EFFECT OF ETHIONINE ON PROTEIN CONTENT OF LIVER IN GROWING RATS*

Transcription

1 EFFECT OF ETHIONINE ON PROTEIN CONTENT OF LIVER IN GROWING RATS* BY MELVIN LEVINE AND JOHN V. FOPEANO, JR. (From the Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor, Michigan) (Received for publication, December 4, 152) Previous work on the effect of ethionine on the liver lipide of rats has shown that injection of large amounts of nn-ethionine into rats results in the development of fatty livers in females within 24 hours whereas males are variable in their response (1, 2). The addition of ethionine to the diet seems to have no effect on the liver lipide of male rats (3) and under some conditions (4) ethionine appears to be lipotropic. No data are available on the effect of feeding ethionine to females. Tarver and his colleagues interpret their data on the inhibition of labeled methionine and glycine incorporation in rats by ethionine (5) as indicating an interference with protein synthesis. Stekol and Weiss believe that, to some extent, the toxicity of ethionine is due to its conversion to triethylcholine (TEC) (6, 7). TEC has been shown to be lipotropic by McArthur and Lucas (8) and others. Levine and Tarver () found evidence to indicate that ethionine may be taken up into the proteins in viva to form abnormal products. It was believed that paired feeding experiments, in which a concentration of ethionine which would allow slow growth without a choline or methionine supplement (3) was used, might indicate how ethionine exerted its effect, and whether the results obtained in long term feeding experiments could be reconciled with those obtained by injection of ethionine. The data presented below indicate (1) that ethionine is not lipotropic in male rats, (2) that ethionine is antilipotropic in female rats, (3) that it induces the formation of abnormal protein in the liver, and (4) that the lowered caloric intake and the increased water content of the liver of animals receiving ethionine account for the apparent lipotropism of this amino acid. Methods Young male and female albino rats from several litters obtained from the Department of Pathology were grouped according to weight and sex * This work was supported in part by a grant from the Faculty Research Fund of the Horace H. Rackham School of Graduate Studies (Project No. 827), University of Michigan. 57

2 58 ETHIONINE AND LIVER COMPOSITION and housed in individual screen bottom cages. Groups receiving ethionine were fed the diet ad lib&m. Food was weighed daily and an equal amount of the basal diet was given to paired controls. A second set of controls received the basal diet ad libitum. Several animals which refused the diet during the course of the experiment were discarded. TABLE Growth of Rats on 6 Per Cent Casein Diets, with and without Supplemental Ethionine and Choline The values given are averages for groups of nine to twelve rats maintained on the diet for 24 days. For composition of the diet, see the text. Ethionine and choline, when present, made up and per cent of the diet, respectively. Eight of the twelve groups of rats were fed ad libitum. Rats in Groups 2, 5, 8, and 11 were paired to rats in Groups 1,4, 7, and, respectively. GgtP _- S&X M. F. I M. F. I I - Dietary Ethionine None Ethionine None I Ethionine, Choline I Ethionine, Choline supplements choline choline I I1 Weight?ood per day Initial Final Per day.i- 1?er gm. food eaten b-m. gm. m. gm f 0.02* f i f f f * f z!z f i O f * Standard error of the mean = (2(X - M)2/n(n - l))t, where X is the individual observation and M is the mean of n observations. T Gain The composition of the basal diet used approximated that of Stekol and Weiss (3) and contained vitamin-free casein (Labco) 25, sucrose 15, cornstarch 28, salts () 2.5, corn oil (Mazola) 20, cod liver oil 5, Ruffex 3.5, and vitamin mixture 1 per cent. The vitamin mixture consisted of cornstarch 5.5 gm., thiamine hydrochloride 37 mg., pyridoxine hydrochloride 37 mg., riboflavin 75 mg., calcium pantothenate 300 mg., p-aminobenzoic acid 300 mg., nicotinic acid 375 mg., and inositol 2.5 gm. The ethionine diet contained per cent DL-ethionine (U. S. Industrial Chemicals, Inc.), replacing an equal amount of corn-starch in the basal diet. This amount was used, since it allowed slow growth without methionine or choline supplementation (3). Choline hydrochloride, when

3 M. LEVINE AND J. V. FOPEANO, JR. 5 present, was equimolar to ethionine and constituted per cent of the diet. The rats were allowed water ad libitum and were weighed every 4 days. Growth on the various diets is presented in Table I. At the end of 24 days the rats were killed by decapitation and exsanguinated. The livers were quickly removed, blotted with filter paper, weighed, cut up, and dropped into absolute alcohol. Lipides were extracted by the method of Best, Channon, and Ridout (11). The lipide-free, finely ground residue was dried at 5 for several hours and allowed to equilibrate with air at room temperature overnight before being weighed. The sum of the weights of the lipide fraction and of the lipide-free residue was used as the dry liver weight. The difference between this figure and that for wet weight of liver was used as the water content of liver. The lipidefree residue was extracted once with cold per cent trichloroacetic acid (TCA) and twice with cold 5 per cent TCA. The residue was then treated with hot 5 per cent TCA to remove nucleic acids (12) and washed again with hot 5 per cent TCA. Although lipide-free, the residue was washed twice with boiling absolute alcohol, centrifuged while still hot, and dried by washing twice with ether. The second hot TCA extraction and the two hot alcohol extractions were found to be necessary to remove glycogen which, as preliminary investigations had indicated, could not be removed by extraction with cold TCA. The residue was heated to 5 for 3 hours allowed to equilibrate in air overnight, and weighed. This preparation is referred to as liver protein. Results Table I indicates that the inhibition of growth in rats receiving ethionine is, in part, due to the lower food intake, since paired controls grow at a lower rate than controls fed ad libitum. The effect is quite marked in animals which receive no choline, when the paired controls (Groups 2 and 5) grow at about one-half the rate of controls fed ad libitum (Groups 3 and 6). In males receiving choline the difference in rate of growth is smaller (Groups 8 and ), while in females receiving choline (Groups 11 and 12) there is no real difference. This is reflected in the protein content of the liver which is discussed below. In all cases, animals receiving ethionine utilized food less efficiently than paired controls, as shown by the smaller gain in weight per gm. of food consumed. Male and female rats receiving ethionine unsupplemented by choline had consistently higher amounts of water in the liver than controls. Males receiving ethionine (Group 1) had a liver water content of 73.0 f 0.6 per cent as compared to 6.1 f 0.4 per cent for paired controls (Group 2) and 67.7 f 0.7 per cent for controls fed ad Z&turn (Group 3). The value for Group 1 is significantly higher than both control groups,

4 600 ETHIONINE AND LIVER COMPOSITION since t is found to be 5.40 (p < 0.01) and 5.75 (p < 0.01) when the mean of Group 1 is compared with the means of Groups 2 and 3, respectively. Females receiving ethionine but no choline (Group 4) had 72.1 f 0.5 per cent water in the liver. This figure is significantly higher than the 6.2 f 0.3 per cent found for paired controls (Group 5) (t = 4.7, p < 0.01) and also significantly higher than the 6.4 f 0.3 per cent water found for controls fed ad Zibitum (Group 6) (t = 4.63, p < 0.01). t values were calculated according to Fisher (13) by the statistical method described in Table II. In contrast to these results, the water content of the livers of rats which received choline as well as ethionine did not differ significantly from those of the paired controls or those fed ad Zibitum. The livers of animals of Groups 7 to had contained 71.1 f 0.5, 70.1 f 0.4, and 6.3 f 0.7 per cent water, respectively, with no significant differences between the means. Groups to 12 all contained 70 per cent water in the livers. The greater amount of water in the livers of rats receiving ethionine without a choline supplement is accompanied by a relative increase in the protein content of the liver. This will be discussed below. The data in Table II show the effect of the various dietary treatments on the content of liver lipide, as per cent of both wet and dry weight of liver. It is evident that at least two factors must be considered in an interpretation of the effect of ethionine on liver lipide, the caloric intake and the changed water content of the livers of rats receiving ethionine. The latter is reflected in several significant differences observed in a comparison of liver lipide as per cent of wet and dry liver weight. On a wet weight basis, male rats receiving ethionine and no choline (Group 1) have a significantly lower liver lipide content than controls fed ad liibitum (Group 3). However, when computed on a dry liver weight basis, there is no significant difference between the two groups. This is also noted in female rats which received ethionine (Group 4) when compared with controls fed ad Z&turn (Group 6). On the basis of fresh liver weight, there is no significant difference between the liver lipide levels, whereas, on the basis of dry liver weight, the ethionine-fed animals have a significantly higher liver lipide content. Such differences in the per cent liver lipide content, when calculated on the basis of wet and dry weights of liver, are not seen among rats receiving a choline-supplemented diet (Groups 7 to 12). In the latter groups, the smaller differences in growth rate found between animals receiving ethionine and their controls (Table I) indicate an improved nutritional state over rats not receiving choline. This improved nutritional state apparently reverses the tendency toward increased water content in the livers of ethionine-receiving animals. Computed on a wet liver basis, the lipide content of the livers of male

5 M. LEVINE AND J. V. FOPEANO, JR. 601 rats receiving ethionine but not choline (Group 1) is lower, but not significantly so, than the lipide content of paired controls (Group 2). When the lipide content is calculated on the basis of dry liver weight, Group 1 is TABLE Effect of Feeding DL-Ethionine on Liver Lipide Content of Male and Female Rats See Table I for explanation of diets and supplements. II I;, Corn arison of differences g etween means* lo. of Total li ides of Total Ii ides of Its wet t! wer p;~;&~;h Ethiotnzfed dry?iv,r Paired with ad libitum fed and Zibitsm fed fed and ethionine-fed ethionine-fed - per Cenf t t *er cmit 7.35 f 0.31t f 0.7; (p < 0.01) 7.88 zk f (p < 0.02 (p < 0.05 > 0.01) > 0.02) Comparison of differences between means* Eth$ksfed lib&m t t f & f f 1.0 (p < 0.02 > 0.01) 6.57 f f f f 0.7 I 7.54 f f (p < 0.02 (p < 0.01) > 0.01) 6.50 i zt f f 1.8 I 5.55 zt f 0. (p < 0.01) (p < O.Ol)l f ; 6.35 i 0.25,035 / 21.2 f fed (p < 0.01) (p < 0.01) (p < O.Ol).(p < 0.01) 0.78 * t values were calculated by the expression, t = (MI - M2)(n1nJn1 + n#/ (@(XI - Md2 + Z(XZ - M#)l(nl + n2-2))+, wh ere MI and M* are the means of the two groups of observations, Xr and X2 are the individual observations, and nr and n2 are the number of rats in each group (13). t Standard error of the mean. slightly, but not significantly, higher than Group 2. From these results and the previous comparison of ethionine-fed animals (Group 1) with the controls fed ad Zibitum (Group 3), it is clear that in male rats, under the conditions of this experiment, the caloric intake is of importance in determining the content of liver lipide. Further, it is evident that any apparent tendency to a lowering of the liver lipide by ethionine is due to

6 602 ETHIONINE AND LIVER COMPOSITION restricted food intake as well as to significant differences in the water content of the livers. Further evidence that ethionine is not lipotropic when fed in the diet, but rather tends to raise liver lipide, is given by a comparison of Groups 7 to, comprising males which received the choline supplement. The amount of lipide in the livers of rats in Group 7, which received ethionine, is significantly higher than that of Group 8, the paired controls, both on a wet and dry liver basis, but is not significantly higher than that of Group, the controls fed ad libitum. It is noteworthy that male rats on the ad libitum basal diet, both with and without choline, do not respond in a consistent manner, as shown by the relatively large standard error of the mean. Other investigators (1, 2) have observed the same inconsistency when ethionine is injected into males. Feeding experiments with diets containing ethionine have, in the past, been restricted to male rats. Tarver and his colleagues have found (1, 2) that female rats given large doses of ethionine intraperitoneally developed fatty livers within 24 hours. In the present experiment it is seen that ethionine fed in moderate amounts in the diet induces fatty livers in females which are significantly higher in lipide content than livers of paired contros (Groups 4 to 6, to 12). A comparison of Group 4, which received ethionine but not choline, with Group, which received ethionine and choline, shows that in females choline enhances the tendency toward fatty liver. As a result, animals in Group have a significantly higher liver lipide content than paired controls of Group 11 or controls of Group 12 fed ad libitum. This is true whether lipide content is calculated on the basis of wet or dry weights of liver. It should not be overlooked that animals receiving choline in the diet ate more and grew better than those which received no choline supplement. Thus the higher content of liver lipide in animals in Group as compared to that in rats in Group 4 may be merely a reflection of an improved nutritional state. Stekol and Weiss interpret their results with male rats (3) as indicating no interference by ethionine with the turnover of liver lipides. The results obtained under the present experimental conditions show that in both males and females receiving choline in amounts equimolar to the ethionine of the diet the liver lipide content is significantly higher than that in controls receiving an equal amount of the basal diet. In females, which develop fatty livers more easily than males on an ethionine diet, the lipide content is also significantly higher than that in the controls fed ad libitum. Various investigators (14-16) have noted that the liver protein of rats, calculated as per cent body weight, depends upon the amount of protein in the diet. Fasting results in a lowering of the liver protein as per cent of the body weight. Kosterlitz (16) has shown that a computation of

7 M. LEVINE AND J. V. FOPEANO, JR. 603 protein as per cent of fresh liver will tend to hide protein changes due to variations in lipide, nucleic acid, and water. In Table III are given the calculations of liver protein as per cent of final body weight. It is clear that ethionine-fed animals which received no dietary choline (Groups 1 and 4) have significantly higher liver. protein contents calculated as per cent of body weight than do the paired controls (Groups 2 and 5). On the TABLE III Effect of Supplemental Dietary DL-Ethionine on Liver Protein Content of Male and Female Rats See Table I for diets and supplements and Table II for calculations of t. Group No No. of Liver protein of rats body weight fier cent 0.4 zk f f f f f & f * f f f 0.03 I_ Comparison of differences between means Ethionine-fed with paired controls 2.40 (p < 0.05 > 0.02) t 3.04 (p < 0.01) thionineted with controls fed ad libihm Controls fed ad ibitum with paired controls 4.58 (p < 0.01) 4.40 (p < 0.01) 6.21 (p < 0.01) 1.31 t other hand, rats receiving choline along with the ethionine (Groups 7 and ) have contents of liver protein which do not differ significantly from those of their paired controls (Groups 8 and 11). A comparison of paired controls (Groups 2, 5, 8, and 11) with controls fed ad Zibitum (Groups 3, 6,, and 12) shows that, except for females receiving choline (Group ll), all groups which were restricted in diet have a significantly lower content of liver protein than controls fed ad Zibitum. It is noteworthy that female controls fed ad libitum and receiving choline (Group 12) had a daily food intake only 11 per cent greater than that of the paired controls in Group 11

8 604 ETHIONINE AND LIVER COMPOSITION (Table I), whereas other controls fed ad libitum consumed from 27 to 66 per cent more food per day than their respective paired controls. The fact that there is no significant difference between the means of Groups 11 and 12 is presumably correlated with the almost equal food consumption. This is in agreement with the findings of I$osterlitz (16) and Addis et al. (14, 15). All groups receiving ethionine have contents of protein which do not vary significantly from those of controls fed ad l&turn, despite the restricted food consumption, and lower efficiency of food utilization. DISCUSSION The results obtained in the present experiment with the paired feeding technique show differences which have been obscured in earlier experiments (3,4). The mode of action of an inhibitor such as ethionine may not become apparent if due regard is not given to the caloric intake of the experimental animals. The results reported here indicate that any tendency toward lipotropism or lack of interference with lipide turnover by ethionine may have been due chiefly to differences in food intake. It has been shown by Grifhth and Wade (17) that the development of a fatty liver depends, under certain conditions, on the rate of growth of the experimental animal. The relative increase of lipide in livers of rats receiving both ethionine and choline over those receiving only ethionine or only choline indicates that ethionine interferes to some extent with the turnover of liver lipide. Farber et al. (1) found that choline did not reverse the effect of ethionine injections upon the induction of fatty livers, whereas large doses of sucrose or glucose, injected at about the same time as the ethionine, resulted in a marked decrease in liver lipide. These investigators concluded that the fatty liver was a type induced by fasting. Ackermann (18) observed that methionine reversed the inhibition of the multiplication of influenza virus by ethionine, although choline, as well as betaine, was inactive. Levine and Tarver found () that, in animals prefed a diet containing ethionine, less labeled ethionine was taken up into the protein than in animals receiving the same ration without ethionine. The latter investigators postulated a saturation with abnormal protein products. The present evidence concerning the effect of ethionine on the protein of liver is consistent with the view of the formation of abnormal protein which is not readily broken down or passed into the blood stream. Since choline supplementation of the ethionine diet gives rise to a significant increase in the liver lipide of female rats, as well as a slight but insignificant rise in the liver lipide of male rats receiving ethionine, it must be concluded that the role of choline, under the conditions of this experiment, is to spare methionine for purposes of protein synthesis. The increased concentration of methi-

9 M. LEVINE AND J. V. FOPEANO, JR. 605 onine is then available to the animal to reverse the effect of ethionine on liver protein. Whether this effect on liver protein is limited to ethionine or may be exerted by other compounds is at present under investigation. SUMMARY 1. Paired feeding experiments with diets containing nn-ethionine show that a tendency toward lowered liver lipide content in male rats receiving ethionine and no choline is due to lowered food intake and increased water content of the liver. The addition of choline to the diet results in an increase in liver lipide above that of paired controls. 2. Female rats receiving ethionine, both with and without choline, have higher liver lipide contents than paired controls. 3. The liver protein content of male and female rats receiving ethionine and no choline is significantly higher than that of paired controls. The addition of choline to the diet abolishes this difference. 4. It is concluded that the increased liver protein content in animals receiving ethionine and no choline in the diet is in harmony with the postulate that ethionine in the diet results in the formation of abnormal protein which accumulates in the liver. BIBLIOGRAPHY 1. Farber, E., Simpson, M. V., and Tarver, H., J. BioZ. Chem., 182, 1 (150). 2. Jensen, D., Chaikoff, I. L., and Tarver, H., J. BioZ. Chem., 12, 35 (151). 3. Stekol, J. A., and Weiss, K., J. BioZ. Chem., 17, 4 (14). 4. Hardwick, V. L., and Winzler, R. J., Proc. See. Exp. Biol. and Med., 6, 217 (148). 5. Simpson, M. V., Farber, E., and Tarver, H., J. BioZ. Chem., 182, 81 (150). 6. Stekol, J. A., and Weiss, K., J. BioZ. Chem., 186, 577 (150). 7. Stekol, J. A., and Weiss, K., J. BioZ. Chem., 186, 585 (150). 8. McArthur, C. S., and Lucas, C. C., Biochem. J., 46, 226 (150).. Levine, M., and Tarver, H., J. BioZ. Chem., 12,835 (151).. Hubbell, R., Mendel, L. B., and Wakeman, A. J., J. Nutr., 14, 273 (137). 11. Best, C. H., Channon, H. J., and Ridout, J. H., J. Physiol., 81, 40 (134). 12. Schneider, W. C., J. BioZ. Chem., 161, 23 (145). 13. Fisher, R. A., Statistical methods for research workers, London, th edition (146). 14. Addis, T., Poo, L. J., and Lew, W., J. BioZ. Chem., 116, 111 (136). 15. Addis, T., Poo, L. J., and Lew, W., J. BioZ. Chem., 115, 117 (136). 16. Kosterlitz, H. W., J. Physiol., 6, 14 (147). 17. Griffith, W. H., and Wade, N. J., J. BioZ. Chem., 132, 627 (140). 18. Ackermann, W. W., J. Exp. Med., 3, 333 (151).

10 EFFECT OF ETHIONINE ON PROTEIN CONTENT OF LIVER IN GROWING RATS Melvin Levine and John V. Fopeano, Jr. J. Biol. Chem. 153, 202: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1