Inhibition of human pancreatic lipase by tetrahydrolipstatin: Further kinetic studies showing its reversibility

Tetrahydrolipstatin (THL, Orlistat) is a potent inhibitor of gastrointestinal lipases. Using the pH-stat technique we report that, in the absence of substrate, THL (at a molar excess of 100) inhibits rapidly (after few minutes of incubation) human pancreatic lipase (HPL). Bile salts over their critical micellar concentration (CMC) were found to accelerate the inhibition process.At variance with the generally accepted model of a covalent and quasi-irreversible acyl-lipase complex, we showed here that the inhibition of HPL could be rapidly and partially reversed in the presence of an emulsion of short- or long-chain triacylglycerols, as indicated by a kinetic reactivation process. The presence of bile salts in the incubation medium, containing THL and HPL, was found to stabilise the covalent complex as reflected by a decrease in the reactivation rate. Paradoxically, the presence of bile salts in the lipase assay enhanced this reactivation process probably by forming mixed micelles between bile salts and THL, which accelerates the deacylation phenomenon.On the basis of this kinetic study, a general model is proposed to describe the inhibition of lipases by THL in the aqueous phase as well as its partial reactivation process at the lipid–water interface.     

Human gastric lipase. The effect of amphiphiles

Human gastric lipase (HGL) activity on tributyrin emulsion was detected only in the presence of amphiphiles such as bile salts, proteins (serum albumin, beta-lactoglobulin or ovalbumin) or phosphatidylcholine. These findings are contrary to the strong inhibitory effect of amphiphiles observed on pure pancreatic lipase. To reveal HGL activity, amphiphiles should be added prior to HGL. This may prevent irreversible interfacial denaturation. HGL activity was found to be restricted to a triacylglycerol/water surface tension ranging from 8 dyn/cm to 13 dyn/cm. All amphiphiles, which decrease the interfacial tension below 8 dyn/cm, act as irreversible inhibitors of HGL in the absence and in the presence of bile salts. Our results confirm that HGL is capable of hydrolysing triacylglycerol in the presence of the physiological concentration of bile salts prevailing in the upper small intestine and in the presence of alimentary proteins. These observations could explain the high dietary lipid absorption observed under pancreatic lipase deficiency.     

Surface behaviour of bile salts and tetrahydrolipstatin at air/water and oil/water interfaces.

The surface behaviour of two bile salts, sodium deoxycholate (NaDC) and sodium taurodeoxycholate (NaTDC), as well as that of tetrahydrolipstatin (THL), a potent gastrointestinal lipase inhibitor, was studied at air/water and oil/water interfaces, using interfacial tensiometry methods. The surface behaviour of NaDC and NaTDC was comparable at both oil/water and air/water interfaces. A fairly compact interfacial monolayer of bile salts is formed well below the critical micellar concentration (CMC) and can help to explain the well-known effects of bile salts on the kinetic behaviour of pancreatic lipases. Using the Wilhelmy plate technique, the surface pressure-molecular area curves recorded with THL at the air/water interface showed a collapse point at a surface pressure of 24.5 mN.m(-1), corresponding to a molecular area of 70 A(2). Surprisingly, using the oil drop method, a limiting molecular area of 160 A(2) was found to exist at the oil/water interface. On the basis of the above data, space-filling models were proposed for bile salts and THL at air/water and oil/water interfaces.     

Digestive lipases: from three-dimensional structure to physiology

Human gastric lipase (HGL) is a lipolytic enzyme that is secreted by the chief cells located in the fundic part of the stomach. HGL plays an important role in lipid digestion, since it promotes the subsequent hydrolytic action of pancreatic lipase in duodenal lumen. Physiological studies have shown that HGL is able of acting not only in the highly acid stomach environment but also in the duodenum in synergy with human pancreatic lipase (HPL). Recombinant HGL (r-HGL) was expressed in the baculovirus/insect cell system in the form of an active protein with a molecular mass of 45 kDa. The specific activities of r-HGL were found to be similar to that of the native enzyme when tested on various triacylglycerol (TG) substrates. The 3-D structure of r-HGL was the first solved within the mammalian acid lipase family. This globular enzyme (379 residues) shows a new feature, different from the other known lipases structures, which consists of a core domain having the alpha/beta hydrolase fold and a cap domain including a putative 'lid' of 30 residues covering the active site of the lipase (closed conformation). HPL is the major lipolytic enzyme involved in the digestion of dietary TG. HPL is a 50 kDa glycoprotein which is directly secreted as an active enzyme. HPL was the first mammalian lipase to be solved structurally, and it revealed the presence of two structural domains: a large N-terminal domain (residues 1-336) and a smaller C-terminal domain (residues 337-449). The large N-terminal domain belongs to the alpha/beta hydrolase fold and contains the active site. A surface loop called the lid domain (C237-C261) covers the active site in the closed conformation of the lipase. The 3-D structure of the lipase-procolipase complex illustrates how the procolipase might anchor the lipase at the interface in the presence of bile salts: procolipase binds to the C-terminal domain of HPL and exposes the hydrophobic tips of its fingers at the opposite site of its lipase-binding domain. These hydrophobic tips help to bring N-terminal domain into close conformation with the interface where the opening of the lid domain probably occurs. As a result of all these conformational changes, the open lid and the extremities of the procolipase form an impressive continuous hydrophobic plateau, extending over more than 50 A. This surface might able to interact strongly with a lipid-water interface. The biochemical, histochemical and clinical studies as well as the 3-D structures obtained will be a great help for a better understanding of the structure-function relationships of digestive lipases.     

Effects of tetrahydrolipstatin, a lipase inhibitor, on absorption of fat from the intestine of the rat

Tetrahydrolipstatin (THL) derived by hydrogenation from lipstatin, a lipase inhibitor produced by Streptomyces toxytricini, has been shown to inhibit in vitro the activity of all three lipases secreted to the gastro-intestinal tract; gastric lipase, pancreatic lipase and carboxylester lipase (cholesterol ester hydrolase). The effects of THL on intestinal absorption of fat (transport to the thoracic duct chyle) has now been investigated after intraduodenal infusion in a rat model. Absorption of label from oleic acid when administered with monoolein in micellar bile salt solution was not affected by THL in concentrations up to 10(-4) M calculated on the volume of the aqueous phase. Absorption of free cholesterol in micellar bile salt solution of the lipolytic products of triolein; oleic acid and monoolein, is not significantly affected at a concentration of THL of 10(-4) M. Absorption of cholesterol from cholesteryl oleate under the same conditions is almost completely inhibited. The results indicate that absorption of free cholesterol is not dependent on the activity of pancreatic cholesterol ester hydrolase. The absorption of emulsified triolein was not significantly affected by 10(-5) M THL but decreased to around 30% of the controls by a concentration 10-times higher. There was no significant decrease of cholesterol absorption when administered in emulsified triolein while absorption of cholesteryl oleate was reduced at both concentrations of THL and almost completely at 10(-4) M. Radioactivity from [2-14C]THL when administered emulsified in triolein was recovered in urine, bile and thoracic duct lymph to 10-14, 8-13 and 1-3%, respectively, largely independent on dose administered. Label from [1"-14C] THL was recovered in the same amounts in lymph but much less in bile and urine indicating that the amino acid moiety has been split off early in the absorption process.     

Inhibition of dog and human gastric lipases by enantiomeric phosphonate inhibitors: a structure-activity study

The crystal structures of gastric lipases in the apo form [Roussel, A., et al. (1999) J. Biol. Chem. 274, 16995-17002] or in complex with the (R(P))-undecyl butyl phosphonate [C(11)Y(4)(+)] [Roussel, A., et al. (2002) J. Biol. Chem. 277, 2266-2274] have improved our understanding of the structure-activity relationships of acid lipases. In this report, we have performed a kinetic study with dog and human gastric lipases (DGL and HGL, respectively) using several phosphonate inhibitors by varying the absolute configuration of the phosphorus atom and the chain length of the alkyl/alkoxy substituents. Using the two previously determined structures and that of a new crystal structure obtained with the other (S(P))-phosphonate enantiomer [C(11)Y(4)(-)], we constructed models of phosphonate inhibitors fitting into the active site crevices of DGL and HGL. All inhibitors with a chain length of fewer than 12 carbon atoms were found to be completely buried in the catalytic crevice, whereas longer alkyl/alkoxy chains were found to point out of the cavity. The main stereospecific determinant explaining the stronger inhibition of the S(P) enantiomers is the presence of a hydrogen bond involving the catalytic histidine as found in the DGL-C(11)Y(4)(-) complex. On the basis of these results, we have built a model of the first tetrahedral intermediate corresponding to the tristearoyl-lipase complex. The triglyceride molecule completely fills the active site crevice of DGL, in contrast with what is observed with other lipases such as pancreatic lipases which have a shallower and narrower active site. For substrate hydrolysis, the supply of water molecules to the active site might be achieved through a lateral channel identified in the protein core.     

Modulating the activity of avian pancreatic lipases by an alkyl chain reacting with an accessible sulfhydryl group

Both turkey (TPL) and chicken (CPL) pancreatic lipases possess only one exposed sulfhydryl residue (Cystein114). After preincubation with the lipase, the sulfhydryl reagent C12 -TNB was found to be a powerful inhibitor of TPL whereas it had no effect on the CPL activity. Based on the 3D structure modelling and the molecular dynamics, the bulky dodecyl chain might hamper the lid movement of the TPL leading to the lipase inhibition upon reaction with C12 -TNB. Meanwhile, the predicted position of the C12 chain linked to Cystein114 of CPL could not block the lid opening mechanism which explains the absence of inhibition by C12 -TNB. Surprisingly, when added during the substrate hydrolysis, C12 -TNB activated the TPL but not the CPL that was slightly inhibited under these conditions. The 3D structure model generated for the open forms of C12 -TPL and C12 -CPL complexes showed that Cystein114 is still accessible and might react with C12 -TNB. Our models clearly explain the activation of TPL and the partial inhibition of CPL after the binding of the C12 chain to the enzyme.     

Role of a sulfhydryl group in gastric lipases. A binding study using the monomolecular-film technique

Native human and rabbit gastric lipases (HGL and RGL, respectively) were inactivated after modification of one sulfhydryl group/enzyme molecule. HGL and RGL were covalently labeled using 5,5'-dithiobis(2-nitro-[14C]benzoic acid) and the interaction of 2-nitro-5-thio-[14C]benzoic-acid-labeled lipases ([14C]Nbs-lipases) with monomolecular lipid films was investigated. Our results show that [14C]Nbs-lipases bind to lipid films as efficiently as native HGL or RGL. The critical surface pressure pi c and the maximal surface pressure (delta pi max) of [14C]Nbs-lipases were enhanced in comparison with those of native RGL and HGL. These changes in behavior were probably due to an increase in hydrophobicity brought about, directly or indirectly, by the binding of the Nbs radical. This chemical modification thus blocks the hydrolysis site and reinforces the hydrophobic character of the gastric lipases.     

The Effects of Serum Albumin and Related Amino Acids on Pancreatic Lipase and Bile Salts Inhibited Microbial Lipases

The activities of microbial lipases were inhibited by bile salts in a non-emulsifying assay system. To protect lipase activities from inactivation, the effects of proteins and amino acids were investigated. Bovine serum albumin (BSA) and α-lactalbumin (α-LA) stored the bile salts inhibited microbial lipases. Among N-end amino groups contained in BSA, L-histidine restored the activities of the bile salts inhibited microbial lipases. On the other hand, pancreatic lipase activity was stimulated by not only BSA, but L-histidine and L-aspartic acid as N-end amino groups of BSA and additionally accelerated it in combination with bile salts.     

In vitro lipolysis by human pancreatic lipase is specifically abolished by its inactive forms

In human adults, the enzymatic hydrolysis of dietary fat along the digestive tract is sequentially catalyzed by two main enzymes, human gastric lipase (HGL) and human pancreatic lipase (HPL). Both a chemically inhibited form of HPL as well as an inactive HPL mutant with a glycine residue substituted for its catalytic serine were found to be strong inactivators of HPL activity. In the presence of bile salts, this inhibition was clearly due to competition for colipase. We established that the chemically inhibited HPL, probably in its open conformation, had a much greater affinity for colipase than the closed native form of HPL. These inhibitory effects are quite substantial, because a 0.2-M excess of the chemically inhibited HPL form relative to HPL reduced the catalytic lipolytic activity by 50% in the presence of an equimolar amount of colipase.     

Biochemical and molecular characterization of Staphylococcus xylosus lipase

The Staphylococcus xylosus strain secretes a non-induced lipase in culture medium: S. xylosus lipase (SXL). Pure SXL is a monomeric protein (43 kDa). The 23 N-terminal amino acid residues were sequenced. This sequence is identical to that of Staphylococcus simulans lipase (SSL); in addition, it exhibits a high degree of homology with Staphylococcus aureus lipase (SAL NCTC 8530) sequences. The cloning and sequencing of gene part encoding the mature lipase shows one nucleotide difference with SSL, which corresponds to the change of one residue at a position 311. The lipase activity is maximal at pH 8.2 and 45 degrees C. SXL is able to hydrolyse triacylglycerols without chain length specificity. The specific activity of about 1900 U/mg was measured using tributyrin or triolein as substrate at pH 8.2 and at 45 degrees C in the presence of 2 mM CaCl2. In contrast to some previously characterized staphylococcal lipases, Ca2+ is not required to trigger the activity of SXL. SXL was found to be stable between pH 5 and pH 8.5. The enzyme maintains 50% of its activity after a 15-min incubation at 60 degrees C. Using tripropionin or vinyl esters as substrates, SXL does not present the interfacial activation phenomenon. Unlike many lipases, SXL is able to hydrolyse its substrate in the presence of bile salts or amphiphilic proteins. SXL is a serine enzyme, which is inhibited by THL.     

The effects of the broad-specificity lipase inhibitor, tetrahydrolipstatin, on the growth, development and survival of the larvae of Epiphyas postvittana (Walker) (Tortricidae, Lepidoptera)

The effects of the lipase inhibitor, tetrahydrolipstatin (THL), on neonate Epiphyas postvittana (Walker) (Lepidoptera, Tortricidae) larvae were investigated by feeding on control artificial diets (with and without 2% ethanol) and diets containing 2% ethanol and one of three concentrations of THL (0.011%, 0.037% and 0.11%). Small but significant reductions in growth rate, percent pupation and time to pupation were observed for larvae feeding on 2% ethanol control diet compared with standard control diet, but larger reductions in all parameters occurred with increasing THL concentration. Third instar larvae fed 0.011% THL in the diet had 40% of the midgut lipase activity in the relevant control larvae and showed up-regulation of gene expression of the gastric lipase-like family but not the pancreatic lipase-like family of midgut lipases.     

Purification and biochemical characterization of the LIP2 lipase from Yarrowia lipolytica

The LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) was obtained from two genetically modified strains with multi-copies of the lip2 gene and further purified using gel filtration and cation exchange chromatography. Four YLLIP2 isoforms were identified and subjected to N-terminal amino-acid sequencing and mass spectrometry analysis. These isoforms differed in their glycosylation patterns and their molecular masses ranged from 36,874 to 38,481 Da, whereas the polypeptide mass was 33,385 Da. YLLIP2 substrate specificity was investigated using short (tributyrin), medium (trioctanoin) and long (olive oil) chain triglyceride substrates at various pH and bile salt concentrations, and compared with those of human gastric and pancreatic lipases. YLLIP2 was not inhibited by bile salts at micellar concentrations with any of the substrates tested, and maximum specific activities were found to be 10,760+/-115 U/mg on tributyrin, 16,920+/-480 U/mg on trioctanoin and 12,260+/-700 U/mg on olive oil at pH 6.0. YLLIP2 was found to be fairly stable and still active on long chain triglycerides (1590+/-430 U/mg) at pH 4.0, in the presence of bile salts. It is therefore a good candidate for use in enzyme replacement therapy as a means of treating pancreatic exocrine insufficiency.     

Biochemical characterization, cloning, and molecular modelling of chicken pancreatic lipase

Chicken pancreatic lipase (CPL) was purified from delipidated pancreas. Pure CPL was obtained after ammonium sulphate fractionation, then DEAE-cellulose, Sephacryl S-200 gel filtration, and FPLC Mono-Q Sepharose columns. The pure lipase is a glycosylated monomer having a molecular mass of about 50kDa. The 23 N-terminal amino acid residues of CPL were sequenced. The sequence is similar to those of avian and mammalian pancreatic lipases. CPL presents the interfacial activation phenomenon tested with tripropionin or vinyl ester. When CPL was inhibited by synthetic detergent (TX-100) or amphipathic protein (BSA), simultaneous addition of bile salts and colipase was required to restore the full CPL activity. In the absence of colipase and bile salts, CPL was unable to hydrolyse tributyrin emulsion. This enzyme can tolerate, more efficiently than HPL, the accumulation of long-chain free fatty acids at the interface when olive oil emulsion was used as substrate in the absence of bile salts and colipase. The CPL activity, under these conditions, was linear whereas that of HPL decreased rapidly. Anti-TPL polyclonal antibodies cross-reacted specifically with CPL. The gene encoding the mature CPL was cloned and sequenced. The deduced amino acid sequence of the mature lipase shows a high degree of homology with the mammalian pancreatic lipases. A 3D structure model of CPL was built using the HPL structure as template. We have concluded that a slight increase in the exposed hydrophobic residues on the surface of CPL, as compared to HPL, could be responsible for a higher tolerance to the presence of long-chain free fatty acids at the lipid/water interface.     

Inactivation of gastric and pancreatic lipases by diethyl p-nitrophenyl phosphate

Reacting gastric and pancreatic lipases with mixed diethyl p-nitrophenyl phosphate/bile salt micelles resulted in a stoichiometric inactivation of these enzymes as tested on emulsified tributyroylglycerol and trioleoylglycerol as substrates. Diethyl p-nitrophenyl phosphate treated gastric lipases were also inactive on water-soluble p-nitrophenyl acetate, whereas the modified pancreatic lipase was still able to hydrolyze this water-soluble substrate. The binding of diethyl p-nitrophenyl phosphate modified pancreatic and gastric lipases to tributyroylglycerol/water interface was comparable to that of native lipases. The essential free sulfhydryl group of gastric lipases underwent no chemical changes due to the reaction with micellar diethyl p-nitrophenyl phosphate. All in all, these results indicate that, in both gastric and pancreatic lipases, the essential serine residue which was stoichiometrically labeled by this organophosphorus reagent is involved in catalysis and not in lipid binding.     

A comparative kinetic study on human pancreatic and Thermomyces lanuginosa lipases: Inhibitory effects of tetrahydrolipstatin in the presence of lipid substrates

The inhibitory effects of tetrahydrolipstatin (THL) on the hydrolytic activity of human pancreatic lipase (HPL) and T. lanuginosa lipase (TLL) on various lipidic substrates ‘poisoned’ with THL as previously described was studied, using either the pH-stat, monomolecular film or oil drop technique.Prior to adding lipase (method C), an ethanolic solution of THL was injected in a tributyrin (TC4) or a purified soybean oil (PSO) emulsion prepared in a pH-stat vessel. Under these conditions, THL was found to be a potent HPL inhibitor. After being dissolved in the pure triglyceride phase (method D), THL also strongly inhibited HPL. However, with TC4 as substrate TLL was efficiently inhibited by THL only when method C was used and not method D. The very different inhibitory effects on HPL and TLL recorded with method D and PSO as substrate were confirmed using the monomolecular film and oil drop techniques.With a monomolecular film of dicaprin (di-C10) as substrate, 1 molecule of THL embedded in 400 000 molecules of di-C10 sufficed to reduce the HPL activity to half of its initial value.HPL was therefore efficiently inhibited by THL with all the methods and substrates tested here. Paradoxically, TLL was inhibited by THL molecules transiently present in the aqueous phase and not by the THL molecules present at the triglyceride/water interface. It should therefore be stressed that the inhibitory effects of THL on each lipase depend strongly on the method and the substrate used.     

Potential human cholesterol esterase inhibitor design: benefits from the molecular dynamics simulations and pharmacophore modeling studies

Human pancreatic cholesterol esterase (hCEase) is one of the lipases found to involve in the digestion of large and broad spectrum of substrates including triglycerides, phospholipids, cholesteryl esters, etc. The presence of bile salts is found to be very important for the activation of hCEase. Molecular dynamic simulations were performed for the apoform and bile salt complexed form of hCEase using the co-ordinates of two bile salts from bovine CEase. The stability of the systems throughout the simulation time was checked and two representative structures from the highly populated regions were selected using cluster analysis. These two representative structures were used in pharmacophore model generation. The generated pharmacophore models were validated and used in database screening. The screened hits were refined for their drug-like properties based on Lipinski's rule of five and ADMET properties. The drug-like compounds were further refined by molecular docking simulation using GOLD program based on the GOLD fitness score, mode of binding, and molecular interactions with the active site amino acids. Finally, three hits of novel scaffolds were selected as potential leads to be used in novel and potent hCEase inhibitor design. The stability of binding modes and molecular interactions of these final hits were re-assured by molecular dynamics simulations.     

A highly stable Yarrowia lipolytica lipase formulation for the treatment of pancreatic exocrine insufficiency

Yarrowia lipolytica lipase has been assumed to be a good candidate for the treatment of fat malabsorption in patients with pancreatic insufficiency. Nevertheless, no systematic studies on its stability under physiological conditions pertaining to the human GI (gastrointestinal) tract have been published. Stability of various Y. lipolytica lipase powder formulations at various physiological pH values as well as the effect of digestive proteases and bile salts on enzyme activity were investigated. Results were compared with those obtained from another competing fungal lipase sourced from Candida rugosa. Among the studied formulations, Y. lipolytica lipase stabilized with gum arabic and skimmed milk powder was the most promising powder formulation. Under acidic conditions (pH 3-5), this formulation showed higher stability than those observed with the other Y. lipolytica lipase formulations and C. rugosa lipase. In addition, in the presence of gum arabic and skimmed milk powder as additives, Y. lipolytica lipase exhibited markedly higher resistance to pepsin, trypsin and chymotrypsin actions. Resistance to proteolytic degradation by digestive proteases was also by far higher than that observed with C. rugosa lipase. Similar behaviour was, however, observed when these two fungal lipases were incubated with increased concentrations of bile salts. Residual lipase activity of both fungal lipases showed a slight decrease in NaTDC (sodium taurodeoxycholate) concentration above 4 mM. Consequently, Y. lipolytica lipase formulated with gum arabic and milk powder seemed to have great potential for use as a therapeutic tool for patients with pancreatic insufficiency.     

Green tea extract (AR25) inhibits lipolysis of triglycerides in gastric and duodenal medium in vitro

In this study, we aimed to evaluate in vitro the inhibitory activity of a green tea extract (AR25 standardized at 25% catechins) on gastric and pancreatic lipase activities. We first used tributyrin as a substrate to evaluate the capability of AR25 to induce digestive lipase inhibition. Gastric lipase was totally inhibited by 40 mg AR25/g tributyrin whereas pancreatic lipase inhibition was maximum (78.8 +/- 0.7%) with 80 mg AR25/g tributyrin. We then used triolein, a long-chain triglyceride, to check whether AR25 could alter lipase activities on a physiologic substrate. AR25 60 mg/g triolein induced a dramatic inhibition of gastric lipase (96.8 +/- 0.4%) whereas pancreatic lipase activity was partially reduced (66.50 +/- 0.92%). Finally, the concerted action of gastric and pancreatic lipases was studied with an excess of enzymes to mimic the physiologic conditions observed in vivo. Incubation of AR25 with an excess of digestive lipases resulted in a drastic decrease in gastric lipolysis but the inhibitory effect on pancreatic lipase was less marked. On the whole, as compared to the control, lipolysis of triolein under the successive action of the two digestive lipases was reduced by 37 +/- 0.6% in the presence of AR25. Because a lipid/water interface is necessary for lipolysis to occur, lipid emulsification and emulsion droplet size were measured in gastric and duodenal media in the presence of AR25. In gastric and duodenal conditions, AR25 inhibited the lipid emulsification process. From these data we conclude that (1) in vitro, fat digestion is significantly inhibited by 60 mg AR25/g triolein, and (2) gastric as well as pancreatic lipase inhibition could be related to altered lipid emulsification in gastric or duodenal media. The green tea extract AR25 exhibiting marked inhibition of digestive lipases in vitro is likely to reduce fat digestion in humans.