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1 Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan
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Abstract |
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(Received 9 November 2006;
accepted after revision 16 March 2007; first published online 23 March 2007)
Corresponding author T. Hira: Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan. Email: hira{at}chem.agr.hokudai.ac.jp
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Introduction |
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Using non-digestible sucrose fatty acid esters (Noker et al. 1997) in PBD rats, the present study sought to determine whether the inhibitory effects of fat on the pancreatic secretion depend on free fatty acids or esterified fatty acids. We used a stearate-rich digestible triacylglycerol, cocoa butter, as a control fat because the fatty acid moiety of the sugar esters tested is also rich in stearic acid. In order to confirm that there was no degradation of the sucrose esters used, a lipase inhibitor was applied with the sucrose fatty acid ester. Guanidinated casein, whose lysine residues were converted to homoarginine, was used as a potent stimulant of pancreatic exocrine secretion (Hara et al. 1995). We also examined effects of the sucrose ester on secretion of the gastrointestinal hormone, peptide YY (PYY), which is known to inhibit pancreatic secretion (Jin et al. 1993; Naruse et al. 2002) in anaesthetized PBD rats.
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Methods |
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Guanidinated casein was prepared by a previously described method (Hara et al. 1995). The conversion rate of lysyl residues to homoarginine was 96%. Briefly, guanidinated casein (55 g l–1) was hydrolysed with pepsin (0.55 g l–1; Sigma) at pH 1.8 for 10 min at 37°C, and the hydrolysate was then neutralized and desalted (guanidinated casein hydrolysate; HGC). Sucrose fatty acid esters were provided by Dai-Ichi Kogyo Seiyaku Co. (Kyoto, Japan). The water-insoluble sucrose fatty acid ester F-10 is a mixture of di-, tri- and polyesters without monoesters, and the water-soluble F-160 contains 70% monoesters and 30% polyesters. These sucrose fatty acid esters contain stearic acid (70%) and palmitic acid (30%) as esterified fatty acids. The lipase inhibitor (Schwizer et al. 1997) orlistat (tetrahydrolipstatin) was a gift from F. Hoffmann-La Roche Ltd. (Basel, Switzerland). This drug inhibits pancreatic lipase, gastric lipase, carboxyl ester lipase (cholesterol esterase) of pancreatic origin and the bile-salt-stimulated lipase of human milk (Borgstrom, 1988).
Animals and diets in experiments 1 and 2
Male Sprague–Dawley rats (8 weeks old, Japan SLC Inc., Hamamatsu, Japan) were fed a semipurified, sucrose-based diet containing 25% casein for 5 days (American Institute of Nutrition, 1977; Reeves, 1989). After a 24 h fast, cannulae were implanted into the common bile–pancreatic duct, duodenum and upper ileum under pentobarbitone anaesthesia (sodium pentobarbitone, 40 mg (kg body weight)–1 i.p.; Abbott Co., North Chicago, IL, USA), as previously described (Hira et al. 1997; Hara et al. 2000). Briefly, the small tip (7–8 mm) of a polyethylene catheter (SP 28; i.d. 0.4 mm, o.d. 0.8 mm; Natsume Seisakusyo, Tokyo, Japan) was inserted into the common bile–pancreatic duct. The other end of the catheter was connected to silicone tubing (Silascon no. 00, i.d. 0.5 mm, o.d. 1.0 mm; Dow Corning Co., Kanagawa, Japan). A silicone catheter (Silascon no. 00) for returning BPJ to the ileal lumen was placed through a fistula 45 cm distal to the ligament of Treitz, i.e. in the middle of the small intestine. These catheters were tunnelled subcutaneously and connected to each other at the back of the neck to maintain the BPJ flow. Another silicone catheter (Silascon no. 00), for administration of the test solution, was inserted into the duodenal lumen through a gastric fistula. In the PBD rats, BPJ flow bypasses the proximal small intestine through the catheters and flows into the ileal lumen. The rats were allowed to recover for 6 days with free access to the semipurified diet described above. The common bile–pancreatic duct was examined after experiments, and rats with a swollen duct resulting from occlusion of the catheters were excluded from the analysis. The test solution or emulsion was administered into the duodenum through the catheter by a bolus injection (1 ml for 1 min) after twice sampling the BPJ in a fasting state. For BPJ sampling, the bile–pancreatic duct catheter was extended with a polyethylene tube (SP 28; Natsume Seisakusyo), and BPJ was collected from the polyethylene tube via an outlet placed 5 cm from the bottom of the cage. The BPJ was collected for 3 min at each time point shown in Figs 1 and 2, and was recirculated continuously into the ileum through the ileal catheter except during the 3 min sampling periods. Rats were allowed to move freely in the cages throughout the experimental period. The experiments were performed in a room controlled at 23 ± 2°C, with a 12 h–12 h light–dark cycle (08.00–20.00 h, light period). All rats were killed by exsanguination at the end of experiment under anaesthesia (sodium peptobarbitone, 40 mg (kg body weight)–1, i.p.). The study was approved by the Hokkaido University Animal Committee, and the animals were maintained in accordance with the guidelines for the care and use of laboratory animals of Hokkaido University.
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The PBD rats were divided into three groups on the basis of body weight so that average body weight of each group have the same range, after a 6 day recovery period and a 24 h fast. One group received 1 ml of HGC (150 mg ml–1) solution containing sodium caseinate (10 mg ml–1) as an emulsifying agent. The second group received this solution supplemented with cocoa butter (100 mg ml–1), and the third group received this solution supplemented with fatty acid ester F-10 (100 mg ml–1). The test solution was administered into the duodenum of PBD rats through the duodenal catheter. The BPJ was collected before and after the administration as described above.
Experiment 2: effect of the water-soluble sucrose fatty acid ester F-160 on protein-induced pancreatic secretion in the presence of a lipase inhibitor
Three groups of PBD rats were prepared as in experiment 1. The three test solutions contained HGC (150 mg ml–1) with sucrose (100 mg ml–1), HGC (150 mg ml–1) with the sucrose fatty acid ester F-160 (100 mg ml–1), or HGC (150 mg ml–1) with F-160 (100 mg ml–1) and the lipase inhibitor orlistat (0.4 mg ml–1), respectively. One millilitre of the test solution was administered into the duodenum, and BPJ was collected before and after administration as described above.
Experiment 3: effect of the sucrose fatty acid ester F-160 on protein-induced pancreatic secretion and plasma PYY in anaesthetized PBD rats
Two groups of PBD rats were prepared as in experiment 1, with some modifications. The catheter redirecting the BPJ from the common bile–pancreatic duct to the distal intestine was left in the abdominal cavity for a 6 day recovery period to prevent physical damage by the rats. On the day of the experiment, another catheter was implanted into the jugular vein to collect blood samples under anaesthesia (sodium pentobarbitone, 40 mg (kg body weight)–1 i.p.). Through a small mid-line abdominal incision, the catheter circulating the BPJ in the abdominal cavity was cut in half, and an extension catheter (20 cm) was connected to the end of the bile–pancreatic catheter to collect BPJ in the basal state for 3 min. At the same time, blood samples (100 µl) for PYY measurement were drawn into a syringe containing aprotinine (final concentration 200 kIU ml–1) and heparin (final concentration 50 IU ml–1) through the jugular catheter. After BPJ collection for 3 min, the extension catheter was replaced with a short (1 cm) polyethylene tube to reconnect the catheters between the bile–pancreatic duct and the ileum. Then, 2 ml of HGC (150 mg ml–1) solution containing either sucrose (100 mg ml–1) or F-160 (100 mg ml–1) was directly injected into the duodenum over 2 min. We injected 2 ml of the test solutions in experiment 3 because intestinal motility is known to be suppressed under anaesthesia. The BPJ (for 3 min) and blood samples (100 µl) were collected at 30, 60 and 120 min after the duodenal injection and were kept on ice until the end of the experiment. During the experiment, additional pentobarbitone (20 mg (kg body weight)–1 i.p. was injected to maintain anaesthesia, and body temperature was maintained using a heating pad. Plasma was separated from blood samples by centrifugation at 2500g for 15 min at 4°C, and then frozen at –80°C until PYY measurement. Plasma PYY concentrations were measured using a commercial enzyme immunoassay (EIA) kit (Yanaihara Institute Inc., Shizuoka, Japan). The antiserum cross-reacts 100% with intact PYY(1–36) (rat), 0% with PYY(19–36) (human), 0.7% with neuropeptide Y (NPY, human), 0% with NPY(1–19) (human) and 0% with pancreatic polypeptide (human).
Analyses
The volume of BPJ was measured gravimetrically, with 1 µl of BPJ taken as 1 mg as the basis for the measurement of pooled BPJ (100 µl = 100 mg). Trypsinogen in BPJ diluted with 0.9% NaCl containing 0.1% Triton X-100 was activated by purified enterokinase (Sigma) at 30°C for 20 min in a 15 mM Tris buffer (pH 8.1). Trypsin activities were estimated photometrically (Rick, 1976) using the synthetic substrate, N-
-p-toluene-sulphonyl-L-arginine methyl ester (TAME). The protein concentration in BPJ was quantified with a modified version of Lowry's method (Lowry et al. 1951; Sugawara, 1975).
Calculations and statistical analysis
One unit of trypsin was defined as the activity necessary to hydrolyse 1 µmol of substrate for 1 min at 30°C. Values for the basal state (0 min) were calculated as the average of two samples before the administration of the test solution. The influence of administration and time on the secretion profiles was determined by two-way ANOVA. The significance of differences among means was determined by least significant difference (LSD) test (P < 0.05).
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Results |
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In experiment 2, only HGC containing sucrose induced significant increases in the volume of BPJ secretion at each time point after the duodenal administration (Fig. 2A). The administration of HGC with sucrose induced increases in protein and trypsin secretion of two- to threefold higher than basal values (Fig. 2B and C), but neither protein nor trypsin secretion were changed after the duodenal administration of HGC containing F-160. In the presence of the lipase inhibitor orlistat, HGC containing F-160 did not induce any increases in either pancreatic protein or trypsin secretion. Values for protein and trypsin secretion in both the HGC + F-160 and the HGC + F-160 + orlistat groups were much lower than values in the HGC group at almost all time points.
In experiment 3 (Fig. 3A and B), basal BPJ volume and protein secretion were lower than those in experiment 1 and 2, possibly owing to the anaesthesia. Trypsin secretion was not measured in experiment 3 because the secretory pattern was found always to be similar to that of protein secretion in experiment 1 and 2, as well as in the results of previous studies (Hira et al. 1997, 2003). Intraduodenal injection of HGC + sucrose tended to increase BPJ volume, though not significantly. Protein secretion was increased in the HGC + sucrose-treated group at 60 and 90 min compared with the basal value (at 0 min). However, HGC + F-160 did not cause any significant increase in protein secretion throughout the experimental period. This result is consistent with that of experiment 2, in which pancreatic protein secretion was lower in the HGC + F-160 group than in the HGC + sucrose group.
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Discussion |
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In experiment 1, we emulsified test liquids because the sucrose ester and cocoa butter were not water soluble. It is possible that physicochemical masking of HGC by its dispersion into the particles is responsible for the suppression. To clarify this issue, in experiment 2 we used another sucrose ester, F-160, which is soluble in water and does not require emulsification. Non-emulsified, water-soluble sucrose ester F-160 completely suppressed the enhancement of the pancreatic secretion induced by HGC (Fig. 2) as well as by the emulsified fatty acid ester. This finding reveals that the sucrose fatty acid ester itself, not the masking of HGC, is responsible for the suppression. In addition, we assessed the effect of sucrose itself on protein-induced pancreatic secretion because sucrose is another fundamental component of sucrose fatty acid esters. We observed that HGC with sucrose still strongly stimulated pancreatic secretion, showing that sucrose is not involved in the suppression.
Sucrose esters are resistant to lipase; however, previous reports showed that some sucrose esters can be digested in the intestine (Shigeoka et al. 1984; Noker et al. 1997). If the suppression depends on fatty acids released from sucrose esters, inhibition of lipase activity should reverse the sucrose ester-induced inhibition of pancreatic secretion. However, the sucrose ester continued to suppress the protein-induced pancreatic secretion in the presence of the lipase inhibitor orlistat. These results indicate that protein-induced pancreatic secretion is inhibited by sucrose esters, not by free fatty acids. The F-10 and F-160 sucrose esters contained 2.2 and 1.7% (w/w) free fatty acids, respectively. These sucrose esters liberated only 1.9 (F-10) and 0.2% (F-160) of the free fatty acids after in vitro digestion in Tris buffer (pH 8.2) containing fresh BPJ for 60 min at 37°C, whereas triolein released 20% (w/w) fatty acids. These amounts of free fatty acids released from the sucrose esters are thought to be too small to induce significant PYY release, since previous reports have shown that large amounts of fatty acids (> 100 mg per rat) are required to induce significant PYY release in rats (Anini et al. 1999).
Administration of HGC strongly stimulated pancreatic secretion in PBD rats, which is associated with CCK release independent of proximal luminal proteases as described in our previous studies (Hara et al. 1994, 1997). Peptide YY is released by fatty acids in the distal intestine (Fu-Cheng et al. 1995; Dumoulin et al. 1998; Onaga et al. 2002) and inhibits pancreatic secretion (Jin et al. 1993; Naruse et al. 2002). To investigate the involvement of PYY in the suppression mechanism, plasma PYY levels were measured in PBD rats after the intraduodenal injection of HGC with sucrose or F-160. We used anaesthetized rats after chronic BPJ diversion in order to reduce physical stress on rats during the collection of BPJ and blood. In the case of conscious rats, four catheters for BPJ collection and return, duodenal infusion and blood collection would need to be implanted. These would be difficult to maintain during the recovery period, and are also difficult to handle during the experiment. In the present experiment 3, the F-160 sucrose ester suppressed pancreatic secretion induced by HGC (Fig. 3) under anaesthesia to the same degree as in experiment 2. Duodenal administration of F-160 with HGC induced higher PYY release than did HGC with sucrose. This is the first finding that a sucrose ester stimulates PYY secretion in vivo. Fatty acids induce PYY release, as demonstrated in previous papers (Aponte et al. 1989; Anini et al. 1999). Sucrose esters are indigestible, however, and we confirmed that the suppression of pancreatic secretion is triggered by sucrose ester itself in experiment 2. These results suggest that sucrose esters themselves, possibly the esterified fatty acid moieties, stimulate PYY release in the mid- and distal intestine. A recent study has demonstrated that PYY-producing L cells are located not only in the ileum but also in the jejunum in rats (Mortensen et al. 2003).
It is unclear how sucrose fatty acid esters stimulate PYY release from enteroeondocrine L cells. The mechanism for free fatty acid-induced PYY release from L cells is also poorly understood. It has been shown that carboxyl group and carbon chain length are responsible for fatty acid sensing in CCK releasing-enteroendocrine I cells (McLaughlin et al. 1998; Hira et al. 2004). However, free fatty acids were found not to be responsible for PYY release in the present study, since sucrose esters contain only small amounts of free fatty acids and do not liberate free fatty acids by luminal digestion, as described above. Further investigations on the mechanism involved in sucrose ester-induced PYY release are necessary.
Interestingly, HGC + sucrose also induced transient PYY secretion, which peaked at 60 min after injection. This result agrees with those from previous reports showing peptone-induced PYY release in the rat ileum (Dumoulin et al. 1998) and in humans (Calbet & Holst, 2004). Our findings show that HGC also stimulates PYY release in the mid- and distal intestine, and also show that the effects of esterified fatty acids are more potent than and additive to the effects of protein.
The present results suggest that undigested lipids activate PYY release to inhibit pancreatic secretion. Pancreatico-biliary diversion eliminates pancreatic enzymes from the proximal small intestine, so this could be a model for digestive dysfunction, such as dyspepsia. Excessive amounts of medium- and long-chain fatty acids in the distal intestine could increase epithelial permeability, permitting drug, allergen, toxin and virus passage through the intestinal barriers (Usami et al. 2001; Yata et al. 2001; Soderholm et al. 2002; Mine & Zhang, 2003). Therefore, the physiological relevance of the suppression of the pancreatic secretion triggered by fat in the distal small intestine may be that it reduces the risk of the adverse effects of fat.
In conclusion, the duodenal administration of sucrose fatty acid esters stimulates PYY release and suppresses pancreatic secretion without hydrolysis by lipase. The inhibitory mechanism may be triggered by the esterified fatty acids themselves.
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References |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, n
= 5), or HGC with either 100 mg cocoa butter (
, n
= 6) or 100 mg sucrose fatty acid ester F-10 (•, n
= 9)]. The value at each time represents BPJ volume, protein concentration or trypsin activity in BPJ secreted for 3 min. Two-way ANOVA P values of BPJ volume, protein, and trypsin secretion were 0.014, < 0.001 and < 0.001 for administration (A), 0.054, < 0.001 and < 0.001 for time (T), and 0.934, 0.024 and 0.033 for A x T, respectively. + Significantly different from the value at 0 min in each group (LSD, P < 0.05). Values at the same time point indicated by different letters differ significantly (LSD, P < 0.05).
