The lipase inhibitor tetrahydrolipstatin binds covalently to the putative active site serine of pancreatic lipase

Tetrahydrolipstatin (THL) is a selective inhibitor of fat absorption. In animal models, it has anti-obesity and anti-hypercholesterolemic activity and is presently in clinical trials for these indications. THL binds covalently to pancreatic lipase. Complete inhibition of lipolytic activity is obtained concomitant with the incorporation of 1 mol of THL/mol of enzyme. Pancreatic lipase is the best studied lipase, but published results concerning its catalytic mechanism are still controversial. In order to learn more about the inhibitory mechanism of THL, a selective lipase inhibitor interacting at or near the catalytic site, and therefore, to obtain more information on the catalytic mechanism of lipase, we have determined the amino acid residue to which THL is bound. After proteolytic degradation of porcine pancreatic lipase inhibited with radioactively labeled THL, the labeled peptides were isolated and analyzed by quantitative amino acid analysis, N-terminal sequencing, and by mass spectrometry with fast atom bombardment ionization. The data clearly show that THL is bound as an ester to the serine 152 of the lipase.     

Mode of action of tetrahydrolipstatin: a derivative of the naturally occurring lipase inhibitor lipstatin

Tetrahydrolipstatin is a specific lipase inhibitor derived from lipstatin, a lipid produced by Streptomyces toxytricini. In addition to pancreatic lipase, it is shown in the present study that tetrahydrolipstatin also inhibits human gastric lipase, carboxyl ester lipase (cholesterol esterase) of pancreatic origin and the closely related bile-salt-stimulated lipase of human milk. It does not inhibit the exocellular lipase from Rhizopus arrhizus or a lipase recently isolated from Staphylococcus aureus. In the presence of a water-insoluble substrate, such as tributyrin, the inhibition has the characteristics of an irreversible inactivation of the uncompetitive type, thus indicating that an enzyme.substrate.inhibitor complex is formed, which cannot undergo further reaction to yield the normal product. This reaction probably takes place at the aqueous/oil interface of the substrate. In aqueous solution, in the absence of substrate, the inhibition of carboxyl ester lipase by tetrahydrolipstatin has the characteristics of being reversible, and finally becomes of a temporary nature analogues to the trypsin-trypsin inhibitor system. It is suggested that an enzyme-inhibitor complex of an acyl-enzyme type is formed that is slowly hydrolysed, with water as the final acceptor, leaving an intact enzyme and an inactive form of the inhibitor. The enzyme thus consumes the inhibitor, which undergoes a chemical conversion, as indicated by a change in mobility in an appropriate thin-layer chromatographic system, indicating an increase in hydrophilicity. Evidence is presented that the reaction product is an acid and that the functional group of tetrahydrolipstatin is the beta-lactone reacting with the active site of the enzyme.     

Inhibition of pancreatic lipase in vitro by the covalent inhibitor tetrahydrolipstatin.

Tetrahydrolipstatin inhibits pancreatic lipase from several species, including man, with comparable potency. The lipase is progressively inactivated through the formation of a long-lived covalent intermediate, probably with a 1:1 stoichiometry. The lipase substrate triolein and also a boronic acid derivative, which is presumed to be a transition-state-form inhibitor, retard the rate of inactivation. Therefore, in all probability, tetrahydrolipstatin reacts with pancreatic lipase at, or near, the substrate binding or active site. Tetrahydrolipstatin is a selective inhibitor of lipase; other hydrolases tested were at least a thousand times less potently inhibited.     

Effects of the lipase inhibitors, Triton WR-1339 and tetrahydrolipstatin, on the synthesis and secretion of lipids by rat hepatocytes

The lipase inhibitors, Triton WR-1339 and tetrahydrolipstatin, were incubated with rat hepatocytes. Triton WR-1339 increased the recovery of triacylglycerol in the hepatocytes and incubation medium by 31% and 38%, respectively. Tetrahydrolipstatin decreased the accumulation of newly synthesized, and of total triacylglycerol in the medium. This compound might be useful in determining mechanisms involved in intracellular triacylglycerol metabolism and the secretion of very low density lipoproteins.     

Electrical charge on protein regulates its absorption from the rat small intestine

The effect of the electrical charge on the intestinal absorption of a protein was studied in normal adult rats. Chicken egg lysozyme (Lyz), a basic protein with a molecular weight of 14,300, was selected and several techniques for chemical modification were applied. Then the intestinal absorption of Lyz derivatives was evaluated by measuring the radioactivity in plasma and tissues, after the administration of an (111)In-labeled derivative to an in situ closed loop of the jejunum. After the administration of (111)In-Lyz, the level of radioactivity in plasma was comparable with the lytic activity of Lyz, supporting the fact that the radioactivity represents intact Lyz. (111)In-cationized Lyz showed a 2-3 times higher level of radioactivity in plasma, whereas the radioactivity of (111)In-anionized Lyz was much lower. The absorption rate of (111)In-Lyz derivatives calculated by a deconvolution method was correlated for the strength of their positive net charge. A similar relationship was observed using superoxide dismutase. These findings indicate that the intestinal absorption of a protein is, at least partially, determined by its electrical charge.     

Effects of ischemia-reperfusion on the absorption and esterase metabolism of diltiazem in rat intestine

Intestinal ischemia-reperfusion (I/R) is a serious clinical condition that triggers a complex inflammatory response. Inflammatory processes affect some enzymatic systems related to intestinal drug metabolism and bioavailability. Diltiazem (DTZ) is a calcium channel blocker, which is extensively metabolised in the intestine by esterases and different CYP450 isoforms. The main biotransformation pathway of DTZ in rats is desacetylation by esterases. This study analysed the effect of I/R on intestinal absorption and metabolism of DTZ, focusing on esterase activity, through different methodologies, after 60 min of superior mesenteric artery occlusion and 30 min of reperfusion or sham surgical procedures. The rate of DTZ appearance in blood during in situ studies increased significantly in the I/R group (0.094+/-0.014 10(-5) cm/s vs 0.271+/-0.110 10(-5) cm/s) and the calculated metabolised fraction of DTZ decreased significantly, showing an important reduction in the desacetylase activity in the I/R group. These results were supported by microsomal incubations, where desacetylase activity was related to esterases by specific inhibition, using paraoxon and bis-nitrophenylphosphate, and also by studies in everted rings. DTZ metabolism was higher in the jejunum than in the ileum, the esterase activity being affected by I/R in both regions. The present findings suggest that I/R injury clearly affects the esterases' activity and modifies the amount of DTZ and its metabolites in blood during in situ perfusion. This modification of intestinal esterase activity could be important for the pharmacokinetic behaviour of other drugs and prodrugs after intestinal pathologies involving inflammation and oxidative stress.     

The absorption of insulin from various regions of the rat intestine

The absorption of intact, biologically active insulin from the ileum, or the ascending colon was measured by the resulting changes in blood glucose concentration. One hour after injection of the ascending colon with a 1 ml volume containing 12 u insulin and 2 mg DOC the blood glucose level was reduced to 50% of the initial value, i.e. 31±2.0 mg%.When insulin was injected directly into the lumen of the ileum, the addition of 3 mg soybean trypsin inhibitor boosted the insulin effect. Direct injection of the ileum with 12 u insulin and 3 mg soybean trypsin inhibitor resulted in a significant drop in blood glucose: 69±5.0 and 85±8.1% of the initial concentration, following 1 and 2 hours, respectively.In the presence of soybean trypsin inhibitor, it was found that the endogenous bile salts in the ileum aid in the absorption of biologically active insulin.     

Lipoprotein lipase secretion from isolated rat fat cells of different size

Lipoprotein lipase(LPL) release from isolated small fat cells from young rats and large fat cells from older, fatter rats was compared during in vitro incubation at 30 degrees C. Although large fat cells had nearly three times higher cellular LPL activity, they secreted similar amounts of LPL activity into the incubation medium under both basal conditions (Krebs Ringer bicarbonate buffer containing 4% albumin and 6mM glucose) and after stimulation of LPL release by 5% human serum, or serum plus 1 U/ml heparin. These data suggest that previous observations of an altered tissue distribution of LPL in adipose tissue containing large fat cells can be at least in part explained by an alteration at the level of LPL secretion.     

Effect of dietary fat on pancreatic lipase levels in the rat

The effect of dietary fat on levels of lipase and other enzymes in rat pancreas has been studied. It was possible to raise levels of lipase in animals by supplementing their commercial chow diet with added fat or by raising the level of fat in semipurified diets from 4% to 22%. Pancreatic amylase levels decreased in rats fed the high fat diets, whereas levels of chymotrypsinogen and trypsinogen were unaffected. The type of carbohydrate in the semipurified diets made no difference. Thus, the levels of enzymes in rats fed dextrose-containing diets or cornstarch-containing diets were similar. On the basis of the present data, and results of others, it would appear that levels of pancreatic lipase are increased when the fat content of the diet is raised from about 5% to 15-22%, but that little or no additional increase in lipase levels can be attained by any further increase in the amount of dietary fat.     

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.     

Nickel absorption and distribution from rat small intestine In situ

The aim of this work was to study the absorption of nickel chloride in rats by means of the intestinal perfusion in situ technique at nickel concentrations of 1, 5, 10, 25, and 100 mg/L. Active transport and facilitated diffusion seem to play an important role in the intestinal absorption of nickel at concentrations≤10 mg/L. At higher concentrations, the absorption rate would be limited by saturation of the carriers. The distribution of the absorbed nickel was studied by intestinal perfusion of a 10-mg Ni/L solution for 30 or 60 min. Both in concentration and amount, the jejunum showed the higher values of absorbed nickel, followed by the kidneys and liver. When all of the collected organs (brain, heart, liver, lungs, spleen, kidneys, and testicles) and blood, but not the small intestine, are analyzed following a 60-min perfusion, it was found that 1% of the initial concentration had passed through the intestinal barrier.     

Stimulatory release of lipoprotein lipase activity from rat fat pads by vanadate

Vanadate stimulated the release of lipoprotein lipase (LPL) activity from rat fat pads into the medium in a time- and dose-dependent manner. It exerted the synergetic effect with heparin. The stimulatory effects of vanadate and heparin were decreased by incubation in Na+- or Ca2+-free media but were well preserved in K+-free medium. Amiloride inhibited the vanadate-stimulated release of LPL activity in a dose-dependent manner, but did not inhibit the heparin-stimulated release of LPL activity. Colchicine, antimycin A, and carbonyl cyanide m-chlorophenylhydrazone suppressed the stimulatory effect of vanadate, but cycloheximide did not. Preincubation of the fat pads with the tetrakis (acetoxymethyl) ester of quin 2 (quin 2-AM) inhibited the vanadate-stimulatory release of LPL activity without affecting basal activity. The concentration required for half-maximal inhibition of the action of vanadate by quin 2-AM was calculated to be 39 microM, suggesting that the action of vandate was dependent on intracellular Ca2+ concentration. The heparin-stimulated release, on the other hand, was not inhibited even at higher concentrations of quin 2-AM (up to 200 microM). These findings suggest that vanadate stimulates the release of LPL activity through mechanisms of action involving amiloride-sensitive and calcium-dependent pathways with a requirement of metabolic energy.     

普伐他汀大鼠在体肠吸收动力学

目的:研究普伐他汀在大鼠小肠的吸收情况。方法:采用大鼠在体小肠回流实验装置,利用HPLC紫外检测的方法测定肠循环液中酚红和普伐他汀的含量。采用XTerra@MS C-18色谱柱(5μm,150mm×2.1 mm.ID),流动相为3.5 mmol/L磷酸二氢钠溶液—乙腈(70:30,用磷酸调至pH 3.0),流速为0.2ml/min;结果:普伐他汀在大鼠小肠全肠段的吸收速率常数和吸收百分率分别为0.110±0.023(h~(-1))和18.21±2.50%。普伐他汀在小肠中吸收量与时间呈线性关系,但吸收速率较低。结论:普伐他汀可以通过增加药物的脂溶性,进而提高药物的生物利用度。