deficient adult mice were
stained as described under "Materials and Methods." A,
PLRP2-deficient pancreas stained with antibody against mouse PLRP2.
B, wild-type pancreas stained with antibody against mouse
PLRP2. C, PLRP2-deficient pancreas stained for
-galactosidase activity. The arrowhead marks an
islet.
-Galactosidase activity could also be detected in the acinar cells
of the PLRP2-deficient animals (Fig. 2C). Staining was not
present in the islets. The acinar cell-specific expression of
-galactosidase was consistent with proper targeting of the PLRP2
gene. Like the staining pattern of the antibody, -galactosidase activity varies over regions of the pancreas. Not all acinar cells stain to the same extent, demonstrating that individual acinar cells
express PLRP2 at different levels.
-galactosidase mRNA
because the targeted deletion was downstream from the region
complementary to the probe. Representative gels are shown in Fig.
3C. No expression of PTL was detected in newborn and
10-day-old mice even with overexposure of the gel (Fig. 3C,
far right part). Importantly, these results
demonstrate that the temporal pattern of expression for the genes
encoding PTL, PLRP1, and PLRP2 are the same in the mouse as in the rat and that expression of PTL and PLRP1 was not altered in the
PLRP2-deficient animals.
View larger version (64K):
[in a new window]
Fig. 3.
Temporal expression of mRNA encoding
PLRP1, PLRP2, and PTL. The levels of mRNA encoding PLRP1
(white bars), PLRP2 (gray
bars), and PTL (black bars) are shown
for wild-type pancreas (A) and for PLRP2-deficient pancreas
(B). Duplicate assays on 1 and 2 µg of total RNA were
performed for each mRNA species and each genotype. The results were
averaged and normalized for 1 µg of total RNA and to arbitrary
cyclophyllin units. C, representative RNase protection gels
are shown. The mRNA species is given below each gel, and
the age of the animal is shown above each lane in
the respective gels. The exposure times for each of the gels
is not equivalent. The third and fourth
gels show two different exposure times, 1 min (gel 3) and 15 min (gel 4), for the same PTL samples.
Stool weight of 10-day suckling mice
View larger version (17K):
[in a new window]
Fig. 4.
Fecal fat in PLRP2-deficient mice at various
ages. Feces were collected, and fats were extracted as described
under "Materials and Methods." The results are expressed as the
percentage of fat comprising the total stool dry weight. The ages of
the animals are given on the x axis. The percentage of
calories as fat is given for the adults. Black
bars, wild type; white bars,
heterozygotes; gray bars, PLRP2-deficient. The
error bars are one S.D. Heterozygote adults were
not studied. The fecal fat is significantly different for the 10-day
animals compared with the corresponding genotype in the other groups
(p < 0.05). In the 10-day group, the value for the
PLRP2-deficient mice is significantly different from the wild-type and
heterozygote animals; p < 0.0001 for both.
View larger version (58K):
[in a new window]
Fig. 5.
Separation of lipid classes by thin layer
chromatography. Feces were collected and fats extracted as
described under "Materials and Methods." Thin layer chromatography
was done in a one-dimensional two-solvent system as described under
"Materials and Methods." The fats extracted from 100 mg of dried
feces were dissolved in 1 ml of chloroform, and the indicated volume
was spotted on a TLC plate. M, standards; lanes
1-4 are fecal fats from 10-day pups. Lane
1, wild type; lane 2, heterozygote;
lane 3, PLRP2-deficient, 5.0 µl;
lane 4, PLRP2-deficient 0.5 µl; lane
5, adult wild type; lane 6, adult
PLRP2-deficient. The adults samples were collected while the animals
were eating regular chow. TG, triglyceride;
1,3-DG, diglyceride; FFA, free fatty acid;
MG, monoglyceride.
View larger version (21K):
[in a new window]
Fig. 6.
Weight gain of PLRP2-deficient mice compared
with wild-type and heterozygote mice. All pups from six different
heterozygote breeding pairs were weighed starting at 4 days of life.
Only litters of four or more pups were included. After weaning, tail
DNA was prepared, and the genotype of the animals was determined by
polymerase chain reaction as described under "Materials and
Methods." Filled circles, wild type;
open circles, heterozygotes (het);
filled inverted triangles,
PLRP2-deficient (hom). The data was analyzed by two-way
ANOVA.
View larger version (13K):
[in a new window]
Fig. 7.
Cytotoxicity in PLRP2-deficient mice.
A, wild-type (circles) and PLRP2-deficient
(diamonds) mice were immunized with P815 tumors. Ten days
after immunization, splenocytes were removed and incubated at indicated
dilutions with 51Cr-labeled target cells for 4 h.
Supernatants were collected and counted in a -counter.
Symbols represent the mean ± S.E. of triplicate
determinations and are representative of several independent
experiments. B, splenocytes from mince immunized as in
A were stimulated in vitro with mitomycin
C-treated P815 cells for 6 days. Cultures were then used as effector
cells as in A. C, in vitro cultured
splenocytes were used as effectors in a cytotoxic assay as described in
A with P815 cells that had been labeled with
[3H]oleic acid. Supernatants were collected after 3 h for scintillation counting.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-galactosidase histochemistry revealed
patchy expression of PLRP2 by acinar cells, suggesting that acinar
cells contain varying amounts of PLRP2. The notion of heterogeneous
stores of pancreatic exocrine enzymes is not new (23, 24). Earlier
studies demonstrated that enzyme contents differ among individual
zymogen granules and that enzyme secretion is nonparallel. The
heterogeneity of exocrine proteins in acinar cells provides a potential
explanation for the nonparallel, adaptive changes in relative amounts
of digestive enzymes in response to diet.
FOOTNOTES
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Alves, J. Leong, D. L. Tamang, V. Elliott, M. Lowe, and D. Hudig Hydrolysis of tumor cell lipids after CTL-mediated death Int. Immunol., May 1, 2009; 21(5): 543 - 553. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Pang, C. Li, Y. Zhao, S. Wang, W. Dong, P. Jiang, and J. Zhang The Environmental Light Influences the Circulatory Levels of Retinoic Acid and Associates with Hepatic Lipid Metabolism Endocrinology, December 1, 2008; 149(12): 6336 - 6342. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Miller and M. E. Lowe Carboxyl Ester Lipase from Either Mother's Milk or the Pancreas Is Required for Efficient Dietary Triglyceride Digestion in Suckling Mice J. Nutr., May 1, 2008; 138(5): 927 - 930. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Gilham, E. D. Labonte, J. C. Rojas, R. J. Jandacek, P. N. Howles, and D. Y. Hui Carboxyl Ester Lipase Deficiency Exacerbates Dietary Lipid Absorption Abnormalities and Resistance to Diet-induced Obesity in Pancreatic Triglyceride Lipase Knockout Mice J. Biol. Chem., August 24, 2007; 282(34): 24642 - 24649. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Eydoux, J. De Caro, F. Ferrato, P. Boullanger, D. Lafont, R. Laugier, F. Carriere, and A. De Caro Further biochemical characterization of human pancreatic lipase-related protein 2 expressed in yeast cells J. Lipid Res., July 1, 2007; 48(7): 1539 - 1549. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Carrier, G. Deblois, C. Champigny, E. Levy, and V. Giguere Estrogen-related Receptor {alpha} (ERR{alpha}) Is a Transcriptional Regulator of Apolipoprotein A-IV and Controls Lipid Handling in the Intestine J. Biol. Chem., December 10, 2004; 279(50): 52052 - 52058. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. D'Agostino and M. E. Lowe Pancreatic Lipase-related Protein 2 Is the Major Colipase-Dependent Pancreatic Lipase in Suckling Mice J. Nutr., January 1, 2004; 134(1): 132 - 134. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. W. Huggins, L. M. Camarota, P. N. Howles, and D. Y. Hui Pancreatic Triglyceride Lipase Deficiency Minimally Affects Dietary Fat Absorption but Dramatically Decreases Dietary Cholesterol Absorption in Mice J. Biol. Chem., October 31, 2003; 278(44): 42899 - 42905. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. E. Aldwell, I. G. Tucker, G. W. de Lisle, and B. M. Buddle Oral Delivery of Mycobacterium bovis BCG in a Lipid Formulation Induces Resistance to Pulmonary Tuberculosis in Mice Infect. Immun., January 1, 2003; 71(1): 101 - 108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Lowe The triglyceride lipases of the pancreas J. Lipid Res., December 1, 2002; 43(12): 2007 - 2016. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. W. Huggins, A. C. Boileau, and D. Y. Hui Protection against diet-induced obesity and obesity- related insulin resistance in Group 1B PLA2-deficient mice Am J Physiol Endocrinol Metab, November 1, 2002; 283(5): E994 - E1001. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. D'Agostino, R. A. Cordle, J. Kullman, C. Erlanson-Albertsson, L. J. Muglia, and M. E. Lowe Decreased Postnatal Survival and Altered Body Weight Regulation in Procolipase-deficient Mice J. Biol. Chem., February 22, 2002; 277(9): 7170 - 7177. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. V. Hooper, M. H. Wong, A. Thelin, L. Hansson, P. G. Falk, and J. I. Gordon Molecular Analysis of Commensal Host-Microbial Relationships in the Intestine Science, February 2, 2001; 291(5505): 881 - 884. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. N. Howles, G. N. Stemmerman, C. M. Fenoglio-Preiser, and D. Y. Hui Carboxyl ester lipase activity in milk prevents fat-derived intestinal injury in neonatal mice Am J Physiol Gastrointest Liver Physiol, September 1, 1999; 277(3): G653 - G661. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |