Biosci. Biotechnol. Biochem., 69 (9), 1726–1732, 2005
Hydrolysis of Micellar Phosphatidylcholine Accelerates Cholesterol Absorption in Rats and Caco-2 Cells Tadateru H AMADA,1; y Ikuo I KEDA,2 Kayoko T AKASHIMA,1 Makoto K OBAYASHI,1 Yoko K ODAMA,1 Takashi I NOUE,1 Ryosuke M ATSUOKA,1 and Katsumi I MAIZUMI1 1
Laboratory of Nutrition Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan 2 Laboratory of Food and Biomolecular Science, Department of Food Function and Health, Division of Bioscience and Biotechnology for Future Bioindustries, Graduate School of Agricultural Science, Tohoku University, Miyagi 981-8555, Japan Received April 11, 2005; Accepted May 26, 2005
Lymphatic recovery of cholesterol infused into the duodenum as bile salt micelles containing phosphatidylcholine (PC) was accelerated by the co-administration of phospholipase A2 in bile and pancreatic juice diverted rats. Previously we observed that cholesterol esterase, which has the ability to hydrolyze PC, caused the same effect under a similar experimental condition (Ikeda et al., Biochim. Biophys. Acta, 1571, 34–44 (2002)). Accelerated cholesterol absorption was also observed when a part of micellar PC was replaced by lysophosphatidylcholine (LysoPC) and oleic acid. Phospholipase A2 facilitated the incorporation of micellar cholesterol into Caco-2 cells in a dose-dependent manner. There was a highly negative correlation between the incorporation of cholesterol into Caco-2 cells and the content of micellar PC remaining in the culture medium. The release of cholesterol as a monomer from bile salt micelles was enhanced when a part of micellar PC was replaced with LysoPC and oleic acid. These results strongly suggest that the release of monomer cholesterol from bile salt micelles is accelerated by hydrolysis of PC in bile salt micelles and hence that cholesterol absorption is enhanced. Key words:
phospholipase A2 ; cholesterol esterase; phosphatidylcholine; intestinal cholesterol absorption
Pancreatic cholesterol esterase, which is now predominantly called carboxyl ester lipase, is a nonspecific lipolytic enzyme capable of hydrolyzing cholesterol esters, tri-, di-, and mono-acylglycerols, phospholipids, lysophospholipids, and ceramide.1) Because cholesterol esters are not directly absorbed, cholesterol esterase is thought to be important for intestinal absorption of
cholesterol esters. Although some research has pointed out that cholesterol esterase can facilitate intestinal absorption of unesterified cholesterol,2–4) other observations did not show acceleration of cholesterol absorption.5–7) In addition, Howles et al. have reported that cholesterol esterase is not essential for absorption of unesterified cholesterol using cholesterol esterase knockout mice.8) In contrast to these observations, we showed recently that cholesterol esterase facilitates intestinal absorption of cholesterol dissolved in a bile salt micelle containing phosphatidylcholine (PC), but not in a PC-depleted micelle in thoracic duct-cannulated rats.9) In addition, cholesterol esterase accelerated the incorporation of micellar cholesterol into differentiated Caco-2 cells, a model of intestinal epithelial cells, only when micelles contained PC.9) The effect was found to occur through reduction of the affinity between cholesterol and a bile salt micelle containing PC. Since cholesterol esterase has the ability to hydrolyze PC,1) we suggested that cholesterol esterase induces reduction of the affinity of cholesterol to bile salt micelles through hydrolysis of micellar PC.9) If acceleration of the incorporation of micellar cholesterol into intestinal cells occurs through hydrolysis of PC, phospholipase A2 , which has a primary role in PC hydrolysis in the intestinal lumen,10) must have a similar effect to cholesterol esterase. In the present study, the effect of porcine pancreatic phospholipase A2 on absorption of cholesterol was examined in vivo and in vitro.
Materials and Methods Materials. Egg yolk PC, egg yolk lysophosphatidylcholine (LysoPC), oleic acid, porcine pancreatic phos-
y To whom correspondence should be addressed. Fax: +81-92-642-3005; E-mail:
[email protected] Abbreviations: PC, phosphatidylcholine; LysoPC, lysophosphatidylcholine; DMEM, Dulbecco’s Modified Eagle Medium; FBS, fetal bovine serum; LPDS, lipoprotein deficient serum; PBS, phosphate buffered saline; TLC, thin layer chromatography; ACAT, acyl-CoA cholesterol acyltransferase
Hydrolysis of Micellar Phosphatidylcholine and Cholesterol Absorption
pholipase A2 , and cholesterol (purity < 99%) were purchased from Sigma (Sigma-Aldrich Japan, Tokyo, Japan). Human recombinant cholesterol esterase was kindly provided by Meiji Milk Products Co., Tokyo. Sodium taurocholate (purity > 97%) was purchased from Nacalai Tesque (Kyoto, Japan). [4-14 C] cholesterol was purchased from Amersham Pharmacia Biotech (Tokyo, Japan). Preparation of micellar solutions. A series of micellar solutions were prepared in a culture medium or a phosphate buffer contained 6.6 mM sodium taurocholate, 0.6 mM PC, 0.05 mM [4-14 C] cholesterol, and 1 mM oleic acid.9) The micelles, prepared by sonication, were passed through a 0.2 mm syringe filter (25 mm, GD/X, Whatman, Inc., Clifton, NJ, U.S.A.) and kept at 37 C until use. Where indicated, micellar solutions contained hydrolysis products of PC. One micellar solution contained 0.45 mM PC, 0.15 mM LysoPC, 0.15 mM oleic acid, and 0.05 mM cholesterol and the other contained 0.3 mM PC, 0.3 mM LysoPC, 0.3 mM oleic acid, and 0.05 mM cholesterol. Animals. Adult male SD rats (Seac Yoshitomi, Fukuoka, Japan) weighing 300–330 g were housed under a normal (12 h) light cycle and allowed free access to laboratory chow (NMF, Oriental Yeast Co., Tokyo, Japan) and water ad libitum prior to use. All animal studies were carried out under the guidelines for animal experiments of the Faculty of Agriculture, Graduate School, Kyushu University, and Law 105 and Notification 6 of the government of Japan. Cannulations of thoracic and bile ducts. Bile and pancreatic juice were drained by an indwelling bile duct catheter inserted into the common bile duct near the junction to the duodenum.9) The left thoracic lymphatic duct cephalad to the cisterna chyli was also cannulated. A third indwelling catheter was placed in the duodenum for administration of test micellar solutions. After surgery, the animals were placed in restraining cages and used for cholesterol absorption study. Effect of phospholipase A2 on the lymphatic absorption of micellar cholesterol. The rats cannulated in the thoracic and bile ducts were intraduodenally given a continuous infusion of a mixed micellar solution composed of 0.6 mM PC, 0.05 mM cholesterol, 1 mM oleic acid, and 6.6 mM sodium taurocholate prepared with 15 mM sodium phosphate buffer (pH 7.4) containing 62 mM NaCl and 139 mM glucose at a rate of 3.4 ml/ h until the end of the experiment. The next morning, animals with a constant lymph flow rate were administered 2 ml of the test micellar solution containing [4-14 C] cholesterol with or without porcine pancreatic phospholipase A2 (188 ng: 0.3 U). Preliminary studies showed that 0.3 U of phospholipase A2 had almost the same PC-hydrolyzing activity as 7.5 U of cholesterol
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esterase under our experimental condition. Previously we found that 7.5 U of cholesterol esterase accelerated lymphatic absorption of cholesterol.9) Lymph was collected in ice-chilled tubes containing EDTA and the radioactivity was measured. The rats were not allowed drinking water in order to avoid dilution of the micellar solution during the course of lymph collection. Effect of hydrolysis products of PC on lymphatic recovery of cholesterol. To investigate the effect of hydrolysis products of PC on lymphatic cholesterol recovery, three types of micellar solutions were prepared and intraduodenally infused to cannulated rats during the course of experiment. As the control micelle, 0.6 mM PC, 0.05 mM cholesterol, and 6.6 mM sodium taurocholate were sonicated in a 15 mM sodium phosphate buffer (pH 7.4) containing 62 mM NaCl and 139 mM glucose. In the PC:LysoPC:oleic acid = 0.45:0.15:0.15 micelle, 0.45 mM PC, 0.15 mM LysoPC, and 0.15 mM oleic acid were contained instead of the 0.6 mM PC in the control micelle. In the PC:LysoPC:oleic acid = 0.3:0.3:0.3 micelle, 0.3 mM PC, 0.3 mM LysoPC, and 0.3 mM oleic acid were contained instead of the 0.6 mM PC in the control micelle. The next morning, 2 ml of each micelle containing [4-14 C] cholesterol were infused and the radioactivities of lymph collected for 24 h were measured by liquid scintillation counter. Cell culture. Caco-2 cells were grown on 100 mm plastic petri dishes at 37 C in air and 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO, Invitrogen Co., Carlsbad, CA, U.S.A.), supplemented with 10% fetal bovine serum (FBS, GIBCO), 2 mM Lglutamine, 10 mg/ml insulin, 100 mg/ml streptomycin, 100 IU/ml penicillin, 1% non essential amino acids (ICN Biomedicals, Inc., Aurora, OH, U.S.A.), and 3.7 g/l NaHCO3 .11) Cells were subcultured and grown to confluency on the apical side of presoaked membrane filters (Biocoat cell culture collagen type I, FALCON, Nippon Becton Dickinson Co., Tokyo, Japan). At 14–16 days after reaching confluency, cells were subjected to experimentation. At 24 h prior to commencing the experiments, the culture medium containing 10% FBS was replaced with the culture medium supplemented with 10% lipoprotein deficient serum (LPDS) prepared from FBS.9) A mixed micellar solution containing 0.6 mM PC, 0.05 mM [4-14 C] cholesterol, 1 mM oleic acid, and 6.6 mM sodium taurocholate was prepared in culture medium. After filtration through a 0.2 mm filter, the micellar solution supplemented with 10% LPDS was added on the apical side of differentiated Caco-2 cells and culture medium was added on the lower well (basolateral side). In the case where the effect of human cholesterol esterase or porcine pancreatic phospholipase A2 was examined, they were added on the apical side. To investigate the dose dependency of phospholipase A2 on cholesterol absorption, various
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Fig. 1. Effect of Porcine Pancreatic Phospholipase A2 (PLA2 ) and Hydrolysis Products of Micellar Phosphatidylcholine (PC) on Lymphatic Recovery of Micellar Cholesterol in Thoracic Duct-Cannulated Rats Drained of Pancreatic Juice and Bile. A, Bile and pancreatic juice-diverted rats were infused with a micellar solution to the duodenum overnight. The micellar solution contained 6.6 mM sodium taurocholate, 1 mM oleic acid, 0.6 mM PC, 0.05 mM cholesterol, 62 mM NaCl, and 139 mM glucose in 15 mM sodium phosphate buffer at pH 7.4. The next morning, the rats were administered 2 ml of a test micellar solution containing [4-14 C] cholesterol with or without porcine pancreatic PLA2 (188 ng protein, 0.3 U). Lymph was collected for 24 h. One unit porcine pancreatic PLA2 was defined to be equivalent to 1 mmol PC hydrolyze to lysophosphatidylcholine (LysoPC)/min (627 ng protein). The lymph flow was 38:5 2:5 and 37:1 1:6 ml/24 h in the PLA2 () and PLA2 (þ) groups respectively. Data are means SE for eight rats. *, Significantly different from the PLA2 () group at P < 0:05. B, The control micelle contained 6.6 mM sodium taurocholate, 0.6 mM PC, 0.05 mM cholesterol, 62 mM NaCl, and 139 mM glucose. The PC:LysoPC:oleic acid = 0.45:0.15:0.15 micelle contained 0.45 mM PC, 0.15 mM LysoPC, and 0.15 mM oleic acid instead of the 0.6 mM PC in the control micelle. The PC:LysoPC:oleic acid = 0.3:0.3:0.3 micelle contained 0.3 mM PC, 0.3 mM LysoPC, and 0.3 mM oleic acid instead of the 0.6 mM PC in the control micelle. The lymph flow was 36:2 2:44 ml/24 h in the control micelle, 40:2 2:64 ml/24 h in the PC:LysoPC:oleic acid = 0.45:0.15:0.15 micelle, and 38:9 1:55 ml/24 h in the PC:LysoPC:oleic acid = 0.3:0.3:0.3 micelle. Data are means SE of five to eight rats. a, b, Different letters show significant difference at P < 0:05.
amounts of phospholipase A2 (6.27, 18.8, and 37.6 mg) were added. After 24 h of the incubation, the culture medium on the apical and basolateral sides was withdrawn and the cells were washed twice with phosphate buffered saline (PBS) and scraped. ACS II (Amersham Biosciences, Tokyo, Japan) was added to the mediums and the cells were solubilized by NCS II (Amersham). The levels of radioactivity were counted with a liquid scintillation counter. The amount of cholesterol incorporated into Caco-2 cells was calculated by the sum of cholesterol associated with cells and secreted to the basolateral side. Lipid extraction and separation. Total lipids of Caco2 cells, micellar solutions, and culture medium collected from the apical side of Caco-2 cells at the end of the incubation with a micellar solution were extracted with chloroform:methanol = 2:1.12) Free and esterified cholesterol were separated by thin layer chromatography (TLC, hexane:diethyl ether:acetic acid = 83:16:1 as the developing solvent) and visualized with I2 vapor. Radioactivities of free and esterified cholesterol were measured with a liquid scintillation counter. PC and
LysoPC were separated with TLC (chloroform: methanol:water = 60:40:5 as the developing solvent) and the amounts of inorganic phosphorus of PC were determined.13) Transfer of cholesterol from micellar solutions to triolein. The effect of hydrolysis products of micellar PC on the affinity of cholesterol for bile salt micelles was examined, as described previously.14) One and a half milliliters of a micellar solution and 0.5 ml of triolein were placed in a plastic tube, flushed with N2 , and sealed. Mixed micellar solution composed of 6.6 mM sodium taurocholate, 0.6 mM PC, 0.1 mM cholesterol, and 132 mM NaCl in 15 mM sodium phosphate buffer (pH 7.4) was prepared. To investigate the effect of the hydrolysis products of micellar PC, 25% of micellar PC was replaced with LysoPC and oleic acid (PC:LysoPC: oleic acid = 0.45:0.15:0.15 micelle). The tubes were incubated at 37 C in an oscillating water bath for 6 h (190 oscillations/min) at which time the rate of cholesterol transfer from micellar solutions to triolein was linear. At the end of the incubation, the contents of each tube were transferred to Beckman Quick Seal tube, and
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the oil and aqueous phases were separated by ultracentrifugation (Optima TL Ultracentrifuge, Beckman, Palo Alto, CA, U.S.A.) at 100;000 g for 1 h at 37 C. The oil and aqueous phases were collected and analyzed for cholesterol concentration by gas-liquid chromatography using 5-cholestane as an internal standard.14) Statistical analysis. All data were expressed as means SE. Student’s t-test or Bonferroni/Dunn test were used, and P values of
