Some effects of excess methionine on lipide metabolism in the rat

ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS 70, 392-400 (1957)

Some Effects of Excess Methionine on Lipide Metabolism in the RatI. Jay From

S. Roth

and

W.

Milstein

the William Goldman Isotope Laboratory, Division of Biological Hahnemann Medical College, Philadelphia, Pennsylvania Received

It has been demonstrated an additional animals

pair-fed

experiments pressions

of lipide metabolism. metabolism carcass

below

of the effects was

similarly

Contrary

content.

volved

in the observed

the incorporation

as fatty

methionine-fed

acids

of the

and control

the extra

to expectations,

and

adipose

ex-

aspects

of the decrease cholesterol

however,

in

might

feeding

increased

to investigate

The

quantitative

acid on cholesterol

that

rat significantly in cholesterol

when compared

on various

since, in view

it was expected

bal-

30 days the

methionine.

to obtain

of the excess amino

of acetate-2-Cl4 liver

in nitrogen After

excess methionine

In order

increase

(l-3).

in fat deposits

designed

interest

to the Wistar

cholesterol

fat levels,

of feeding

observed,

plasma

depleted

diet without

The action

be reduced.

excess methionine

could be maintained

were

of particular

fat previously

12, 1956

to lose weight

visibly

the same

described

Chemistry,

that rats fed a 12 % casein diet containing

they continued

were, on autopsy,

to controls

November

4.8 % DL-methionine

ance even though

well

Seymour

4.8%

carcass

and

the mechanisms

in-

and

decrease

into hepatic tissue,

was

in carcass

cholesterol,

as

measured

in

animals. EXPERIMENTAL

Methods A total of four experiments was run in which excess methionine was fed for 28 days. In each experiment male Wistar rats weighing from 169 to 290 g. were fed,

1This study was supported by funds provided under Contract with the U. S. Air Force School of Aviation Medicine, Randolph 4 A preliminary account of this work was presented at the 128th American Chemical Society, Minneapolis, Minnesota, September, 392

AF 18(690)-928 Field, Texas. meeting of the 1955.

EXCESS

METHIONINE

AND

LIPIDE

393

METABOLISM

ad libitum, a 12% casein diet containing 4.8% excess nn-methionine. Control rats equal in number and total starting weights were fed 12% casein alone. Since the methionine-fed rats voluntarily restrict their food consumption, the food intake of the controls was adjusted to that eaten by the rats given methionine. The composition and preparation of the diets has been described (2). In the isotope experiments, at the end of the 28-day feeding period, the animals were either injected with acetate-2-Cl4 and then sacrificed at intervals thereafter, or sacrificed and liver slices incubated with acetate-2-C’“, as described below. In those experiments not utilizing labeled acetate, the animals were sacrificed by exsanguination at the end of the feeding period. Total lipide content was determined by the method of Haven et al. (4), and liver, carcass, and serum cholesterol were assayed by the method of MacIntyre and Ralston (5) using aliquots of petroleum ether extracts of the saponified tissues, in the case of liver and carcass. Serum phospholipide was determined by a modification of the method of Youngsburg and Youngsburg (6). RESULTS

Effect of Feeding Various Diets to Rats In this first experiment two ad l&turn fed groups of animals, which are described in Table I, were included in addition to the methioninefed and control rats. On autopsy, the livers and kidneys were removed and weighed, and then dried at 80” for 1 week to determine water content. The results with these organs confirmed those previously reported (2,3) and are not included. The changes in weight and the results of the TABLE I The Effects of Feeding Various Diets to Rats Cholesterol Group

and diet

aT$,$s

Weight change

in

Csrcsss

%r

p~dii-

Tot%ziY

Serum %

I. 12% caseina

9

II. 12% casein +4.8yo DLmethionine III. 12oJ,casein ad lib. IV. Stock diete ad lib.

9

-17.0 (+11.8)b -24.0 (-16.4)

w. %

mg./100 g.

ms .%

g./lOO g.

163 (106) 295 (347) 6.0

5.94 (8.73)

227 (175) 373 (401) 7.7

5.24 (5.55)

10

+20.7

137

507

6.4

14.3

8

+29.0

104

313

5.8

5.6

0 Pair-fed against group II. b Figures in parentheses refer to values obtained using eviscerated carcasses. = Purina chow.

in a second experiment

394

ROTH

AND

MILSTEIN

lipide assays for the various groups are given in Table I. Consideration of the weight changes listed in col. 3 illustrates the loss of weight effected by feeding 4.8 % nn-methionine. Due to the voluntary restriction of food intake by this group, the pair-fed controls lost weight, although less than the methionine-fed animals. In this experiment the total carcass3 lipide of the methionine-fed rats was 12 % less than the pair-fed controls. In a second experiment (values in parentheses in Table I), the rats fed excess methionine restricted their food intake less, and consequently the controls gained weight in this case. The greater difference between total carcass lipides of the control and methionine-fed animals in this second experiment (36%) may be a reflection of the increased food intake of both groups. Another possible cause is the fact that, in the second experiment, eviscerated carcasses were used for total lipide determinations, while in the first experiment the entire carcass (minus liver and kidneys) was saponified. The results in both experiments offer direct confirmation of the conclusion that feeding excess methionine gives rise to greater carcass fat loss than that observed in pair-fed controls. This conclusion was reached previously, indirectly, by consideration of weight changes and nitrogen balances in similar groups of rats (2). Examinaation of the cholesterol values in Table I indicates that feeding 4.8% methionine significantly increased serum and carcass cholesterol content. The higher serum cholesterol values in the first experiment, in contrast to the second, are not explained but are probably not related to decreased food consumption since it has been shown (7) that fasting rats for 2-3 weeks did not change the serum cholesterol level. Animals fed 12 % casein alone ad Z&turn (Group III) showed considerably elevated total carcass lipide and cholesterol contents; the former is in keeping with the visual observation that animals kept on this diet for 4-5 months become obese. Serum phospholipide levels showed a rough direct correlation with serum cholesterol levels in all groups: Increase in cholesterol was accompanied by an increase in phospholipide. Isotope Studies Incorporation of Acetate-d-C14 into Liver Lipides in Methionine-Fed and Control Rats. After 28 days of feeding, six methionine-fed rats and six pair-fed controls were injected intraperitoneally with 1 ml. of a saline solution containing about 6 microcuries (PC.) of acetate-2-CY4. A total of 5 mmoles of acetate was contained in 1 ml. of the injected solution in 3 Carcass refers to the entire animal minus the liver and kidneys.

EXCESS

METHIONINE

AND

LIPIDE

395

METABOLISM

this and other experiments. The animals were deprived of food and water and sacrificed 6 hr. later under ether. Previous experiments had shown that high levels of incorporation of Cl4 are reached in from 4 to 6 hr. after injection. The livers were rapidly removed and weighed, and 2-g. samples were saponified and extracted by a modification of the method of Masoro, Chaikoff, and Dauben (8). Cholesterol was precipitated as the digitonide from a suitable aliquot of the appropriate extracts, and, after washing, was plated from pyridine solution and counted in a gas-flow counter. Fatty acids, extracted from the acidified saponification solutions, were plated directiy and counted under identical conditions. The results are shown in Table II. Increase in liver size, in comparison to the controls, and elevated lipide content have been previously reported (10). The increases in the incorporation of acetate-2-Cl4 into cholesterol and fatty acids over the control values are not significantly influenced by dilution effects due to body weight differences since calculation of the incorporation on this basis did not influence the results. Consideration of the data in the last two columns of Table II indicates a greatly increased incorTABLE

II

Incorporation ofAcetate&Cl4

into Liver Lipides and Control Rats

in Methionine-Fed

Liver lipides/g. liver

Counts/min./g. recovered in liver

Rat

Body wt. g.

Liver/i00 g. g.

Cholesterol w.

Fatty acids mg.

Cholesterol

Fatty acids

M la M2 M3 M4 M5 M6

140 153 165 141 149 148 -

4.5 4.4 4.8 3.8 4.1 4.1 -

2.9 3.4 5.2 3.1 4.4 2.4 -

42 40 56 39 39 36 -

113 140 118 73 185 177 -

7075 4725 7650 3925 3650 5425

149

4.3

3.6

42

134

5408

168 186 171 165 179 185 -

3.9 3.8 3.6 3.9 3.7 3.7 -

2.5 2.9 2.1 2.9 2.5 2.3 -

40 50 35 36 47 32 -

30 29 28 40 33 30 -

1975 1575 1525 1725 2200 1500

Average 176 3.8 2.5 a M = Methionine-fed; C = Controls.

40

32

1750

Average

c c2 c3 c4 c5 C6

la

396

ROTH

AND

MILSTEIN

poration of acetate into hepatic cholesterol and fatty acids by methior&e-fed rats. Incorporation of Acetate-&P4 into Hepatic Cholesterol and Adipose Tissue Fatty Acids in Methionine-Fed and Control Rats at Di$erent Time Intervals. After 28 days of feeding 4.8 % excess methionine, ten control and ten methionine-fed animals were injected intraperitoneally with 1 ml. of a saline solution containing 12.2 PC. of acetate-2-CY4. Four hours later four rats from each group were sacrificed by a sharp blow on the head. Livers and testicular adipose tissue (11) were extracted and saponified as described above. Two additional animals from each group were sacrificed 24,48, and 72 hr. after the first four, and the tissues were worked up in a similar manner.The results obtained are depicted in Fig. 1A. It is again evident that hepatic tissue of the methionine-fed animals incorporates acetate-2-Cl4 into cholesterol to a much greater extent than controls. The level of radioactivity in the cholesterol obtained from livers of the experimental rats declines rapidly, so that, by 28 hr. after injection of the labeled acetate, it is almost the same as in the controls. The incorporation of acetate-2-Cl4 into adipose tissue fatty acids is shown in

HOURS

1. A : Incorporation of acetate-2-CY into liver cholesterol in methioninefed and control rats. B: Incorporation of acetate-2-C” into adipose tissue fatty acids in methionine-fed and control rats. Zero time is 4 hr. after intraperitoneal injection of 12.2 PC. of acetate-2-O’. H Animals fed 4.8% nL-methionine for 28 days; q Controls pair-fed against methionine rats. The first bar in each group represents the average of four animals; other bars represent the average of two animals. FIG.

EXCESS

METHIONINE

AND

LIPIDE

METABOLISM

397

Fig. 1B. Methionine feeding reduced the radioactivity of the adipose tissue fatty acids almost to zero, whereas the control adipose tissue fatty acids contained considerable activity which declined slowly during the three days of measurement. The reason for this lack of activity in adipose tissue fatty acids of rats fed 4.8 % methionine is not clear. It could be the result of failure of transport of fats from the liver, or a local decrease in synthesis, or greatly increased catabolism. To extend the observations obtained in wivo, the effects of feeding excess methionine on cholesterol metabolism were investigated by in vitro techniques. Oxidation of Acetate-N?* and Incorporation into Cholesterol by Liver Slices from Methionine-Fed and Control Rats. Male Wistar rats fed excess methionine for 28 days and pair-fed control rats were used in the following experiment. At the end of the feeding period five animals in the methionine group and two controls were sacrificed by a blow on the head, the livers were removed, and 600 mg. of slices was prepared from each liver using a Stadie-Riggs slicer. The tissue was added to 125-ml. Erlenmeyer flasks containing a center well. Each flask contained 5 ml. of Krebs-Henseleit buffer (gassed with 95 % 02 , 5 % COZ), 1 ml. of acetate solution containing 0.5 PC. of acetate-2-C14, and a total of 5 mmoles of sodium acetate. The center well contained KOH absorbed on filter paper, and the gas phase was oxygen. Incubation with shaking was carried out for 3 hr. at 37”, after which the reactions were stopped by the addition of 0.5 ml. of 6 N HCl to the flasks, followed by shaking for another 30 min. to insure complete absorption of the CO2 . Aliquots of the evolved COz were removed for the determination of Cl4 by the method of Entenman et al. (9). The liver slices were filtered through glass wool, and the filtrate was extracted with petroleum ether. The extract and tissue fragments were combined and saponified, and cholesterol was isolated and plated as previously described. The results which are listed in Table III also give the values for two ad Zibitum (stock diet) fed controls for comparative purposes. The liver slices of the methioninefed animals showed a considerable depression in the oxidation of acetate when compared to either the pair-fed or ad l&turn controls, but the incorporation of acetate-2-Cl4 into hepatic cholesterol was greatly increased by methionine feeding. Thus these in vitro experiments confirm the data obtained in wivo which also indicated an increased incorporation of acetate-2-P into hepatic cholesterol of methionine-fed rats. In this same experiment two animals in each group were injected intraperitoneally with 1 ml. of saline containing 5 PC. of acetate-2-U4, and

398

ROTH

AND

TABLE Cl402 Production Cholesterol

MILSTEIN

III

from Acetate-2474 and Incorporation by Liver Slices from Methionine-Fed

into

Radioactivityrecovered/100 mg. of liver slices C’OZ Cholesterol c0un1s/mis. cowls/mitt.

Animaland diet” 4.8% DL-methionine Rat No. 1

of Acetate-%Cl4 and Control Rats

j- 12% casein

n

; 4 5 Pair-fed controls (12% casein) 3 2 Ad lib. controls (stock diet) 1 ” L

4058 4509 4818 5451 4837

4397 1923 2263 1666 4085

7393

567 508

7613 9272

903

8619

688

5 Diet was fed for a period of 28 days.

the animals were placed in a gas-metabolism chamber. Expired COz was collected hourly for 6 hr. Although a small increase (19 %) was observed in the respiratory C1*02 of the methionine-fed animals, further experiments are necessary to establish the significance of this increase. DISCUSSION

From the evidence presented in this and previous reports, it appears fairly clear that the feeding of excess methionine accelerates the loss of fat from the eviscerated carcass of the rat. Concurrently, there is a greatly increased incorporation of acetate-2-Cl4 into liver cholesterol, accompanied by hypercholesterolemia and increased total carcass cholesterol. Liver lipides appear to be slightly higher in the methioninefed animal, than in controls, while incorporation of acetate into adipose tissue fatty acids is reduced to very low levels by methionine feeding. The data in Table II indicate increased incorporation of acetate-2-Cl4 into hepatic fatty acids, just the reverse of what occurs with adipose tissue fatty acids, as illustrated in Fig. 1. These facts, taken in conjunction with the decreased C402 production from acetate-2-W by liver slices from methionine-fed rats, suggest that quite different mechanisms are operating in the two tissues. It is possible that in the liver there is decreased fat catabolism. This could be reconciled with the observed hypercholesterolemia and continued carcass fat loss if the

EXCESS METHIONINE

AND LIPIDE METABOLISM

399

catabolism of fats by adipose, and possibly other nonhepatic tissues, was greatly accelerated in the methionine-fed animals. This could account for continued fat loss and the production of large quantities of acetoacetate and acetate to be funneled into cholesterol production in the liver. Unfortunately, the experimental design has not tested this possibility but it would appear to be an important point. The low incorporation of acetate-2-Cl4 into adipose tissue fatty acids, while offering no proof for this explanation is at least consistent with it. It is of interest to mention that some of the effects in rats, brought about by feeding excess methionine are similar to those observed in diabetes. In this disease there is greatly increased catabolism of fat, accompanied by hypercholesterolemia. Furthermore, incorporation of acetate or glucose into fatty acids of adipose tissue is significantly reduced in the diabetic rat (11) , The similarities between methionine feeding and diabetes exist only in regard to certain aspects of lipide metabolism, however. Thus, preliminary tests have failed to indicate associated carbohydrate disturbances such as glycosuria, hyperglycemia, or pathology of the endocrine portion of the pancreas in animals fed excess methionine. In view of the hypercholesterolemia and increased carcass cholesterol content produced in rats by feeding excess methionine, it would be of interest to determine whether prolonged feeding of this amino acid would give rise to atherosclerosis in animals susceptible to this disease such as the guinea pig or rabbit. As studies of this nature might furnish a clue to the mechanism of the methionine-induced hypercholesterolemia of the rat, they are currently under investigation. ACKNOWLEDGMENT The authors wish to express their gratitude cal examination

of the pancreas

to Dr. J. H. Van Dyke of methionine-fed rats.

for histologi-

SUMMARY

Wistar rats fed a synthetic diet containing 4.8 % excess nn-methionine for 28 days showed increased serum and carcass cholesterol, and decreased total carcass lipide compared to pair-fed controls. The incorporation of acetate-2-Cl4 into hepatic cholesterol in tivo and in vitro was significantly greater in the methionine-fed animals, but incorporation of acetate-2-Cl4 into fatty acids of adipose tissue was much less than in controls. In methionine-fed animals, liver fatty acid was unchanged, and liver cholesterol increased in comparison to the levels observed in pair-

400

ROTH AND

MILSTEIN

fed controls. Methionine-fed and control rats produced respiratory CPOZ from injected labeled acetate at approximately the same rate; however, liver slices from animals fed excess amino acid showed a depression in the production of CY402 from acetate-2-Cl4 when compared to control slices. The results were discussed with reference to some similarities to the derangements in fat metabolism in the diabetic rat. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

BROWN, J. H., AND ALLISON, J. B., Proc. Sot. Exptl. Biol. Med. 69,196 (1948) ROTH, J. S., AND ALLISON, J. B., J. Biol. Chem. 188, 173 (1950). ROTH, J. S., AND ALLISON, J. B., PTOC.Sot. Exptl. Biol. Med. 70,327 (1949). HAVEN, F. L., BLOOR, W. R., AND RANDALL, C., Cancer Research 9,511 (1949). MACINTYRE, I., AND RALSTON, M., Biochem. J. 66, xliii (1954). YOUNQSBURO, G. E., AND YOUNOSBURO, M. V., J. Lab. Clin. Med. 16, 158 (1930). SURE, B., KIH, M. C., AND CHURCH, A., J. Biol. Chem. 103, 417 (1933). MASORO, E. J., CHAIHOFF, I. L., AND DAUBEN, W. G., J. Biol. Chem. 179,1117 (1949). ENTENMAN, C., LERNER, S. R., CHAIKOFF, I. L., AND DAUBEN, W. G., PTOC. Sot. Exptl. Biol. Med. 70, 364 (1949). ROTH, J. S., ALLISON, J. B., AND MILCH, L. J., J. Biol. Chem. 186, 113 (1950). HAUSBERQER, F. X., MILSTEIN, S. W., AND RUTMAN, R. J., J. Biol. Chem. 208, 431 (1954).