Mechanisms of Inhibition of Triacylglycerol Hydrolysis by Human Gastric Lipase
2002; Elsevier BV; Volume: 277; Issue: 31 Linguagem: Inglês
10.1074/jbc.m202839200
ISSN1083-351X
AutoresYan Pafumi, Denis Lairon, P. Lechène de la Porte, Christine Juhel, Judith Storch, Margit Hamosh, Martine Armand,
Tópico(s)Diet and metabolism studies
ResumoIn the human stomach, gastric lipase hydrolyzes only 10 to 30% of ingested triacylglycerols because of an inhibition process induced by the long chain free fatty acids generated, which are mostly protonated at gastric pH. The aim of this work was to elucidate the mechanisms by which free fatty acids inhibit further hydrolysis.In vitro experiments examined gastric lipolysis of differently sized phospholipid-triolein emulsions by human gastric juice or purified human gastric lipase, under close to physiological conditions. The lipolysis process was further investigated by scanning electron microscopy, and gastric lipase and free fatty acid movement during lipolysis were followed by fluorescence microscopy. The results demonstrate that: 1) free fatty acids generated during lipolysis partition between the surface and core of lipid droplets with a molar phase distribution coefficient of 7.4 at pH 5.40; 2) the long chain free fatty acids have an inhibitory effect only when generated during lipolysis; 3) inhibition of gastric lipolysis can be delayed by the use of lipid emulsions composed of small-size lipid droplets; 4) the release of free fatty acids during lipolysis induces a marked increase in droplet surface area, leading to the formation of novel particles at the lipid droplet surface; and 5) the gastric lipase is trapped in these free fatty acid-rich particles during their formation. In conclusion, we propose a model in which the sequential physicochemical events occurring during gastric lipolysis lead to the inhibition of further triacylglycerol lipolysis. In the human stomach, gastric lipase hydrolyzes only 10 to 30% of ingested triacylglycerols because of an inhibition process induced by the long chain free fatty acids generated, which are mostly protonated at gastric pH. The aim of this work was to elucidate the mechanisms by which free fatty acids inhibit further hydrolysis.In vitro experiments examined gastric lipolysis of differently sized phospholipid-triolein emulsions by human gastric juice or purified human gastric lipase, under close to physiological conditions. The lipolysis process was further investigated by scanning electron microscopy, and gastric lipase and free fatty acid movement during lipolysis were followed by fluorescence microscopy. The results demonstrate that: 1) free fatty acids generated during lipolysis partition between the surface and core of lipid droplets with a molar phase distribution coefficient of 7.4 at pH 5.40; 2) the long chain free fatty acids have an inhibitory effect only when generated during lipolysis; 3) inhibition of gastric lipolysis can be delayed by the use of lipid emulsions composed of small-size lipid droplets; 4) the release of free fatty acids during lipolysis induces a marked increase in droplet surface area, leading to the formation of novel particles at the lipid droplet surface; and 5) the gastric lipase is trapped in these free fatty acid-rich particles during their formation. In conclusion, we propose a model in which the sequential physicochemical events occurring during gastric lipolysis lead to the inhibition of further triacylglycerol lipolysis. human gastric lipase phospholipid oleic acid fluorescein isothiocyanate 5-dimethylaminonaphthalene-1-sulfonyl tetramethyl rhodamine isothiocyanate triolein Dietary fat digestion and absorption is a complex process involving enzyme activities and physicochemical changes (1Carey M.C. Small D.M. Bliss C.M. Annu. Rev. Physiol. 1983; 45: 651-677Crossref PubMed Scopus (643) Google Scholar, 2Brockman H.L. Borgstrom B. Brockman H.L. Lipases. Elsevier Science Publishers B. V., Amsterdam1984: 3-46Google Scholar, 3Armand M. Borel P. Dubois C. Senft M. Peyrot J. Salducci J. Lafont H. Lairon D. Am. J. Physiol. 1994; 266: G372-G381PubMed Google Scholar, 4Armand M. Borel P. Pasquier B. Dubois C. Senft M. André M. Peyrot J. Salducci J. Lairon D. Am. J. Physiol. 1996; 34: G172-G183Google Scholar, 5Hernell O. Staggers J.E. Carey M.C. Biochemistry. 1990; 29: 2041-2056Crossref PubMed Scopus (449) Google Scholar). In humans, hydrolysis of dietary triacylglycerols starts in the stomach where it is catalyzed by an acid-stable gastric lipase, a globular protein of about 50 kDa with a broad pH range (6Hamosh M. Lingual and Gastric Lipases: Their Role in Fat Digestion. CRC Press, Boca Raton, FL1990Google Scholar, 7Roussel A. Canaan S. Egloff M.P. Rivière M. Dupuis L. Verger R. Cambillau C. J. Biol. Chem. 1999; 274: 16995-17002Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Triacylglycerol hydrolysis continues in the duodenum, by the synergetic actions of gastric and colipase-dependent pancreatic lipases and bile secretion (1Carey M.C. Small D.M. Bliss C.M. Annu. Rev. Physiol. 1983; 45: 651-677Crossref PubMed Scopus (643) Google Scholar). A characteristic feature of these lipases is their specificity to act on insoluble emulsified substrates (1Carey M.C. Small D.M. Bliss C.M. Annu. Rev. Physiol. 1983; 45: 651-677Crossref PubMed Scopus (643) Google Scholar, 2Brockman H.L. Borgstrom B. Brockman H.L. Lipases. Elsevier Science Publishers B. V., Amsterdam1984: 3-46Google Scholar). A fewin vitro and in vivo experiments have shown that the extent of lipid emulsification, which directly affects the lipid/water interface area, modulates the activity of digestive lipases (8Borel P. Armand M. Ythier P. Dutot G. Melin C. Senft M. Lafont H. Lairon D. J. Nutr. Biochem. 1994; 5: 124-133Crossref Scopus (104) Google Scholar, 9Armand M. Borel P. Ythier P. Dutot G. Melin C. Senft M. Lafont H. Lairon D. J. Nutr. Biochem. 1992; 3: 333-341Crossref Scopus (173) Google Scholar, 10Armand M. Pasquier B. André M. Borel P. Senft M. Peyrot J. Salducci J. Portugal H. Jaussan V. Lairon D. Am. J. Clin. Nutr. 1999; 70: 1096-1106Crossref PubMed Scopus (377) Google Scholar). Dietary lipids are organized mainly in the form of droplets in the aqueous digestive system (1Carey M.C. Small D.M. Bliss C.M. Annu. Rev. Physiol. 1983; 45: 651-677Crossref PubMed Scopus (643) Google Scholar, 3Armand M. Borel P. Dubois C. Senft M. Peyrot J. Salducci J. Lafont H. Lairon D. Am. J. Physiol. 1994; 266: G372-G381PubMed Google Scholar, 4Armand M. Borel P. Pasquier B. Dubois C. Senft M. André M. Peyrot J. Salducci J. Lairon D. Am. J. Physiol. 1996; 34: G172-G183Google Scholar). The lipid droplets consist of a hydrophobic core containing the majority of the triacylglycerol molecules, esterified cholesterol, and fat-soluble vitamins, surrounded by an amphipatic surface monolayer of phospholipids, free cholesterol, and a few triacylglycerol molecules (11Miller K.W. Small D.M. J. Colloid Interface Sci. 1982; 89: 466-478Crossref Scopus (69) Google Scholar, 12Borel P. Grolier P. Armand M. Partier A. Lafont H. Lairon D. Azais-Braesco V. J. Lipid Res. 1996; 37: 250-261Abstract Full Text PDF PubMed Google Scholar). Earlier studies on lipoprotein models (11Miller K.W. Small D.M. J. Colloid Interface Sci. 1982; 89: 466-478Crossref Scopus (69) Google Scholar, 13Miller K.W. Small D.M. Gotto A.M. Plasma Lipoproteins. Elsevier Science Publishers B.V., Amsterdam1987: 1-74Google Scholar, 14Miller K.W. Small D.M. J. Biol. Chem. 1983; 258: 13772-13784Abstract Full Text PDF PubMed Google Scholar) and a recent investigation using dietary emulsions (12Borel P. Grolier P. Armand M. Partier A. Lafont H. Lairon D. Azais-Braesco V. J. Lipid Res. 1996; 37: 250-261Abstract Full Text PDF PubMed Google Scholar) have shown that 2–5 mol % of the droplet surface lipid is triacylglycerol, thereby enabling lipase action at the surface of the lipid droplet. In healthy humans, gastric lipolysis leads to the hydrolysis of 10–30% (3Armand M. Borel P. Dubois C. Senft M. Peyrot J. Salducci J. Lafont H. Lairon D. Am. J. Physiol. 1994; 266: G372-G381PubMed Google Scholar, 4Armand M. Borel P. Pasquier B. Dubois C. Senft M. André M. Peyrot J. Salducci J. Lairon D. Am. J. Physiol. 1996; 34: G172-G183Google Scholar, 10Armand M. Pasquier B. André M. Borel P. Senft M. Peyrot J. Salducci J. Portugal H. Jaussan V. Lairon D. Am. J. Clin. Nutr. 1999; 70: 1096-1106Crossref PubMed Scopus (377) Google Scholar, 15Carrière F. Barrowman J.A. Verger R. Laugier R. Gastroenterol. 1993; 105: 876-888Abstract Full Text PDF PubMed Scopus (384) Google Scholar) of ingested triacylglycerols, generating mainly free fatty acids and diacylglycerols (1Carey M.C. Small D.M. Bliss C.M. Annu. Rev. Physiol. 1983; 45: 651-677Crossref PubMed Scopus (643) Google Scholar, 16Patton J.S. Rigler M.W. Liao T.H. Hamosh P. Hamosh M. Biochim. Biophys. Acta. 1982; 712: 400-407Crossref PubMed Scopus (49) Google Scholar, 17Gargouri Y. Piéroni G. Rivière C. Lowe P.A. Saunière J.F. Sarda L. Verger R. Biochim. Biophys. Acta. 1986; 879: 419-423Crossref PubMed Scopus (142) Google Scholar). This facilitates subsequent triacylglycerol hydrolysis by pancreatic lipase by allowing fat emulsification (3Armand M. Borel P. Dubois C. Senft M. Peyrot J. Salducci J. Lafont H. Lairon D. Am. J. Physiol. 1994; 266: G372-G381PubMed Google Scholar, 4Armand M. Borel P. Pasquier B. Dubois C. Senft M. André M. Peyrot J. Salducci J. Lairon D. Am. J. Physiol. 1996; 34: G172-G183Google Scholar) and promoting enzyme activity (8Borel P. Armand M. Ythier P. Dutot G. Melin C. Senft M. Lafont H. Lairon D. J. Nutr. Biochem. 1994; 5: 124-133Crossref Scopus (104) Google Scholar, 17Gargouri Y. Piéroni G. Rivière C. Lowe P.A. Saunière J.F. Sarda L. Verger R. Biochim. Biophys. Acta. 1986; 879: 419-423Crossref PubMed Scopus (142) Google Scholar). Furthermore, in physiological (preterm or full-term infants) (18Armand M. Hamosh M. Mehta N.R. Angelus P.A. Philpott J.R. Henderson T.R. Dwyer N.K. Lairon D. Hamosh P. Pediatr. Res. 1996; 40: 429-437Crossref PubMed Scopus (193) Google Scholar) and pathological (cystic fibrosis, pancreatitis) (19Abrams C.K. Hamosh M. Hubbard V.S. Dutta S.K. Hamosh P. J. Clin. Invest. 1984; 73: 374-382Crossref PubMed Scopus (91) Google Scholar, 20Balasubramanina K. Zentler-Munro P.L. Batten J.C. Northfield T.C. Pancreas. 1992; 7: 305-310Crossref PubMed Scopus (29) Google Scholar, 21Roulet M. Weber A.M. Paradis Y. Roy C.C. Chatrand L. Lassalle R. Morin C.L. Pediatr. Res. 1980; 14: 1360-1367Crossref PubMed Scopus (54) Google Scholar) pancreatic insufficiencies, gastric lipolysis plays a key role in the digestion of dietary fat by hydrolyzing 10–40% of fat in the stomach (18Armand M. Hamosh M. Mehta N.R. Angelus P.A. Philpott J.R. Henderson T.R. Dwyer N.K. Lairon D. Hamosh P. Pediatr. Res. 1996; 40: 429-437Crossref PubMed Scopus (193) Google Scholar, 19Abrams C.K. Hamosh M. Hubbard V.S. Dutta S.K. Hamosh P. J. Clin. Invest. 1984; 73: 374-382Crossref PubMed Scopus (91) Google Scholar, 20Balasubramanina K. Zentler-Munro P.L. Batten J.C. Northfield T.C. Pancreas. 1992; 7: 305-310Crossref PubMed Scopus (29) Google Scholar, 21Roulet M. Weber A.M. Paradis Y. Roy C.C. Chatrand L. Lassalle R. Morin C.L. Pediatr. Res. 1980; 14: 1360-1367Crossref PubMed Scopus (54) Google Scholar), as well as acting more effectively in the duodenum because of acid pH conditions (19Abrams C.K. Hamosh M. Hubbard V.S. Dutta S.K. Hamosh P. J. Clin. Invest. 1984; 73: 374-382Crossref PubMed Scopus (91) Google Scholar). The relatively limited extent of lipolysis by the gastric lipase under physiological or pathological conditions suggested that a feedback inhibition by the products of lipolysis probably occurs (1Carey M.C. Small D.M. Bliss C.M. Annu. Rev. Physiol. 1983; 45: 651-677Crossref PubMed Scopus (643) Google Scholar, 22Hamosh M. Ganot D. Hamosh P. J. Biol. Chem. 1979; 254: 12121-12125Abstract Full Text PDF PubMed Google Scholar). It has been hypothesized that the inhibition of gastric lipase activity may be due to the progressive release of protonated free fatty acids (8Borel P. Armand M. Ythier P. Dutot G. Melin C. Senft M. Lafont H. Lairon D. J. Nutr. Biochem. 1994; 5: 124-133Crossref Scopus (104) Google Scholar, 23Gargouri Y. Pieroni G. Rivière C. Sauniére J.F. Lowe P.A. Sarda L. Verger R. Gastroenterology. 1986; 91: 919-925Abstract Full Text PDF PubMed Google Scholar) that might accumulate at the lipid droplet surface (1Carey M.C. Small D.M. Bliss C.M. Annu. Rev. Physiol. 1983; 45: 651-677Crossref PubMed Scopus (643) Google Scholar, 16Patton J.S. Rigler M.W. Liao T.H. Hamosh P. Hamosh M. Biochim. Biophys. Acta. 1982; 712: 400-407Crossref PubMed Scopus (49) Google Scholar, 22Hamosh M. Ganot D. Hamosh P. J. Biol. Chem. 1979; 254: 12121-12125Abstract Full Text PDF PubMed Google Scholar). At present, however, the mechanism by which free fatty acids inhibit gastric lipase action in the stomach is unknown. It can be suggested from the literature that long chain free fatty acids prevent further gastric lipase lipolysis by modifying the physicochemical properties of the lipid/water interface, especially the interfacial tension or the surface pressure (2Brockman H.L. Borgstrom B. Brockman H.L. Lipases. Elsevier Science Publishers B. V., Amsterdam1984: 3-46Google Scholar, 13Miller K.W. Small D.M. Gotto A.M. Plasma Lipoproteins. Elsevier Science Publishers B.V., Amsterdam1987: 1-74Google Scholar, 16Patton J.S. Rigler M.W. Liao T.H. Hamosh P. Hamosh M. Biochim. Biophys. Acta. 1982; 712: 400-407Crossref PubMed Scopus (49) Google Scholar, 24Gargouri Y. Piéroni G. Ferrato F. Verger R. Eur. J. Biochem. 1987; 169: 125-129Crossref PubMed Scopus (30) Google Scholar, 25Gargouri Y. Piéroni G. Ferrato F. Verger R. Eur. J. Biochem. 1986; 156: 305-310Crossref PubMed Scopus (80) Google Scholar); they could prevent the interfacial binding of gastric lipase or promote its release from the droplet surface (2Brockman H.L. Borgstrom B. Brockman H.L. Lipases. Elsevier Science Publishers B. V., Amsterdam1984: 3-46Google Scholar), or they could limit the number of triacylglycerol molecules located at the droplet surface by steric hindrance (13Miller K.W. Small D.M. Gotto A.M. Plasma Lipoproteins. Elsevier Science Publishers B.V., Amsterdam1987: 1-74Google Scholar). However, thus far no study has provided direct evidence for the mechanisms involved. A few previous studies have examined the effect of free fatty acid on gastric lipase activity (8Borel P. Armand M. Ythier P. Dutot G. Melin C. Senft M. Lafont H. Lairon D. J. Nutr. Biochem. 1994; 5: 124-133Crossref Scopus (104) Google Scholar, 23Gargouri Y. Pieroni G. Rivière C. Sauniére J.F. Lowe P.A. Sarda L. Verger R. Gastroenterology. 1986; 91: 919-925Abstract Full Text PDF PubMed Google Scholar) or the distribution of fatty acids in model systems (26Spooner P.J.R. Bennett Clark S. Gantz D.L. Hamilton J.A. Small D.M. J. Biol. Chem. 1988; 263: 1444-1453Abstract Full Text PDF PubMed Google Scholar, 27Spooner P.J.R. Gantz D.L. Hamilton J.A. Small D.M. J. Biol. Chem. 1990; 265: 12650-12655Abstract Full Text PDF PubMed Google Scholar, 28Ekman S. Derksen A. Small D.M. Biochim. Biophys. Acta. 1988; 959: 343-348Crossref PubMed Scopus (13) Google Scholar), however, they were not performed under physiological conditions. In the present work we have performed several in vitro experiments using conditions close to those occurring physiologically, to understand the mechanism of inhibition of gastric lipolysis in vivo, and to begin to elucidate conditions that will enable modulation of the extent of gastric lipolysis. Human gastric juice was collected from healthy adult patients for diagnostic purposes after pentagastric stimulation (6 μg/kg) (a generous gift from Dr. J. Peyrot and Pr. J. Salducci, Gastroenterology Department, Nord Hospital, Marseille, France). Pure human gastric lipase (HGL)1 with a specific activity of 910 units/mg on tributyrin was obtained according to Thiruppathi and Balasubramanian (29Tiruppathi C. Balasubramanian K.A. Indian J. Biochem. Biophys. 1985; 22: 111-114PubMed Google Scholar). Gastric lipase activity of gastric juice (100–200 μl) or purified lipase was determined using a pH-stat titrator (Metrohm, Herisau, Switzerland) at pH 5.40 and 37 °C with tributyrin as substrate (ICN Biomedicals Inc., OH) as previously described (3Armand M. Borel P. Dubois C. Senft M. Peyrot J. Salducci J. Lafont H. Lairon D. Am. J. Physiol. 1994; 266: G372-G381PubMed Google Scholar, 23Gargouri Y. Pieroni G. Rivière C. Sauniére J.F. Lowe P.A. Sarda L. Verger R. Gastroenterology. 1986; 91: 919-925Abstract Full Text PDF PubMed Google Scholar). One lipase unit corresponds to the release of 1 μmol of fatty acid per min. The relative proportion of lipids used was chosen in accordance with human daily dietary intake (1Carey M.C. Small D.M. Bliss C.M. Annu. Rev. Physiol. 1983; 45: 651-677Crossref PubMed Scopus (643) Google Scholar). The lipid mixture contained 93.5% triolein (w/w) (ICN), 6% phospholipids (PL) (w/w) (l-α-phosphatidylcholine, XVI-E from egg yolk), and 0.5% free cholesterol (w/w) (both from Sigma, La Verpillière, France). Lipids were solubilized in chloroform/methanol (2:1, v/v), mixed, dried under nitrogen, and dessicated using a rotavapor under vacuum at 30 °C. The lipid mixture was stored at −20 °C. For lipolysis experiments, the mixture contained [3H]- or [14C]triolein (3 × 104 dpm/μmol of triolein or 1.5 × 106 dpm/μmol of triolein when a low HGL/TO ratio was used) and [14C]l-α1-palmitoyl–2-oleoyl phosphatidylcholine (3.5 × 104 dpm/μmol PL) (PerkinElmer Life Science, Dreiech, Belgium). Lipid mixtures enriched with different concentrations of oleic acid (OA) (2 and 6.9% oleic acid (w/w), i.e. 0.063 and 0.216 μmol of OA/μmol of TO) contained [14C]triolein (PerkinElmer Life Sciences, 3 × 104 dpm/μmol of triolein) and [3H]oleic acid (1.2 × 104 dpm/μmol of OA) (Amersham International plc, UK). These OA/TO ratios were selected to mimic the amount of free fatty acids released after 2 or 25 min of gastric lipolysis under present physiological conditions, respectively. To study the distribution of lipolysis-generated free fatty acids between the core and surface of the lipid droplet, the lipid mixture was radiolabeled with [14C]triolein (106dpm/μmol of triolein), [3H]l-α-dipalmitoyl phosphatidylcholine (106 dpm/μmol PL), and [3H]cholesterol (107 dpm/μmol of free cholesterol) (PerkinElmer Life Sciences). The lipid mixture for fluorescence studies was labeled with dansyl cholesterol (gift from Drs. A. Misharin and C. Alquier). A fine emulsion (about 0.7 μm in median diameter) was prepared by sonication of 100 mg of lipid mixture in 6 ml of distilled water for 10 min at 95% power level and a frequency of 20,178 Hz (Sonoreactor, Undatim, Japan), in ice/ethanol. A medium-size emulsion (about 2 μm) was obtained by sonicating 100 mg of lipid mixture in 3 ml of distilled water for 5 min at 25 watts power in an ice/ethanol cooling bath using a microtip probe (Brandson 250 W sonifier, Osi, France). A coarse emulsion (about 15 μm) was prepared by mechanical stirring of 100 mg of lipid mixture in 1 ml of distilled water for 1.5 min at room temperature. The emulsions obtained were collected after concentration and removal of excess phospholipids as follow: the coarse emulsion was allowed to stand for 10 min in ice, and the medium-size and fine ones were centrifuged 10 min or 1 h at 4,000 rpm and 10 °C, respectively. The resultant triacylglycerol/phospholipid ratios (w/w) were found to be 50/1, 40/1, and 14/1 for the coarse, medium, and fine emulsions, respectively. The emulsion droplet sizes were determined as previously reported (3Armand M. Borel P. Dubois C. Senft M. Peyrot J. Salducci J. Lafont H. Lairon D. Am. J. Physiol. 1994; 266: G372-G381PubMed Google Scholar) using a particle-size analyzer (Capa-700, Horiba, Kyoto, Japan). The results are given in the form of a frequency distribution graph (Fig. 1). Emulsion median diameter (μm) and emulsion surface area (Sw, m2/g emulsified fat) were calculated by the particle-sizer software from the droplet size distribution. The coarse emulsion (Fig. 1 A) was composed of lipid droplets sizing from 1 to 40 μm with a majority of particles between 8 and 30 μm (about 70% total particles by volume). For the medium-size emulsion (Fig. 1 B), lipid droplets sized from 0.1 to 40 μm and about 80% of total particles ranged from 1 to 4 μm. The fine emulsion (Fig. 1 C) was mainly composed of small size droplets from 0.1 to 2 μm, with 75% of total particles sizing between 0.1 and 1 μm. Emulsion surface area varied inversely with the emulsion median diameter. Both parameters were significantly different for the three emulsions (ANOVA, p < 0.05). Experiments were carried out at 37 °C and pH 5.40, using polycarbonate test tubes (13 × 51 mm, Beckman Instruments, Palo Alto, CA) to limit the loss of lipid molecules on the inner surface of the test tube during lipolysis. The reaction medium was a 2.5-ml mixture containing 100 mm sodium acetate, 150 mm NaCl, 6 mm CaCl2 (buffer L), 1.5 μm bovine serum albumin, and 25 μmol of triolein emulsified as described above. The HGL/TO ratio was selected to mimic physiological conditions (3Armand M. Borel P. Dubois C. Senft M. Peyrot J. Salducci J. Lafont H. Lairon D. Am. J. Physiol. 1994; 266: G372-G381PubMed Google Scholar, 10Armand M. Pasquier B. André M. Borel P. Senft M. Peyrot J. Salducci J. Portugal H. Jaussan V. Lairon D. Am. J. Clin. Nutr. 1999; 70: 1096-1106Crossref PubMed Scopus (377) Google Scholar, 15Carrière F. Barrowman J.A. Verger R. Laugier R. Gastroenterol. 1993; 105: 876-888Abstract Full Text PDF PubMed Scopus (384) Google Scholar), i.e. excess enzyme, and was 2.5 units (53.2 pmol)/μmol of triolein. Samples (200 μl) were collected at intervals from 0 to 100 min, and lipids were extracted immediately by the Folch method (30Folch J. Lees M. Stanley J.H.G. J. Biol. Chem. 1957; 226: 498-509Abstract Full Text PDF Google Scholar). Lipids were separated by thin-layer chromatography (TLC) on silica gel (Ready plastic sheet F1500, Schleider and Schuell, Germany) according to Bitman and Wood (31Bitman J. Wood D.L. J. Liq. Chromatogr. 1981; 4: 1023-1034Crossref Scopus (44) Google Scholar). After exposure to iodine vapors, individual lipid spots were scraped and the radioactivity was measured by scintillation counting (1600TR, Packard, Meriden, CT). The reaction medium was a 0.8 ml of mixture of buffer L with medium-size [3H]triolein emulsion, at pH 5.4 and 37 °C. Three HGL/TO ratios were used corresponding to high (53.2 pmol/μmol) and moderate (10.6 pmol/μmol) physiological ratios, and to a large excess substrate (0.21 pmol/μmol). In the first experiment, a large amount of exogenous OA (1.4 μmol of OA/μmol of triolein) (final ethanol concentration: 1.2–2.5%, v/v) was added prior to the initiation of lipolysis or 5 min after. In addition, a physiological amount of OA, corresponding to the amount of free fatty acid generated during a 60-min lipolysis of the emulsion (0.3 μmol of OA/μmol of triolein), was added 5 min after lipolysis started. For the second and third experiments, 0.3 μmol of OA/μmol of triolein was added before or 5 min after the beginning of lipolysis. Samples (100 μl) were collected at intervals from 0 to 60 min. [3H]OA produced were separated by liquid-liquid partition (32Belfrage P. Vaughan M. J. Lipid Res. 1969; 10: 341-344Abstract Full Text PDF PubMed Google Scholar) and radioactivity was measured as described above. The reaction medium was a 0.8-ml mixture of buffer L with [3H]OA (2 or 6.9%) and [14C]TO-PL emulsions of 2.5 or 1.3 μm, respectively, at pH 5.4 and 37 °C. The HGL/TO ratio was 53.2 pmol/μmol. Samples (100 μl) were collected at intervals from 0 to 60 min and the amount of [14C]OA released was determined as described above. The reaction medium was a 1.1-ml mixture of buffer L and [3H]triolein medium-size emulsion at pH 5.4 and 37 °C, with a HGL/TO ratio of 53.2 pmol/μmol. Samples (100 μl) were collected from 0 to 60 min. After 60 min lipolysis, a new dose of HGL was added, thus doubling the initial concentration of enzyme, and lipolysis was carried out for an additional 60 min. Samples (100 μl) were collected then from 65 to 120 min after lipolysis. The reaction medium was a 1.5-ml mixture of buffer L and [3H]triolein medium-size emulsion, at pH 5.4 and 37 °C. Pure HGL or gastric juice, with a gastric lipase equivalent/triolein ratio of 53.2 pmol/μmol were used. Samples (100 μl) were collected from 0 to 90 min. At 90 min, a [14C]triolein medium-size emulsion was added, thus doubling the initial concentration of triolein. Samples (100 μl) were then collected at 95 to 180 min lipolysis. The amounts of [3H]- and [14C]OA released were determined as described above. Rates of lipolysis were calculated as the percent of triacylglycerols hydrolyzed, from micromoles of released OA at a given time (OA t ) relative to the total micromoles of physiologically releasable OA (S n-1 andS n-3 positions), based on the initial amount of triolein, using the following equation: [OA t /(TO × 2)] × 100. Samples of a radiolabeled medium-size emulsion collected before or after 10, 25, and 60 min lipolysis by pure HGL were transferred to sealed glass disposable micro-sampling pipettes (inner diameter (1.1–1.2 mm) × L (75 mm)) (Corning, New York) and the surface and core phases of the lipid droplets were separated by centrifugating at 20,000 rpm for 18 h in a Beckman SW 40 Ti swinging bucket rotor (Beckman Instruments, Inc.) using a Beckman Ultracentrifuge (model number L7) according to Miller and Small (11Miller K.W. Small D.M. J. Colloid Interface Sci. 1982; 89: 466-478Crossref Scopus (69) Google Scholar). Lipid classes of the two phases were analyzed as described above. Samples of a lipolysed medium-size emulsion collected at intervals from 0 to 80 min were mixed volume/volume with 1% OsO4 in distilled water at pH 5.4 and room temperature. The mixture was gently shaken, put on a microscope cover glass, and fixed overnight in a moist chamber at room temperature. The emulsion deposit was then gently washed with distilled water, first dried with filter paper followed by drying 1 day in a silica gel dessicator. Preparations were gold-palladium coated then examined at magnification ×4,800 to 6,600 with a JSM-35CF scanning electron microscope (JEOLS, Paris, France) operated at 35 kV accelerating potential. An aliquot (50 μl) of a medium-size emulsion previously incubated with HGL for 60 min was mixed in a shaking bath at 37 °C with a medium-size emulsion labeled with dansyl cholesterol to distinguish from the first emulsion, at pH 5.40 for 15 min. Then the mixture was incubated for 30 min on ice with 10 μl of purified specific polyclonal anti-HGL antibodies (6.2 mg/ml) from rabbit (diluted 10 times in buffer L) followed by incubation for 30 min on ice with 10 μl of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Zymed Laboratories Inc., South San Francisco, CA) (diluted 10 times in buffer L). High levels of antibodies were used to obtain sufficient labeling at acid pH and cold temperature. A negative control was performed without the enzyme. HGL was mixed with TRITC (Molecular Probe, Inc.) (25 μg/mg of protein) in 25 mm sodium carbonate buffer, pH 9.0, at room temperature for 1 h, then quickly neutralized to pH 6.0, dialyzed against sodium acetate 50 mm, 150 mm NaCl, pH 6.0, at cold temperature overnight and concentrated on PEG-6000 at 4 °C. Under these conditions HGL retained about 60% of its initial activity. Based on the method of Holczinger (33Holczinger L. Acta Histochem. 1959; 8: 167-175PubMed Google Scholar), a copper acetate solution (Sigma) (0.15% final concentration) was mixed carefully into the reaction medium after 90 min lipolysis of a medium-size emulsion. The copper-free fatty acid soaps formed were then visualized with FITC-Gly-Gly-His (Molecular probe) (38 μg/ml final concentration), a marker with high selectivity and sensitivity for Cu2+ due to the presence of the tripeptide commonly called copper-binding peptide (34Lau S.J. Kruck T.P.A. Sarkar B. J. Biol. Chem. 1974; 249: 5878-5884Abstract Full Text PDF PubMed Google Scholar). All specimens were examined under a Leitz Dialux 20 microscope (Jena, Germany) equipped with a Ploemopak 3.1 epifluorescence system using filters specific for FITC (filter bloc Leitz model L3), dansyl cholesterol (filter block Leitz model A2), or TRITC (filter block Leitz model N2), at a final magnification of ×1,250. Photomicrographs were taken using a CCD color Camera (DC 100, Leica, Switzerland). A [14C]triolein, [3H]PL, free [3H]cholesterol medium-size emulsion was incubated with HGL for 90 min. The lipid particles generated during lipolysis were isolated by FPLC using a Superose 6 column (6 × 57 cm) at room temperature with a flow rate of 0.3 ml/min with buffer L as eluent. The lipid droplets are retained on the column. The fractions obtained were analyzed for lipid composition by TLC and radioisotope counting as described above. The size of the lipid particles was determined with a quasielastic light-scattering detector (SEMAtech, Nice, France). The presence of HGL was ascertained by immunoblotting (35Burnette W.N. Anal. Biochem. 1981; 112: 195-203Crossref PubMed Scopus (5927) Google Scholar) by depositing 20 μl of the various fractions on a polyvinylidene difluoride membrane; the membrane was soaked for 30 min at room temperature in 5% skim milk in a TBS Tween buffer, washed, and incubated with HGL-polyclonal rabbit antiserum (final dilution 1:5000); immunodetection was carried out with alkaline phosphatase-labeled goat anti-rabbit IgG (final dilution 1:5000) (Sigma). Statistical significances were analyzed by one-way analysis of variance (ANOVA) and the differences were determined by the Fisher's test at a probability of 95%. Correlation coefficient was obtained from linear regression (StatView II; Abacus, Berkeley, CA) (36Winer B.J. Statistical Principles in Experimental Design. McGraw-Hill, New York1971Google Scholar). The amounts of free fatty acid released
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