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Originally published In Press as doi:10.1074/jbc.M303422200 on August 12, 2003

J. Biol. Chem., Vol. 278, Issue 44, 42899-42905, October 31, 2003
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Pancreatic Triglyceride Lipase Deficiency Minimally Affects Dietary Fat Absorption but Dramatically Decreases Dietary Cholesterol Absorption in Mice*

Kevin W. Huggins{ddagger}, Lisa M. Camarota, Philip N. Howles, and David Y. Hui§

From the Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

Received for publication, April 2, 2003 , and in revised form, July 30, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
This study generated pancreatic triglyceride lipase (PTL)-null mice to test the hypothesis that PTL-mediated hydrolysis of dietary triglyceride is necessary for efficient dietary cholesterol absorption. The PTL–/– mice grew normally and displayed similar body weight as their PTL+/+ littermates. Plasma lipid levels between animals of various PTL genotypes were similar when they were maintained on either a basal low fat diet or a western-type high fat/high cholesterol diet. Although the lack of a functional PTL delayed fat absorption during the initial hour of feeding a bolus load of olive oil containing [3H]triolein and [14C]cholesterol, the rate of [3H]triolein absorption was similar between PTL+/+ and PTL–/– mice after the initial 1-h period. Importantly, comparison of fecal fat content revealed similar overall fat absorption efficiency between PTL+/+ and PTL–/– mice. In contrast, the PTL–/– mice displayed significant decrease in both the rate and the amount of cholesterol absorbed after a single meal. The plasma appearance of [14C]cholesterol was found to be 75% lower (p < 0.0005) in PTL–/– mice compared with PTL+/+ mice after 4 h. The total amount of [14C]cholesterol excreted in the feces was 45% higher (p < 0.0004) in PTL–/– mice compared with PTL+/+ mice over a 24-h period. These results indicate that the delayed fat digestion due to PTL deficiency results in a significant reduction in cholesterol absorption, although other enzymes in the digestive tract may compensate for the lack of PTL in PTL–/– mice in fat digestion and absorption.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The digestion and absorption of dietary lipids is a process involving a multitude of steps in several different tissues. Fat digestion begins in the stomach, with a small amount of dietary triglyceride being hydrolyzed by preduodenal lipases and peristalsis (1, 2). The undigested lipids are delivered to the intestinal lumen as crude lipid emulsion particles (3–5). These emulsion particles consist of a core of dietary triglyceride with a monolayer of phospholipids, cholesterol, and free fatty acids (1). Within the lumen of the intestine, hydrolysis of the lipid emulsion particles proceeds via the action of pancreatic enzymes. The digested products are mixed with biliary phospholipids and bile salts to form micellar structures or complexed with phospholipids in vesicles. Solubilization of the lipolytic products of fat digestion, cholesterol, and other dietary components is essential because all are minimally soluble in aqueous systems and are dependent upon the detergent properties of bile acids for dispersion in the intraluminal environment. Once solubilized, the digestion products are taken up by the enterocyte and packaged into lipoprotein particles for delivery throughout the body.

Numerous biochemical and genetic studies suggested that cholesterol absorption is a protein-mediated process. Most of these studies have focused on the process of cholesterol transport across the brush-border membrane of the enterocyte (6). The role of proteins that mediate intraluminal lipid digestion in influencing cholesterol absorption is less understood. Earlier studies have documented that the removal of the pancreas resulted in dramatic reduction of cholesterol absorption by the intestine (7). These studies suggested the importance of pancreatic lipolytic enzymes in dietary cholesterol absorption. Of these enzymes, cholesterol ester lipase (CEL)1 and Group 1B phospholipase A2 (PLA2) have been proposed to have a direct role in controlling cholesterol absorption via their enzymatic activities (8–11). However, data from CEL and PLA2 knockout mice showed that intestinal cholesterol absorption was similar to wild-type mice indicating that these enzymes do not have a major physiological role in mediating cholesterol absorption in vivo (12, 13).

In addition to CEL and PLA2, the pancreas also secretes pancreatic triglyceride lipase (PTL) in response to a lipid meal (14). This protein is a member of the lipase gene family and has been shown in association with colipase to be the primary enzyme in the digestive tract capable of hydrolyzing triglycerides in emulsified lipid particles (15). Whether PTL also participates in cholesterol absorption has not been explored. Previous in vitro experiments using an intestinally derived cell line have suggested that PTL-mediated triglyceride hydrolysis was necessary for cholesterol transport from emulsions to intestinal cells (16). Evidence supporting an in vivo role for PTL in cholesterol absorption is provided by reports of decreased cholesterol absorption in animals fed the lipase inhibitor tetrahydrolipstatin (16–18). Unfortunately, this reagent is nonspecific and is capable of inhibiting several lipases, including PTL, CEL, and other related enzymes (19, 20). Nevertheless, these results support the hypothesis that remodeling of core neutral lipids of lipid carriers may be necessary prior to absorption of dietary cholesterol from the gastrointestinal tract.

The goal of the present study is to determine the role of PTL in dietary lipid absorption in vivo. Specifically, we generated mice containing a null mutation of PTL to test the hypothesis that fat and cholesterol absorption efficiency is correlated to the efficiency of luminal lipid digestion, which is in turn dependent on the activity of PTL in the intestinal lumen.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Targeting Construct and Generation of Pancreatic Triglyceride Lipase-null Mice—A rat PTL cDNA clone was obtained from Dr. Patsy Brannon (Cornell University) and used to screen a 129 mouse genomic library made in {lambda}-DASH phage vector that was obtained from Dr. Thomas Doetschman (University of Cincinnati). A positive clone that also hybridized with probes corresponding to 5'- and 3'-flanking regions of the rat PTL gene showed that the mouse PTL gene spans over 20 kb in length. Genomic clones that hydridized with oligonucleotide primers with specific sequences for the authentic PTL gene, but not the sequences for pancreatic lipase-related protein-1 and -2 (21), were selected for propagation and further characterization.

A 4-kb BamHI-digested DNA fragment, encoding sequences from intron 1 to intron 6 of the mouse PTL gene, was obtained from the original {lambda} clone and inserted into a similarly digested PTZ18U plasmid to generate PTZ-mPTL. Restriction digest of the recombinant plasmid vector revealed the absence of an XhoI restriction site. A specific XhoI recognition sequence was introduced into exon 4 of the mouse PTL gene by the site-specific oligonucleotide-directed mutagenesis technique of Zoller and Smith (22), as described previously (23). A 1.1-kb XhoI-SalI-digested DNA fragment, containing a thymidine kinase promoter-driven neomycin-resistant gene, was then isolated from pMC1Neo-PolyA (Stratagene; Cedar Creek, TX) and inserted into the mutagenized PTZ-mPL plasmid at the unique XhoI site in exon 4 of the mouse PTL gene. This procedure resulted in a disrupted mouse PTL gene with 2.4-kb PTL sequence upstream of the insertion site and 1.6-kb PTL sequence downstream from the neomycin gene cassette insert (Fig. 1A). The insertion of the TK-neomycin-resistant gene into exon 4 resulted in the disruption of the PTL gene preceding domains required for PTL enzymatic activity (24, 25).



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  		FIG. 1.
Strategy for generating pancreatic triglyceride lipase-deficient mice. A, the schematic representations of the endogenous PTL allele, targeting construct, and mutant PTL allele is shown. The restriction enzymes indicated are PstI (P) and BamHI (B). The PTL gene contains 6 exons (filled boxes). The construct used for targeting contained a neo cassette at the BstI site in exon 4 of the 4-kb BamHI fragment. Location of primers are indicated as follows: P1, primer 1; P2, primer 2; and P3, primer 3. B, a representative polyacrylamide gel of a PCR analysis of ear punch DNA from PTL–/–, PTL+/–, and PTL+/+ mice. Arrows show the location of the 500- (mutant allele) and 250-bp (wild-type allele) bands.

 

Targeted inactivation of the PTL gene in mouse embryonic stem (ES) cells were performed using R1 ES cells as described previously (12). Approximately 5.5 x 107 ES cells in 0.5 ml of culture medium were electroporated in the presence of 3 pmol of the PTL-targeting DNA construct using an IBI GeneZapper 450. Surviving cells were placed in selection medium containing 400 µg/ml of the neomycin analog G418. Resistant colonies were picked after 10 days and expanded individually in 96-well dishes. Genomic DNA from the targeted ES cells was digested with PstI, radiolabeled with 32P, and hybridized with a 5' probe from the targeting DNA. This hybridized with a 5.4-kb fragment in wild-type DNA. Homologous recombination at the endogenous PTL gene locus yielded a positive signal at 4.7 kb due to the insertion at the PstI site present in the thymidine kinase promoter of the neomycin-resistant gene cassette (data not shown). The PTL gene-targeted ES cells were microinjected into 4.5-day blastocysts collected from C57BL/6 mice. The embryos were transferred into the uterine horn of (C57BL/6 x C3H/HeN) F1 pseudopregnant females to generate chimeric mice. Chimeras were identified by PCR. The positive chimeric mice were mated with Black Swiss mice to test for germ line transmission of the ES cell genome. Agouti pups were screened by PCR analysis. Ear punch tissue DNA was obtained by proteinase K digestion. Two µl of the DNA sample was used for PCR analysis. Primers used for the PCR (Fig. 1A) are as follows: primer 1, 5'-GCTACTGGAAGAAACGCGAC-3'; primer 2, 5'-GCCTTTGTCCTCCTCAGTTG-3'; primer 3, 5'-CTCGTCCTGCAGTTCATTCA-3'. Each reaction contained 2.5 µM of each primer, 200 µM of each dNTP (Invitrogen), and 1 unit of Taq polymerase (Invitrogen) in a total volume of 20 µl. All reactions were prepared on ice, preheated to 94 °C for 5 min, and subjected to 30 cycles of amplification consisting of 1 min at 94 °C, 1 min at 60 °C, 3 min at 72 °C, and 10 min at 72 °C. Samples were analyzed on polyacrylamide gels (Fig. 1B). The predicted size from the PCR primers is 250 bp for the wild-type exon 4 and 500 bp for the exon 4 containing the neo insert. Heterozygotes from different parents were mated to obtain mice with homozygous disruption of the PTL gene.

Animals and Diets—All animals used in these studies were backcrossed five times into the C57BL/6 background. The mouse colony was maintained in a temperature- and humidity-controlled room with a 12:12-h light-dark cycle and fed a rodent chow (LM485; Harlan-Teklad, Madison, WI) with free access to water. In some experiments, mice were fed a western-type high fat/high cholesterol diet containing 21% fat and 0.15% cholesterol by weight (TD88137, Harlan Teklad) for 4 weeks. Body weights were recorded at weaning (3 weeks of age) and again at 6 weeks of age. Food intake measurements were performed as described previously (26). All animal protocols used in this study were approved by the Institutional Animal Care and Use Committee at the University of Cincinnati.

Pancreatic Triglyceride Lipase Determination—Mice were euthanized by cervical dislocation and the abdominal cavities opened. The pancreas was removed and homogenized on ice in a solution containing 10 mM sodium phosphate, pH 6.2, 0.1 M NaCl, 1 mM EDTA, 0.02% sodium azide, 1.5% glycerol, and 0.2% soybean trypsin inhibitor (Sigma). The homogenate was centrifuged at 14,000 x g for 10 min at 4 °C. The supernatant was divided into aliquots and stored at –80 °C. Total pancreatic protein concentrations were determined by the method of Lowry et al. (27).

Pancreatic triglyceride lipase protein levels were determined by immunoblot analysis. Forty µg of pancreatic protein was separated by SDS-PAGE (10%) using the method of Laemmli (28). Protein was transferred to polyvinylidene difluoride (PVDF) membranes (0.45 µm, Amersham Biosciences) in transfer buffer (0.025 M Tris, 0.19 M glycine, 20% methanol). The PVDF membranes were then blocked with Blotto (5% non-fat dry milk in 10 mM Tris-HCl, pH 7.4, 120 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature. The primary PTL antibody, produced against a synthetic peptide corresponding to residues 38–59 of mouse PTL, was diluted 1:1000 in Blotto and incubated with the PVDF membrane for 1 h at room temperature. The blots were washed three times for 10 min each with Blotto. The secondary antibody (goat anti-mouse IgG conjugated to horseradish peroxidase; Amersham Biosciences) was diluted 1:5000 in Blotto and incubated with the PVDF membrane for 1 h at room temperature. The blots were then washed and developed using enhanced chemiluminescence (Pierce).

Triglyceride hydrolysis activity in the pancreatic extracts was determined as described previously (29). Briefly, 10 µCi of [3H]triolein (Amersham Biosciences) was dried under a stream of nitrogen in a glass tube and resuspended in 5 ml of assay buffer (30 mM Tris-HCl, pH 8.0, 1 mM CaCl2, 4 mM taurodeoxycholate) containing 1.56 µmol of triolein (final concentration = 0.312 mM). The mixture was vortexed and sonicated for 3 min to generate an emulsion. Forty five µl of the radiolabeled substrate emulsion was added to separate glass tubes on ice. Five µl of pancreatic extract from PTL+/+, PTL+/–, and PTL–/– mice were then added to each tube. Reactions were carried out for 10 min at room temperature. The reaction was stopped by adding 15 volumes of chloroform/methanol/heptane (125:140:100; v/v/v), followed by 5 volumes of 50 mM sodium carbonate, pH 10.5. The mixture was vortexed and centrifuged for phase separation. The amount of radiolabeled fatty acid liberated from [3H]triolein hydrolysis was determined by liquid scintillation counting of the aqueous phase. In selected experiments, taurodeoxycholate was replaced with taurocholate in the assay buffer.

Plasma Chemistries—Plasma was obtained by low speed centrifugation of the blood samples after a 5-h fast (beginning at 9:00 a.m.). Plasma triglyceride and cholesterol concentrations were determined using a colorimetric assay kit from Wako Chemicals (Richmond, VA).

Rate of Postprandial Fat and Cholesterol Absorption—Male mice (6–8 weeks old) consuming a low fat chow diet were fasted overnight. The following morning, the mice were injected with 12.5 mg of Triton WR-1339 (Sigma) to block lipolysis (30). Ten minutes later, the mice received an intragastric load of 100 µl of olive oil containing 1 µCi of [3H]triolein, 2 µCi of [14C]cholesterol, and 2 mg/ml cholesterol (Sigma). The mice were allowed access to water but not food during the course of the experiment. Blood samples were taken 1, 2, and 4 h post-gavage by tail bleeding to detect the appearance of radioactivity in the plasma. The mice were sacrificed by cervical dislocation at the end of the 4-h period. The small intestines were removed, divided into 4 equal pieces, and flushed with excess saline. The intestinal segments were homogenized in saline, and an aliquot was taken for radioactivity determination.

Total Cholesterol Absorption from Bolus Meal—Cholesterol absorption studies were performed on 6–8-week-old male mice consuming a basal low fat diet by using the procedure described previously (12, 31). Briefly, the mice were transferred to metabolic cages 24 h prior to the beginning of experiments. Each mouse received, by stomach gavage, a bolus test meal (100 µl) of 2 µCi of [14C]cholesterol, 2 mg/ml cholesterol, and 0.5 µCi of [3H]sitostanol (Amersham Biosciences) in olive oil or 100 µl of phospholipid vesicles prepared as described previously (13). The mice were returned to their metabolic cages, where they had free access to food and water. Feces were collected over a 24-h period. After collection, the feces were homogenized in water and then extracted with an equal volume of chloroform/methanol (2:1, v/v). An aliquot of the organic phase from each sample was used for scintillation counting to determine the amount of radioactive sterols excreted in the feces. Cholesterol absorption efficiency was determined as a percentage of the administered dose absorbed after determining the amount of nonabsorbed cholesterol present in the feces, corrected for recovery based on the amount of [3H]sitostanol present in each sample, using the formula described by Grundy et al. (32), as follows: {1 –((3H dpm)/(14C dpm) in feces)/((3H dpm)/(14C dpm) in administered dose)} x 100. In a separate set of experiments, cholesterol absorption experiments were performed as stated above, except that the fecal samples were collected for 24 or 96 h and saponified as described previously (33).

Fecal Cholesterol Excretion—Feces were collected over a 72-h period from individually housed mice fed a chow diet. The feces were combined, dried, weighed, and ground into a powder. A 1-g aliquot was saponified with 5 ml of alcoholic KOH at 65 °C for 2 h. Five ml of water were added, and lipids were extracted with 15 ml of petroleum ether containing 1.0 mg of stigmastanol (Sigma) as an internal standard. The amount of cholesterol was quantitated by gas chromatography (GC) analysis as described previously (34).

Fecal Lipid Analysis—Feces were collected from mice fed the low fat chow and western diet (4 weeks) over a 24-h period. The stool samples were dried to a constant weight in a vacuum oven, and the lipids were extracted from 100 mg of dried feces (35) and analyzed by thin layer chromatography (36, 37).

Biliary Cholesterol Output, Bile Acid Composition, and Hepatic Cholesterol Concentration—To examine biliary cholesterol output, gallbladders were cannulated as described previously (38). Briefly, mice were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg body weight; Fort Dodge Laboratories, Inc., Fort Dodge, IA) and xylazine (16 mg/kg; The Butler Co., Columbus, OH) diluted in 0.9% NaCl. An upper midline incision was made, and the gallbladder was exposed. The common bile duct was ligated, and the gallbladder was cannulated with a polyethylene catheter. Hepatic bile was collected for 1 h by gravity. Bile was stored at –20 °C until analysis. At the end of the 1-h collection period, the livers were removed, and ~250-mg samples were saponified as described above to determine hepatic cholesterol concentrations. Biliary cholesterol and hepatic cholesterol were determined by gas chromatography as described previously (34). Total and individual bile salt concentrations were determined by high pressure liquid chromatography as described previously (39).

Statistical Analysis—All results are presented as means ± S.D. Differences between genotypes were determined by Student's t test. p < 0.05 was accepted as a statistically significant difference between two groups. All statistical analysis was completed using the SigmaStat software from Jandel (San Rafael, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Appropriate gene targeting for ablation of PTL protein expression was confirmed by analysis of pancreatic homogenates from PTL+/+, PTL+/–, and PTL–/– mice for the presence of PTL. Western blot analysis indicated no PTL expression in the homogenates of the PTL–/– mice. The levels of PTL protein in pancreatic extracts of the PTL+/– mice were approximately half those of the PTL+/+ mice (Fig. 2A). When the pancreatic extracts were assayed for triglyceride hydrolytic activity, significant decreased [3H]triolein hydrolytic activity was observed in extracts of PTL+/– and PTL–/– mice compared with those of PTL+/+ mice (Fig. 2B). However, significant triglyceride hydrolytic activity was still observed in the extract of PTL-null mice when the assay was performed in the presence of taurocholate (Fig. 2B, open bars). Because CEL in the pancreas is also capable of triglyceride hydrolysis, in a manner that is dependent on the presence of trihydroxylated bile salt, [3H]triolein hydrolysis was also assayed in the presence of taurodeoxycholate instead of taurocholate to determine specifically the contribution of PTL to triglyceride hydrolytic activity in the pancreas of the genetically modified mice. The results showed that hydrolysis of radiolabeled triglyceride was decreased ~90% in the PTL–/– mice (Fig. 2B, filled bars). The PTL+/– mice had ~50% PTL activity compared with the PTL+/+ mice. These results confirmed the successful generation of PTL-deficient mice.



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  		FIG. 2.
PTL immunoblot and activity analysis. A, 40 µg of total pancreatic protein from each PTL genotype was run on a 10% SDS-polyacrylamide gel, transferred to PVDF membrane, and blotted with anti-mouse PTL. B, total pancreatic protein was analyzed for PTL activity. The enzyme reaction was carried out by incubating 5 µl of pancreatic protein from each PTL genotype with 45 µl of lipid emulsion containing 1.56 µmol of triglyceride and [3H]triolein in the presence (open bars) or absence (closed bars) of taurocholate. The reaction was carried out for 10 min at room temperature. The liberated free fatty acid was counted for radioactivity, and data were normalized for total protein. Data are means ± S.D. for 3 mice per genotype.

 

Crosses between heterozygous PTL+/– mice yielded offsprings at the expected 1:2:1 ratio of wild-type to heterozygous to homozygous knockout genotypes. There were no differences in body weight at 3 or 6 weeks of age (Fig. 3), indicating normal growth and development in these animals. Food intake studies showed no difference between PTL+/+ and PTL–/– mice (3.1 ± 0.5 versus 3.3 ± 0.2 g/day). There were also no differences in plasma triglyceride and cholesterol concentrations between PTL+/+ and PTL–/– mice when they were fed either the low fat chow or after 4 weeks of feeding the western-type high fat/high cholesterol diet (Table I). Hepatic cholesterol concentrations were similar between the two groups of mice (Table II).



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  		FIG. 3.
Body weights of male PTL+/+, PTL+/–, and PTL–/– mice. Data points represent mean ± S.D. of body weight at 3 (filled bars) and 6 (open bars) weeks of age. Mice were bred from heterozygous matings. The numbers of mice from each genotype were as follows: PTL+/+, 13; PTL+/–, 19; PTL–/–, 10. There was no difference between body weights of PTL mice at 3 or 6 weeks of age.

 

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  		TABLE I
Plasma lipid measurements in PTL+/+ and PTL–/– mice

Measurements (mg/dl) are means ± S.D. for n = 6-10 mice per group.

 

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  		TABLE II
Sterol and bile acid parameters in PTL+/+ and PTL–/– mice

Data represent the mean ± S.D. of n = 3-4 mice per PTL genotype. Biliary bile acid parameters and compositions were measured from individual hepatic bile collected during the 1-h biliary secretion studies as described under "Experimental Procedures." bw, body weight; TC, taurocholate; T-{beta}-MC, tauro-{beta}-muricholate; TCDC, taurochenodeoxycholate; TDC, taurodeoxycholate; TUDC, tauroursodeoxycholate.

 

The principal role of PTL is generally believed to be the hydrolysis of dietary fat and mediating fat absorption in the intestinal lumen. Therefore, we investigated the effect of PTL gene deletion in dietary lipid absorption efficiency. Dietary triglyceride and cholesterol absorption was assessed by determining postprandial appearance of radiolabeled lipids in plasma after supplying the animals with a fatty meal containing [3H]triolein and [14C]cholesterol by stomach gavage. Mice fed the basal low fat diet were injected with Triton WR-1339 to inhibit lipolysis and suppress lipoprotein clearance from circulation. A bolus load of olive oil containing [3H]triolein and [14C]cholesterol was then given by gastric gavage. The appearance of 3H and 14C radioactivities in plasma at various times was determined as measurements for dietary fat and cholesterol absorption, respectively. As shown in Fig. 4A, the results indicated decreased appearance of the 3H radiolabel in the plasma of PTL–/– mice compared with that observed in PTL+/+ mice. Interestingly, the difference in plasma appearance was observed only at the 1-h time point, and the rate of 3H appearance in plasma between the two groups of mice was similar after the initial 1-h period. In contrast, the rate of appearance of [14C]cholesterol in plasma was decreased at all time points in the PTL–/– mice. At 4 h, there was a 75% decrease (p < 0.0005) in the amount of [14C]cholesterol absorbed from the diet to the circulation in PTL–/– mice compared with that observed in PTL+/+ mice (Fig. 4B).



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  		FIG. 4.
Postprandial fat and cholesterol absorption. Fat and cholesterol absorption was determined by measuring the appearance of [3H]triglyceride (A) and [14C]cholesterol (B) after intragastric gavage of [3H]triolein and [14C]cholesterol in olive oil. After an overnight fast, male PTL+/+ (filled circles) and PTL–/– (open circles) were injected via the retroorbital plexus with 12.5 mg of Triton WR-1339. Ten minutes after injection, the mice received an intragastric load of 1 µCi of [3H]triolein, 2 µCi of [14C]cholesterol, and 2 mg/ml cholesterol in 100 µl of olive oil. Blood samples were taken at 1, 2, and 4 h after gavage by tail bleeding. Radioactivity appearing in plasma was determined by liquid scintillation counting. Data are expressed as means + S.D. from 8 animals in each group. *, significant difference from wild-type animals, p < 0.05.

 

The data showed that PTL-deficient mice have reduced fat absorption during the 1st h after the lipid gavage. However, comparing the slope of the fat absorption curve after the initial 1-h period indicated a similar rate of fat absorption. This observation suggested that there may be a delay in the absorption process and that fat absorption may be shifted from predominantly a proximal intestinal event in the PTL+/+ mice to a more distal site in the PTL–/– mice. To test this hypothesis, intestines were collected from the mice 4 h post-gavage, and uptake of the radiolabeled lipids in the intestinal segments was determined. The results in Fig. 5A showed that the majority of fat absorption in the PTL+/+ mice occurred in proximal intestinal segments (segments 1 and 2). In contrast, fat absorption in the PTL–/– mice was shifted to more distal segments of the intestine (segments 2 and 3), with a significant (p < 0.01) level of fat absorption occurring in the terminal ileum (segment 4) in the PTL–/– mice.



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  		FIG. 5.
Postprandial fat and cholesterol distribution in intestinal mucosa. Small intestines were removed from PTL+/+ (filled bars) and PTL–/– (open bars) mice, divided into 4 equal sections, and flushed with 19 mM taurodeoxycholate after receiving a lipid meal as described in Fig. 4. Intestinal segments were homogenized, and radioactivity was determined by liquid scintillation counting. Data are expressed as means ± S.D. from 4 animals in each group. * denotes significant difference from wild-type animals, p < 0.05.

 

When intestinal uptake of cholesterol radioactivity was determined, the PTL+/+ mice had the majority of radiolabel associated with the proximal portions of the intestine (segments 1 and 2) with a smaller amount in the distal portions (segments 3 and 4). Interestingly, uptake of radiolabeled cholesterol in the PTL–/– mice was significantly decreased in segments 1 (p < 0.003) and 2 (p < 0.04) with no changes in the distal portions of the intestine (segments 3 and 4) when compared with PTL+/+ mice.

We next investigated the overall efficiency of fat and cholesterol absorption in the PTL+/+ and PTL–/– mice. Overall fat absorption efficiency was assessed by analysis of feces collected over a 24-h period from PTL+/+ and PTL–/– mice fed the low fat and high fat diets. There were no differences in fecal fat content regardless of diet between the PTL+/+ and PTL–/– mice (Fig. 6). Fecal cholesterol concentrations were also similar between PTL+/+ and PTL–/– mice (Table II). There were also no apparent differences in the amount of various lipids between the two groups of animals when the fecal lipids were analyzed by thin layer chromatography (data not shown). To assess the overall efficiency of cholesterol absorption from a single lipid-rich meal, the amount of radiolabeled cholesterol excreted in the feces over a 24-h period after gastric infusion of a bolus dose of [14C]cholesterol in olive oil was determined. The results showed a 45% decrease (p < 0.0004) in the amount of radiolabeled cholesterol absorbed in the PTL–/– mice compared with the PTL+/+ (Fig. 7A, treatment A). The PTL+/– mice had similar cholesterol absorption efficiency as the PTL+/+ mice (63.5 ± 6.1%). Recently, Wang and Carey (33) demonstrated that cholesterol absorption studies performed without saponification may contain radiolabeled bile salts potentially overestimating cholesterol absorption values. In order to determine that our initial cholesterol absorption values were not suspect, we performed cholesterol absorption studies in which the feces were collected for 24 h or over a 96-h period and saponified with alcoholic KOH. The results obtained were very similar to our initial studies using the CHCl3/MeOH extraction procedure (Fig. 7A, comparing treatments B and C with Treatment A). These data indicated that ablation of the PTL gene results in a decreased absorption of cholesterol through the intestine with only minimal effects on dietary fat absorption.



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  		FIG. 6.
Fecal fat in PTL-deficient mice fed a low fat and high fat/high cholesterol diet. Feces were collected over a 24-h period from PTL+/+ (filled bars) and PTL–/– (open bars) mice fed a low fat chow and high fat/high cholesterol diet (4 weeks). Fat content was determined after lipid extraction, and results are expressed as percentage of fat per total stool dry weight. Data are expressed as mean ± S.D. for 4 mice per each PTL genotype.

 


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  		FIG. 7.
Cholesterol absorption from an oil emulsion and phosphatidylcholine vesicles. Male mice (6–8 weeks old) were fed by stomach gavage a 100-µl bolus lipid test meal consisting of 2 µCi of [14C]cholesterol, 0.5 µCi of [3H]sitostanol, 2 mg/ml cholesterol in olive oil (A) or phospholipid vesicles (B). Data in A represent studies in which feces were collected and analyzed by three different treatments. Treatment A, 24-h collection and lipid extraction in CHCl3/MeOH (2:1); treatment B, 24-h collection and saponification in alcoholic KOH; treatment C, 96-h collection and saponification in alcoholic KOH. The percentage of cholesterol absorbed was determined based on the amount of nonabsorbed [14C]cholesterol excreted in the feces after normalizing for recovery with the nonabsorbable sterol [3H]sitostanol. The data represent the mean ± S.D. from n = 8 PTL+/+ and n = 8 PTL–/– for mice fed olive oil in Treatment A; n = 4 PTL+/+ and n = 3 PTL–/– in Treatments B and C; or n = 4 PTL+/+ and n = 4 PTL–/– from mice fed vesicles in B. * denotes significant difference from wild-type animals, p < 0.05.

 

The decreased cholesterol absorption from a lipid-rich meal observed in PTL–/– mice is consistent with our in vitro observation that cholesterol absorption from triglyceride-rich emulsions requires prior triglyceride hydrolysis and remodeling of the cholesterol carrier (16). This hypothesis was further examined in vivo by comparing cholesterol absorption efficiency between PTL+/+ and PTL–/– mice when the radiolabeled cholesterol substrate was supplied as a phosphatidylcholine vesicle preparation that did not contain triglyceride. Interestingly, the results showed that there was no significant difference between PTL+/+ and PTL–/– mice in absorption of the radiolabeled cholesterol when the substrate was provided as a vesicular complex with phospholipids (Fig. 7B). These data demonstrated that the difference in cholesterol absorption from lipid-rich substrates between PTL+/+ and PTL–/– mice is not due to defects in cholesterol transport across the brush border membrane, but is likely due to the inaccessibility of the emulsion-bound cholesterol to be absorbed in the proximal intestine.

Additional experiments were also performed to determine whether the decrease in cholesterol absorption in the PTL–/– mice was due to increased biliary cholesterol output or altered bile acid metabolism. Flowing bile collected over a 1-h period from PTL+/+ and PTL–/– mice was analyzed for cholesterol and bile acid contents. There were no differences in the amounts of cholesterol and total bile acids secreted in the bile of these animals (Table II). When bile acid composition in the mice was analyzed (Table II), the major bile acids present were tauro-{beta}-muricholate and taurocholate, and there were no differences in their concentrations between the PTL+/+ and PTL–/– mice. However, there was a notable difference in the minor bile acids with the PTL–/– mice displaying a decrease in the amount of taurochenodeoxycholate (p < 0.003) and an increase in taurodeoxycholate (p < 0.005) when compared with the PTL+/+ mice (Table II). Although the mechanism by which PTL deficiency alters the composition of the minor bile acids is unknown at this time, importantly, the data indicate that the cholesterol/bile acid ratio in the bile juice is similar between PTL+/+ and PTL–/– mice. Thus, the decrease in cholesterol absorption in PTL–/– mice is not due to increased biliary cholesterol output or altered bile acid metabolism in these animals.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The purpose of the present study was to examine the role of PTL in dietary lipid absorption in vivo. We have shown previously (16) that PTL-mediated triglyceride hydrolysis was necessary before cholesterol can be transported from lipid emulsions to intestinally derived cells in vitro. Also, studies have shown decreased absorption of dietary fat and cholesterol in animals fed the nonspecific lipase inhibitor tetrahydrolipstatin (16–18). We therefore hypothesize that fat and cholesterol absorption efficiency is correlated to the efficiency of luminal lipid digestion, which is in turn dependent on the activity of PTL in the intestinal lumen. To test our hypothesis, we generated mice containing a null mutation of PTL. The results show that PTL-deficient mice had the following: 1) normal growth and development with no difference in body weight and plasma lipid levels; 2) delayed triglyceride absorption after feeding, but no discernible difference in overall fat absorption; and 3) a significant decrease in the rate of cholesterol absorption as well as the amount of cholesterol absorbed from a single meal.

The absorption of dietary fat is an extremely efficient process resulting in >98% of dietary triglyceride being hydrolyzed and absorbed by the intestine. This has been attributed mainly to the action of PTL in the intestinal lumen. In fact, humans with PTL and colipase deficiency suffer from steatorrhea and deficiencies of fat-soluble vitamins and essential fatty acids (40, 41). Therefore, it has generally been assumed that the high level of PTL release in response to a lipid-rich meal is sufficient to completely digest dietary fat. Interestingly, when radiolabeled triglyceride was fed to the PTL–/– mice in the form of an oil emulsion, there was a delay in postprandial appearance of [3H]triglyceride into plasma due to a decreased absorption at the 1-h time point (Fig. 4A). However, the rate of [3H]triglyceride uptake subsequent to the 1st h was similar between the two groups of mice. Additional experiments showed that the delay in fat absorption was due to a shift in [3H]triglyceride absorption from the proximal to more distal regions of the small intestine (Fig. 5A). Importantly, the delayed triglyceride absorption had no impact on the total amount of dietary triglyceride absorbed from a single meal. These data indicate that while PTL deficiency delays dietary fat absorption early in the digestive process, compensatory mechanisms exist in the intestinal lumen of PTL–/– mice that allow for triglyceride hydrolysis and the subsequent absorption of fatty acids in the absence of PTL. This was confirmed when we did not detect any increase in fecal lipids in the PTL–/– mice even when they were fed a high fat diet (Fig. 6).

The results of delayed but nearly normal overall triglyceride absorption efficiency in PTL–/– mice were quite different from the lipid absorption characteristics observed in colipase-deficient animals. Colipase is a coenzyme required for overcoming the normal inhibitory properties of bile salts, phospholipids, and dietary proteins on PTL enzymatic activity (42). Therefore, it has been assumed that dietary fat digestion is dependent upon the presence of colipase as well as PTL (15). Interestingly, mice lacking colipase had decreased postnatal survival rate, and the survivors displayed substantial steatorrhea on a high fat diet when compared with wild-type mice (43). These characteristics were not observed in the current study of PTL–/– mice (Figs. 3 and 6). The difference between PTL–/– and colipase-null mice is most likely due to the fact that PTL is not fully expressed until day 21 after birth and therefore is not required for postnatal development (21). In fact, Lowe et al. (37) showed that fat digestion in the neonate is controlled by another protein called pancreatic lipase-related protein 2. This protein is also expressed in adults, albeit at a lower level (21), suggesting that the pancreatic lipase-related protein 2 may account for the nearly normal fat digestion and absorption efficiency observed in the PTL–/– mice. Because the enzymatic activity of pancreatic lipase-related protein 2 is also dependent on colipase, the difference in fat absorption efficiency between PTL–/– and colipase-null mice is consistent with this hypothesis.

Another possible candidate for triglyceride hydrolysis in the intestinal lumen in the absence of PTL is CEL. This lipolytic enzyme is capable of hydrolyzing cholesteryl esters, phospholipids, lysophospholipids, and triacylglycerol (44, 45). When pancreatic extracts were assayed for triglyceride hydrolytic activity from the PTL–/– mice in the presence of taurodeoxycholate (which does not activate CEL activity), minimal triglyceride hydrolytic activity was observed. However, when taurodeoxycholate was replaced with taurocholate, a natural activator of CEL activity, there was an increase in triglyceride hydrolysis observed in all animals, especially in the PTL–/– mice (Fig. 2B). Based on this observation, we speculate that CEL is another major enzyme that can account for the triglyceride hydrolysis in the intestinal lumen of PTL–/– mice. Although CEL activity may be sufficient to mediate triglyceride absorption in the absence of PTL, it must be noted that the role of CEL and pancreatic lipase-related protein 2 as compensatory enzymes for PTL may not be mutually exclusive as both of these enzymes may serve similar roles. The cross-breeding of PTL-deficient mice generated in this study with CEL-deficient mice previously produced in our laboratory (12) and with the pancreatic lipase-related protein-2-deficient mice (37) will allow us to examine directly the relative contribution of each of these digestive enzymes in dietary fat absorption.

In contrast to the effects of PTL deficiency on fat absorption, cholesterol was markedly influenced by the absence of PTL. When cholesterol absorption was measured, there was a decrease in the rate of the plasma appearance of radiolabeled cholesterol in the PTL–/– mice over a 4-h period compared with the PTL+/+ mice (Fig. 4B). Moreover, the total amount of cholesterol absorbed from a single fatty meal was also decreased in the absence of PTL (Fig. 7A). Consistent with results reported by other investigators (46, 47), our data showed that dietary cholesterol is absorbed predominantly in the proximal intestine of wild-type mice. The lack of PTL-mediated triglyceride hydrolysis resulted in the inaccessibility of the dietary cholesterol in lipid emulsions for absorption by the proximal gut. Although compensatory enzyme(s) can assist in triglyceride digestion in the PTL–/– mice, the reduced digestive efficiency prolonged the digestion process, delaying fat absorption to the distal portion of the intestine. However, cholesterol liberated from lipid emulsions after triglyceride hydrolysis in the distal intestine is less efficiently taken up by the distal intestine and is excreted into feces as nonabsorbed cholesterol.

The precise mechanism governing the difference in cholesterol absorption efficiency between proximal and distal gut remains to be determined. This may be related to cholesterol solubility in the lumen of proximal versus distal intestine. The partitioning of cholesterol into bile salt-containing micelles has been shown to be necessary for cholesterol entry into the mucosa (48, 49). However, bile salts are primarily reabsorbed by a receptor-mediated process in the ileum (50, 51). Thus, cholesterol liberated from emulsions after lipid digestion in the distal gut is expected to be less well solubilized than cholesterol in the proximal intestine and is unlikely to be absorbed. This hypothesis is supported by results of Tsuchiya et al. (52) who studied the effects of ileal transposition into the upper jejunum on lipid absorption. These authors found that ileal transposition decreased absorption of cholesterol because of the premature absorption of bile salts. In addition, there may also be an alternative hypothesis, including possible difference in expression of protein(s) responsible for cholesterol absorption in proximal versus distal regions of the intestine. The distinction between these possibilities will require additional experimentation and the identification of the putative cholesterol transporter in intestine.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Program Project Grant DK54504 (to D. Y. H.) and National Research Service Award DK10065 (to K. W. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Dept. of Nutrition and Food Science, 328 Spidle Hall, Auburn University, Auburn, AL 36849. Back

§ To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0529. Tel.: 513-558-9152; Fax: 513-558-2141; E-mail: huidy{at}email.uc.edu.

1 The abbreviations used are: CEL, carboxyl ester lipase (cholesterol esterase); PLA2, phospholipase A2; PTL, pancreatic triglyceride lipase; ES cells, embryonic stem cells; PVDF, polyvinylidene difluoride. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Laura Woollett and Patrick Tso for valuable discussions and Dr. Norman Granholm and Scott Street for mouse genotype analysis. Nick Schildmeyer, Tara Riddle, and Heather Branam provided excellent technical assistance to this study.



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 RESULTS
 DISCUSSION
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