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Fatty liver induced by the addition of excess cystine to a soya-bean protein diet in rats
Camp.
Eiochem.
Physiol
0~%29i92 so0 + 0.00 0 1992Pergamon Press plc
Vol. 102A,No. 1, pp. 185489, 1992
Printed in Great Britain
FATTY LIVER INDUCED BY THE ADDITION OF EXCESS CYSTINE TO A SOYA-BEAN PROTEIN DIET IN RATS YORITAKA AOYAMA,*
HIDEKI MATSUMOTO,ERI HITOMI-OHMURAand AKIRA YOSHIDA
Department of Agricultural Chemistry, School of Agriculture, Nagoya University, Furo-cho, Chikusa, Nagoya 464-01, Japan. Telephone: (052) 781-5111 (Receiued 27 August 1991) Abstracb-1.
The effects of excess cystine added to diets with casein, egg protein, soya-bean protein and wheat gluten as protein source on liver and serum lipids of rats were compared. 2. The addition of excess cystine to a soya-bean protein diet produced lipid accumulation in the liver. 3. The addition of excess cystine to casein, egg protein and soya-bean protein diets, but not a wheat gluten diet, increased serum cholesterol. -- _
INTRODUCTION Feeding on an excess cystine diet, but not on a diet cont~ning excess methionine, valine or histidine for 2 months, resulted in a rise of serum cholesterol (Serougne and Rukaj, 1983; Rukaj and Serougne, 1983; Serougne et al., 1984,1987). However, excessive addition of lysine and tryptophan caused a decrease in serum cholesterol (Serougne and Rukaj, 1983). Furthermore, except for absorption and fecal excretion, all the other flows involved in cholesterol turnover were increased when excess cystine was added (Rukaj and Serougne, 1983) and a significant linear correlation exists between individual values of cholesterolemia and those of internal secretion of cholesterol (Serougne er al., 1987). The addition of excess cystine caused an increase in, not only serum cholesterol, but also serum phospholipid within 6 days of feeding, and the addition of either konjac mannan or pectin to the cystine excess diet prevented the hypercholesterolemia (Aoyama et al., 1987). In the course of the study concerning the effects of cystine on the formation of h~rcholesterolemia in rats, we found that rats given the soya-bean protein diet with excess cystine produced fatty liver. This did not occur in rats given the diet cointaining casein as a protein source (Aoyama et al., 1987). The present studies were undertaken to determine whether this effect can be consistently elicited and to compare the lipid composition in the liver and serum with that of rats when cystine was added to the diet containing proteins other than soya-bean protein. MATERIALS
AND METHODS
Animals Male rats of the Wistar strain (Japan SLC., Hamamatsu, Shizuoka, Japan) weighing about 140 g were housed individually in screen-bottomed cages in an air-conditioned animal room at a temperature of approximately 23°C. Light was regulated so as to provide equal periods of light (8 a.m.-8 p.m.) and dark (8 p.m.-8 a.m.). *To whom correspondence
should be addressed,
Diets The composition of the diets without excess cystine are shown in Table 1. The cystine-excess diet had the same composition as the corresponding diet except that 35 g of maize starch per kg of the diet was replaced by 35g of I.-cystine (Ajinomoto Co., Tokyo, Japan). Casein (813 g as crude protein per kg) (Katayama Chemical Industries Co., Osaka, Japan), egg protein (747 g as crude protein per kg) (prepared by washing twice with hot ethyl alcohol and then twice with n-hexane to remove fats from whole egg powder, Taiyo~agaku Co., Yokkaichi, Japan), soya-bean protein (soybean protein isolate) (791 g as crude protein per kg) (Fujipro R, Fuji Oil Co., Osaka, Japan) and wheat gluten (737 g as crude protein per kg) (Katayama Chemical Co.) were used as dietary protein sources. In order to supply the diet with 200g of protein (N x 6.25) per kg of diet, casein, egg protein, soybean protein isolate and wheat protein were added in this amount, respectively. The rats had free access to food and water throughout the experimental periods. Analysis At the end of the experimental period (6 days), blood was removed from the heart of the rats which had been anesthetized with diethyl ether between 9:30 and 10: 30 a.m. The livers were immediately excised and weighed. The lipid contents of the livers were determined gravimetrically after extraction and purification (Folch et al., 1957). Lipids soluble in chloroform: methanol (2: 1, v/v) were. used for the determination of triacylglycerol r&l cholesterol (total, free plus ester&d). Triacylglycerol (Wahlefeld, 1974) and cholesterol (Siedel el al., 1983) in the liver were determined by the enzymatic methods, respectively. Phospholipid was determined by difference [total lipids - (triacylglycerol + cholesterol)]. Triacylglycerol (Wahlefeld, 1974), cholesterol (Siedel et al., 1983) and phospholipid (Takayama et al., 1977) in serum were determined by the enzymatic methods. Statistics Data were subjected to Student’s r-test (Snedecor and Cochran, 1967) and Duncan’s multiple range test (Duncan, 1955) to determine if differences in means are significant. RESULTS
The effects of the addition of excess cystine to a diet containing various proteins on food intake, changes 185
Y. AOYAMA et al.
186
Table 1. Composition of the diets without excess cystine’
h?i3
Caseint Egg proteint Soybean protein isolate@ Wheat glutent AIN vitamin mixturelill Choline chloridet AIN mineral mixture11 Maize oil** Maize starchtt
Casein
protein
246 10 2 35 50 657
268 10 2 35 50 635
k/k)
Soya-bean protein 253 IO 2 35 50 650
Wheat gluten 271 IO 2 35 50 632
*The cystine-excess diet had the same composition as the corresponding diet except that 35 g of maize starch was replaced by 35 g of L-cystine per kg of diet. tKatayama Chemical Industries Co., Osaka, Japan. IPrepared from whole egg powder produced by Taiyoukagaku Co., Yokkaichi, Mie, Japan. §Fujipro R, Fuji Oil Co., Osaka, Japan. IlAmerican Institute of Nutrition (1977) J. Nutr. 107, 1340-1348. TAmerican Institute of Nutrition (1980) .I. Nutr. 110, 1726. l*Nihon Syokuhin Kako Co., Fuji, Shizuoka, Japan. ttChuo Syokuryo Co., Inazawa, Aichi, Japan.
in body weight and liver weight are summarized in Table 2. Food intake and gain in body weight were suppressed by the addition of excess cystine to all four diets tested. The increase in body weight on the wheat gluten diet was the lowest. Hepatomegaly resulted from the addition of cystine to the casein and the soya-bean protein diets, but not to the egg protein and the wheat gluten diets. When the soya-bean protein diet was not supplemented with cystine, liver weight of rats was significantly lower than in rats on the casein, egg protein and wheat gluten diets which had similar values. When diets were supplemented with cystine, liver weights in rats on the casein, egg protein and soya-bean protein diets were heavier than those of rats on the wheat gluten diet. Total liver lipids and their components for rats are summarized in Table 3. The addition of excess cystine to the soya-bean protein diet, but not to the casein, egg protein and wheat gluten diets produced lipid accumulation in the liver. Total liver lipid content of rats was similar for the casein, egg protein, soya-bean protein and wheat gluten diets without excess cystine. When fed the diet supplemented with cystine, the total liver lipid level of rats on the soya-bean protein diet was significantly higher than those of rats on the casein, egg protein and wheat gluten diets which had similar values. When excess cystine was not added, liver triacylglycerol of rats fed the casein diet was
lower than that of the wheat gluten diet, and higher than in the egg protein and soya-bean protein diet. When excess cystine was added, changes in liver triacylglycerol were essentially similar to those in liver lipids. The addition of excess cystine to either the egg protein or wheat gluten diet increased liver cholesterol. When cystine was not added, liver cholesterol for the soya-bean protein diet was significantly higher than for the egg protein or wheat gluten diet. Liver cholesterol for the casein and egg protein diets was higher than for the wheat gluten diet. When cystine was supplemented, liver cholesterol was not affected by the kinds of proteins in the diet. The addition of excess cystine to either the egg protein or soya-bean protein diet lowered liver phospholipid. When cystine was not supplemented, phospholipid in the liver of rats fed either the egg protein or soya-bean protein diet was higher than in rats fed either the casein or wheat gluten diet. On the cystine excess diet, the liver phospholipid of rats was not influenced by the kinds of protein in the diet. The effects of excess cystine added to the diets on serum lipids are summarized in Table 4. The addition of excess cystine lowered or tended to lower serum triacylglycerol. Of the proteins tested, the lowering of serum triacylglycerol was highest in the soya-bean protein group. When cystine was not added, serum triacylglycerol of rats on the egg protein diet was higher than those on the casein and wheat gluten diets. When cystine was added, serum triacylglycerol of rats on the egg protein diet was significantly higher than on the casein, soya-bean protein and wheat gluten diets, which had similar values. Excess cystine added to the casein diet increased serum phospholipid. When cystine was not added, serum phospholipid for the soya-bean protein diet was lower than for the other three groups, which had similar values. With diets supplemented with cystine, the feeding of the casein diet resulted in higher phospholipid in serum as compared to those for the other three groups. Serum phospholipid for the soya-bean protein and wheat gluten diets was significantly lower than for the egg protein diet. The addition of excess cystine to the casein, egg protein and soya-bean protein diets caused hypercholesterolemia. When cystine was not added, serum cholesterol of rats on the soya-bean protein diet was significantly lower than for the three other groups. When cystine was added, serum cholesterol for the egg protein diet was significantly lower than for the casein diet, and higher
Table 2. Effects of excess cvstine on food intake. changes in body weight and liver weight Cystine added (g/kg) Food intake (g/6 days)
0 35
Changes in body weight (g/6 days) Liver weight (g/kg body weight)
0 35
P
P
0 35 P
Casein 88 k 2 50 + I
Diet Egg protein 87 + 3 47 * 1
Soya-bean protein 92 f 2 45 * 1
Wheat gluten 85 + 2 43 * 2
Data represents means + SEM for six rats. a.b.r.ysMeans within the same horizontal column that do not share a common superscript letter were significantly different: P < 0.05. NS: not significant.
Cystine-induced
fatty liver
187
Table 3. Effects of excess cystine on total lipids, triacylglycerol, cholesterol and phospholipid of the liver
Liver lipids
Cystine added k/kg)
Total lipids (mg/g)
0 35
Triacylglycerol (mg/g)
0 35
Cholesterol (mgN
0 35
Phospholipid (mg/g)
0 35
P
P
P
P
Casein 43.4 f 0.7 42.8 f 2.2’ NS 21.7+ I.Ib 20.8 + I .9’ NS 2.09 5 0.3Pb 2.08 k 0.05 NS 19.5 5 l.lb 19.9 + 1.3 NS
Diet Eg-&t protein
Soya-bean urotein
Wheat &ten
47.3 * 0.9 43.7 * 1.1’ NS 17.3 f 0.8’ 18.3 f 1.1’ NS 1.96 + 0.03b 2.18 + 0.04
47.1 + 1.7 71.3 + 7.4y NS 17.0 f 0.8’ 47.9 f 8.gy
46.9 f 2.0 48.3 f 3.5’ NS 25.5 f I .7’ 20.5 + I .8’ NS 1.69 k 0.06’ 2.18 * 0.05
Data represents means f SEM for six rats. “~b~‘~Y~‘Means within the same horizontal column that do not share a common superscript letter were significantly different: P < 0.05. NS: not significant.
than for the soya-bean protein diets, which had similar values.
and wheat gluten
DISCUSSION It is apparent from Table 3 that the excess cystine in the soya-bean protein diet resulted in the accumulation of liver lipids, although the mechanism remains obscure. Analysis of liver lipids revealed that the increase is almost entirely due to the triacylglycerol in common with nutritional fatty livers previously reported (Creasey et al., 1961; Hevia and Visek, 1980; Hevia et al., 1980; Lombardi et al., 1966; Milner, 1978; Narayan et al., 1975). As is classically reported, the amount of liver fat was doubled by the addition of cystine to a choline-deficient diet low in casein and high in fat (Beeston and Channon, 1936), and cystine supplement of a choline-deficient diet containing adequate amounts of casein increased the accumulation of liver fat (Mulford and Griffith, 1942). These phenomena are, to some extent, similar to the present findings. However, they used the casein diet deficient in choline and the soya-bean protein diet supplemented with an adequate amount of choline chloride was used for the present report. The methionine content of casein, egg protein, soybean protein isolate and wheat gluten is 3 1, 33, 14 and 18 mg/g protein, respectively (Resources Council, Science and Technology Agency, Japan). Cystine content in casein, egg protein, soybean protein isolate and wheat gluten is 5, 26, 14 and 22 mg/g protein, respectively (Resources Council, Science and Tech-
nology Agency, Japan). Since methionine content is least in soybean protein isolate, the addition of either 3 g of methionine or 2 g of choline chloride completely prevented lipid accumulation in the liver due to excess cystine added to the soya-bean protein diet (Aoyama et al., unpublished). Thus, it is reasonable to conclude that excess cystine in a soya-bean protein diet might cause the increase in the requirement of methionine and thereafter induce a mild cholinedeficiency. The development of nutritional fatty livers in rats on a choline-deficient diet (Aoyama et al., 1971; Lombardi et al., 1966), a diet deficient in either threonine or lysine (Aoyama and Ashida, 1972), an arginine-devoid diet (Aoyama et al., 1981; Milner, 1978), a lysine-excess diet (Aoyama et al., 1983; Hevia et al., 1980; Hevia and Visek, 1980), a fructose diet (Aoyama et al., 1973) and a glycerol diet (Aoyama et al., 1977; Narayan et al., 1975) has been demonstrated. The impaired release of triacylglycerol as serum lipoproteins from liver, the increased fatty acid mobilization from adipose tissue into liver, the enhanced fatty acid synthesis in the liver and the impaired fatty acid oxidation in the liver have all been implicated as causative factors in the hepatic triacylglycerol accumulation occurring with excess cystine added to the soya-bean protein diet. In fact, serum triacylglycerol decreased when excess cystine was added to the soya-bean protein diet (Table 4). The lowered serum triacylglycerol due to excess cystine was the highest in rats fed the soya-bean protein diet (0.85 vs 1.75 as compared to 111 vs 152, 151 vs 214
Table 4. Serum lipids in rats fed diets with or without excess cystine
Serum lipids
Cystine added (g/kg)
Triacylglycerol (mgiml)
0 35
Phospholipid (mg/mU
0 35
Cholesterol (mgiml)
0 35
P
P
P
Casein 1.52k0.10b 1.11 f0.15Z co.05 I .98 * 0.05” 2.55 f 0.09’
1.13*0.03* 1.58 f 0.07”
Diet Egg protein 2.14 f 0.27’ 1.51 fO.lOY NS I .95 + 0.05a 2.14 + O.lOY
Soya-bean protein 1.75 f 0.14Ob 0.85 + 0.10’
NS
NS
I.11 f0.05’ 1.35 f 0.07’ <0.05
0.90 f 0.02b 1.18*0.02’
Wheat gluten 1.34 f 0.03’ 0.88 * 0.05’
Data represents means k SEM for six rats. B.b.r.x.Y.zMeans within the same horizontal column that do not share a common superscript letter were significantly different: P < 0.05. NS: not significant.
188
Y. AOYAMAer al.
and 88 vs 134). Therefore, one of the factors for the production of the fatty liver might be due to the decreased transport of t~acylgly~rol from the liver into blood. However, since the addition of excess cystine to the casein, egg protein and wheat gluten diets also decreased or tended to decrease the serum triacylglycerol (Table 4), the possibility of impaired transport of triacylglycerol from the liver into blood might be ruled out. An ambitious experiment attempting to elucidate the mechanism for the fatty liver induced by the addition of excess cystine to a soya-bean protein diet is now in progress. As shown in Table 4, without excess cystine, serum cholesterol of rats on a soya-bean protein diet was the lowest of all proteins tested. A marked cholesterollowering effect of dietary soya-bean protein in comparison with casein has been reported with several animal models (Carroll and Hamilton, 1975; Nagata et al., 1981, 1982; Sirtori et al., 1984; Yadav and Liener, 1977). The mechanism by which the administration of soya-bean protein can reduce serum cholesterol levels is poorly understood, but the following reports may be helpful. A major factor contributing to the cholesterol lowering effect of plant proteins must reside in differences in their amino acid profiles compared to that of animal proteins. The binding of a cholesterol-rich lipoprotein fraction to hepatic membranes is normal when a soya-bean protein-~hoIestero1 diet was administered, and markedly reduced with a casein-cholesterol diet (Sirtori et al., 1984). The activity of HMG-CoA reductase in the liver of rats on a casein diet was lower than on a soya-bean protein diet. It is surmised that the loss of steroids from the body exceeds the amount of cholesterol synthesis from the results of reduced absorption and enhanced fecal excretion of steroids (Nagata er ai., 1982). The addition of excess cystine to the wheat gluten diet failed to produce hypercholesterolemia. Since lysine in the wheat gluten is the most limiting (Munaver and Harper, 1959) and its protein quality is poor, the effects of dietary addition of both iysine and excess cystine on serum cholesterol must be examined. When rats were fed a diet without cholesterol, cystine had no effect on serum cholesterol level when 3 or 6 g of cystine per kg of diet was added, and only elevated it at 12 g of cystine per kg of diet, whereas, methionine reduced it at 24 g per kg of diet (Yagasaki et al., 1986). The response of cystine to serum cholesterol is similar to the results shown in the present paper. Excess cystine (10 g per kg of diet) added to a diet with cholesterol and sodium cholate reduced plasma cholesterol, and excess methionine (10 g per kg of diet) added to this diet markedly increased plasma cholesterol (Su~yama et al., 1986). The effect of excess cystine on serum cholesterol is likely to vary from a high cholesterol diet to a non-cholesterol diet. SUMMARY
The effects of excess cystine added to diets with casein, egg protein, soya-bean protein and wheat gluten as protein sources on liver and serum lipids of rats were examined. Liver lipid contents among four
groups tested were not changed when cystine was not added. The addition of excess cystine to a soya-bean protein diet, but not to a casein, egg protein and wheat gluten diet, caused lipid accumulation in the liver. The increase in liver lipids was due to triacylglycerol. When cystine was added, the liver lipid level was similar among the three groups except for the soya-bean protein diet. When cystine was not added, serum triacylgly~rol of rats fed the egg protein diet was si~i~cantly higher than in rats fed the casein and wheat gluten diets. When cystine was added, serum triacylglycerol of rats fed the egg protein diet was significantly higher than those of the other three groups. The addition of excess cystine to the diets lowered or tended to lower serum triacylgly~rol. When cystine was not added, serum phospholipid and cholesterol of rats fed the soya-bean protein diet were lower than those for the other three groups which had similar respective values. When cystine was added, serum phospholipid and cholesterol in rats fed the egg protein diet were lower than that of the casein diet, and higher than those of the soyabean protein and wheat gluten diets. The addition of cystine to the casein diet resulted in the increase in serum phospholipid. Serum cholesterol was increased by the addition of excess cystine to the casein, the egg protein and the soya-bean protein diets. These data suggest that one of the factors for the formation of fatty liver due to excess cystine added to the soyabean protein diet might be responsible for the decreased transport of triacylglycerol from liver into blood, since the effect lowering serum triacylglycerol induced by excess cystine was the highest in rats fed the soya-bean protein diet. REFERENCES
Aoyama Y. and Ashida K. (1972) Effect of excess and deficiency of individual essential amino acids in diets on the liver lipid content of growing rats. J. Nutr. 102, 1025-1032. Aoyama Y. and Ashida K. (1973) Effect of various carbohydrates in a repletion diet after protein depletion on liver lipid content of rats. J. Nurr. 103, 225-230. Aoyama Y., Ohmura E., Yoshida A. and Ashida K. (1987) Effect of dietary composition on lipids in serum and liver of rats fed a cystine-excess diet. Agric. Biol. Chem. 51, 3125-3131. Aoyama Y., Sakaida K., Yoshida A. and Ashida K. (1983) Effects on liver and serum lipids of dietary supplements of methionine and excess lysine given to previouslystarved rats. Br. J. Nurr. Jo, 627-636. Aoyama Y., Yasui H. and Ashida K. (1971) Effect ofdietary protein and amino acids in a choline-deficient diet on lipid accumulation in rat liver. J. Nuir. 101, 739-745. Aoyama Y., Yoshida A. and Ashida K. (1977) Effects of dietarv fats and fattv acids on the liver lipid accumulation induced by refeeding a diet containing giycerol to starved rats and its inhibition bv linoleic acid. J. Nurr. 107, 1120-l 125. Aoyama Y., Yoshida A. and Ashida K. (1981) Effect of some dietarv additions to either an arginine-devoid diet or a diet supplemented with erotic acid &fed after starvation on liver lipid content during essential fatty acid deficiency in rats. .I. Nutr. 111, 895-906. Beeston A. L. and Channon H. J. (1936) Cystine and the dietary production of fatty livers. J. biol. Chem. 30, 280-284.
Cystine-induc ed fatty liver Carroll K. K. and Hamilton R. M. G. (1975) Effect of dietary protein and carbohydrate on plasma cholesterol levels in relation to atherosclerosis. J. Food Sci. 40, 18-23.
Creasey W. A., Hankin L. and Hanschmacher R. E. (1961) Fatty livers induced by erotic acid. I. Accumulation and metabolism of lipids. J. biol. Chem. 236, 20642070. Duncan D. B. (1955) Multiple range and multiple F tests. Biometrics 11, 142. Folch J., Lees M. and Sloane-Stanley G. H. (1957) A simple method for the isolation and purification of total lipides from animal tissues. J. bioi. Chem. 226, 497-509. Hevia P.. Kari F. W.. Ulman E. A. and Visek W. J. (1980) Serum’ and liver l&ids in growing rats fed caseinwith L-lysine. J. Nutr. li0, 122cl230.Hevia P. and Visek W. J. (1980) Liver and serum linids and lipoproteins of rats fed‘5% i-lysine. Lipiak 15, 95-99. Lombardi B., Vgazio G. and Raick A. N. (1966) Cholinedeficiency fatty liver: relation of plasma phospholipids to liver triglycerides. Am. J. Physiol. 210, 31-36. Milner J. A. and Perkin E. G. (1978) Liver lipid alterations in rats fed arginine deficient diets. Lipids 13, 563-565. Mulford D. J. and Griffith W. H. (1942) Choline metabolism. VIII. The relation of cystine and of methionine to the requirement of choline in young rats. J. Nufr. 23, 91-100. Munaver S. M. and Harper A. E. (1959) Amino acid balance and imbalance. II. Dietary level of protein and lysine requirement. J. Nurr. 69, 58-64. Nagata Y., Ishikawa N. and Sugano M. (1982) Studies on the mechanism of antihypercholesterolemic action of soy protein and soy protein-type amino acid mixtures in relation to the casein counterparts in rats. J. Nutr. 112, 16141625.
Nagata Y., Tanaka K. and Sugano M. (1981) Further studies on the hypocholesteroleamic effect of soya-bean protein in rats. Br. J. Nutr. 45, 233-241. Narayan K. A., McMullan J. J., Butler D. P., Wakefield T. and Calhoun J. J. (1975) Dietary glycerol-induced fat accumulation in rat livers. Nulr. Rep. Int. 12, 21 l-219. Resources Council, Science and Technology Agency, Japan (1986) Standard Tables of Food Composition in Japan. Amino Acid Composition of Fooa!s. Revised edition.
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Rukaj A. and Serougne C. (1983) Effect of excess dietary cystine on the biodynamics of cholesterol in the rat. Biochim. biophys. Acia 735, l-5. Serougne C., Ferezou J. and Rukaj A. (1984) Effect of excess dietary L-cystine on the rat plasma lipoproteins. Ann. Nutr. Merab. 28, 311-320.
Serougne C., Ferezou J. and Rukaj A. (1987) A new relationship between cholesterolemia and cholesterol synthesis determined in rats fed an excess of cystine. Biochim. biophys. Acta 921, 522-530.
Serougne C. and Rukaj A. (1983) Plasma and lipoprotein cholesterol in rats fed L-amino acid-supplemented diets. Ann. Nutr. Merab. 27, 386-395.
Siedel J., Hagele E. O., Ziegenhorn J. and Wahlefeld A. (1983) Reagent for the enzymatic determination of serum total cholesterol with improved lipolytic efficiency. Chin. Chem. 29, 1075-1080. Sirtori C. R., Galli G., Lovati M. R., Carrara P., Bosiso E. and Kienle M. G. (1984) Effects of dietary proteins on the regulation of liver lipoprotein receptors in rats. J. Nutr. 114, 1493-1500. Snedecor G. W. and Cochran W. G. (1967) Statistical Methods, 6th edition Iowa State University Press, Ames, IA. Sugiyama K., Mizuno M. and Muramatsu K. (1986) Effect of individual amino acids on plasma cholesterol level in rats fed a high cholesterol diet. J. Nurr. Sci. Vitaminol. 32, 623633.
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Eiochem.
Physiol
0~%29i92 so0 + 0.00 0 1992Pergamon Press plc
Vol. 102A,No. 1, pp. 185489, 1992
Printed in Great Britain
FATTY LIVER INDUCED BY THE ADDITION OF EXCESS CYSTINE TO A SOYA-BEAN PROTEIN DIET IN RATS YORITAKA AOYAMA,*
HIDEKI MATSUMOTO,ERI HITOMI-OHMURAand AKIRA YOSHIDA
Department of Agricultural Chemistry, School of Agriculture, Nagoya University, Furo-cho, Chikusa, Nagoya 464-01, Japan. Telephone: (052) 781-5111 (Receiued 27 August 1991) Abstracb-1.
The effects of excess cystine added to diets with casein, egg protein, soya-bean protein and wheat gluten as protein source on liver and serum lipids of rats were compared. 2. The addition of excess cystine to a soya-bean protein diet produced lipid accumulation in the liver. 3. The addition of excess cystine to casein, egg protein and soya-bean protein diets, but not a wheat gluten diet, increased serum cholesterol. -- _
INTRODUCTION Feeding on an excess cystine diet, but not on a diet cont~ning excess methionine, valine or histidine for 2 months, resulted in a rise of serum cholesterol (Serougne and Rukaj, 1983; Rukaj and Serougne, 1983; Serougne et al., 1984,1987). However, excessive addition of lysine and tryptophan caused a decrease in serum cholesterol (Serougne and Rukaj, 1983). Furthermore, except for absorption and fecal excretion, all the other flows involved in cholesterol turnover were increased when excess cystine was added (Rukaj and Serougne, 1983) and a significant linear correlation exists between individual values of cholesterolemia and those of internal secretion of cholesterol (Serougne er al., 1987). The addition of excess cystine caused an increase in, not only serum cholesterol, but also serum phospholipid within 6 days of feeding, and the addition of either konjac mannan or pectin to the cystine excess diet prevented the hypercholesterolemia (Aoyama et al., 1987). In the course of the study concerning the effects of cystine on the formation of h~rcholesterolemia in rats, we found that rats given the soya-bean protein diet with excess cystine produced fatty liver. This did not occur in rats given the diet cointaining casein as a protein source (Aoyama et al., 1987). The present studies were undertaken to determine whether this effect can be consistently elicited and to compare the lipid composition in the liver and serum with that of rats when cystine was added to the diet containing proteins other than soya-bean protein. MATERIALS
AND METHODS
Animals Male rats of the Wistar strain (Japan SLC., Hamamatsu, Shizuoka, Japan) weighing about 140 g were housed individually in screen-bottomed cages in an air-conditioned animal room at a temperature of approximately 23°C. Light was regulated so as to provide equal periods of light (8 a.m.-8 p.m.) and dark (8 p.m.-8 a.m.). *To whom correspondence
should be addressed,
Diets The composition of the diets without excess cystine are shown in Table 1. The cystine-excess diet had the same composition as the corresponding diet except that 35 g of maize starch per kg of the diet was replaced by 35g of I.-cystine (Ajinomoto Co., Tokyo, Japan). Casein (813 g as crude protein per kg) (Katayama Chemical Industries Co., Osaka, Japan), egg protein (747 g as crude protein per kg) (prepared by washing twice with hot ethyl alcohol and then twice with n-hexane to remove fats from whole egg powder, Taiyo~agaku Co., Yokkaichi, Japan), soya-bean protein (soybean protein isolate) (791 g as crude protein per kg) (Fujipro R, Fuji Oil Co., Osaka, Japan) and wheat gluten (737 g as crude protein per kg) (Katayama Chemical Co.) were used as dietary protein sources. In order to supply the diet with 200g of protein (N x 6.25) per kg of diet, casein, egg protein, soybean protein isolate and wheat protein were added in this amount, respectively. The rats had free access to food and water throughout the experimental periods. Analysis At the end of the experimental period (6 days), blood was removed from the heart of the rats which had been anesthetized with diethyl ether between 9:30 and 10: 30 a.m. The livers were immediately excised and weighed. The lipid contents of the livers were determined gravimetrically after extraction and purification (Folch et al., 1957). Lipids soluble in chloroform: methanol (2: 1, v/v) were. used for the determination of triacylglycerol r&l cholesterol (total, free plus ester&d). Triacylglycerol (Wahlefeld, 1974) and cholesterol (Siedel el al., 1983) in the liver were determined by the enzymatic methods, respectively. Phospholipid was determined by difference [total lipids - (triacylglycerol + cholesterol)]. Triacylglycerol (Wahlefeld, 1974), cholesterol (Siedel et al., 1983) and phospholipid (Takayama et al., 1977) in serum were determined by the enzymatic methods. Statistics Data were subjected to Student’s r-test (Snedecor and Cochran, 1967) and Duncan’s multiple range test (Duncan, 1955) to determine if differences in means are significant. RESULTS
The effects of the addition of excess cystine to a diet containing various proteins on food intake, changes 185
Y. AOYAMA et al.
186
Table 1. Composition of the diets without excess cystine’
h?i3
Caseint Egg proteint Soybean protein isolate@ Wheat glutent AIN vitamin mixturelill Choline chloridet AIN mineral mixture11 Maize oil** Maize starchtt
Casein
protein
246 10 2 35 50 657
268 10 2 35 50 635
k/k)
Soya-bean protein 253 IO 2 35 50 650
Wheat gluten 271 IO 2 35 50 632
*The cystine-excess diet had the same composition as the corresponding diet except that 35 g of maize starch was replaced by 35 g of L-cystine per kg of diet. tKatayama Chemical Industries Co., Osaka, Japan. IPrepared from whole egg powder produced by Taiyoukagaku Co., Yokkaichi, Mie, Japan. §Fujipro R, Fuji Oil Co., Osaka, Japan. IlAmerican Institute of Nutrition (1977) J. Nutr. 107, 1340-1348. TAmerican Institute of Nutrition (1980) .I. Nutr. 110, 1726. l*Nihon Syokuhin Kako Co., Fuji, Shizuoka, Japan. ttChuo Syokuryo Co., Inazawa, Aichi, Japan.
in body weight and liver weight are summarized in Table 2. Food intake and gain in body weight were suppressed by the addition of excess cystine to all four diets tested. The increase in body weight on the wheat gluten diet was the lowest. Hepatomegaly resulted from the addition of cystine to the casein and the soya-bean protein diets, but not to the egg protein and the wheat gluten diets. When the soya-bean protein diet was not supplemented with cystine, liver weight of rats was significantly lower than in rats on the casein, egg protein and wheat gluten diets which had similar values. When diets were supplemented with cystine, liver weights in rats on the casein, egg protein and soya-bean protein diets were heavier than those of rats on the wheat gluten diet. Total liver lipids and their components for rats are summarized in Table 3. The addition of excess cystine to the soya-bean protein diet, but not to the casein, egg protein and wheat gluten diets produced lipid accumulation in the liver. Total liver lipid content of rats was similar for the casein, egg protein, soya-bean protein and wheat gluten diets without excess cystine. When fed the diet supplemented with cystine, the total liver lipid level of rats on the soya-bean protein diet was significantly higher than those of rats on the casein, egg protein and wheat gluten diets which had similar values. When excess cystine was not added, liver triacylglycerol of rats fed the casein diet was
lower than that of the wheat gluten diet, and higher than in the egg protein and soya-bean protein diet. When excess cystine was added, changes in liver triacylglycerol were essentially similar to those in liver lipids. The addition of excess cystine to either the egg protein or wheat gluten diet increased liver cholesterol. When cystine was not added, liver cholesterol for the soya-bean protein diet was significantly higher than for the egg protein or wheat gluten diet. Liver cholesterol for the casein and egg protein diets was higher than for the wheat gluten diet. When cystine was supplemented, liver cholesterol was not affected by the kinds of proteins in the diet. The addition of excess cystine to either the egg protein or soya-bean protein diet lowered liver phospholipid. When cystine was not supplemented, phospholipid in the liver of rats fed either the egg protein or soya-bean protein diet was higher than in rats fed either the casein or wheat gluten diet. On the cystine excess diet, the liver phospholipid of rats was not influenced by the kinds of protein in the diet. The effects of excess cystine added to the diets on serum lipids are summarized in Table 4. The addition of excess cystine lowered or tended to lower serum triacylglycerol. Of the proteins tested, the lowering of serum triacylglycerol was highest in the soya-bean protein group. When cystine was not added, serum triacylglycerol of rats on the egg protein diet was higher than those on the casein and wheat gluten diets. When cystine was added, serum triacylglycerol of rats on the egg protein diet was significantly higher than on the casein, soya-bean protein and wheat gluten diets, which had similar values. Excess cystine added to the casein diet increased serum phospholipid. When cystine was not added, serum phospholipid for the soya-bean protein diet was lower than for the other three groups, which had similar values. With diets supplemented with cystine, the feeding of the casein diet resulted in higher phospholipid in serum as compared to those for the other three groups. Serum phospholipid for the soya-bean protein and wheat gluten diets was significantly lower than for the egg protein diet. The addition of excess cystine to the casein, egg protein and soya-bean protein diets caused hypercholesterolemia. When cystine was not added, serum cholesterol of rats on the soya-bean protein diet was significantly lower than for the three other groups. When cystine was added, serum cholesterol for the egg protein diet was significantly lower than for the casein diet, and higher
Table 2. Effects of excess cvstine on food intake. changes in body weight and liver weight Cystine added (g/kg) Food intake (g/6 days)
0 35
Changes in body weight (g/6 days) Liver weight (g/kg body weight)
0 35
P
P
0 35 P
Casein 88 k 2 50 + I
Diet Egg protein 87 + 3 47 * 1
Soya-bean protein 92 f 2 45 * 1
Wheat gluten 85 + 2 43 * 2
Data represents means + SEM for six rats. a.b.r.ysMeans within the same horizontal column that do not share a common superscript letter were significantly different: P < 0.05. NS: not significant.
Cystine-induced
fatty liver
187
Table 3. Effects of excess cystine on total lipids, triacylglycerol, cholesterol and phospholipid of the liver
Liver lipids
Cystine added k/kg)
Total lipids (mg/g)
0 35
Triacylglycerol (mg/g)
0 35
Cholesterol (mgN
0 35
Phospholipid (mg/g)
0 35
P
P
P
P
Casein 43.4 f 0.7 42.8 f 2.2’ NS 21.7+ I.Ib 20.8 + I .9’ NS 2.09 5 0.3Pb 2.08 k 0.05 NS 19.5 5 l.lb 19.9 + 1.3 NS
Diet Eg-&t protein
Soya-bean urotein
Wheat &ten
47.3 * 0.9 43.7 * 1.1’ NS 17.3 f 0.8’ 18.3 f 1.1’ NS 1.96 + 0.03b 2.18 + 0.04
47.1 + 1.7 71.3 + 7.4y NS 17.0 f 0.8’ 47.9 f 8.gy
46.9 f 2.0 48.3 f 3.5’ NS 25.5 f I .7’ 20.5 + I .8’ NS 1.69 k 0.06’ 2.18 * 0.05
Data represents means f SEM for six rats. “~b~‘~Y~‘Means within the same horizontal column that do not share a common superscript letter were significantly different: P < 0.05. NS: not significant.
than for the soya-bean protein diets, which had similar values.
and wheat gluten
DISCUSSION It is apparent from Table 3 that the excess cystine in the soya-bean protein diet resulted in the accumulation of liver lipids, although the mechanism remains obscure. Analysis of liver lipids revealed that the increase is almost entirely due to the triacylglycerol in common with nutritional fatty livers previously reported (Creasey et al., 1961; Hevia and Visek, 1980; Hevia et al., 1980; Lombardi et al., 1966; Milner, 1978; Narayan et al., 1975). As is classically reported, the amount of liver fat was doubled by the addition of cystine to a choline-deficient diet low in casein and high in fat (Beeston and Channon, 1936), and cystine supplement of a choline-deficient diet containing adequate amounts of casein increased the accumulation of liver fat (Mulford and Griffith, 1942). These phenomena are, to some extent, similar to the present findings. However, they used the casein diet deficient in choline and the soya-bean protein diet supplemented with an adequate amount of choline chloride was used for the present report. The methionine content of casein, egg protein, soybean protein isolate and wheat gluten is 3 1, 33, 14 and 18 mg/g protein, respectively (Resources Council, Science and Technology Agency, Japan). Cystine content in casein, egg protein, soybean protein isolate and wheat gluten is 5, 26, 14 and 22 mg/g protein, respectively (Resources Council, Science and Tech-
nology Agency, Japan). Since methionine content is least in soybean protein isolate, the addition of either 3 g of methionine or 2 g of choline chloride completely prevented lipid accumulation in the liver due to excess cystine added to the soya-bean protein diet (Aoyama et al., unpublished). Thus, it is reasonable to conclude that excess cystine in a soya-bean protein diet might cause the increase in the requirement of methionine and thereafter induce a mild cholinedeficiency. The development of nutritional fatty livers in rats on a choline-deficient diet (Aoyama et al., 1971; Lombardi et al., 1966), a diet deficient in either threonine or lysine (Aoyama and Ashida, 1972), an arginine-devoid diet (Aoyama et al., 1981; Milner, 1978), a lysine-excess diet (Aoyama et al., 1983; Hevia et al., 1980; Hevia and Visek, 1980), a fructose diet (Aoyama et al., 1973) and a glycerol diet (Aoyama et al., 1977; Narayan et al., 1975) has been demonstrated. The impaired release of triacylglycerol as serum lipoproteins from liver, the increased fatty acid mobilization from adipose tissue into liver, the enhanced fatty acid synthesis in the liver and the impaired fatty acid oxidation in the liver have all been implicated as causative factors in the hepatic triacylglycerol accumulation occurring with excess cystine added to the soya-bean protein diet. In fact, serum triacylglycerol decreased when excess cystine was added to the soya-bean protein diet (Table 4). The lowered serum triacylglycerol due to excess cystine was the highest in rats fed the soya-bean protein diet (0.85 vs 1.75 as compared to 111 vs 152, 151 vs 214
Table 4. Serum lipids in rats fed diets with or without excess cystine
Serum lipids
Cystine added (g/kg)
Triacylglycerol (mgiml)
0 35
Phospholipid (mg/mU
0 35
Cholesterol (mgiml)
0 35
P
P
P
Casein 1.52k0.10b 1.11 f0.15Z co.05 I .98 * 0.05” 2.55 f 0.09’
1.13*0.03* 1.58 f 0.07”
Diet Egg protein 2.14 f 0.27’ 1.51 fO.lOY NS I .95 + 0.05a 2.14 + O.lOY
Soya-bean protein 1.75 f 0.14Ob 0.85 + 0.10’
NS
NS
I.11 f0.05’ 1.35 f 0.07’ <0.05
0.90 f 0.02b 1.18*0.02’
Wheat gluten 1.34 f 0.03’ 0.88 * 0.05’
Data represents means k SEM for six rats. B.b.r.x.Y.zMeans within the same horizontal column that do not share a common superscript letter were significantly different: P < 0.05. NS: not significant.
188
Y. AOYAMAer al.
and 88 vs 134). Therefore, one of the factors for the production of the fatty liver might be due to the decreased transport of t~acylgly~rol from the liver into blood. However, since the addition of excess cystine to the casein, egg protein and wheat gluten diets also decreased or tended to decrease the serum triacylglycerol (Table 4), the possibility of impaired transport of triacylglycerol from the liver into blood might be ruled out. An ambitious experiment attempting to elucidate the mechanism for the fatty liver induced by the addition of excess cystine to a soya-bean protein diet is now in progress. As shown in Table 4, without excess cystine, serum cholesterol of rats on a soya-bean protein diet was the lowest of all proteins tested. A marked cholesterollowering effect of dietary soya-bean protein in comparison with casein has been reported with several animal models (Carroll and Hamilton, 1975; Nagata et al., 1981, 1982; Sirtori et al., 1984; Yadav and Liener, 1977). The mechanism by which the administration of soya-bean protein can reduce serum cholesterol levels is poorly understood, but the following reports may be helpful. A major factor contributing to the cholesterol lowering effect of plant proteins must reside in differences in their amino acid profiles compared to that of animal proteins. The binding of a cholesterol-rich lipoprotein fraction to hepatic membranes is normal when a soya-bean protein-~hoIestero1 diet was administered, and markedly reduced with a casein-cholesterol diet (Sirtori et al., 1984). The activity of HMG-CoA reductase in the liver of rats on a casein diet was lower than on a soya-bean protein diet. It is surmised that the loss of steroids from the body exceeds the amount of cholesterol synthesis from the results of reduced absorption and enhanced fecal excretion of steroids (Nagata er ai., 1982). The addition of excess cystine to the wheat gluten diet failed to produce hypercholesterolemia. Since lysine in the wheat gluten is the most limiting (Munaver and Harper, 1959) and its protein quality is poor, the effects of dietary addition of both iysine and excess cystine on serum cholesterol must be examined. When rats were fed a diet without cholesterol, cystine had no effect on serum cholesterol level when 3 or 6 g of cystine per kg of diet was added, and only elevated it at 12 g of cystine per kg of diet, whereas, methionine reduced it at 24 g per kg of diet (Yagasaki et al., 1986). The response of cystine to serum cholesterol is similar to the results shown in the present paper. Excess cystine (10 g per kg of diet) added to a diet with cholesterol and sodium cholate reduced plasma cholesterol, and excess methionine (10 g per kg of diet) added to this diet markedly increased plasma cholesterol (Su~yama et al., 1986). The effect of excess cystine on serum cholesterol is likely to vary from a high cholesterol diet to a non-cholesterol diet. SUMMARY
The effects of excess cystine added to diets with casein, egg protein, soya-bean protein and wheat gluten as protein sources on liver and serum lipids of rats were examined. Liver lipid contents among four
groups tested were not changed when cystine was not added. The addition of excess cystine to a soya-bean protein diet, but not to a casein, egg protein and wheat gluten diet, caused lipid accumulation in the liver. The increase in liver lipids was due to triacylglycerol. When cystine was added, the liver lipid level was similar among the three groups except for the soya-bean protein diet. When cystine was not added, serum triacylgly~rol of rats fed the egg protein diet was si~i~cantly higher than in rats fed the casein and wheat gluten diets. When cystine was added, serum triacylglycerol of rats fed the egg protein diet was significantly higher than those of the other three groups. The addition of excess cystine to the diets lowered or tended to lower serum triacylgly~rol. When cystine was not added, serum phospholipid and cholesterol of rats fed the soya-bean protein diet were lower than those for the other three groups which had similar respective values. When cystine was added, serum phospholipid and cholesterol in rats fed the egg protein diet were lower than that of the casein diet, and higher than those of the soyabean protein and wheat gluten diets. The addition of cystine to the casein diet resulted in the increase in serum phospholipid. Serum cholesterol was increased by the addition of excess cystine to the casein, the egg protein and the soya-bean protein diets. These data suggest that one of the factors for the formation of fatty liver due to excess cystine added to the soyabean protein diet might be responsible for the decreased transport of triacylglycerol from liver into blood, since the effect lowering serum triacylglycerol induced by excess cystine was the highest in rats fed the soya-bean protein diet. REFERENCES
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