The phospholipase A(2) inhibitor methyl indoxam suppresses diet-induced obesity and glucose intolerance in mice.

BACKGROUND AND PURPOSE: Previous results have shown that mice lacking in the group 1B phospholipase A(2) (Pla2g1b) are resistant to obesity and diabetes induced by feeding a diabetogenic high-fat/high-carbohydrate diet. This study examined the potential of using the Pla2g1b inhibitor methyl indoxam as therapy to suppress diet-induced obesity and diabetes. EXPERIMENTAL APPROACH: Male C57BL/6 mice were fed the diabetogenic diet with or without methyl indoxam supplementation. Body weight gain, fasting plasma glucose levels, glucose tolerance and postprandial lysophospholipid absorption were compared. KEY RESULTS: Wild-type C57BL/6 mice fed the diabetogenic diet without Pla2g1b inhibitor showed 31 and 69% body weight gain after 4 and 10 weeks respectively. These animals also showed elevated plasma glucose levels and were glucose intolerant. In contrast, C57BL/6 mice fed the diabetogenic diet with 90 mg.kg(-1) of methyl indoxam gained only 5% body weight after 10 weeks. These animals were also euglycaemic and displayed normal glucose excursion rates in glucose tolerance test. Methyl indoxam suppression of diet-induced body weight gain and glucose intolerance was correlated with the inhibition of Pla2g1b-mediated postprandial lysophospholipid absorption. CONCLUSIONS AND IMPLICATIONS: These results show that oral supplementation of a diabetogenic diet with the Pla2g1b inhibitor methyl indoxam effectively suppresses diet-induced obesity and diabetes in mice. This suggests that Pla2g1b inhibition may be a potentially effective oral therapeutic option for treatment of obesity and diabetes.

Development of an indirect method for measuring porcine pancreatic lipase in human duodenal fluid.

Patients with exocrine pancreatic insufficiency are usually treated with porcine pancreatic enzymes but the bioavailability of these enzymes in the gut remains a matter of discussion. In order to determine the duodenal availability of porcine pancreatic lipase (PPL) present in pancreatic extracts (PE) taken orally, we developed a method for quantifying PPL in samples containing both PPL and human pancreatic lipase (HPL). Total pancreatic lipase activity measurements using the pH-stat technique and tributyrin as substrate were combined with an HPL-specific ELISA. Based on the known specific activity of the purified HPL, its activity was deduced from the ELISA measurements, and the PPL activity was obtained by subtracting the HPL activity from the total pancreatic lipase activity. This assay was established and validated using various samples containing pure PPL and recombinant HPL or PE, mixed or not with human duodenal juice. Samples collected in vivo from patients treated with PE were also tested. It was found that PPL did not affect the HPL ELISA, and the indirect PPL assay gave a measurement accuracy of 6.6% with the samples containing pure PPL and 10% with those containing PE. This assay was also used successfully to discriminate between PPL and the endogenous HPL present in the duodenal contents of patients with severe pancreatic insufficiency treated with PE. This method might provide a useful means of assessing the availability of PEs at their site of action, in the absence of a PPL-specific ELISA.

Effect of nonionic surfactants on Rhizopus homothallicus lipase activity: a comparative kinetic study.

Based on amino-terminal sequencing and mass spectrometry data on the Rhizopus homothallicus lipase extracted using solid (SSF) and submerged state fermentation (SmF) methods, we previously established that the two enzymes were identical. Differences were observed, however, in terms of the specific activity of these lipases and their inhibition by diethyl p-nitrophenyl phosphate (E600). The specific activity of the SSF lipase (10,700 mumol/min/mg) was found to be 1.2-fold that of SmF lipase (8600 mumol/min/mg). These differences might be the result of residual Triton X-100 molecules interacting with the SSF lipase. To check this hypothesis, the SmF lipase was incubated with submicellar concentrations of Triton X-100. The specific activity of the lipase increased after this treatment, reaching similar values to those measured with the SSF lipase. Preincubating SSF and SmF lipases with E600 at a molar excess of 100 for 1 h resulted in 80% and 60% enzyme inhibition levels, respectively. When the SmF lipase was preincubated with Triton X-100 for 1 h at a concentration 100 times lower than the Triton X-100 critical micellar concentration, the inhibition of the lipase by E600 increased from 60% to 80%. These results suggest that residual detergent monomers interacting with the enzyme may affect the kinetic properties of the Rh. homothallicus lipase.

Egg yolk lipoproteins as substrates for lipases.

Egg yolk emulsions containing phospholipids (about 31%, w/w) are classically used as substrates for measuring phospholipase A2 activity using the pH-stat method. Here we investigated the susceptibility of egg yolk lipoproteins to lipolysis by various highly purified lipases of animal or microbial origin. Egg yolk lipoproteins, which contain up to 65% triacylglycerols, were found to be effective substrates for all the lipases tested. The specific activities measured on egg yolk lipoproteins using the pH-stat technique were found to be 8000, 1000, 1250 and 1700 U/mg in the case of human pancreatic lipase, horse pancreatic lipase, porcine pancreatic lipase and Humicola lanuginosa lipase, respectively. No activity was detected in the absence of colipase with any of the pancreatic lipases tested. Consequently, the classical egg yolk assay cannot be considered as a specific phospholipase A2 assay.

Surface properties of unsaturated non-oxidized and oxidized free fatty acids spread as monomolecular films at an argon/water interface.

The interfacial properties of monomolecular films of stearic acid (SA) oleic acid (OA), linoleic acid (LA), ricinoleic acid (RA), 13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid (13-HPODE) and 13(S)-hydroxyoctadeca-9Z,11E-dienoic acid (13-HODE) were studied by recording the changes occurring in response to monomolecular film compression in their surface pressure and surface potential at the argon/water interface. The oxidized free fatty acids are more expanded than the parent non-oxidized free fatty acids, reflecting a higher hydrophilic-lipophilic balance. The lift-off values of the molecular area of 13-HODE, 13-HPODE and RA were 68, 74 and 106 A2 molecule(-1), respectively, as compared to 47 and 40 A2 molecule(-1) in the case of LA and OA, respectively. Variations in the molecular orientation of free fatty acids can result in large changes in the dipole moment which are not accompanied by appreciable changes in the surface pressure. In the case of the oxidized free fatty acids, the spontaneous desorption into the aqueous phase was found to increase at increasing surface pressures. The desorption rates of OA and LA increased dramatically in the presence of beta-cyclodextrin (beta-CD); whereas the presence of beta-CD only slightly increased the desorption rates of the oxidized free fatty acids.

Functional expression in insect cells, one-step purification and characterization of a recombinant phospholipase D from cowpea (Vigna unguiculata L. Walp).

Phospholipase D (PLD) is an important enzyme involved in signal transduction, vesicle trafficking and membrane metabolism. In this study, large amounts of a recombinant plant PLD alpha were secreted into the culture medium of baculovirus-infected insect cells and purified to homogeneity in the form of a fully active enzyme. The transient production of recombinant PLD alpha yielded a protein (rPLD alpha a, 88 kDa) together with a shorter form (rPLD alpha b, 87 kDa), which accumulated in the medium. N-Terminal amino acid sequencing of the rPLD alpha a and rPLD alpha b showed that rPLD alpha b resulted from proteolytic cleavage at Gly8-Ile9. Immunoblotting showed that both rPLD alpha a and rPLD alpha b are recognized by a polyclonal antibody previously raised against native soybean PLD alpha. One-step calcium-dependent octyl-Sepharose chromatography was used to obtain the two highly purified forms of rPLD alpha, as attested by gel electrophoresis, N-terminal amino acid sequence and mass spectrometry. The N-terminal region of PLD alpha is homologous with the C2 domains which are present in a number of enzymes known to be involved in signal transduction and/or phospholipid metabolism. The truncated rPLD alpha b lacks the first acidic amino acid in its N-terminus, which is probably involved in the calcium binding site. The rPLD alpha b was thus easily eluted from the octyl-Sepharose column by decreasing the calcium concentration of the buffer from 50 to 30 mM, whereas, the rPLD alpha a was eluted after chelating calcium ions with EDTA. The purified rPLD alpha yield reached a level of 10 mg per liter of serum-free culture medium. The availability of baculovirus-derived rPLD alpha constitutes a valuable source of enzyme for future crystallographic studies to determine its three-dimensional structure.

Study of fatty acid specificity of sunflower phospholipase D using detergent/phospholipid micelles.

The fatty acid specificity of phospholipase D purified from germinating sunflower seeds was studied using mixed micelles with variable detergent/phospholipid ratios. The main advantage of this approach is that since the substrate is integrated in the detergent micelles, comparisons can be made between the kinetic constants of a wide range of phosphatidylcholine (PtdCho) compounds with various fatty acid contents. Phospholipase D is subject to interfacial activation as it is most active on water-insoluble substrates. It is not active on sphingomyelin and only slightly on lysophosphatidylcholine. By fitting the curves based on the experimental kinetic data, the interfacial dissociation constant of phospholipase D, the maximum hydrolysis rate Vm and the kinetic constant Km(B), were determined with the micellar substrate. The specificity of various substrates was examined by comparing the Vm/Km(B) values, and it was noted that sunflower phospholipase D is most active on medium-chain fatty PtdCho compounds. With long-chain natural phospholipids, the specificity of phospholipase D was slightly dependent on the level of fatty acid unsaturation. The pure enzyme was able to hydrolyse the sunflower phospholipids present in mixed detergent micelles but not the phospholipids integrated in the natural sunflower oil body structure. We concluded, however, that during the germination of sunflower seeds, phospholipase D might be involved in the degradation of oil bodies, since other factors present in crude seed extracts may make phospholipids accessible to the enzyme.

Cell-permeable ceramides prevent the activation of phospholipase D by ADP-ribosylation factor and RhoA.

The mechanism of inhibition of phospholipase D (PLD) by ceramides was determined using granulocytes differentiated from human promyelocytic leukemic (HL-60) cells. In a cell-free system, hydrolysis of phosphatidylcholine by membrane-bound PLD depended upon phosphatidylinositol 4,5-bisphosphate, guanosine 5'-3-O-(thio)triphosphate) (GTPgammaS), and cytosolic factors including ADP-ribosylating factor (ARF) and RhoA. C2-(N-acetyl-), C8- (N-octanoyl-), and long-chain ceramides, but not dihydro-C2-ceramide, inhibited PLD activity. Apyrase or okadaic acid did not modify the inhibition of PLD by ceramides, indicating that the effect in the cell-free system was unlikely to be dependent upon a ceramide-stimulated kinase or phosphoprotein phosphatases. C2- and C8-ceramides prevented the GTPgammaS-induced translocation of ARF1 and RhoA from the cytosol to the membrane fraction. In whole cells, C2-ceramide, but not dihydro-C2-ceramide, inhibited the stimulation of PLD by N-formylmethionylleucylphenylalanine and decreased the amounts of ARF1, RhoA, CDC42, Rab4, and protein kinase C-alpha and -beta1 that were associated with the membrane fraction, but did not alter the distribution of protein kinase C-epsilon and -zeta. It is concluded that one mechanism by which ceramides prevent the activation of PLD is inhibition of the translocation to membranes of G-proteins and protein kinase C isoforms that are required for PLD activity.

"Cross talk" between the bioactive glycerolipids and sphingolipids in signal transduction.

Hydrolysis of phosphatidylcholine via receptor-mediated stimulation of phospholipase D produces phosphatidate that can be converted to lysophosphatidate and diacylglycerol. Diacylglycerol is an activator of protein kinase C, whereas phosphatidate and lysophosphatidate stimulate tyrosine kinases and activate the Ras-Raf-mitogen-activated protein kinase pathway. These three lipids can stimulate cell division. Conversely, activation of sphingomyelinase by agonists (e.g., tumor necrosis factor-alpha) causes ceramide production that inhibits cell division and produces apoptosis. If ceramides are metabolized to sphingosine and sphingosine 1-phosphate, then these lipids can stimulate phospholipase D and are also mitogenic. By contrast, ceramides inhibit the activation of phospholipase D by decreasing its interaction with the G-proteins, ARF and Rho, which are necessary for its activation. In whole cells, ceramides also stimulate the degradation of phosphatidate, lysophosphatidate, ceramide 1-phosphate, and sphingosine 1-phosphate through a multifunctional phosphohydrolase (the Mg(2+)-independent phosphatidate phosphohydrolase), whereas sphingosine inhibits phosphatidate phosphohydrolase. Tumor necrosis factor-alpha causes insulin resistance, which may be partly explained by ceramide production. Cell-permeable ceramides decrease insulin-stimulated glucose uptake in 3T3-L1 adipocytes after 2-24 h, whereas they stimulate basal glucose uptake. These effects do not depend on decreased tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 or the interaction of insulin receptor substrate-1 with phosphatidylinositol 3-kinase. They appear to rely on the differential effects of ceramides on the translocation of GLUT1-and GLUT4-containing vesicles. It is concluded that there is a significant interaction and "cross-talk" between the sphingolipid and glycerolipid pathways that modifies signal transduction to control vesicle movement, cell division, and cell death.

A new method for determining phospholipase D activity using the monomolecular film technique.

A versatile and continuous assay for phospholipase D (PL D) activity was developed using the monomolecular film technique. For this purpose, a two-step enzymatic reaction was used. First, PL D hydrolysis of stable 1,2-diacyl-sn-glycero-3-phosphocholine (PC) films by PL D generated a stable 1,2-diacyl-sn-glycero-3-phosphate (PA) film and water-soluble choline. Secondly, the latter acidic phospholipid, in contrast to the initial PC molecule, was further hydrolysed under the action of porcine pancreatic lipase (PPL) in order to give rise to lysophosphatidic acid and fatty acid, which were rapidly desorbed from the interface. With this new procedure, it is possible to obtain continuous and accurate kinetic measurements of the PL D-catalyzed reaction with phospholipid monolayers as substrates. The PLD kinetics were linear with time and the velocities recorded were directly dependent upon the amount of PL D used. In a preliminary study, we investigated the effects of the surface pressure on the PL D activity.

Phospholipase D from soybean (Glycine max L.) suspension-cultured cells: purification, structural and enzymatic properties.

Phospholipase D (phosphatidylcholine phosphatidohydrolase EC 3.1.4.4) from soybean (Glycine max L.) suspension-cultured cell was purified around 1,200-fold to homogeneity by acetone precipitation, Macro-Prep High Q anion exchange, and octyl-Sepharose CL-4B affinity chromatography. The purified enzyme released 1,600 mumol of choline per min per mg of protein. The enzyme is monomeric with a molecular mass of 92 kDa, as estimated by SDS-PAGE. One of the most interesting characteristics of the purified soybean phospholipase D was the dependence of the pH optimum on the Ca2+ ion concentration in the assay. With 10 mM, 20 mM and 40 mM Ca2+ ions, the optima were at pH 7.5, 6 and 5.5, respectively. The specific adsorption of phospholipase D onto octyl-Sepharose gel suggests that the molecule becomes more hydrophobic in the presence of Ca2+ ions. The amino acid sequence of the first 18 N-terminal residues of soybean phospholipase D revealed a high degree of homology with those previously published for cabbage leaf and castor bean endosperm enzymes. Western blots of the soybean phospholipase D showed an immunoreactivity with antibodies raised against a synthetic peptide corresponding to the 15 N-terminal amino acid residues of phospholipase D from cabbage leaves.

Human pancreatic lipase. Importance of the hinge region between the two domains, as revealed by monoclonal antibodies.

Several monoclonal antibodies (mAbs) were prepared against human pancreatic lipase (HPL). Two enzyme-linked immunosorbent assay (ELISA) procedures were set up for screening hybridomas producing specific antibodies. Four mAbs (81-23, 146-40, 315-25, and 320-24) of the IgG1 isotype were found to react with HPL in both simple sandwich and double sandwich ELISAs, while mAb 248-31, of the IgG2b isotype, reacted only with HPL in a double sandwich ELISA. The results of Western blot analysis carried out with native and SDS-denatured HPLs indicated that mAb 248-31 recognized only native HPL, while all the other mAbs recognized both forms of HPL. Since mAb 248-31 did not recognize SDS-denatured HPL, it was not possible to localize its epitope. To carry out epitope mapping along the primary sequence of HPL, four fragments (14, 26, 30, and 36 kDa) resulting from a limited chymotryptic cleavage of HPL were characterized by Western blotting as well as N-terminal amino acid sequence analysis. Of the above five anti-HPL mAbs, four (81-23, 248-31, 315-25, and 320-24) were found to inhibit the lipolytic activity of HPL (in both the presence and absence of bile salts and colipase), while mAb 146-40 had no inhibitory effects. The epitope recognized by mAb 146-40 was found to be located in the N-terminal domain (Lys1-Phe335). Combined immunoinactivation and epitope mapping studies showed that three inhibitory mAbs (81-23, 315-25, and 320-24) recognize overlapping epitopes from the hinge region between the N- and C-terminal domains of HPL, belonging to the 26-kDa fragment. In the presence of lipids, a significant decrease has been observed in the bending angle between the N- and C-terminal domains of the HPL tertiary structure (van Tilbeurgh, H., Egloff, M. P., Martinez, C., Rugani, N., Verger, R. and Cambillau, C. (1993) Nature 362, 814-820). From the present immunochemical data, we further propose that locking the hinge movement with mAbs may induce lipase immunoinactivation.

Improved purification and biochemical characterization of phospholipase D from cabbage.

Phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) was purified from cabbage leaves. The two step purification procedure involved hydrophobic chromatography on Octyl-Sepharose followed by a Mono-Q/FPLC-column with a total yield of 23% and a purification factor of 1000. A zymographic assay was used to detection of PL D activities at various stages of purification under non denaturing PAGE. The molecular mass was determined to be 90 kDa using the SDS/PAGE method, and 90,200 Da as calculated from the amino acid analysis. The isoelectric point of the enzyme is acidic (pI = 4.7). The amino-acid composition and 29 residues of the NH2-terminal amino-acid sequence were determined.

Uncoupling of catalysis and colipase binding in pancreatic lipase by limited proteolysis.

In the intestine, the hydrolysis of triglycerides by pancreatic lipase is performed only in the presence of colipase, whose function is to anchor lipase to the bile-salt-coated lipid interface. Biochemical and crystallographic data on porcine and human lipases have shown that the molecule is made of two well-delimited domains. In order to get more information on the role of the domains in catalysis and colipase binding, we performed limited proteolysis on lipase from various species and obtained different patterns of cleavage. In the case of porcine and human lipases, only the C-terminal domain (12 kDa) could be obtained after chymotryptic attack, whereas in the horse enzyme the cleavage of the Leu410-Thr411 bond gave rise to a large N-terminal (45 kDa) and a small C-terminal (4 kDa) fragment. The isolated porcine and human C-terminal domains were completely inactive towards emulsified tributyrin, though were able to bind colipase. Conversely, the horse 45 kDa fragment retained the lipase activity but failed to correctly bind colipase. This work definitely proves that catalysis and colipase binding are separate events involving topographically distinct regions of the molecule and focuses attention on the role of the C-terminal domain in colipase binding.