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-Polylysine Inhibits Pancreatic Lipase Activity and Suppresses Postprandial Hypertriacylglyceridemia in Rats
Department of Food Sciences and Nutritional Health, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto, 606-8522, Japan and * Research Laboratory, Nisshin Pharma Incorporated, Oi-machi, Iruma, Saitama, 356-8511, Japan
1To whom correspondence should be addressed. E-mail: kido{at}kpu.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-Polylysine (-PL) has been used as a food additive in Japan for many years. In this study, it inhibited human and porcine pancreatic lipase activity in substrate emulsions containing bile salts and phosphatidylcholine, in the concentration range of 10–1000 mg/L. At the same concentrations, it also destroyed the emulsifying activity, suggesting that lipase inhibitory activity and emulsion breakdown activity were associated. -PL inhibited porcine pancreatic lipase activity and destroyed emulsion breakdown activity at 1000 mg/L in the substrate containing bile salts and phosphatidylcholine alone. -PL did not inhibit lipase activity or affect emulsifying activity at 1000 mg/L in the substrates containing arabic gum and polyvinyl alcohol. A comparison of lipase inhibitory activity between -PL and three types of 
-PL with differing polymerization rates was performed. The lipase inhibitory activity of -PL was not different from that of -PL (44 lysine residues). -PL maintained its inhibitory activity after incubation with trypsin, -chymotrypsin and pepsin, whereas -PL did not. The effect of -PL on postprandial hypertriacylglyceridemia was investigated in rats. The plasma triacylglycerol concentration in rats intragastrically administered 
15 mg/kg of both fat emulsion and -PL was significantly lower at 2 and 3 h after administration than that in rats administered fat emulsion alone (P < 0.05). These results strongly suggest that -PL is able to suppress dietary fat absorption from the small intestine by inhibiting pancreatic lipase activity.
KEY WORDS: • -polylysine • lipase inhibitory activity • emulsion breakdown activity • hypotriacylglyceridemic activity • rats
Obesity is a serious disease that can lead to numerous health problems including diabetes, hypertension and atherosclerosis (1). The etiology of obesity is complex and involves the interplay of numerous environmental and genetic factors. However, obesity is essentially the consequence of a long-term positive energy balance in which energy intake exceeds energy expenditure. Dietary intake and composition, together with physical activity, are the primary modifiable factors that influence energy balance. In particular, excessive consumption of dietary fat may play a major contributory role in the development of obesity (2,3). Dietary fat has a higher energy density than other macronutrients. Moreover, fat has only a weak effect on postprandial satiety. Fat also tends to be very palatable, and studies have shown that both obese men and women seem to have definite preferences for high fat foods (4). This, together with the high energy density and low satiating effect of fat, can result in the overeating or passive overconsumption of high fat foods. The primary treatment for preventing obesity is a low fat diet; another possible treatment is ingestion of a natural product that selectively limits intestinal absorption of dietary fat.
Pancreatic lipase plays a crucial role in lipid absorption from the intestine (5). This enzyme acts at the surface of emulsified lipid droplets, formed from bile salts and phospholipids in the small intestine, to hydrolyze triacylglycerols to fatty acids and monoacylglycerols. The resulting fatty acids and monoacylglycerols are incorporated into bile salt-phospholipid micelles. These micelles are absorbed into the brush border of the small intestine and eventually enter the bloodstream as chylomicrons.
Natural products that inhibit the activity of pancreatic lipase may suppress dietary fat absorption from the small intestine. Potential candidates include basic peptides and proteins, which destroy the lipid emulsion by binding to bile salts, thus suppressing the activity of pancreatic lipase.
In the course of our screening program for pancreatic lipase inhibitors from naturally occurring basic proteins and peptides, -polylysine (-PL, 25–30 lysine residues) was found to inhibit pancreatic lipase. -PL has been used as a food additive for >10 y in Japan, and its safety has been confirmed (6,7).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-PL, purchased from Wako Pure Chemical (Tokyo, Japan), was used in all experiments except the in vivo study in which a 500 mg/g powder of -PL in dextrin from Chisso (Tokyo, Japan) was used. Three types of -polylysine (-PL), which differed in polymerization rate (11, 44 and 222 lysine residues), were purchased from Sigma Chemical (St. Louis, MO). Human and type II porcine pancreatic lipase (EC 3.1.13), porcine trypsin (EC 3.4.21.1) and pepsin (EC 3.4.23.1), bovine -chymotrypsin (EC 3.4.21.1) and L--phosphatidylcholine (type X-E) from dried egg yolk were purchased from Sigma. All other chemicals were of analytical grade.
Pancreatic lipase activity.
Determination of pancreatic lipase activity was based on the amount of free fatty acids liberated from emulsified olive oil. Substrate emulsions containing bile salts, cholesterol and phospholipid were selected to mimic in vivo conditions. In particular, the concentration of the bile salts was formulated to approximate that of duodenal aspirates (8). Lipase activity was determined by measuring free fatty acids liberated from emulsified olive oil. The substrate emulsion (0.6 mL in a 10-mL centrifuge tube) was prepared by ultrasonification of olive oil (48 g/L) in a solution containing 1 mmol/L taurochenodeoxycholate, 9 mmol/L taurocholate, 0.1 mmol/L cholesterol, 0.8 g/L L--phosphatidylcholine, 1 mmol/L calcium acetate and 100 mmol/L Tris-HCl (pH 8.0). After addition of the test compound dissolved in 75 µL of 100 mmol/L-Tris-HCl (pH 8.0), or vehicle alone, the assay tube was preincubated for 5 min at 37°C. The enzyme reaction was started by the addition of 75 µL lipase solution containing 0.1 g/L of 100 mmol/L Tris-HCl (pH 8.0). After incubation for 30 min at 37°C, the concentration of free fatty acids in the reaction mixture was measured according to the method of Duncombe (9), using oleic acid as the standard. In all lipase assays, porcine pancreatic lipase was used and in one experiment, human pancreatic lipase was also used to compare the inhibitory activity of -PL against lipase from different species. When 0.1 g/L of human and porcine lipase solution was used, the amount of fatty acids liberated was 0.13 and 0.30 mmol/(min · L), respectively. The inhibitory activity of each sample was reported as the relative fatty acid concentration liberated compared with the control value. Unless otherwise stated, this assay system (standard assay) was used for determination of lipase inhibitory activity of -PL and other compounds.
In some experiments, lipase activity was determined using other emulsifying reagents; the substrate emulsion was prepared by ultrasonification of olive oil (48 g/L) in a solution containing a fixed concentration of each emulsifying reagent listed in Table 1, 1 mmol/L calcium acetate and 100 mmol/L Tris-HCl (pH 8.0); the assay was performed in a manner similar to that described above, except that in substrates C and D, the concentration of added lipase solution into the assay tube was 1.0 g/L and 10 g/L, respectively. The assay conditions are summarized in Table 1.
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-PL and -PL by trypsin, -chymotrypsin and pepsin.
Trypsin, -chymotrypsin and pepsin were used for digestion of -PL and three types of -PL (11, 44 and 222 lysine residues). When trypsin and -chymotrypsin were used as digestive enzymes for each compound, the following procedure was performed: buffer solution containing 1 mmol/L of test compound and 1 g/L of digestive enzyme (trypsin and -chymotrypsin) in 100 mmol/L Tris-HCl (pH 8.0) was incubated for 1 h at 37°C, followed by 15 min at 85°C, to inactivate the enzyme. The pancreatic lipase assay in the presence of a digestive enzyme–treated sample (75 mL/0.75 L of lipase reaction mixture) was performed, and inhibitory activity was determined. When using pepsin, the procedure was the same as that described above except that the enzyme reaction was performed in 100 mmol/L KCl-HCl (pH 2.0) buffer solution, and 1 mol/L NaOH was added to neutralize the reaction mixture after the enzyme reaction was complete.
Emulsion breakdown activity.
The emulsifying activity of the lipase reaction mixture was determined according to the turbidimetric method of Jackman et al. (10). The emulsion (6 µL) was carefully drawn from the bottom of the centrifuge tube, in which a lipase reaction had been performed for 30 min at 37°C and diluted in 1 mL of 1 g/L SDS in 100 mmol/L Tris-HCl (pH 8.0). The absorbance of the diluted emulsion was measured against a buffer blank at 500 nm and was used as a measure of emulsifying activity.
Effect of -PL on plasma triacylglycerol concentrations in rats.
This study was performed under the guidelines for animal experimentations of Kyoto Prefectural University. Male Sprague-Dawley rats, 7 wk old and weighing 
200 g, were purchased from Japan SLC (Shizuoka, Japan) and acclimated for several days to the animal room before the start of the experiment. The animal room was maintained at 23 ± 2°C on a 12-h light:dark cycle.
The lipid emulsion contained 200 g/L soybean oil, 12 g/L egg yolk lecithin and 22.5 g/L glycerol. The lipid emulsion was administered intragastrically using a bulbed needle to rats that had been food deprived for 18 h at a dose of 10 mL/kg. This treatment was followed by intragastric administration of 1 mL/kg of several different concentrations of -PL dissolved in distilled water (treated group) or distilled water only (control group). Blood was drawn from a tail vein before (0 h) and 1, 2, 3, 4 and 8 h after administration of the lipid emulsion. The concentrations of triacylglycerol, total cholesterol and phospholipid in plasma were determined by an automatic analyzer (CL-8000, Shimazu, Kyoto, Japan). The area under the triacylglycerol concentration curve (AUC) for 8 h was calculated using the method described for determining the AUC of glucose (11).
Statistics.
Results are expressed as means ± SEM. Changes in triacylglycerol concentration from initial levels in each treated group were analyzed by repeated-measures ANOVA, followed by Dunnet’s multiple comparison test. One-way ANOVA was used for the comparison of triacylglycerol concentration at each time after fat administration and AUC among tested groups. If the F-test was significant, Dunnet’s multiple comparison test was used to detect significantly different means. A P-value of <0.05 was considered to be significant. These statistical calculations were performed with GraphPad Prism for windows version 3.0 (GraphPad Software, San Diego, CA).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-PL.
In the standard lipase assay, 10–1000 mg/L -PL inhibited both porcine and human lipase activity (Fig. 1a). The inhibitory activity of -PL against both human and porcine pancreatic lipase reached a maximum at 100 mg/L (93% inhibition against human lipase and 80% against porcine). The inhibitory activity of -PL was approximately the same at 1000 and 100 mg/L. In addition, 10–1000 mg/L -PL destroyed the stability of the substrate emulsions, with maximum activity at 100 mg/L (Fig. 1b).
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-PL at 1000 mg/L against porcine pancreatic lipase was determined in substrates with (A and B) and without (C, D and E) bile salts (Table 1). -PL inhibited lipase activity in substrates containing bile salts (A, B) and phosphatidylcholine (E), but not in the other substrates tested (C and D) (Figure 2a). The emulsion activities of substrates A, B and E were almost completely destroyed by the addition of -PL 1000 mg/L, but substrates C and D were not affected (Fig. 2b).
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-PL on porcine pancreatic lipase activity and emulsion stability in substrates A and E were investigated. At concentrations 10 mg/L, -PL inhibited lipase activity in substrate A (Figure 3a); however, in substrate E (Fig. 3b), inhibitory activity did not occur at 10 mg/L. The maximum inhibitory activity of -PL against porcine pancreatic lipase was higher in substrate A than in substrate E (A: 85% inhibition, and E: 63% inhibition). A breakdown of the substrate emulsions A and E occurred in the presence of the same concentration of -PL at which lipase was inhibited i.e., -PL destroyed emulsifying activity in substrate A at 10 mg/L and in substrate E at 100 mg/L (Fig. 3b).
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-PL and other lysine homopolymers.
The inhibitory activities of L-lysine, -PL and three types of -PL, which differed in their polymerization rate, were determined using porcine lipase (Fig. 4a). L-Lysine did not inhibit porcine lipase, even at 1 mmol/L. The 50% inhibitory concentrations (IC50) for the other 4 lysine homopolymers were: -PL (222 lysine residues, 3.1 µmol/L) < -PL (25–35 lysine residues, 6.8 µmol/L) 
-PL (44 lysine residues, 7.6 µmol/L) < -PL (11 lysine residues, 37 µmol/L). The IC50 of -PL was approximately equal to that of -PL (44 lysine residues).
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-PL against porcine pancreatic lipase was not affected by treatment with the digestive enzymes, pepsin, trypsin and -chymotrypsin (Fig. 4b). However, after trypsin treatment, the -PL did not inhibit porcine pancreatic lipase.
Effect of -PL on plasma triacylglycerol concentrations in rats.
Intragastric administration of fat emulsion to rats significantly increased plasma triacylglycerol concentration 1–4 h after fat administration (P < 0.05). A maximum was reached 3 h after administration, followed by a gradual decrease to the baseline level at 8 h (Fig. 5). The plasma triacylglycerol concentrations in rats administered both fat emulsion and -PL (15–100 mg/kg) were significantly lower 2 and 3 h after administration than those in rats administrated fat emulsion alone (15 mg/kg -PL; P < 0.05 at 2 and 3 h, 25–50 mg/kg -PL; P < 0.05 at 2 h, P < 0.01 at 3 h, and 100 mg/kg -PL; P < 0.01 at 2 and 3 h; Fig. 5). Plasma cholesterol and phospholipid concentrations were not affected by -PL (15–100 mg/kg) at any time after fat administration (data not shown).
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-PL significantly suppressed the AUC of plasma triacylglycerol at 15, 25 and 100 mg/kg (P < 0.05) (Figure 6). At these doses, the AUC was decreased by >95%.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-PL, a polybasic peptide originally isolated from the culture broth of Streptomyces albulus (12–14), has been used as a food preservative for >10 y in Japan. This substance has been reported to have antimicrobial and antiviral activities (15,16). We describe the first report of lipase inhibitory activity and suppressive effect on postprandial hypertriacylglyceridemia of -PL.
Recently, protamine, purothionin and histone, which belong to a class of basic proteins, were reported to inhibit lipase (16). These substances inhibit lipase in substrates containing phosphatidylcholine, but not arabic gum. From these results, it was suggested that these compounds inhibit lipase activity by interaction with triolein-phosphatidylcholine. In the present study, the breakdown of emulsifying activity by -PL was measured directly. In addition, it was shown that lipase inhibition by -PL always accompanied the breakdown of substrate emulsion (Figs. 123). We demonstrated that -PL suppressed pancreatic lipase activity by destroying the emulsifying activity of the substrate. The inhibition of lipase by protamine, purothionin and histone may be due to similar activity. -PL inhibited lipase activity in the substrates containing bile salts (A and B in Table 1) and phosphatidylcholine (E in Table 1) as emulsifying reagents. In the substrate containing sodium taurocholate, inhibitory activity of -PL was 10 times higher compared with the substrate containing phosphatidylcholine (Fig. 3), but was comparable to the standard substrate. This result suggests that the breakdown of the substrate emulsion by interaction of -PL with bile salts was mainly responsible for lipase inhibition. -PL may also inhibit pancreatic lipase in the digestive tract by the same mechanism because the concentration of bile salts in the digestive tract was estimated to be approximately the same as that in the standard substrate (8). -PL has a very unique structure; it is a homopolymer of L-lysine connected via 25–30 residues in an isopeptide linkage (12). We sought to determine whether the number of amino groups or the position of the amino group (- or -) in the molecule played the more important role in the inhibitory activity against lipase. In the present study, the lipase inhibitory activity of -PL in the standard substrate was equal to that of -PL (44 lysine residues), suggesting that the number of amino groups in the molecule is important for the inhibitory activity of -PL, but that the position of the amino group is not. -PL differed from the other -PL in that its inhibitory activity against pancreatic lipase was maintained after incubation with digestive enzymes. These results suggested that -PL would inhibit lipase in the digestive tract, whereas other basic proteins showing inhibitory activity in vitro may be degraded in vivo.
After fat digestion, the increase in plasma triacylglycerol levels is thought to be caused by an elevation in chylomicron triacylglycerols. A compound that suppresses postprandial hypertriacylglyceridemia is thought to act by at least two different mechanisms, i.e., inhibition of absorption of fat from the intestine and promotion of catabolism of triacylglycerol present in the bloodstream as chylomicrons. The hypotriacylglyceridemic activity of -PL is likely due to the former mechanism, because -PL strongly inhibited pancreatic lipase in vitro.
Neomycin, a nonabsorptive polybasic antibiotic, similar to -PL, has been reported to induce the precipitation of mixed micelle solutions in vitro and in vivo, and reduce total and LDL cholesterol concentrations in type II hyperlipoproteinemia (17–19). It is expected that -PL will also reduce plasma cholesterol levels in hyperlipemia. Studies in rats examining this possibility are in progress.
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FOOTNOTES |
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-PL, -polylysine; IC50, 50% inhibitory concentration. 
Manuscript received 28 December 2002. Initial review completed 28 January 2003. Revision accepted 10 March 2003.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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