The intestine expresses pancreatic triacylglycerol lipase: regulation by dietary lipid

James T. Mahan, Ghanshyam D. Heda, R. Hanumantha Rao, and Charles M. Mansbach II

Department of Medicine, Division of Gastroenterology, The University of Tennessee, Memphis, Memphis 38163; and The Veterans Affairs Medical Center, Memphis, Tennessee 38104


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We identified the enzyme responsible for alkaline lipolysis in mucosa of rat small intestine. RT-PCR was used to amplify a transcript that, by cloning and sequencing, is identical to pancreatic triacylglycerol lipase. In rats fed normal laboratory chow, pancreatic triacylglycerol lipase mRNA was detected in all four quarters of the small intestine, with the first quarter expressing about three times as much of this transcript as was found in the more distal three-quarters combined. Both acutely and chronically administered dietary fat were shown to regulate pancreatic triacylglycerol lipase mRNA expression and lipase activity. The synthesis of pancreatic triacylglycerol lipase protein by the small intestine was demonstrated by in vivo radiolabeling experiments using [35S]methionine/cysteine followed by immunoprecipitation with an anti-pancreatic triacylglycerol lipase antibody. Immunohistochemical studies suggest that pancreatic triacylglycerol lipase protein expression is restricted to enterocytes throughout the small intestine. To our knowledge, this is the first report identifying rat small intestinal mucosa as a site of pancreatic triacylglycerol lipase synthesis and the first demonstration of its modulation in the mucosa by dietary fat. We propose that pancreatic triacylglycerol lipase is used by the intestine to hydrolyze the mucosal triacylglycerol that is not transported in chylomicrons.

lipid absorption


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE MAMMALIAN LIPASE GENE family includes pancreatic triglyceride (triacylglycerol) lipase (PTL), hepatic lipase, lipoprotein lipase (13), preduodenal (gastric and lingual) lipases (41), and two pancreatic lipase-related proteins (28). Although each of these lipase family members may play a role in fat metabolism, their expression varies both developmentally and by tissue. The preduodenal and pancreatic lipases are known to hydrolyze dietary fats in the lumen of the gastrointestinal tract of adult mammals. Their combined action produces monoacylglycerols (MAGs) and fatty acids that are readily absorbed by enterocytes lining the small intestine. In addition to lipases in the lumen, there is also evidence of a lipase in mammalian enterocytes (9, 38). The exact identity and origin of this intracellular form is unknown.

Our observations indicate that there are two distinct lipase, or lipase-like, enzymes in the rat small intestine (31, 37). One is maximally active under acidic conditions and the other under basic conditions. Theoretically, a number of known enzymes could explain this lipolytic activity. The possibilities include acid lipase, cholesterol esterase, microsomal triacylglycerol hydrolase, and PTL. For the following reasons, we considered PTL as the most likely candidate enzyme that could provide lipolytic activity in the intestine. 1) Lipid hydrolytic activity was expressed in the absence of bile salts, ruling out significant contribution by cholesterol esterase (bile salt-stimulated lipase; see Refs. 5 and 21). 2) Because of the pH profile at which the majority of the lipolytic activity occurred (pH 8.0-8.5; see Ref. 37), acid lipase was ruled out (pH optimum 5.6; see Ref. 30). 3) Microsomal triacylglycerol hydrolase (7) was improbable because of the cytosolic location of mucosal lipolytic activity (31). Because many of the characteristics of the observed lipase in the intestine were consistent with that of PTL, we sought to determine if PTL is synthesized in the intestine and, if so, if it is identical at the molecular level to the PTL made in the pancreas. Furthermore, because many intestinal enzymes are regulated by their substrates, we also investigated whether the mucosal lipase is responsive to acute or chronic fat loading.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and collection of mucosa. Male Sprague-Dawley rats weighing ~230-300 g were maintained on laboratory chow containing 4% fat (wt/wt) before experiments. To collect mucosa for RNA isolation or enzyme assay, the small intestine was first divided into quarters. Each quarter was rinsed two times with cold PBS, split lengthwise, and spread, mucosa up, on a chilled glass plate. The mucosa was collected by scraping with a glass slide.

In experiments to divert pancreatic fluid from reaching the intestine, the pancreatic duct was cannulated with a PE-10 cannula (Clay Adams, Parsippany, NJ). The cannula was introduced in the duodenal lumen through a stab wound in the duodenal wall made by an 18-gauge needle, threaded into the Ampulla of Vater for 2 mm, and tied in place using silk sutures. The common bile duct was cannulated with another PE-10 cannula and secured with silk ties. Both cannulas exited the rat through the right subcostal incision used to expose the duodenum and common bile duct. The pancreatic duct cannula drained clear fluid, and the bile duct cannula drained yellow fluid. A duodenal feeding cannula was also placed (PE-50) through which the rat received Vivonex as a protein and calorie source for 48 h. At the conclusion of the infusion period, the rats had only 6% of the tryptic activity in their intestinal lumens compared with noncannulated control rats.

RNA isolation. Mucosa from each quarter of the small intestine was homogenized in guanidine thiocyanate buffer, and total RNA was isolated by CsCl density gradient ultracentrifugation followed by phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1) extractions (39). RNA quality and concentration were determined by electrophoresis on agarose gels with ethidium bromide staining. Only samples with intact 28S and 18S RNA were used. mRNA was isolated from each total RNA using the Promega (Madison, WI) PolyATtract system. All mRNA samples were suspended in 100 µl of sterile nuclease-free water containing 75 units recombinant RNasin RNase inhibitor (Promega) and stored at -80°C until use.

Identification of PTL mRNA in small intestinal mucosa. First-strand cDNA was synthesized from small intestinal mRNA using RT (AMV-RT from Promega) and oligo(dT) primers. Gene-specific PTL primers, 5'-ATGCTGATGCTGTGGACATTTGCA-3' and 5'-CTAACATGCAGACAGTGTAAGCAGGAC-3' (from GenBank, accession no. M58369), were used to amplify the cDNA. The amplified DNA was ligated into the pGEM-Easy vector (Promega), which was then used to transform DH5alpha Escherichia coli. Plasmid DNA was purified from selected colonies using QIAprep and QIAtip columns (Qiagen, Valencia, CA). The plasmid insert was initially sequenced in both directions by using universal vector primers [M13(-21) forward, M13 reverse, T7, and SP6]. This generated partial sequences that were extended by using a series of internal primers to produce contiguous sequences covering the entire coding region. All sequencing was performed by the dideoxynucleotide dye terminator method. A consensus cDNA sequence, identified using DNasis software (DNasis 2.0; Hitachi Software, San Bruno CA), was found to be 99.6% identical to that reported for pancreatic PTL (pPTL) cDNA (GenBank accession no. M58369). Alignment of this consensus sequence [referred to in this report as intestinal PTL (iPTL)] with the published sequence for PTL from rat pancreas (referred to in this report as pPTL) was also accomplished using the DNasis 2.0 program.

Transfection of COS cells with iPTL transcript. The complete coding region of rat iPTL was subcloned downstream of the cytomegalovirus promoter into the eukaryotic expression vector pCR 3.1-Uni (Invitrogen, San Diego, CA). COS-7 cells were transfected using a lipofectamine procedure (Life Technologies, Gaithersburg, MD) with either the vector containing the iPTL insert or the vector alone. Expression was analyzed by measuring lipase activity in the medium and cells at 72 h using a sensitive radiometric assay that has been described in detail previously (31).

Semiquantitative comparison of iPTL mRNA levels by RT-PCR. Relative quantitation of iPTL mRNA levels was achieved by RT-PCR. First, equivalent amounts of mRNA were determined for all samples on the basis of their beta -actin mRNA levels. This was accomplished by producing beta -actin cDNA from the sample mRNAs using Promega's Access RT-PCR kit with rat beta -actin intron-spanning primers (Clontech Laboratories, Palo Alto, CA). The RT step (AMV-RT) was performed at 48°C for 45 min. Next, a two-step PCR program was used to amplify the 764-bp beta -actin sequence. The reactions were first incubated at 94°C for 2 min followed by 17 amplification cycles by using the following two-step program: 94°C for 30 s and 67°C for 90 s. This was followed by an additional elongation step of 8 min at 70°C. Finally, the beta -actin PCR product was electrophoresed and quantified (as described separately later). These experiments were performed in triplicate, and their mean values were used to determine equivalent amounts of sample mRNAs.

Using the iPTL gene-specific internal primers, iPTL/607 (5'-ACGCGGCTGAACCTTACTTCC-3') and iPTL/831 (5'-GT- CGCGAGTTCCTTCCCAGAT-3'), with a Sigma (St. Louis, MO) avian RT-PCR kit, we performed RT-PCR with equivalent amounts of mRNA as template. In these experiments, a Sigma enhanced avian myeloblastosis virus RT (AMV-RT) was used for reverse transcription at 60°C for 35 min. The reactions were then incubated at 94°C for 2 min followed by 34 amplification cycles using the following PCR program: 94°C for 30 s, 63°C for 30 s, and 69°C for 30 s. The last cycle was followed by an 8-min additional elongation step at 70°C. The 245-bp PCR product was electrophoresed and quantified (as described separately later). These experiments were performed in triplicate, and the mean values were used for comparison of samples.

A series of preliminary experiments established that, at 17 and 34 PCR cycles for actin and iPTL, respectively, the amount of RT-PCR product was linearly related to the amount of starting mRNA (Fig. 1). Control reactions were routinely conducted in which the RT step was omitted to test for genomic DNA amplification. Additional RT-PCR controls included omitting mRNA as another test for contamination (Fig. 2). To ensure the specificity of the iPTL 607/831 primer pair, the amplified product (245 bp) was cloned and sequenced. The iPTL 607/831 product was identical to the nucleotide sequence pPTL (M58369).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1.   Determination of optimal number of PCR cycles for use in semiquantitative RT-PCR experiments. In these preliminary experiments, a 10-µl aliquot was removed from each PCR tube at the completion of the indicated number of cycles. These samples were electrophoresed on agarose gels, stained with SYBR Green I, and quantitated as described in MATERIALS AND METHODS. Based on these curves, we decided to use 17 cycles for beta -actin quantitation and 34 for intestinal pancreatic triglyceride lipase (iPTL) quantitation in all subsequent experiments. A: actin; B: iPTL.



View larger version (65K):
[in this window]
[in a new window]
 
Fig. 2.   Evidence that the 245-bp band produced by RT-PCR using iPTL 607/831 primers and mRNA from the fourth quarter of the small intestine is not due to amplification of genomic DNA or a pancreatic contaminant. The mRNA samples in this experiment were obtained from rats in which only the lower part of the abdomen was opened for removal of the distal-most segment of the small intestine. This surgical approach was used to preclude any chance of contamination with pancreatic tissue. Next, RT-PCR was performed using the iPTL primers both with and without RT. The cycle number was increased to 45 to maximize the chance of detecting a faint band upon electrophoresis. Lane on right contains amplified RT-PCR product from pancreatic duct-diverted rats (see MATERIALS AND METHODS) using the same primers as in gel. The agarose gel was stained with ethidium bromide and photographed under UV illumination. Lanes 1, 4, and 8, DNA ladder with the indicated fragment sizes; lanes 2 and 3, mRNA from the fourth quarter of rat 1 (lane 2, -RT and lane 3, +RT); lanes 5 and 6, mRNA from the fourth quarter of rat 2 (lane 5, -RT and lane 6, +RT); lane 7, no RNA added, +RT.

Quantification of specific RT-PCR products. RT-PCR products were electrophoresed in 2% agarose gels in 1× Tris-borate-EDTA. The gels were soaked in SYBR Green I (Molecular Probes, Eugene OR) at 1:10,000 dilution for 3 h, and the intensity of the bands was determined by scanning laser densitometry (STORM 860 and ImageQuant software; Molecular Dynamics, Sunnyvale CA).

Affinity purification of antibody to pancreatic lipase. An IgG fraction of sheep anti-human pPTL (Biodesign International, Kennebunk, ME) was affinity purified on a column consisting of pig pPTL (Sigma) coupled to AminoLink Plus agarose beads (Pierce Chemical, Rockford, IL). By Western blotting, this anti-PTL antibody preparation identifies a single band [relative molecular mass (Mr) = 49 kDa] in extracts of rat small intestine or pancreas (data not shown).

Immunodepletion of lipolytic activity from intestinal cytosol. Intestinal cytosol was prepared (19) and treated with affinity-purified anti-pPTL antibody or an equivalent amount of sheep IgG. Cytosol (39 µg protein) was incubated with either 10 or 500 µg of lipase antibody (or control IgG) overnight at 4°C and centrifuged at 12,000 g for 15 min. The supernatant was used for the lipase assay, as previously described (31).

Synthesis of iPTL by the small intestinal mucosa. The first quarter of the small intestine in an anesthetized rat was flushed with a bolus of saline to clear the lumen of partially digested food and secretions. Ligatures were placed around the small intestine at 2 and 12 cm below the entrance of the common bile duct to prevent pancreatic secretions from reaching the intestine. Next, 5.5 µCi of [35S]methionine/cysteine were introduced into the isolated lumen. One hour later, the animal was killed, and the mucosa was collected and homogenized at 4°C in HEPES buffer containing protease inhibitors. The radiolabeled extract was precleared with protein G beads (Sigma) to reduce the concentration of proteins that might bind nonspecifically. Next, an aliquot of affinity-purified sheep anti-human pPTL IgG and fresh protein G beads was added to the precleared extract. After 1 h of incubation at 4°C the beads were collected by centrifugation and washed extensively with 1 M NaCl. The beads were boiled for 5 min in reducing SDS-PAGE sample buffer, and the resulting supernatant containing the immunoprecipitated radiolabeled protein was separated on an 8.5% SDS-PAGE. The gel was dried and placed against Kodak X-Omat film until an image developed.

iPTL immunohistochemistry. Segments of small intestine and pancreas were fixed in 10% buffered formalin and routinely processed in paraffin. Sections (8 µm) were cut and collected on poly-L-lysine-coated glass slides. The primary antibody, affinity-purified sheep anti-human pPTL IgG, was used at a final concentration of 12 µg/ml. IgG from a nonimmunized sheep was used at 33 µg/ml as a negative control. The secondary antibody was a monoclonal anti-sheep IgG peroxidase conjugate (Sigma) diluted 1:1,000. Diaminobenzidine-urea-hydrogen peroxide (Sigma) was used as the substrate for color development.

Data analysis. Contiguous nucleotide sequences were aligned and analyzed using DNasis 2.0. All primers were designed with Oligo 5.0 software (National Biosciences, Plymouth, MN). Quantitative data are expressed as means ± SE. Variation among group means was determined by one-way ANOVA and P values by the Student-Newman-Keuls test (GraphPad InStat Software, San Diego, CA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We used RT-PCR to identify and clone a PTL transcript from the intestinal mucosa. On sequencing, the PTL cDNA from rat intestine (iPTL) was found to be 99.6% identical to pPTL (GenBank accession no. M58369). The few nucleotide differences that exist are the following: 1) iPTL has a C at position 473 rather than the G reported for pPTL and 2) CT at positions 501-502 and 503-504 in iPTL rather than TC at these two positions in pPTL (Fig. 3). We sequenced through this region of iPTL cDNA eight times (four in each direction), and each time a unique internal primer was used to be certain that the sequence generated was correct.


View larger version (7K):
[in this window]
[in a new window]
 
Fig. 3.   Alignment of discrepant region of the rat intestinal cDNA sequence that we derived with the corresponding region of the published sequences for pancreatic triglyceride lipase (PTL) in rat pancreas and brain. Nonidentical nucleotides are enclosed in box. NCBI GenBank accession numbers are in parentheses.

At the amino acid level, iPTL and pPTL are 99.4% identical. The predicted amino acid sequence for iPTL differs from pPTL (M58369) as follows: 1) iPTL would have an F rather than L at position 148 and 2) YS rather than HP at positions 158-159 (Fig. 4). To determine whether the sequence we found coded for an enzymatically active protein, we transfected COS-7 cells with the full-length iPTL coding sequence and later assayed the medium and cells for lipase activity. At 72 h posttransfection, the iPTL-transfected cells had produced considerable lipase activity (2.19 µmol oleic acid released · min-1 · well-1). The amount of lipase activity secreted in the medium was nearly 17 times as much as was retained in the cells. In the mock-transfected cells (vector alone), the total amount of lipase activity (intracellular and secreted) was negligible (<0.03 µmol oleic acid released · min-1 · well-1). These data show that the iPTL sequence we found is enzymatically active even though it differs slightly from the sequence reported for pPTL (M58369).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   Predicted amino acid sequence of iPTL and comparison with other members of the lipase gene family. Differences between pancreas PTL (pPTL; M58369) and iPTL (and the residues at the same positions in the other sequences) are enclosed in boxes. The partial sequences shown here for comparison with rat iPTL are from the NCBI GenBank databases (protein or nucleotide). Accession numbers are in parentheses. Rat PTL sequences from 3 different tissues are in italics.

We then examined iPTL mRNA expression along the length of the small intestine by semiquantitative RT-PCR using a pair of primers (see MATERIALS AND METHODS) that produce a 245-bp product from approximately the middle of the full iPTL transcript. Using this approach, we found that iPTL mRNA was present in the mucosa of all four quarters of the small intestine. In rats fed normal laboratory chow, the first quarter contained ~77% of the total amount of iPTL mRNA present in the small intestine. The remaining amount was distributed over the distal three-quarters in decreasing abundance, from ~12% for the second quarter down to 5% for the fourth quarter (see chow-fed values in Fig. 7). These data demonstrate that transcription of iPTL occurs throughout the small intestine.

The finding of iPTL transcripts throughout the intestine suggests that we were not amplifying a contaminant from the pancreas. To support this contention, we obtained mucosa from 48-h pancreatic duct-diverted rats and repeated the RT-PCR for each of the four quarters of the intestine. On agarose gel electrophoresis, the amplified product migrated at the expected size for iPTL, 245 bp (shown in Fig. 2, right). In summary, the data strongly suggest that a pancreatic contaminant was not the origin of the PTL transcript that we amplified from the intestine.

To establish whether the iPTL transcript was actually translated in vivo by the intestine, we conducted metabolic radiolabeling experiments using [35S]methionine/cysteine in an isolated bowel loop. To concentrate and identify the protein(s) of interest that was radiolabeled, we immunoprecipitated the iPTL from total mucosal proteins using an affinity-purified anti-PTL antibody and identified a protein of Mr 49 kDa, which is appropriate for PTL. The results of these experiments are shown in Fig. 5. We conclude that the intestine synthesizes iPTL in situ.


View larger version (68K):
[in this window]
[in a new window]
 
Fig. 5.   Evidence for iPTL protein synthesis by the mucosa of the first quarter of the small intestine. Mucosal proteins were metabolically labeled in vivo by [35S]methionine/cysteine incorporation in an isolated bowel segment. A mucosal extract containing the newly synthesized radiolabeled proteins was immunoprecipitated with affinity-purified anti-PTL antibodies. The precipitated protein was separated by SDS-PAGE, and autoradiography was performed. Lane 1 contains the 35S-labeled whole extract of mucosa. Lane 2 contains the protein immunoprecipitated from the whole extract by our anti-PTL antibody. Arrowhead indicates a relative molecular mass (Mr) of 49 kDa, the size expected for PTL.

In other experiments, we performed immunohistochemistry to examine the distribution of iPTL protein along the length of the small intestine. These results (Fig. 6) suggest that iPTL immunoreactivity is restricted to enterocytes. The proportion of enterocytes that were iPTL positive was highest in the first quarter of the intestine. The distal three-quarters had considerably fewer enterocytes that stained for iPTL. Thus the distribution of iPTL protein as detected by immunohistochemistry is consistent with the distribution of iPTL mRNA as determined by RT-PCR. No iPTL staining was noted in the lamina propria or smooth muscle of the intestine. Also, the absence of iPTL reactivity in the intercellular spaces between enterocytes and in the basal lamina that underlies the enterocytes suggests that iPTL is not secreted basolaterally. Occasionally, a small amount of additional staining was present at the apical membrane of villus tip enterocytes, but it is not clear whether this PTL is of pancreatic or intestinal origin.


View larger version (128K):
[in this window]
[in a new window]
 
Fig. 6.   Immunohistochemical localization of PTL in the following 4 quarters of the small intestine: first quarter (a), second quarter (b), third quarter (c), and fourth quarter (d). Intense PTL immunoreactivity (brown reaction product) is present in villus enterocytes and cryptal cells. The percentage of villus enterocytes that are PTL positive is highest in the first quarter. The reaction product visible at the villus tips in the third and fourth quarters appears to be on the luminal surface of the cells and is therefore presumed to be of pancreatic origin. a-d: Magnification ×375. Adjacent sections treated with control IgG were completely negative in all four quarters. e: higher magnification (×4,000) of midvillus enterocytes from the first quarter; f: section from the first quarter treated with control IgG instead of anti-PTL IgG (×1,000); g: section from the first quarter of the intestine using anti-PTL IgG preabsorbed with porcine pPTL (see MATERIALS AND METHODS; magnification ×2,500).

Finally, we investigated the possibility that iPTL might be regulated by the amount of fat entering the small intestine. In these experiments, we used semiquantitative RT-PCR to compare the iPTL mRNA levels in the following three groups of rats: 1) rats maintained on normal chow with a fat content of 4% (wt/wt), 2) rats acutely loaded with fat by corn oil gavage, and 3) rats chronically (4 days) fed a high-fat diet (20% wt/wt). We considered the levels present in the chow-fed animals to be the baseline values for comparisons.

Our results for acute loading indicate that iPTL mRNA levels in the first quarter are significantly (P < 0.01) decreased 2.5 h after fat administration by gavage (see gavage values in Fig. 7). Means for the distal three-quarters were not different (P > 0.05) in the fat-gavaged rats compared with chow-fed animals. Recovery of iPTL mRNA levels from the observed decrease that followed the acute 2.5-h fat loading was studied at 3.5 and 17 h after the remaining fat had been removed from the stomach. We found significantly (P < 0.05) elevated levels of iPTL mRNA in all four quarters after 3.5 h of recovery compared with the same quarter of either chow-fed or fat-gavaged animals (Fig. 7). After 17 h of recovery, the iPTL levels in all quarters had returned to the baseline values exhibited by the chow-fed animals (Fig. 7).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of acute fat loading on the amount of iPTL mRNA in the small intestine. The relative amount of iPTL mRNA was determined by RT-PCR for each quarter of the small intestine. Chow-fed rats were used to establish baseline control values for comparison purposes. The remaining 3 groups all received identical 2-ml corn oil gavages followed by a 2.5-h absorption period. iPTL mRNA levels were determined either immediately after the absorption period (gavage group) or after a recovery phase of 3.5 h (Gav + 3.5 h) or 17 h (Gav + 17 h). Each point represents the mean and SE (n = 3). **First quarter in the gavage group is significantly (P < 0.05) different from the first quarter in each of the other 3 treatment groups. *Second, third, and fourth quarters in the gavage group are all different (P < 0.05) from the comparable quarters in the group that recovered for 3.5 h but are not different from the chow-fed controls or from the animals recovered for 17 h.

We have also studied the effect of acute fat loading on lipolytic activity and its relationship to iPTL mRNA levels. To more clearly interpret the lipolytic data, it was first necessary to determine the percentage of total lipolytic activity in intestinal cytosol that was due to iPTL. Affinity-purified pPTL antibody (10 µg) reduced cytosol lipolytic activity by 42%, and 500 µg of antibody reduced activity by 86%, suggesting that most of the lipolytic activity expressed by the intestine was because of iPTL. The data describing the relationship between iPTL activity and mRNA levels (Fig. 8) show that at the early recovery point (3.5 h) lipase enzymatic activity is extremely low, a finding that might be expected since the iPTL mRNA level was found to be decreased 3.5 h earlier. At 17 h postgavage recovery, the lipolytic activity had completely recovered to the baseline values of chow-fed rats (Fig. 8), presumably as a result of the dramatic increase in iPTL mRNA seen earlier at the 3.5-h recovery point.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 8.   Effects of acute fat loading on the levels of iPTL mRNA and lipolytic activity in the small intestine. Small intestine is considered as a whole rather than by quarters for the sake of simplicity. The mRNA values are derived from the data presented in Fig. 7. Lipase activity was determined by a sensitive radiometric assay (see MATERIALS AND METHODS). The treatment groups are the same as described in Fig. 7. **P < 0.01, 3.5-h recovery value is significantly different from the other treatment groups for both mRNA and lipase activity.

Because we have shown previously that acute and chronic fat feeding produces different results with respect to complex lipid synthesis (23, 24), we studied rats fed a high-fat diet for 4 days. We found that iPTL mRNA transcript abundance was not affected by chronic fat feeding in the first quarter of the intestine but that the remaining three more distal quarters showed marked increases (Fig. 9), in concert with our previous findings in the hamster (23). It should be noted that, despite the general increase in PTL transcripts, the proximal-to-distal gradient of PTL mRNA was maintained. As shown in Fig. 10, not only was the iPTL mRNA level increased in response to chronic fat feeding, but lipolytic activity was increased as well. This would be the expected result if the mRNA iPTL transcripts are translated into protein in the small intestine.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 9.   Effect of chronic fat feeding on iPTL mRNA levels in the small intestine. The relative amount of iPTL mRNA was determined by RT-PCR for each quarter of the small intestine. The fat-fed group was maintained on a mixture of their normal chow and 20% corn oil (wt/wt) for 4 days. Each bar represents the mean and SE (n = 3). *Significant difference (P < 0.05) between chow-fed and fat-fed groups for the distal three quarters; the first quarters are not different in the 2 treatment groups.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 10.   Effects of chronic fat feeding on the levels of iPTL mRNA and lipolytic activity in the small intestine. Small intestine is considered as a whole rather than by quarters for the sake of simplicity. The mRNA values are derived from the data presented in Fig. 9. Lipase activity was determined by a sensitive radiometric assay (see MATERIALS AND METHODS). The treatment groups are the same as described in Fig. 9. *Significant difference (P < 0.05) between chow-fed and fat-fed groups for both mRNA and lipase activity. FFA, free fatty acid.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These experiments were undertaken to more clearly define the origin of intestinal lipolytic activity originally found by DiNella et al. (9) and confirmed in our laboratory (29-31, 37). Although we discovered both acid and alkaline lipolytic activities to be present in the intestine (29, 31), it was the alkaline active one that appeared to be physiologically important (37). The finding of no diminution in alkaline lipase activity in mucosal cytosol after tying off the pancreatic duct for 48 h suggested that the activity originated in the intestine (37). The ability to clearly differentiate the lipase described by our laboratory from lipase of pancreatic origin (pPTL) is not a trivial point, since the activities of cholesterol esterase (12), amylase (2), and RNase (1) have all been shown to be present in the intestinal mucosa, but the majority of each protein was found to originate in the pancreas. In this report, using a molecular approach, we show that the intestine does indeed synthesize lipase locally, that this lipase is almost identical to pPTL, and that it is responsive to lipid feeding.

RT-PCR studies clearly identified a transcript in the mucosa that is identical to that reported for PTL cDNA from the pancreas. Because the pancreas expresses far more PTL than does the intestine, we were concerned that we might have amplified a pancreatic contaminant. To lessen this potential, we amplified a transcript of appropriate size for PTL from the distal quarter of the intestine, which was removed without disturbing the pancreas (described in the legend to Fig. 2). From the same animals, we also isolated skeletal muscle from the incision used to obtain the segment of intestine. In each instance with skeletal muscle, no PTL message was found. In a second approach to this point, we were unable to find in the intestine the pancreas-specific transcription factor Nkx6.1 (17, 22, 34). Third, we were able to show directly that the intestine can synthesize a protein that is immunoidentifiable as PTL and that is of the appropriate Mr. This establishes that the PTL transcript is actively translated by the intestine and provides a rationale for the finding of lipolytic activity expressed by the intestine. Indeed, our immunodepletion experiments using purified anti-PTL antibody established that most of the intestinal lipolytic activity was a result of iPTL. Last, we diverted pancreatic juice from the intestine for 48 h and found no diminution in PTL activity in the intestine; we continued to be able to isolate iPTL from the intestine. In summary, the data show that our amplified lipase transcript was most likely of intestinal and not pancreatic origin. Our data confirm and extend those of others who have found PTL outside the pancreas, including extrahepatic peribiliary gland epithelium (36), rat brain (38), ground squirrel heart (3), and Caco-2 cells, a colonocyte cell line (35).

Because the mucosa is comprised of multiple cell types, we employed immunohistochemistry to show that immunoidentifiable lipase was present specifically in the enterocytes throughout the small intestine. As shown by this technique, more PTL was present in the proximal quarter of the intestine compared with the more distal gut. These data are consistent with the lipolytic activity and the quantity of mRNA iPTL transcripts, which also demonstrated a similar proximal-to-distal gradient. These parallel data all support the synthesis of iPTL by the enterocytes of the small intestine.

The DNA sequence that we derived for iPTL differed in only minor respects in two areas from that published as M58369 in the GenBank database. The first deviation is our finding of a nucleotide sequence coding for phenylalanine (F) at amino acid residue 148 instead of leucine (L). Phenylalanine is present at this position in PTL from pig, human, nutria, and ground squirrel, whereas PTL from rat pancreas and brain and pancreas from rabbit, guinea pig, and horse have leucine at the same position (see Fig. 4). The second difference is at positions 158 and 159, where we predict tyrosine-serine (YS) rather than histidine-proline (HP). A comparison of all of the known PTL sequences (14 are known; see Fig. 4) reveals that the YS motif is highly conserved across species. Even the two PTL-related proteins (PLRP-1 and PLRP-2) in rat have the YS motif at this position. Only two do not have the YS motif: rat pancreas which has HP and guinea pig pancreas which has YP.

The differences between iPTL as reported here and rat pancreas and brain could simply be due to sequencing errors. For example, a misreading of the nucleotides CT as TC at two places (nucleotides 500-501 and 503-504) would result in the prediction of amino acids HP rather than YS (amino acids 158 and 159 in Fig. 4).

On the other hand, the three distinct nucleotide sequences may reflect PTL transcript isoforms found in vivo. This possibility could occur if only one of the transcripts is encoded by genomic DNA and the other two by posttranscriptional events such as RNA editing. Further characterization of the rat PTL gene is needed to distinguish between all of the possibilities raised. In any case, these minor changes are not likely to be of physiological importance, since they are not part of the putative lipid recognition site (G-X-S-X-G) centered around the active-site serine 169 or the lid domain at amino acid residues 238-262 (42). Our transfection results indicate that the minor differences in iPTL mRNA sequence discussed above are not sufficient to block the enzymatic activity of this lipase.

Another interesting aspect of this study is the regulation of lipase activity and mRNA by dietary fat. The iPTL response to acute lipid loading was dramatically different from chronic feeding. When oil was given by gavage, lipase activity level fell acutely and then returned to pregavage levels 17 h after oil remaining in the stomach had been removed. mRNA levels followed a similar pattern, with mRNA transcripts increasing before the increase in lipase activity, as would be expected. It is not clear why iPTL levels decreased in response to the acute lipid load. However, these data are consistent with our previous finding of a reduced lipolytic activity in the proximal intestine in response to lipid infusion (31). One potential explanation for our observation is toxic injury from the fatty acids released on hydrolysis by pPTL in the lumen of the intestine. However, the rat intestine can process large amounts of fat quickly and efficiently (27), and the rats quickly recovered to control levels of iPTL mRNA and lipolytic activity, making this speculate unlikely.

Chronically (4 days) feeding a high-lipid load increased both steady-state iPTL mRNA levels and lipolytic activity. These experiments parallel our previous data, which show that the chronic feeding of a high-fat diet increases the activity of diacylglycerol acyltransferase and lysophosphatidylcholine acyltransferase in hamsters (23). Furthermore, other studies have shown an effect of chronic fat feeding on the pancreas in which secretin was the proposed hormone associated with an increase in pPTL secretion (32). We interpret the coordinate upward regulation of iPTL mRNA and lipase activity along the whole length of the bowel to suggest that iPTL plays an important physiological role in intestinal lipid metabolism.

Two possibilities exist to explain our findings with regard to chronic fat feeding. The first involves the peptide tyrosine tyrosine (PYY), which is released from the distal small bowel when lipid reaches the ileal lumen (4, 20). An elevated PYY level in the circulation may increase iPTL expression. PYY has already been shown to increase both intestinal fatty acid binding protein mRNA (14) and apolipoprotein A-IV (18). Alternatively, free fatty acids may enter the nucleus, activate transcription factors, and thus modulate iPTL gene expression (40).

Two actions have been suggested for iPTL in the intestinal mucosa. The first was described by Tsujita et al. (38), who proposed that the reverse, i.e., acylation, activity of PTL is expressed in the intestine. In these studies, MAG was shown to be acylated by an enzyme that they called MAG acyltransferase, even though it reacted with antibodies directed against PTL. However, we predict that, under physiological conditions, hydrolytic activity would predominate, since this is the expected reaction of PTL at intracellular pH (6). In this regard, a potential physiological role for the hydrolytic activity of iPTL was recently reported from our laboratory (25). In that study, we showed that endogenous esterified acyl groups like TAG do not readily cross the endoplasmic reticulum (ER) membrane but remain on its cytosolic face. Although we showed that this triacylglycerol (TAG) could be rapidly hydrolyzed, the hydrolytic activity was provided by exogenous lipase added in vitro. However, we have previously shown that active lipolytic activity exists in mucosal homogenates (37), suggesting that lipase is active under in vivo conditions. As shown in the present study, most of the lipolytic activity in the cytosol is iPTL. We therefore propose that a function of iPTL is to release fatty acids from endogenous acyl groups that do not translocate across the ER membrane. In this manner, the free fatty acids may exit the enterocyte by the basolateral surface to be transported to the liver in the portal vein (26).

There is no evidence that intestinally expressed PTL is secreted in the intestinal lumen by the enterocytes in amounts that are physiologically meaningful. In pancreatic duct-ligated rats, [3H]glyceryltrioleate, infused into the duodenum, was poorly hydrolyzed and absorbed (R. F. Dowell and C. M. Mansbach, unpublished observations). The amount of lipid that can be absorbed under conditions of pancreatic exocrine insufficiency (30%; see Ref. 10) is consistent with the amount predicted from the action of preduodenal lipases, which are known to primarily hydrolyze acyl groups esterified at the sn-3 position of TAG (33). These data are also consistent with the steady-state percentage of TAG remaining (70%) after prolonged hydrolysis by preduodenal lipases in the rat (15). Because the pancreatic secretion of digestive enzymes, including pPTL, is almost exclusively from zymogen granules, it may be that enterocytes are unable to secrete iPTL because they lack the prerequisite zymogen granule apparatus. The intense PTL immunoreactivity (see Fig. 6) that is located near the apical aspect of the nucleus may represent aggregated iPTL that has precipitated in the slightly acidic trans-Golgi network (8, 16) and is therefore unable to be secreted. Prior work has shown that pancreatic secretory proteins, including pPTL, precipitate under these conditions in the absence of zymogen granule membranes (11). We therefore speculate that enterocytes retain the iPTL that they synthesize and that this retention is necessary if iPTL is to play an intracellular role in fat processing, as we propose.


    NOTE ADDED IN PROOF

We have sequenced rat pPTL cDNA and find that positions 500-504 are CTACT, the same as we report for intestinal iPTL and as has been reported for rat brain PTL (Gen Bank no. D88534).


    ACKNOWLEDGEMENTS

This study was supported by The Office of Research and Development Medical Research Service, the Department of Veterans Affairs, and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38760.


    FOOTNOTES

Address for reprint requests and other correspondence: C. M. Mansbach II, The Univ. of Tennessee, Memphis, 951 Court Ave., Rm. 555 Dobbs, Memphis, TN 38163 (E-mail: cmansbach{at}utmem.edu).

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.

Received 17 April 2000; accepted in final form 3 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alpers, DH, and Isselbacher KJ. Protein synthesis by rat intestinal mucosa. J Biol Chem 242: 5617-5622, 1967[Abstract/Free Full Text].

2.   Alpers, DH, and Solin M. The characterization of rat intestinal amylase. Gastroenterology 58: 833-842, 1970[ISI][Medline].

3.   Andrews, M, Squire T, Bowen C, and Rollins M. Low-temperature carbon utilization is regulated by novel gene activity in the heart of a hibernating mammal. Proc Natl Acad Sci USA 95: 8392-8397, 1998[Abstract/Free Full Text].

4.   Aponte, GW, Fink AS, Meyer JH, Tatemoto K, and Taylor IL. Regional distribution and release of peptide YY with fatty acids of different chain lengths. Am J Physiol Gastrointest Liver Physiol 249: G745-G750, 1985[Abstract/Free Full Text].

5.   Barrowman, JA, and Borgstrom B. Specificity of certain methods for the determination of pancreatic lipase. Gastroenterology 55: 601-607, 1968[ISI][Medline].

6.   Borgström, B. Influence of bile salt, pH, and time on the action of pancreatic lipase: physiological implications. J Lipid Res 5: 522-531, 1964[Abstract/Free Full Text].

7.   Coleman, RA, and Haynes EB. Differentiation of microsomal from lysosomal triacylglycerol lipase activities in rat liver. Biochim Biophys Acta 751: 230-240, 1983[ISI][Medline].

8.   Dartsch, H, Kleene R, and Kern HF. In vitro condensation-sorting of enzyme proteins isolated from rat pancreatic acinar cells. Eur J Cell Biol 75: 211-222, 1998[ISI][Medline].

9.   DiNella, RR, Meng HC, and Park CR. Properties of intestinal lipase. J Biol Chem 235: 3076-3081, 1960[ISI].

10.   Fredrikzon, B, and Blackberg L. Lingual lipase: an important lipase in the digestion of dietary lipids in cystic fibrosis. Pediatr Res 14: 1387-1390, 1980[Abstract].

11.   Freedman, SD, and Scheele GA. Regulated secretory proteins in the exocrine pancreas aggregate under conditions that mimic the trans-Golgi network. Biochem Biophys Res Commun 197: 992-999, 1993[ISI][Medline].

12.   Gallo, L, Chiang Y, Vahouny G, and Treadwell C. Localization and origin or rat intestinal cholesterol esterase determined by immunocytochemistry. J Lipid Res 21: 537-545, 1980[Abstract].

13.   Giller, T, Buchwald P, Blum-Kaelin D, and Hunziker W. Two novel human pancreatic lipase related proteins, hPLRP1 and hPLRP2: differences in colipase dependence and in lipase activity. J Biol Chem 267: 16509-16516, 1992[Abstract/Free Full Text].

14.   Hallden, G, and Aponte G. Evidence for a role of the gut hormone PYY in the regulation of intestinal fatty acid-binding protein transcripts in differentiated subpopulations of intestinal epithelial cell hybrids. J Biol Chem 272: 12591-12600, 1997[Abstract/Free Full Text].

15.   Hamosh, M, and Scow RO. Lingual lipase and its role in the digestion of dietary lipid. J Clin Invest 52: 88-95, 1973[ISI][Medline].

16.   Henkel, JR, Popovich JL, Gibson GA, Watkins SC, and Weisz OA. Selective perturbation of early endosome and/or trans-Golgi network pH but not lysosome pH by dose-dependent expression of influenza M2 protein. J Biol Chem 274: 9854-9860, 1999[Abstract/Free Full Text].

17.   Jensen, J, Serup P, Karlsen C, Nielsen T, and Madsen O. mRNA profiling of rat islet tumors reveals Nkx 6.1 as a beta -cell-specific homeodomain transcription factor. J Biol Chem 271: 18749-18758, 1996[Abstract/Free Full Text].

18.   Kalogeris, TJ, Qin X, Chey WY, and Tso P. PYY stimulates synthesis and secretion of intestinal apolipoprotein A-IV without affecting mRNA expression. Am J Physiol Gastrointest Liver Physiol 275: G668-G674, 1998[Abstract/Free Full Text].

19.   Kumar, NS, and Mansbach CM II. Determinants of triacylglycerol transport from the endoplasmic reticulum to the Golgi in intestine. Am J Physiol Gastrointest Liver Physiol 273: G18-G30, 1997[Abstract/Free Full Text].

20.   Lin, HC, Zhao X-T, and Wong H. Fat-induced ileal brake in the dog depends on peptide YY. Gastroenterology 110: 1491-1495, 1996[ISI][Medline].

21.   Lindstrom, MB, Sternby B, and Borgstrom B. Concerted action of carboxyl ester lipase and pancreatic lipase during lipid digestion in vitro: importance of the physicochemical state of the substrate. Biochim Biophys Acta 959: 178-184, 1988[ISI][Medline].

22.   Madsen, OD, Jensen J, Peterson HV, Pederson EE, Oster A, Anderson FG, Jorgensen MC, Jensen PB, Larsson LI, and Serup P. Transcription factors contributing to the pancreatic beta-cell phenotype. Horm Metab Res 29: 265-270, 1977.

23.   Mansbach, CM II. Effect of fat feeding on complex lipid synthesis in hamster intestine. Gastroenterology 68: 708-714, 1975[ISI][Medline].

24.   Mansbach, CM II. The origin of chylomicron phosphatidylcholine in the rat. J Clin Invest 60: 411-420, 1977[ISI][Medline].

25.   Mansbach, CM II, and Dowell R. The effect of increasing lipid loads on the ability of the endoplasmic reticulum to transport lipid to the Golgi. J Lipid Res 41: 605-612, 2000[Abstract/Free Full Text].

26.   Mansbach, CM, II, Dowell RF, and Pritchett D. Portal transport of absorbed lipids in the rat. Am J Physiol Gastrointest Liver Physiol 261: G530-G538, 1991[Abstract/Free Full Text].

27.   Mansbach, CM II, and Nevin P. Intracellular movement of triacylglycerols in the intestine. J Lipid Res 39: 963-968, 1998[Abstract/Free Full Text].

28.   Payne, R, Sims H, Jennens M, and Lowe M. Rat pancreatic lipase and two related proteins: enzymatic properties and mRNA expression during development. Am J Physiol Gastrointest Liver Physiol 266: G914-G921, 1994[Abstract/Free Full Text].

29.   Rao, RH, and Mansbach CM II. Acid lipase in rat intestinal mucosa: physiological parameters. Biochim Biophys Acta 1043: 273-280, 1990[ISI][Medline].

30.   Rao, RH, and Mansbach CM II. Purification and partial characterization of intestinal acid lipase. Biochim Biophys Acta 1046: 19-26, 1990[ISI][Medline].

31.   Rao, RH, and Mansbach CM II. Alkaline lipase in rat intestinal mucosa: physiological parameters. Arch Biochem Biophys 304: 483-489, 1993[ISI][Medline].

32.   Rausch, U, Rodgers K, Vasiloudes P, Kern H, and Scheele G. Lipase synthesis in the rat pancreas is regulated by secretin. Pancreas 1: 522-528, 1986[Medline].

33.   Regalska, E, Ransac S, and Verger R. Stereoselectivity of lipases II: stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases. J Biol Chem 265: 20271-20276, 1990[Abstract/Free Full Text].

34.   Sander, M, and German MS. The beta cell transcription factors and development of the pancreas. J Mol Med 75: 327-340, 1997[ISI][Medline].

35.   Spalinger, J, Seidman E, Menard D, and Levy E. Endogenous lipase activity in Caco-2 cells. Biochim Biophys Acta 1393: 119-127, 1998[ISI][Medline].

36.   Terada, T, Kitamura Y, Ashida K, Matsunaga Y, Kato M, Harada K, Morita T, Ohta T, and Nakanuma Y. Expression of pancreatic digestive enzymes in normal and pathologic epithelial cells of the human gastrointestinal system. Virchows Arch 431: 195-203, 1997[ISI][Medline].

37.   Tipton, AD, Frase S, and Mansbach CM II. The isolation and characterization of a mucosal triacylglycerol pool undergoing hydrolysis. Am J Physiol Gastrointest Liver Physiol 257: G871-G878, 1989[Abstract/Free Full Text].

38.   Tsujita, T, Miyazaka T, Tabei R, and Okuda H. Coenzyme A-independent monoacylglycerol acyltransferase from rat intestinal mucosa. J Biol Chem 271: 2156-2161, 1996[Abstract/Free Full Text].

39.   Turpen, T, and Griffith O. Rapid isolation of RNA by a guanidinium thiocyanate/cesium chloride gradient method. Biotechniques 4: 11-15, 1986.

40.   Van der Lee, K, Vork M, De Vries J, Willemsen P, Glatz J, Reneman R, Van der Vusse G, and Van Bilsen M. Long-chain fatty acid-induced changes in gene expression in neonatal cardiac myocytes. J Lipid Res 41: 41-47, 2000[Abstract/Free Full Text].

41.   Winzell, MS, Lowe ME, and Erlanson-Albertson C. Rat gastric procolipase: sequence, expression, and secretion during high-fat feeding. Gastroenterology 115: 1179-1185, 1998[ISI][Medline].

42.   Yang, Y, and Lowe ME. The open lid mediates pancreatic lipase function. J Lipid Res 41: 48-57, 2000[Abstract/Free Full Text].


Am J Physiol Gastrointest Liver Physiol 280(6):G1187-G1196