Biochemical and Molecular Actions of Nutrients

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1 Biochemical and Molecular Actions of Nutrients Tea Catechins with a Galloyl Moiety Suppress Postprandial Hypertriacylglycerolemia by Delaying Lymphatic Transport of Dietary Fat in Rats Ikuo Ikeda, 1 Koichi Tsuda, Yuko Suzuki,* Makoto Kobayashi,* Tomonori Unno,* Hiroko Tomoyori, Hitomi Goto, Yayoi Kawata, Katsumi Imaizumi, Ayumu Nozawa,* and Takami Kakuda* Laboratory of Nutrition Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Kyushu University, Fukuoka , Japan; *Central Research Institute, ITO EN, Ltd., Shizuoka , Japan; and Department of Environmental and Symbiotic Sciences, Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, Kumamoto , Japan ABSTRACT Tea catechins, ( )-epicatechin (EC), ( )-epigallocatechin (EGC), ( )-epicatechin gallate (ECG), and ( )-epigallocatechin gallate (EGCG), have been shown to be epimerized to ( )-catechin (C), ( )-gallocatechin (GC), ( )-catechin gallate (CG), and ( )-gallocatechin gallate (GCG), respectively, during heat treatment. In this study, we examined the effect of tea catechins rich in ECG and EGCG and heat-treated tea catechins rich in CG and GCG on postprandial hypertriacylglycerolemia in rats. Both tea catechins and heat-treated tea catechins suppressed postprandial hypertriacylglycerolemia. Lymphatic recovery of 14 C-trioleoylglycerol in rats cannulated in the thoracic duct was delayed by the administration of tea catechins and heat-treated tea catechins. Tea catechins and heat-treated tea catechins had the same effect on all variables tested. These catechin preparations dosedependently inhibited the activity of pancreatic lipase in vitro. When purified catechins were used, only those with a galloyl moiety inhibited the activity of pancreatic lipase. These results suggest that catechins with a galloyl moiety suppress postprandial hypertriacylglycerolemia by slowing down triacylglycerol absorption through the inhibition of pancreatic lipase. Because postprandial hypertriacylglycerolemia is a risk factor for coronary heart disease, our results suggest that catechins with a galloyl moiety may prevent this disease. J. Nutr. 135: , KEY WORDS: catechins postprandial hypertriacylglycerolemia pancreatic lipase rats tea Tea catechins consist mainly of 4 derivatives; ( )-epicatechin (EC), 2 ( )-epigallocatechin (EGC), ( )-epicatechin gallate (ECG), and ( )-epigallocatechin gallate (EGCG) (Fig. 1). These catechins, which are contained in green tea, oolong tea, and black tea, have been shown to have hypocholesterolemic, antiatherogenic, antiobesity, antioxidative, and anticarcinogenic activities (1 10). We previously showed that tea catechins inhibit intestinal absorption of cholesterol in rats (11,12). It also has been reported that tea catechins prevent LDL oxidation (13 15) and suppress atheroma formation in apoe knockout mice (5). These observations suggest that tea catechins are effective in preventing coronary heart disease. Because postprandial hypertriacylglycerolemia is a risk factor for coronary heart disease (16), its suppression by food components may effectively prevent the disease. Juhel et al. (17) showed that a green tea extract inhibited gastric and pancreatic lipases in vitro. Although the results suggest that a green 1 To whom correspondence should be addressed. iikeda@agr.kyushu-u.ac.jp. 2 Abbreviations used: C, ( )-catechin; CG, ( )-catechin gallate; EC, ( )- epicatechin; ECG, ( )-epicatechin gallate; EGC, ( )-epigallocatechin; EGCG, ( )-epigallocatechin gallate; GC, ( )-gallocatechin; GCG, ( )-gallocatechin gallate; TES, N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid. tea extract slows or inhibits fat absorption in the intestine and suppresses postprandial hypertriacylglycerolemia, the effect has never been experimentally tested. Tea catechins are thought to be responsible for the lipase inhibitor in green tea extract. However, Han et al. (18) reported that tea saponins, not catechins, inhibited pancreatic lipase activity in vitro. In this study, the effect of tea catechins on postprandial hypertriacylglycerolemia was examined in rats, and the underlying mechanism was clarified in vitro and in vivo. We used rats as a model of postprandial hypertriacylglycerolemia, because the processes of digestion and absorption of dietary triacylglycerols are similar in rats and humans. Canned and bottled tea beverages are widely consumed in Asian countries. For example, L canned and bottled tea beverages were produced in 2000 in Japan. About 50% of tea catechins in these beverages are epimerized by heat treatment to ( )-catechin (C), ( )-gallocatechin (GC), ( )- catechin gallate (CG), and ( )-gallocatechin gallate (GCG) (Fig. 1) (19,20). Although many studies have been conducted on physiological functions of tea catechins, studies on heattreated tea catechins are scarce. In this study, the effect of heat-treated tea catechins on postprandial hypertriacylglycerolemia was also compared with tea catechins /05 $ American Society for Nutritional Sciences. Manuscript received 4 July Initial review completed 22 July Revision accepted 21 October

2 156 IKEDA ET AL. FIGURE 1 Chemical structures of tea catechins and their corresponding epimers. MATERIALS AND METHODS Purified catechins, EC, EGC, ECG, EGCG, C, GC, CG, and GCG were obtained from Wako Pure Chemicals. Purities of all of these catechins were 98%. THEA-FLAN 90S, a decaffeinated green tea powder provided from ITO EN, was used as a mixture of tea catechins. This product was rich in EGCG and ECG. Heat-treated tea catechins were prepared by autoclaving the catechin mixture at 120 C for 5 min. The composition of catechins in tea catechins and heat-treated tea catechins is shown in Table 1. Heat-treated tea catechins prepared from tea catechins contained increased amounts of GCG and CG. Tri[1-14 C]oleoylglycerol (2.0 GBq/mmol) was obtained from Amersham Pharmacia Biotech. Postprandial hypertriacylglycerolemia in rats. Male Wistar rats (8 wk old, SPF, Japan SLC) were fed a commercial nonpurified diet (CE-2; CLEA Japan) for 1 wk and deprived of food overnight. After blood was withdrawn from the jugular vein as 0 time, a solution containing tea catechins or heat-treated tea catechins [100 mg/(5 ml deionized water kg body weight)] was given orally. Deionized water was given in the control group. Immediately after administration, a lipid emulsion containing 200 g/l soybean oil, 12 g/l egg lecithin, and 22.5 g/l glycerin was given orally, at a dose of 10 ml/kg (21). Blood (100 L) was withdrawn at 1, 2, 3, 4, 6, and 8 h after administration via the jugular vein. After the separation of serum, triacylglycerol was measured with a commercial kit (Triglyceride E test Wako; Wako Pure Chemicals). Lymphatic recovery of 14 C-trioleoylglycerol in rats cannulated in the thoracic duct. Nine-wk-old male Sprague Dawley rats cannulated in the thoracic duct were given i.g. 3 ml of a test emulsion containing 14 C-trioleoylglycerol with or without catechins. After administration, lymph was collected for 24 h. The test emulsion (3 ml) contained 200 mg sodium taurocholate (Nacalai tesque), 50 mg fatty-acid-free bovine serum albumin fraction V (Bayer), 200 mg trioleoylglycerol (Sigma), and 37 kbq 14 C-trioleoylglycerol in deionized water. When tea catechins and heat-treated tea catechins were administered, these catechins were added to the emulsion at 100 mg in 3 ml test emulsion, respectively. Operations and maintenance of rats and all other procedures were performed as described previously (22,23). All rat studies were carried out under the guidelines for animal experiments of the Faculty of Agriculture, Graduate School Kyushu University and Central Research Institute, ITO EN, and Law 105 and Notification 6 of the government of Japan. Micellar solubility of hydrolysis products of triacylglycerol in vitro. A micellar solution containing 1 mmol/l oleic acid (Sigma), 0.5 mmol/l 1-monooleoylglycerol (Sigma), 6.6 mmol/l sodium taurocholate, 0.6 mmol/l egg phosphatidylcholine (Sigma), and 132 mmol/l sodium chloride in 15 mmol/l sodium phosphate buffer (ph 6.8) was prepared by sonication and was kept at 37 C for 24 h for stabilization of the micelles. A solution of tea catechins or heattreated tea catechins was added to the micelles (final concentration, 2 g/l micelles) and incubated for 1hat37 C. The micellar solution was passed through a 0.2- m syringe filter (25 mm, GDD/X; Whatman). After lipid extraction from the filtrate, the concentration of total fatty acids was measured by GLC (24). Activity of pancreatic lipase in vitro. The activity of pancreatic lipase was measured according to the method of Han et al. (18). An emulsion (9 ml) containing 80 mg trioleoylglycerol, 10 mg phosphatidylcholine, and 5 mg sodium taurocholate in 0.1 mol/l N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES) buffer (ph 7.0) containing 0.1 mol/l sodium chloride was prepared by sonication and kept at 37 C. A total of 100 L of the emulsion was incubated with 5Uofporcine pancreatic lipase (Sigma) solubilized in 0.1 mol/l TES buffer containing 0.1 mol/l sodium chloride and various amounts of catechin solution (100 L) at 37 C for 30 min. Released fatty acids were extracted with chloroform: heptane: methanol (49:49:2, v:v:v) and colorimetrically measured. Statistical analysis. Data are expressed as means SEM. Statistical analysis of data were performed by two-way ANOVA or two-way repeated-measure ANOVA followed by the Tukey-Kramer test to identify differences among groups. Differences were considered significant at P RESULTS Postprandial hypertriacylglycerolemia in rats. Serum triacylglycerol concentration increased after the administration of a fat emulsion and was highest at 2 h after administration (Fig. 2). The serum triacylglycerol concentration at 1, 2, and TABLE 1 Composition of tea catechins and heat-treated tea catechins1 Catechins Tea catechins Heat-treated tea catechins g/100 g dry weight EGCG GCG ECG CG EGC GC nd 0.1 EC ( )C 0.3 nd Total Gallate esters Caffein nd nd 1 nd, not detected.

3 TEA CATECHINS AND HYPERTRIACYLGLYCEROLEMIA 157 FIGURE 2 Effect of tea catechins and heat-treated tea catechins on postprandial hypertriacylglycerolemia in rats intragastrically administered fat emulsions. Values are means SEM, n 7. Means not sharing a letter at a time point differ, P *Within a group, means differ from that at time 0, P Two-way repeated-measure ANOVA: effect of group, P 0.075; effect of time, P ; interaction between group and time, P h after administration was significantly lower in the tea catechin and heat-treated tea catechin groups than in the control group. There was no difference between the 2 catechin-treated groups. Lymphatic recovery of 14 C-trioleoylglycerol in rats cannulated in the thoracic duct. Lymphatic recoveries of the radioactivity given as 14 C-trioleoylglycerol at 1 h after administration were significantly lower in the tea catechin and heat-treated tea catechin groups than in the control group (Fig. 3). The tea catechins and heat-treated tea catechins had similar effects on the lymphatic absorption of trioleoylglycerol. Total 24-h recovery of the radioactivity was the same among the 3 groups. Micellar solubility of hydrolysis products of triacylglycerol in vitro. When bile-salt micelles containing oleic acid, 1-monooleoylglycerol, and phosphatidylcholine were incubated with tea catechins or heat-treated tea catechins in vitro, no precipitates were observed, and the concentrations of total fatty acids originating from oleic acid, monooleoylglycerol, and phosphatidylcholine in bile-salt micelles were not affected by the addition of tea catechins or heat-treated tea catechins (data not shown). Activity of pancreatic lipase in vitro. The activity of pancreatic lipase was dose-dependently inhibited by the addition of tea catechins and heat-treated tea catechins (Fig. 4). There was no difference between the 2 catechin preparations. Catechins with a galloyl moiety dose-dependently inhibited the activity of pancreatic lipase (Fig. 5). CG and GCG more effectively inhibited the lipase activity than ECG and EGCG at 1 and 2 mmol/l. Catechins without a galloyl moiety, C, EC, GC, and EGC did not inhibit pancreatic lipase activity (data not shown). DISCUSSION Our results showed, for the first time, that tea catechins and heat-treated tea catechins suppressed postprandial hypertriacylglycerolemia to almost the same extent in rats (Fig. 2). Because lymphatic recovery of trioleoylglycerol was delayed by the ingestion of tea catechins and heat-treated tea catechins (Fig. 3), one cause of the suppression of postprandial hypertriacylglycerolemia by the catechin preparations may be the slower absorption of triacylglycerol in the intestine. Both tea catechins and heat-treated tea catechins dose-dependently inhibited the activity of pancreatic lipase in vitro (Fig. 4). The results strongly suggest that the delayed transport of trioleoylglycerol to the lymph is caused by the inhibition of pancreatic lipase. In the study of suppression of postprandial hypertriacylglycerolemia (Fig. 2), catechins were administered at a concentration of 30 mg/g trioleoylglycerol. If we assume that one human meal contains g of fats, mg of catechins may be effective doses to inhibit postprandial hypertriacylglycerolemia in humans. Green tea beverages in Japan contain mg of heat-treated tea catechins per serving. Therefore, ingestion of a sufficient amount of catechins is possible. In a preliminary human study, we observed that postprandial hypertriacylglycerolemia in normal subjects was suppressed by the administration of a tea beverage containing about 700 mg/d of heat-treated catechins (unpublished results). In our previous study, the effect of tea catechins rich in EGCG and ECG on lymphatic transport of fatty acids after intragastric administration of coconut oil or palm olein was examined in rats cannulated in the thoracic duct (11). When coconut oil was given, tea catechins delayed lymphatic recovery of fatty acids and the result was consistent with the observation in this study. When palm olein was given, there was no delayed lymphatic recovery, but total recovery for 24 h of fatty acids was lower by the administration of tea catechins. Therefore, the effect of tea catechins on fat absorption was FIGURE 3 Effect of tea catechins and heat-treated tea catechins on lymphatic recovery of [4-14 C] trioleoylglycerol in rats intragastrically administered fat emulsions. Values are means SEM, n 7. Means not sharing a letter at a time differ, P Two-way repeatedmeasure ANOVA: effect of group, P 0.14; effect of time, P ; interaction between group and time, P 0.46.

4 158 IKEDA ET AL. FIGURE 4 Effect of tea catechins and heat-treated tea catechins on the activity of pancreatic lipase in vitro. The enzyme activity at 0 g/l was considered 100%. Values are means SEM, n 3. Within a catechin preparation, means not sharing a letter differ, P Two-way ANOVA: effect of type of catechin, P 0.11; effect of catechin concentration, P ; interaction between type of catechin and catechin concentration, P equivocal in our previous work (11). In that study, lymphatic recovery of fatty acids was analyzed by GLC. Because endogenous fatty acids were contained in lymph fluids, quantitative estimation of absorbed fatty acids was impossible. In the present study, because lymphatic recovery of fatty acids was quantified using radiolabeled trioleoylglycerol, we think that precise information on the effect of tea catechins on fat absorption was obtained. Han et al. (25) showed that tea saponin affected pancreatic lipase activity in vitro. However, they did not report the data, indicating that tea catechins did not inhibit the activity of pancreatic lipase. Although we followed their method to measure the inhibitory effect of tea catechins on pancreatic lipase activity, both tea catechins and heat-treated tea catechins dose-dependently inhibited pancreatic lipase activity (Figs. 4 and 5). Causes of the discrepancy are not clearly explained at present. It is also not evident if tea catechins and heat-treated tea catechins inhibit pancreatic lipase activity in vivo. However, because our study simultaneously showed the suppression of postprandial hypertriacylglycerolemia and delayed recovery of triacylglycerol in lymph by these catechin preparations, it is possible that tea catechins and heat-treated tea catechins inhibit pancreatic lipase activity. Both tea catechins and heat-treated tea catechins contain unidentified components other than catechins (Table 1), likely polymerized or degradated catechins. It is possible that unknown components in catechin preparations also influence the activity of pancreatic lipase. In the present study, major components of heat-treated tea catechins GCG and CG more effectively inhibited the lipase activity than those of tea catechins EGCG and ECG (Fig. 5). Although these results suggest that heat-treated tea catechins that are rich in GCG and CG may more effectively inhibit pancreatic lipase than tea catechins that are rich in EGCG and ECG, different inhibitory effects on the lipase activity were not observed between tea catechins and heat-treated tea catechins (Fig. 4). We also showed that inhibitory effects by tea catechins and heattreated tea catechins on postprandial hypertriacylglycerolemia and on lymphatic absorption of triacylglycerol were comparable (Figs. 2 and 3). These results suggest that unidentified components contained in the preparations of tea catechins and heat-treated tea catechins may partly contribute to the inhibition of pancreatic lipase. The activation of lipoprotein lipase may be another important determinant of postprandial triacylglycerol concentration in serum. However, because studies have never been conducted on this point, more studies on the effect of tea catechins on the activity of lipoprotein lipase are required. Our previous study showed that tea catechins and heattreated tea catechins precipitated cholesterol from bile-salt micellar solutions and inhibited cholesterol absorption in rats (12). In the present study, the addition of these tea catechins to a bile-salt micellar solution containing the hydrolysis products of triacylglycerols, fatty acids, and monooleoylglycerol did not affect the total fatty acid content in the micelles. The results show that the catechin preparations do not exclude oleic acid and monooleoylglycerol from a bile-salt micellar solution, suggesting that catechin preparations do not suppress triacylglycerol absorption by inhibiting micellar solubility of hydrolysis products of triacylglycerols. Murase et al. (6) showed that long-term feeding of tea catechins, the major component of which was EGCG, reduced the deposition of visceral fat in mice fed a high-fat diet. They ascribed the reduced deposition of visceral fat to enhanced -oxidation in the liver. We suggest that the suppression of postprandial hypertriacylglycerolemia may be another reason for the antiobesity activity of tea catechins. Han et al. (25,26) have pointed out that slower absorption of dietary fat decreases the deposition of visceral fat. After a meal, the increase in blood glucose stimulates the secretion of insulin. Insulin activates peripheral lipoprotein lipase and then hydrolysis of chylomicron triacylglycerols is stimulated. Formed FFAs are mainly incorporated into adipose tissue and deposited as tri- FIGURE 5 Effect of purified catechins with a galloyl moiety on the activity of pancreatic lipase in vitro. The enzyme activity at 0 mmol/l is 100%. Values are means SEM, n 6. Means not sharing a letter at a concentration or within a catechin differ, P Two-way ANOVA: effect of type of catechin, P ; effect of catechin concentration, P ; interaction between type of catechin and catechin concentration, P

5 TEA CATECHINS AND HYPERTRIACYLGLYCEROLEMIA 159 acylglycerols. If the concentration of chylomicron triacylglycerols is higher, it is possible that the deposition of fatty acids hydrolyzed from triacylglycerols can be higher in adipose tissues. Therefore the suppression of postprandial hypertriacylglycerolemia by tea catechins may be a cause of the reduction of fat deposition. In conclusion, our study suggests that both green tea catechins and heat-treated tea catechins suppress postprandial hypertriacylglycerolemia, a risk factor for the development of coronary heart disease. Our results, together with previous observations, such as cholesterol-lowering activity (27,28) and prevention of LDL oxidation in vitro (13) and in human studies (14,15), strongly suggest that the ingestion of tea catechins may prevent coronary heart disease. 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