Introduction. Materials and methods

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1 International Journal of Obesity (1999) 23, 174±179 ß 1999 Stockton Press All rights reserved 0307±0565/99 $ Reduction in fat storage during chitin-chitosan treatment in mice fed a high-fat diet L-K Han 1,Y Kimura 1 and H Okuda* 1 1 Second Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu-cho, Onsen-gun, Ehime , Japan OBJECTIVE: Chitin and chitosan are polymers containing more than 5000 acetylglucosamine and glucosamine units, respectively, and their molecular weights are over one million Daltons. The present study assessed the effects of chitin-chitosan on the activity of pancreatic lipase in vitro and on the degree of fat storage induced in mice by the oral administration of a high-fat diet for nine weeks. DESIGN: Mice were fed a high-fat diet and treated with chitin-chitosan for nine weeks. Experiments were also carried out to clarify whether or not chitin-chitosan inhibited pancreatic lipase activity in assay systems using triolein emulsi ed with lecithin, gum arabic or Triton X-100. RESULTS: Chitin-chitosan prevented the increase of body weight, hyperlipidaemia and fatty liver induced by a high-fat diet. Chitin-chitosan inhibited hydrolysis of triolein, emulsi ed with phosphatidylcholine, but not that of triolein emulsi ed with gum arabic and Triton X-100. These results suggest that the site of inhibitory action of chitin-chitosan may not be the enzyme but its substrate. CONCLUSION: The anti-obesity effects of chitin-chitosan in high-fat diet-treated mice might be partly due to the inhibition of intestinal absorption of dietary fat. Consequently, chitin-chitosan might cause improvement of the fatty liver and hyperlipidaemia in mice fed a high fat diet through inhibiting intestinal absorption of dietary fat. Keywords: chitin-chitosan; anti-obesity; high-fat diet; pancreatic lipase Introduction Chitin-chitosan is a mixture of chitin (20%) and chitosan (80%). Chitin and chitosan are polymers containing more than 5000 acetylglucosamine and glucosamine units, respectively, and their molecular weights are over one million Daltons. Although chitin is widely distributed in natural products such as the protective cuticles of crustaceans and insects, and cell walls of some fungi and micro±organisms, it is usually prepared from shells of crabs and shrimp. Alkaline hydrolysis (45% NaOH, 100 C) of chitin converts it to chitosan. The alkaline hydrolysate, however, contains chitosan (80%) and non-reactive chitin (20%) and is usually called `chitin-chitosan'. Inclusion of chitin-chitosan in the diet, reduced total plasma cholesterol and inhibited the absorption of cholesterol and triacylglycerol from lymph in animal models. 1±4 Previously, we reported that chitin-chitosan reduced blood pressure, elevated by NaCl intake, and augmented cytolytic activity of mouse lymphocytes. 5,6 In the course of those experiments, we found that chitin-chitosan might inhibit intestinal absorption of dietary fat by inhibiting hydrolysis of the fat by pancreatic lipase. To test this *Correspondence: Prof Dr Hiromichi Okuda, 2nd Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu-cho, Onsen-gun, Ehime , Japan. Received 18 May 1998; revised 25 August 1998; accepted 6 October 1998 possibility, we studied the effect of chitin-chitosan on pancreatic lipase activity in vitro and measured fat balance by determination of fat excretion in the faeces of the mice fed a high fat diet or a high fat diet plus chitin-chitosan for three days. Moreover, the present investigation was designed to clarify whether or not chitin-chitosan prevented the obesity induced by feeding a high fat diet for a longer term (nine weeks). Materials and methods Materials Triolein and pancreatic lipase were purchased from Sigma (St Louis, MO). Triglyceride E test, Total Cholesterol E test and NEFA C test kits were purchased from Wako Pure Chemical Co. (Osaka, Japan). Chitin-chitosan was supplied by Fuji-Bio Co. Ltd. (Shizuoka, Japan). Measurement of pancreatic lipase activity Lipase activity was determined by measuring the rate of release of oleic acid from triolein. A suspension of triolein (80 mg), lecithin (10 mg) and taurocholic acid (5 mg) in 9 ml of 0.1 M N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), ph 7.0, containing 0.1 M NaCl was sonicated for 5 min. This sonicated substrate suspension (100 ml) was incubated with 50 ml (10 units) of pancreatic lipase and 100 ml of various concentrations of chitin-chitosan suspension for 30 min at 37 C in a total volume of 250 ml. The amount of oleic acid produced was determined by

2 the method of Zapf et al, 7 with a slight modi cation. 8 The incubation mixtures were added to 3 ml aliquots of a 1:1 (v=v) mixture of chloroform and heptane containing 2% (v=v) methanol and extracted by shaking the tubes horizontally for 10 min in a shaker. The mixture was centrifuged at 2000 g for 10 min, and the upper aqueous phase was removed by suction. Copper reagent (1 ml) was then added to the lower organic phase. The tube was shaken for 10 min, the mixture was centrifuged at 2000 g for 10 min, and 0.5 ml of the upper organic phase, which contained copper salts of the extracted free fatty acids (FFA), was treated with 0.5 ml of 0.1% (v=v) bathocuproine in chloroform containing 0.05% (w=v) 3-(2)-tert-butyl-4- hydroxy-anisol. The absorbance was then measured at 480 nm. In addition, pancreatic lipase activity was determined using gum arabic (acidic polymer) or Triton X-100 (neutral polymer) as emulsi er; 45 mg gum arabic or 2.25 mg Triton X-100, instead of lecithin, were used and the enzyme activity assayed as described above. Lipase activity was expressed as mmol oleic acid released per ml reaction mixture per 1 h. Estimation of fat excretion in faeces of mice fed a high fat diet for three days Female ICR mice (three weeks old) were obtained from CLEA Japan Inc. (Osaka, Japan), and housed individually for seven days in a 12 h=12 h light=dark cycle in a temperature- and humidity-controlled room, and given free access to food and water. After adaptation to the lighting conditions for seven days, the healthy mice were used in the experiments. The control mice were given laboratory pellet chow (CLEA Japan Inc.; protein 24%, lipid 3.5%, carbohydrate 60.5%) and water ad libitum for three days. The mice of high-fat diet-fed s, received the high-fat diet (beef tallow 40%, corn starch 10%, sugar 9%, vitamin mixture (AIN-76) 1%, mineral mixture (AIN-76) 4% and casein 36% w=w) and water for three days ad libitum. The mice in the experimental s, received the high-fat diet containing 3%, 7% or 15% chitin-chitosan for three days. The food intake of each mouse was estimated every day, and samples of faeces obtained from each animal at regular intervals. The triacylglycerol content in faeces were measured by extraction of a 150 mg sample with chloroform-methanol (2:1, v=v, 4 ml), with the extract being concentrated under a nitrogen stream. The fat content of the residue was determined by Triglyceride E-Test Wako kit (Wako Pure Chemical Co.). Estimation of body, liver and parametrial adipose tissue weights, serum FFA, triacylglycerol and total cholesterol, and liver triacylglycerol and total cholesterol in mice fed a high-fat diet Female ICR mice (three weeks old) were obtained from CLEA Japan Inc., and maintained in a 12 h=12 h light- =dark cycle in a temperature- and humidity-controlled room. The animals were given laboratory pellet chow and water ad libitum. Sixty- ve mice were divided into ve s (each, nˆ13), with all s matched for body weight after one week of feeding. The control mice continued to be fed laboratory pellet chow ad libitum. The basic composition of the experimental diet was as follows (wt %); beef tallow 40%, corn starch 10%, sugar 9%, vitamin mixture 1% and mineral mixture 4%. The composition of the diet for each experimental was as follows; high-fat : casein 36% and basic components; chitin-chitosan plus high fat : different amounts of casein (21%, 29% and 33%) and chitin-chitosan (15%, 7% and 3%). The mice of high-fat diet-fed s received the highfat diet and water for nine weeks ad libitum. The mice of the experimental s received the high-fat diet containing 3%, 7% or 15% chitin-chitosan for nine weeks. We examined further the effects of different amounts of casein on body, liver and parametrial adipose tissue weights on the same schedule. Mice were killed under anaesthesia with diethyl ether, and liver and parametrial white adipose tissue were quickly removed and weighed. The liver tissues were stored at 780 C until analysis. The liver triacylglycerol and total cholesterol contents were measured as follows: a portion (0.5 g) of the liver tissue was homogenized in Krebs Ringer phosphate buffer (ph 7.4, 4.5 ml), and the homogenate (0.2 ml) was extracted with chloroformðmethanol (2:1, v=v, 4 ml) and the extract was concentrated under a nitrogen stream. The residue was determined by Triglyceride E-Test Wako and T-Cholesterol E-test Wako kits. Each mouse was weighed once a week and the weight recorded. The total amount of food intake by each animal was recorded at least three times a week. Statistical analysis Results are expressed as means standard error (s.e.m.). Statistical analysis was performed by the Scheffe's test to determine signi cance (P<0.05) using Super ANOVA Software. The statistical analysis for body weight was performed using Super ANOVA, based on the data of body weight measured weekly. Results Effects of chitin-chitosan on pancreatic lipase activity As shown in Figure 1a, chitin-chitosan inhibited the pancreatic lipase activity dose-dependently between the concentrations of 6.25 mg=ml and 200 mg=ml in the assay system, using triolein emulsi ed with lecithin. For characterization of the mechanism for inhibition of pancreatic lipase by chitin-chitosan, the enzyme activity was assayed at various concentrations of lecithin-emulsi ed triolein and in the presence of increasing concentrations of chitin-chitosan. A Lineweaver-Burk plot of the data in Figure 1b, shows that chitin-chitosan was a competitive inhibitor. The K m and V max values of the lipase activity for lecithin- 175

3 176 emulsi ed triolein were 6.06 mg=ml and 8.7 nmol=ml=min, respectively. The K i value of chitin-chitosan on the lipase activity in lecithin-emulsi ed triolein was 17.6 mg=ml. When triolein was emulsi ed with gum arabic instead of lecithin, chitin-chitosan did not inhibit its hydrolysis. This result suggests that gum arabic (acidic polymer) reacts with chitin-chitosan (basic polymer), preventing chitin-chitosan from affecting pancreatic lipase. It was found that chitin-chitosan and gum arabic did not result in both particles losing their charge, aggregating and acting as neutral bre (data not shown). In addition, we examined the effects of chitin-chitosan on the lipase activity using neutral polymer Triton X-100 as emulsi er. When triolein was emulsi ed with Triton X-100 ( nal concentration: 0.25 mg=ml) instead of gum arabic, chitin-chitosan did not inhibit the hydrolysis (Figure 1a). These results suggest that the inhibitory effects of chitin-chitosan on pancreatic lipase activity may be mediated by the interaction between substrate and enzyme through lecithin. Effects of chitin-chitosan on fat excretion in faeces of mice fed a high-fat diet for three days As shown in Table 1, the feeding of a high-fat diet plus 7% or 15% chitin-chitosan, enhanced fat excretion in faeces and inhibited absorption of the ingested fat in the balance study. Effects of various contents of casein on body, parametrial adipose tissue and liver weights in ice fed a high-fat diet for nine weeks As shown in Figure 2 and Table 2, it was found that the weights of body, liver and parametrial adipose tissue were not affected by the reduction of casein contents in a high-fat diet. Effects of chitin-chitosan on body, parametrial adipose tissue and liver weights in mice fed a high-fat diet for nine weeks Figure 3 shows the changes in body weights of the s during the experiments. The nal parametrial adipose tissue and liver weights of the s are shown in Figure 4 and Figure 5. Feeding a high-fat diet containing 40% beef tallow for nine weeks caused signi cant increases in body weight at 4±9 weeks and in nal liver and parametrial adipose tissue weights, compared to the normal diet (laboratory pellet chow) (Figure 3, Figure 4 and Figure 5). Feeding a high-fat diet containing 3%, 7% or 15% chitinchitosan, signi cantly reduced the increase in body weight at 4±9 weeks (Figure 3), and the nal parametrial adipose tissue weight as compared to feeding a high-fat diet (Figure 4). Liver weight was also signi cantly reduced by feeding a high-fat diet containing 7% or 15% chitin-chitosan as compared to feeding a high-fat diet (Figure 5). The mean food consumption per week per mouse during the whole experimental period was signi cantly (P<0.05) different between the laboratory chow and high-fat diet s, being kj in the laboratory chow and kj in the high-fat diet, but not signi cantly different between the high-fat and high-fat plus 3%, 7% or 15% chitinchitosan diet s, being (high-fat diet), (3% chitin-chitosan diet), (7% chitin-chitosan diet) and (15% chitin-chitosan diet), respectively. Figure 1 Effects of chitin-chitosan on pancreatic lipase activity. (a) Results are expressed as means s.e.m. of four experiments. sˆtriolein emulsi ed with gum arabic was used as substrate; uˆ Triolein emulsi ed with Triton X-100 was used as substrate; dˆtriolein emulsi ed with lecithin was used as substrate. (b) Lineweaver-Burk plots of the released oleic acid with lecithin-emulsi ed triolein as substrate in the presence of various concentrations of chitin-chitosan. Chitin-chitosan concentrations used: sˆ 0 mg=ml; dˆ6.25 mg=ml; jˆ12.5 mg=ml.

4 Table 1 Effects of chitin-chitosan on fat excretion into faeces of mice fed a high-fat diet (HF) for three days 177 Day Control HF-treated HF plus 3% chitin-chitosantreated HF plus 7% chitin-chitosantreated HF plus15% chitin-chitosantreated Day 1 Faeces (g) Excretion TG (mmol=g) Day 2 Faeces (g) * Excretion TG (mmol=g) * * * Day 3 Faeces (g) * * Excretion TG (mmol=g) * * Results are expressed as means s.e.m. of four mice. *Signi cantly different from high-fat diet-treated, P<0.05. TGˆ triacylglycerol; HFˆhigh-fat diet. Figure 2 Effects of various contents of casein on body weight in mice fed a high-fat diet for nine weeks. Results are expressed as means s.e.m. of 7±15 mice. *Signi cantly different from highfat diet containing 36% casein, P< Effects of chitin-chitosan on serum triacylglycerol, total cholesterol and FFA, and liver triacylglycerol and total cholesterol in mice fed a high-fat diet for nine weeks As shown in Table 3 and Table 4, it was found that feeding a high-fat diet caused hyperlipidaemia, with elevations of serum triacylglycerol and total cholesterol, and caused fatty liver, with accumulation of triacylglycerol and total cholesterol in the liver. Serum triacylglycerol was signi cantly reduced by feeding a Figure 3 Effects of chitin-chitosan on body weight in mice fed a high-fat diet for nine weeks. Results are expressed as means s.e.m. of 13 mice. *Signi cantly different from high-fat diettreated, P< high-fat diet containing 3%, 7% or 15% chitin-chitosan as compared to feeding a high-fat diet (Table 3). Serum total cholesterol was also reduced by feeding the 7% and 15% chitin-chitosan diets (Table 3). Feeding the 15% chitin-chitosan high-fat diet reduced the serum free fatty acid level as compared to feeding the high-fat diet (Table 3). The oral administration of 3%, 7% or 15% Table 2 Effects of casein on parametrial adipose tissue and liver weights in mice fed a high-fat diet for nine weeks Liver weight Adipose tissue weight (g) (g=100 g body weight) Animal (n) Mean s.e.m. Mean s.e.m. Control (Lab chow) * * High-fat diet containing 22% casein High-fat diet containing 31% casein High-fat diet containing 34% casein High-fat diet containing 36% casein Results are expressed as means s.e.m. of 7±15 mice. *Signi cantly different from high-fat diet containing 36% casein, P<0.05.

5 178 Figure 4 Effects of chitin-chitosan on parametrial adipose tissue weight in mice fed a high-fat diet for nine weeks. 1ˆControl ; 2ˆ High fat diet-treated ; 3ˆ High fat diet plus 3% chitin-chitosan; 4ˆ High fat diet plus 7% chitinchitosan; and 5ˆHigh fat diet plus 15% chitin-chitosan. Results are expressed as means s.e.m. of 13 mice. *Signi cantly different from high-fat diet-treated, P< chitin-chitosan prevented the accumulation of liver triacylglycerol and total cholesterol caused by a highfat diet (Table 4). Discussion It is well known that dietary fat is not absorbed from the intestine unless it has been subjected to the action of pancreatic lipase. 9 Previously, we found that basic proteins such as protamines, histones and purothionine inhibited hydrolysis of triolein emulsi ed with phosphatidylcholine. 10 Based on this, we tested the inhibitory action of chitin-chitosan on pancreatic lipase activity, and found that chitin-chitosan, like protamines, inhibited the action of pancreatic lipase (Figure 1). Chitosan inhibited hydrolysis of triolein emulsi ed with phosphatidylcholine, but not that of triolein emulsi ed with gum arabic and Triton X-100. These results suggest that the site of inhibitory action of chitin-chitosan may not be the enzyme but its substrate. The inhibition by chitosan of hydrolysis of dietary fat may cause a decrease in intestinal absorption of fat and reduce blood chylomicron, an excess of which is known to induce hyperlipidaemia, obesity and fatty liver. In the present study, it was con rmed that chitin-chitosan enhanced fat excretion into faeces of mice fed a high fat diet for three days. There are a number of studies describing high-fat diet-induced obesity. 11±14 In the long term (nine weeks) experiment, chitin-chitosan signi cantly reduced both body and parametrial adipose Figure 5 Effects of chitin-chitosan on liver weight in mice fed a high-fat diet for nine weeks. 1ˆ Control ; 2ˆHigh-fat diettreated ; 3ˆ High-fat diet plus 3% chitin-chitosan; 4ˆ Highfat diet plus 7% chitin-chitosan; and 5ˆHigh-fat diet plus 15% chitin-chitosan. Results are expressed as means s.e.m. of 13 mice. *Signi cantly different from high-fat diet-treated, P< tissue weights at doses of 30 (3%), 70 (7%) and 150 (15%) g=kg food (Figure 3 and Figure 4), whereas no differences in the energy consumed were found among the experimental s (except the lab chow ). This indicates that chitin-chitosan prevents the high-fat diet induced increase of body weight by affecting food absorption. In addition to reducing fat storage, chitin-chitosan was found to improve the fatty liver induced by the high-fat diet and to reduce plasma triacylglycerol, cholesterol and FFA, which were elevated in the high-fat diet-fed control mice. It was reported that the weights of the epididymal and retroperitoneal adipose tissue depots were signi cantly higher in high fat diet than low fat diet fed rats. 15 Furthermore, there are reports that both adipocyte size and number are increased in animals with obesity caused by a high fat diet. 16,17 Tsujita et al 18 reported that basal lipolysis in the absence of lipolytic hormones was usually increased in enlarged fat cells in aged and=or obese animals. Recently, Morimoto et al 19 reported that the levels of insulin, FFA and triacylglycerol in rat sera, increased with age, and the increase in serum FFA levels accompanied both the stimulation of basal lipolysis (that is, lipolysis in the absence of lipolytic agents) in fat cells and the enlargement of the cells. Elevation of basal lipolysis causes an increase in plasma FFA, which are then converted tolipids inliver and secreted intoblood as very low density lipoprotein. Therefore, reduction of the obesity by chitin-chitosan may cause reduction of basal lipolysis in fat cells of the obese mice, followed by a

6 Table 3 weeks Effects of chitin-chitosan on serum triacylglycerol, total cholesterol and free fatty acid (FFA) in mice fed a high-fat diet for nine Triacylglycerol (mm) Total cholesterol (mm) FFA (meq=l) Mean s.e.m. Mean s.e.m. Mean s.e.m. 179 Control (Lab chow) * * * High-fat diet-treated High-fat diet plus 3% chitin-chitosan-treated * * High-fat diet plus 7% chitin-chitosan-treated * * High-fat diet plus 15% chitin-chitosan-treated * * * Results are expressed as means s.e.m. of 13 mice. *Signi cantly different from high-fat diet-treated, P< Table 4 Effects of chitin-chitosan on liver triacylglycerol and total cholesterol in mice fed a high-fat diet for nine weeks Triacylglycerol (mmol=g) Mean s.e.m. Total cholesterol (mmol=g) Mean s.e.m. Control (Lab chow) * * High-fat diet-treated High-fat diet plus 3% chitin-chitosan-treated * * High-fat diet plus 7% chitin-chitosan-treated * * High-fat diet plus 15% chitin-chitosan-treated * * Results are expressed as means s.e.m. of 13 mice. *Signi cantly different from high-fat diet-treated, P< decrease in plasma FFA, liver lipids and plasma triacylglycerol and cholesterol. Summarizing these results, it seems likelythatchitin-chitosan may preventthe high-fat diet-induced increase in body fat content through inhibiting intestinalabsorption ofdietaryfat and cause improvement of the fatty liver and hyperlipidaemia in the obese mice, both through inhibiting intestinal absorption of dietary fat and through decrease in basal lipolysis in fat cells of the obese mice. Acknowledgements This work was supported by Research Grants from Fuji-Bio Co. Ltd, (Shizuoka, Japan). References 1 Ikeda I, Tomari Y, Sugano M. Interrelated effects of dietary ber and fat on lymphatic cholesterol and triglyceride absorption in rats. J Nutr 1989; 119: 1383± Sugano M, Watanabe S, Kishi A, Izume M, Ohtakara A. Hypocholesterolemic action of chitosans with different viscosity in rats. Lipids 1988; 23: 187± Ebihara K, Schneeman BO. Interaction of bile acids, phospholipids, cholesterol and triglyceride with dietary bers in the small intestine of rats. J Nutr 1989; 119: 1100± Razdan A, Petterson D. Effect of chitin and chitosan on nutrient digestibility and plasma lipid concentrations in broiler chickens. Br J Nutr 1994; 72: 277± Zhou A, Matsuura Y, Okuda H. Chitosan augments cytolytic activity of mouse lymphocytes. J Trad Med 1994; 11: 62±64. 6 Kato H, Taguchi T, Okuda H, Kondo M, Takara M. Antihypertensive effect of chitosan in rats and humans. J Trad Med 1994; 11: 198± Zapf J, Schoenle E, Waldvogel M, Sand M, Froesch ER. Effects of trypsin treatment of rat adipocyte on biological effects and binding of insulin and insulin-like growth factors: Further evidence for the action of insulin-like growth factors through the insulin receptor. Eur J Biochem 1981; 133: 605± Tsujita T, Okuda H. Carboxylesterase in rat and human sera and their relationship to serum aryl acylamidases and cholinesterase. Eur J Biochem 1983; 133: 215± Verger R. Pancreatic lipase. In: BorgstroÈm B and Brockman HL (eds). Lipase. Elsevier, Amsterdam, 1984, pp 83± Tsujita T, Matuura Y, Okuda H. Studies on the inhibition of pancreatic and carboxylester lipases by protamine. J Lipid Res 1996; 37: 1481± Flatt JP. The difference in the storage capacities for carbohydrate and for fat, and its implications in the regulation of body weight. Ann NY Acad Sci 1987; 499: 104± Awad AB, Bernardis LL, Fink CS. Failure to demonstrate an effect of dietary fatty acid composition on body weight, body composition and parameters of lipid metabolism in mature rats. J Nutr 1990; 120: 1277± Shimomura Y, Tamura T, Suzuki M. Less body fat accumulation in rats fed a saf ower oil diet than in rats fed a beef tallow diet. J Nutr 1990; 120: 1291± Hill JO, Peters JC, Lin D, Yakubu F, Greene H, Swift L. Lipid accumulation and body fat distribution is in uenced by the type of dietary fat fed to rats. Int J Obes 1993; 17: 223± Hill JO, Lin D, Yakubu F, Peters JC. Development of dietary obesity in rats: in uence of amount and composition of dietary fat. Int J Obes 1992; 16: 321± Lemonnier D. Effect of age, sex and site on the cellularity of the adipose tissue in mice and rats rendered obese by a high-fat diet. J Clin Invest 1972; 51: 2907± Obst BE, Schemmel RA, Czajka-Narins D, Merkel R. Adipocyte size and number in dietary obesity resistant and susceptible rats. Am J Physiol 1981; 240 (Endocrinol Metab 3): E47±E Tsujita T, Morimoto C, Okuda H. Mechanism of increase in basal lipolysis of enlarged adipocytes in obese animals. Obes Res 1995; 3: 633s±636s. 19 Morimoto C, Tsujita T, Okuda H. Antilipolytic actions of insulin on basal and hormone-induced lipolysis in rat adipocytes. J Lipid Res 1998; 39: 957±962.