The Lipase C-terminal Domain

A NOVEL UNUSUAL INHIBITOR OF PANCREATIC LIPASE ACTIVITY*

Laurence AyvazianDagger, Brigitte Kerfelec, Simone Granon, Edith Foglizzo, Isabelle Crenon, Christophe Dubois§, and Catherine Chapus

From the INSERM -U476 Nutrition Humaine et Lipides, 18 Avenue Mozart 13009 Marseille, France and the § Laboratoire LAPHAL-BP7-13718 Allauch cedex, France

Received for publication, November 14, 2000, and in revised form, December 19, 2000




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In vertebrates, dietary fat digestion mainly results from the combined effect of pancreatic lipase, colipase, and bile. It has been proposed that in vivo lipase adsorption on oil-water emulsion is mediated by a preformed lipase-colipase-mixed micelle complex. The main lipase-colipase binding site is located on the C-terminal domain of the enzyme. We report here that in vitro the isolated C-terminal domain behaves as a potent noncovalent inhibitor of lipase and that the inhibitory effect is triggered by the presence of micelles. Lipase inhibition results from the formation of a nonproductive C-terminal domain-colipase-micelle ternary complex, which competes for colipase with the active lipase-colipase-micelle ternary complex, thus diverting colipase from its lipase-anchoring function. The formation of such a complex has been evidenced by molecular sieving experiments. This nonproductive complex lowers the amount of active lipase thus reducing lipolysis. Preliminary experiments performed in rats show that the C-terminal domain also behaves as an inhibitor in vivo and thus could be considered a potential new tool for specifically reducing intestinal lipolysis.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In vertebrates, the digestion of dietary triacylglycerols, although initiated in the upper part of the digestive tract by preduodenal lipases, is driven to completion in the intestine by the combined effects of pancreatic lipase (EC 3.1.1.3), colipase, and bile secretion. Intestinal lipolysis proceeds through an heterogeneous catalysis involving a lipase activation step that requires the presence of pancreatic colipase to circumvent the inhibitory effect of natural amphiphilic compounds. In the intestine, these compounds are coating the oil/water interface thus impeding lipase adsorption on its substrate. The primary function of colipase is, therefore, to anchor lipase on its substrate in the presence of amphiphiles.

Pancreatic lipase is made up of a single polypeptide chain folded in two domains (1, 2). The N-terminal domain (residues 1-336) bears the catalytic triad, which is buried underneath a surface loop (the flap) controlling the access of substrate. The C-terminal domain (residues 337-449) is mainly devoted to colipase binding (3-5). The understanding of the activation process has progressed dramatically thanks to the resolution of several three-dimensional structures of both lipase and lipase-colipase complexes (1, 2, 5, 6). The motion of the flap from a closed (inactive conformation) to an open (active conformation) position unmasks the active site that consequently adopts the catalytically active configuration. Recently, Hermoso et al. (7) proposed that in vivo lipase activation results from the formation in solution of a ternary complex involving lipase, colipase, and a mixed bile lipid micelle. The complex represents the functional unit able to bind the emulsified oil droplets and to perform catalysis. The micelle is located in a cavity delineated by the C-terminal domain and colipase. Although mainly interacting with the concave face of colipase, the disc-shaped micelle is also in close contact with several segments of the lipase C-terminal domain. Thus, the strengthening of the interactions between lipase and colipase observed in the presence of micelles (7, 8) mainly occurs via the C-terminal domain of lipase.

In this paper, we investigate a new strategy to regulate specifically the pancreatic lipase activity based on the ability of the isolated C-terminal domain of lipase to behave as a functional entity toward colipase binding (9). We demonstrated that in vitro this domain behaves as a specific inhibitor of pancreatic lipase under conditions approximating the physiological ones. We also report preliminary results indicating that the C-terminal domain is able to significantly reduce lipolysis in vivo in rats.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Taurodeoxycholic acid (NaTDC),1 glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, oleic acid, L-alpha -phosphatidylcholine and tributyrin were purchased from Sigma.

Purification of Porcine Pancreatic Lipase, Colipase, and Lipase C-terminal Domain-- Porcine pancreatic lipase and colipase were purified from a pancreatic powder as previously described in Refs. 10 and 11, respectively. The C-terminal domain was obtained by limited proteolysis of porcine lipase as reported in Ref. 9. Protein concentrations were determined at 280 nm using a molecular extinction coefficient (epsilon ) of 6.65 × 104 M-1 cm-1, 0.4 × 104 M-1 cm-1 and 1.2 × 104 M-1 cm-1 for lipase, colipase, and C-terminal domain, respectively.

N-terminal Sequence Analysis-- N-terminal sequence analyses were performed by stepwise Edman degradation using a gas phase sequencer (Applied Biosystems, model 470A). The resulting phenylthiohydantoin-derivative amino acids were analyzed by high performance liquid chromatography using a C18 column (Brownlee, 5 µm, 220 × 2.1 mm inner diameter). They were eluted using a gradient from 10 to 46% methanol in 7 mM sodium acetate buffer, pH 4.84.

Mass Spectrometry-- MALDI-MS was performed on a reflection time-of-flight mass spectrometer equipped with delayed extraction (Voyager DE-RP, Perceptive Biosystems Inc.). The sample was first dissolved in 10 µl of acetic acid and then diluted with 90 µl of water. From this sample, 0.7 µl (about 4 picomoles) was directly mixed on the support with an equal volume of sinapinic matrix (saturated solution of sinapinic acid solution in 40% acetonitrile, 60% water, made 0.1% in trifluoroacetic acid). Analysis was conducted in linear mode using apomyoglobin as calibrant.

Fractionation by Gel Filtration of the Different Molecular Species-- Molecular sieving experiments were performed on an Ultrogel Aca54 column (90 × 1 cm) equilibrated in 20 mM Tris-HCl buffer, pH 7.5 containing 0.1 M NaCl and 1 mM benzamidine in the presence or in the absence of 4 mM NaTDC. Blue dextran at a concentration of 1 mg/ml was used to determine the void volume of the column. The flow rate was 6 ml/h. The volume of the fractions was 0.8 ml. The fractions were analyzed by either 12 or 15% SDS-PAGE according to Laemmli (12). To normalize data, the Kav values were calculated according to the equation, Kav = (Ve - Vo)/(Vt - Vo) where Ve, Vo, and Vt correspond to the elution volume, the void volume, and the total volume of the column, respectively.

Lipase Activity Measurements-- Lipase activity was potentiometrically determined at 25 °C using either 0.11 M emulsified tributyrin, pH 7.5 or 10 mM triolein, pH 8.5 in the presence of colipase. The lipase and colipase concentrations were in the range of 10-9 M and the lipase/colipase ratio varied from 0.9 to 1.2. One unit corresponds to the release of 1.0 µmol of fatty acid/min. The lipase activity was measured either in the absence or in the presence of increasing amounts of a stock solution of porcine lipase C-terminal domain (10-4 M). Pure or mixed bile salts were added to the assay from either a 15 mM stock solution of NaTDC or a 180 mM stock solution of a physiological mixture of bile salts (total bile salts) containing 54, 46, 22, 20, 23, and 12 mM of glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, and taurodeoxycholic acid, respectively (13).

In Vivo Experiments-- Adult male Wistar rats (Iffo-Credo, l'Arbresle, France) weighing 250-300 g were housed in wire-bottomed cages in an air-conditioned room with a 12-h light/12-h dark cycle. Water and food were supplied ad libidum for a week. Then, the rats were starved for 24 h before the experiment with only water ad libidum. Thereby, rat stomachs were free of any material.

In a first step of experiments, two groups of rats (n = 10, each) were orally given 1.5 ml of a liquid test meal consisting of a lipid emulsion containing triolein, cholesterol, lecithin (90% pure, from Lipoid (FRG), albumin, sucrose, and NaCl prepared in phosphate-buffered saline, pH 7.2 (Sigma, Table I). [carboxyl-14C]Triolein (4.13 GBq/mmol, 98% pure) was used to quantify the extent of lipolysis by measuring the total radioactivity associated with mono, di, and triolein.


                              
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Table I
Composition of the liquid test meals
The mixture was emulsified with a sonicator (Branson).

For the test group of rats, 1 mg of porcine C-terminal domain in 0.5 ml of water was added to the meal. For the control group, 0.5 ml of water was added to the meal. After 3.5 h of digestion, the rats were anesthetized with diethyl ether and then killed by total exsanguination. The small intestine was rapidly removed, and its content was collected on ice by flushing with 10 ml of ice-cold 2 mM sodium taurocholate. After acidification with HCl to inactivate pancreatic lipase and homogenization with a Potter homogenizer, 1-ml aliquots were taken for radioactivity determination. The mucosa of each intestinal segment was scraped on ice and acidified with HCl. Total lipids were extracted overnight two times with 20 volumes of chloroform/methanol (2:1 v/v). Aliquots of mucosal extracts (4 ml) were evaporated to dryness and counted for radioactivity. The cecum was clamped and opened by incision, and its content was collected after rinsing with 2-ml ice-cold normal saline. Lipids were extracted as mentioned above, and their radioactivity was determined by the liquid scintillation method. To evaluate the gastric emptying, the stomach of the rats was clamped at the cardia and pylorus sphincter and removed. Its content was collected on ice after rinsing with 2 ml of ice-cold normal saline and homogenized with a Potter. Aliquots (500 µl) were used for scintillation counting.

In a second set of experiments, the test meal was directly infused in the duodenum to avoid a potential proteolysis of the C-terminal domain in the stomach. The rats were anesthetized with ketamine-xylazine, their stomach was ligaturated, and a catheter was inserted in their duodenum. The test meal (1.5 ml) was infused into the duodenum for 30 min using a peristaltic pump. For the test group (n = 14) 0.1 mg of porcine C-terminal domain in 0.5 ml of water was mixed with the meal, whereas only 0.5 ml of water was added to the meal of the control group (n = 17). The rats were kept anesthetized during the experiment. They were killed after 2 h of digestion, and their intestinal content was analyzed as above mentioned.

Radioactivity Measurements-- 14C radioactivity was measured by dual scintillation counting in the presence of 15 ml of either Pico-Fluor 40 or Insta-Fluor liquid scintillation (Packard, France) in a scintillation spectrometer (Packard 1600 TR) with an external standard for quench correction. Results were expressed in disintegration per min (dpm).

Statistical Analysis-- The statistical significance of the results was analyzed by the one-way analysis of variance and the difference among treatment groups were assessed by Fischer's test (14). Differences associated with a p value < 0.05 were considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Molecular Properties of the Porcine C-terminal Domain-- The C-terminal domain was obtained from limited proteolysis of native porcine lipase and purified as indicated under "Experimental Procedures." SDS-PAGE analysis, amino acid composition (data not shown), N-terminal sequencing (Ala-Arg-Trp-Arg-Tyr-Lys-Val-Ser) indicated that the purified domain corresponds to the expected intact domain (Ala337-Cys445, using the porcine lipase numbering system, Ref. 5). Based on the molecular mass (12,850 Da) determined by mass spectrometry experiments, the sequence 400FIWYNNNVI408 has been corrected to 400FIWYNNVI407.

The ability of the purified domain to properly bind colipase was checked by spectrofluorometry as previously reported (9). In solution, the isolated domain was shown to display an affinity for colipase similar to that of lipase (Kd ~10-6 M).

Lipase Inhibition by the C-terminal Domain-- The influence of the C-terminal domain on the rate of hydrolysis of emulsified tributyrin by native lipase was investigated using either submicellar or supramicellar NaTDC concentrations. In all the experiments, an equimolar ratio of lipase relative to colipase was used.

As shown in Fig. 1, in the presence of NaTDC micelles, addition of the C-terminal domain leads to a noticeable inhibition of lipase activity. The inhibition efficiency, low around the NaTDC critical micellar concentration (CMC ~1 mM), increases with the NaTDC concentration up to 4 mM. Beyond this concentration, no more significant variation is observed. For each NaTDC concentration, increasing the C-terminal domain concentration results in an hyperbolic inhibition plot from which a plateau value and an apparent inhibition constant (Ki app) corresponding to 50% of the plateau value can be determined. Analysis of the inhibition parameters reported in Table II reveals that the affinity between the C-terminal domain and colipase is increased in the presence of micelles by at least one order of magnitude as compared with the affinity between both partners in the absence of micelles. As well, the plateau value varies with the micelle concentration to reach 70-75% inhibition of the lipase activity at 4 mM NaTDC.



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Fig. 1.   Inhibition of the lipase activity on emulsified tributyrin by the isolated C-terminal domain at different NaTDC concentrations. Lipase activity was potentiometrically determined at 25 °C using 0.11 M emulsified tributyrin, pH 7.5 in the presence of colipase. The lipase and colipase concentrations were in the range of 10-9 M. The lipase activity was measured in the presence of increasing amounts of a stock solution of porcine lipase C-terminal domain (10-4 M) at different NaTDC concentrations: , 1 mM; open circle , 2 mM; black-triangle, 4 mM; triangle , 6 mM; and diamond , 8 mM NaTDC.


                              
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Table II
Influence of the NaTDC concentration on the kinetic parameters of lipase inhibition by the isolated C-terminal domain
The plateau and Ki app values were determined from the inhibition plots (Fig. 1). The plateau values are expressed in percent of lipase inhibition. Ki app represents the C-terminal domain concentration required to reach 50% of the plateau value.

Lipase inhibition by the C-terminal domain can be reversed by addition of a large molar excess of colipase relative to lipase. This finding is consistent with a competition between lipase and the C-terminal domain for colipase.

Below the CMC, no significant inhibition of the lipase activity is observed unless a large amount of C-terminal domain is added. For instance, at 0.5 mM NaTDC, only 9% inhibition is observed for a C-terminal domain concentration of 2 × 10-5 M as compared with 70-75% inhibition at 4 mM NaTDC. The addition order of the C-terminal domain in the kinetic assays (before or after lipase) does not noticeably affect its inhibitory effect.

The influence of the nature of the substrate (short or long chain triglycerides) on the inhibitory efficiency of the C-terminal domain was investigated using triolein as substrate. As reported for tributyrin, the presence of micelles is required to observe a significant lipase inhibition. The plateau and Ki app values obtained for 4 mM NaTDC are close to those determined with emulsified tributyrin (Table III).


                              
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Table III
Effect of simple and mixed micelles on the kinetic parameters of lipase inhibition by the isolated C-terminal domain
The kinetic parameters of inhibition (plateau value and Ki app) were determined as reported in Table II. Inhibition of lipase activity was potentiometrically determined at 25 °C and pH 8.5 using 10 mM emulsified triolein as substrate in the presence of colipase and increasing amounts of C-terminal domain. The lipase and colipase concentrations were in the range of 10-9 M. The lipase activity was measured in the presence of simple or mixed micelles. The composition of the total bile salt mixture is described in "Experimental Procedures."

To approach the physiological conditions of intestinal lipolysis, we tested the inhibitory behavior of the C-terminal domain in the presence of different mixed micelles, using 10 mM emulsified triolein as substrate. The concentration of total bile salts (6 mM), phospholipids (0.5 mM), and/or oleic acid (5 mM) approximated concentrations found in duodenum during digestion.

Hyperbolic inhibition plots were still obtained from which the Ki app and plateau values were determined. Analysis of these parameters reveals that the composition of the micelles has no significant influence on the inhibitory efficiency of the C-terminal domain. This finding is to be correlated to a recent work showing that the size rather than the chemical nature is a critical parameter for the micelle to be accommodated in the cavity of the lipase-colipase-micelle ternary complex (15). Using 0.11 M emulsified tributyrin and mixed micelles made of either 6 mM total bile salts or 6 mM total bile salts and 0.5 mM lecithin, plateau values of inhibition ranging from 75 to 90% were observed. The Ki app is quite unaffected by the composition of the micelles (~2 × 10-7 M).

Evidence of the Formation of a C-terminal Domain-Colipase-Micelle Ternary Complex-- To demonstrate the formation of such a complex, we have performed gel filtration experiments on the C-terminal domain either alone or mixed with colipase in the absence or in the presence of NaTDC micelles. A 1.5 molar excess of C-terminal domain relative to colipase was used in the C-terminal domain/colipase mixtures to compare the chromatographic behavior of complexed and uncomplexed C-terminal domain. As shown in Fig. 2, molecular sieving of colipase/C-terminal domain mixtures, with or without NaTDC micelles, yields two peaks. In each case, using SDS-PAGE analysis, the first eluted peak was shown to contain both proteins, indicating the formation of a colipase-C-terminal domain complex, the molar excess of C-terminal domain being eluted under the second peak. However, the elution volume of the colipase-C-terminal domain complex in the presence of micelles is lower than that observed in the absence of micelles. This finding is consistent with an increase in molecular size of the complex likely caused by the binding to NaTDC micelles. By contrast, the elution volume of the C-terminal domain is not significantly affected by the presence of micelles, indicating that this domain does not interact with a micelle. The same experiments were performed with colipase and lipase. To normalize all the data, the Kav values were determined (Table IV).



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Fig. 2.   C-terminal domain-colipase interactions during gel filtration on Ultrogel AcA 54 in the absence or in the presence of NaTDC micelles. The column (90 cm × 1 cm) was equilibrated and eluted with a 20 mM Tris-HCl buffer, pH 7.6, containing 0.1 M NaCl, 1 mM benzamidine, and either no NaTDC or 4 mM NaTDC. Flow rate, 6 ml/h; fraction volume, 0.8 ml. A, elution profile of the C-terminal domain alone in the absence of NaTDC. B and C, elution profile of a mixture of colipase and C-terminal domain (1.5 molar excess of C-terminal domain) in the absence (B) or in the presence (C) of 4 mM NaTDC. Insets, 15% SDS-PAGE: lane 1, colipase; lane 2, C-terminal domain; lane 3, first peak; lane 4, second peak.


                              
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Table IV
Kav values for colipase, C-terminal domain and lipase
The Kav were calculated from the peak elution volumes using Ultrogel AcA 54 columns (90 cm ×1 cm). The columns were equilibrated and eluted at 4 °C with a 20 mM Tris/HCl buffer, pH 7.6, containing 0.1 M NaCl and 1 mM benzamidine either in the absence or in the presence of NaTDC micelles (4 mM NaTDC). The protein concentration loaded on the column was 10-5 M (volume, 0.8 ml).

Analysis of these values clearly shows that the C-terminal domain-colipase complex has a Kav value significantly lower in the presence of micelles than in the absence of micelles. Moreover, as expected from the respective molecular size of the two complexes, the lipase-colipase-micelle complex has a still lower Kav value than that of the colipase-C-terminal domain complex in the presence of micelles. These findings, together with the well known observation that colipase, in contrast to the C-terminal domain, can bind a micelle, are consistent with the formation of a ternary complex associating the C-terminal domain, colipase, and a micelle.

In Vivo Inhibition of Lipolysis by the C-terminal Domain-- Preliminary in vivo experiments were performed on rats as described under "Experimental Procedures." The porcine C-terminal domain was either given by mouth or infused in the duodenum after ligature of the stomach.

In vitro assays showed that the porcine C-terminal domain is stable for hours at pH values ranging from 2.0 to 9.5 and presents a moderate sensitivity to pepsin (data not shown). Therefore, in a first set of experiments, the C-terminal domain was mixed to the test meal and orally given to the rats. After 3.5 h of digestion, the rats were killed. A 90% gastric emptying was observed after 3.5 h of digestion for both the control rats and the rats treated with 1 mg of C-terminal domain, indicating that the presence of the C-terminal domain has no effect on the gastric emptying.

As shown in Fig. 3A, the total 14C radioactivity (associated with mono, di, and triolein) found in the intestine of the rats treated with the C-terminal domain is significantly higher than that found in the intestine of the rats of the control group. This finding is indicative of a lower extent of triolein hydrolysis in the presence of the C-terminal domain. No radioactivity was found in the cecum, as expected.



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Fig. 3.   Effect of the C-terminal domain on the extent of intestinal lipolysis, in vivo. The extent of lipolysis is expressed as the percentage of [14C]lipids (mono, di, and triolein) recovered in the total intestine relative to the ingested (A) or infused (B) [14C]triolein. A, percent of [14C] lipids recovered after 3.5 h of digestion in rats fed with test meals containing the C-terminal domain (1 mg per rat) and in control rats. The percent is calculated relative to the amount of 14C radioactivity resulting from the gastric emptying (90% of total ingested [14C]triolein). The bar is the mean ± S.E. of 10 individual values for either the control group or the C-terminal-treated group. The differences between the groups are significant (p < 0.05). B, percent of [14C]lipids recovered after 2 h of digestion in control rats and in rats for which the C-terminal domain was infused (0.1 mg per rat) into the duodenum after ligature of the stomach. The bar is the mean ± S.E. of 17 (control group) or 14 (C-terminal-treated group) individual values. The differences between the groups are significant (p < 0.05).

In the second set of experiments, the C-terminal domain was infused in the duodenum. After 2 h of digestion, the total 14C radioactivity recovered in the intestine of the rats treated with the C-terminal domain was again higher than that of the control group (Fig. 3B). It must be stressed that in this case the same extent of inhibition of lipolysis is obtained with a 10-fold lower dose of C-terminal domain (0.1 mg instead of 1 mg per rat). These results clearly demonstrate that the C-terminal domain behaves as a lipase inhibitor in vivo.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

These results clearly demonstrate that in vitro under conditions approximating the physiological conditions, the isolated C-terminal domain of lipase behaves as a potent inhibitor of the enzyme, provided either simple or mixed micelles are present. The C-terminal domain acts as a direct competitor of lipase by binding colipase thus diverting it from its lipase-anchoring function. The presence of micelles triggers the inhibitory efficiency of the C-terminal domain by increasing the affinity between both partners through supplementary interactions. This is consistent with the role of the micelle in the ternary complex described by Hermoso et al. (7). The direct binding of a micelle to the C-terminal domain-colipase complex has been shown by molecular sieving experiments. Based on the decrease of the Kav value of the C-terminal domain-colipase complex in the presence of micelles and on the well established finding that colipase possesses a specific lipase binding site distinct from its micelle binding site, it can be proposed that a ternary complex associating the C-terminal domain, colipase, and a single micelle is formed in solution. The size of the micelle rather than its chemical nature is determinant for the formation of such a complex as previously observed for the lipase-colipase-micelle ternary complex. The finding that the inhibitory effect of the C-terminal domain does not depend upon the nature of the substrate is in agreement with previous experimental data showing that the lipase C-terminal domain/colipase binding does not directly involve the lipid/water interface and further supports the assumption that the ternary complex (C-terminal domain-colipase-micelle) is formed in solution.

Therefore, we propose that this inhibitory effect is likely to proceed through the formation of an inactive C-terminal domain-colipase-micelle complex in which by analogy with the active lipase ternary complex the micelle is located in the cavity delineated by the C-terminal domain and colipase (Fig. 4).



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Fig. 4.   Schematic representation of the mechanism of lipase inhibition by the C-terminal domain. Lipase, colipase, and C-terminal domain models are based on the Calpha tracing of lipase-colipase complexes (4, 5, 7). The figure was prepared using WebLab® Viewer ProTM. The presence of the C-terminal domain decreases the amount of colipase available for lipase and thus reduces the amount of the active lipase ternary complex.

In such a model, the domain behaves as a "protein lure" toward colipase, lowering the amount of active lipase/colipase/micelle ternary complex and thus reducing intestinal fat absorption. Preliminary trials performed on rats appear promising for the use of the C-terminal domain as an inhibitor of lipolysis in vivo. A similar decrease of lipolysis was observed when the animals were treated by this domain either orally or by infusion in the duodenum after ligature of the stomach, thus implying that the C-terminal domain behaves as an effective inhibitor of lipolysis in the intestine rather than in the upper part of the digestive tract. This finding emphasizes the specificity of the domain toward the pancreatic lipase/colipase system. These results are also consistent with a noticeable resistance of the domain to proteolysis by pancreatic proteases in the intestine, as previously observed in vitro (16). Moreover, the inhibitory effect of the porcine C-terminal domain in the rat confirms previous findings showing that the lipase/colipase binding is well conserved among species (17, 18).

Therefore, these in vivo results corroborate the mode of inhibition postulated in vitro, namely the C-terminal domain behaves as a specific noncovalent inhibitor of pancreatic lipase by acting as a "protein lure" toward colipase, in contrast to active site-directed inhibitors, which can act on a large number of secretory and intracellular mammalian lipases (19, 20).

In conclusion, this work describes a new specific mild way to control the activity of the pancreatic lipase/colipase system and further emphasizes the importance of the bile micelles in lipase/colipase binding. Based on the positive preliminary in vivo results, this work might also offer promising perspectives in the health field because reducing the level of fat absorption by controlling the dietary triglyceride digestion by gastrointestinal lipases is one of the concerns of industrialized countries in the campaign against obesity and some hyperlipidemia (21).


    ACKNOWLEDGEMENTS

We thank Jacques Bonicel and Angela Guevara for performing the mass spectrometry and sequence analyses. We also acknowledge Michelle Bonneil and Melitza Maffi for their skillful assistance in the in vivo experiments. We acknowledge Gérard Pieroni for helpful discussions.


    FOOTNOTES

* This research was supported in part by Grant 930303 from the Institut National de la Santé et de la Recherche Médicale and grants from the Conseil Général des Bouches du Rhône and from the Fondation pour la Recherche Médicale.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.

Dagger Recipient of financial support from the Laboratoire LAPHAL.

To whom correspondence should be addressed. Tel.: 33 04 91 75 86 19; Fax: 33 04 91 75 15 62; E-mail: chapus@marseille.inserm.fr.

Published, JBC Papers in Press, January 11, 2001, DOI 10.1074/jbc.M010328200


    ABBREVIATIONS

The abbreviations used are: NaTDC, sodium taurodeoxycholate; CMC, critical micelle concentration; PAGE, polyacrylamide gel electrophoresis.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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