- Email: [email protected]
Fatty liver induced by injection of l -tryptophan
BIOCHIMICA
ET BIOPHYSICA
233
ACTA
BBA 55365
FATTY
LIVER
YUKIKO
HIRATA,
INDUCED
TAKASHI
BY
INJECTION
KAWACHI
AND
OF L-TRYPTOPHAN
TAKASHI
SUGIMURA
Biochemistry Division, National Cancer Center Research Institute, Tsukiji, Chtso-ku, Tokyo (Japalz) {Received
March moth, rg67)
SUMMARY
r_-Tryptophan caused the accumulation of neutral lipids in liver within 2.5 h after its intraperitoneal injection into rats. This accumulation of neutral lipids continued for about 24 h. Peripheral fatty liver was diagnosed histologically by Sudan III staining. The minimal effective dose was 0.5 mg/g of body weight. The level of cholesterol and phospholipids in liver did not alter, 3-Hydroxyanthranilic acid and r.-kynurenine were as effective as r.-tryptophan in inducing the accumuIation of neutral lipids, but other metabolites of tryptophan, kynurenic acid, anthr~i~c acid, quinolinic acid, nicotinic acid and nicotinamide did not produce the lipid accumulation. The administration of other amino acids, such as L-leucine, L-lysine, L-tyrosine, L-threonine and L-methionine, did not increase the amount of total lipids in liver. Simultaneous administration of ATP, ADP, AMP, adenosine or folic acid with L-tryptophan prevented the fatty liver. A marked decrease in the concentration of ATP in the liver was shown by the administration of L-tryptophan or L-kynurenine. These observations support the concept that L-tryptophan-induced fattyliveris due to the decreased level of ATP. A possible mechanism of ATP depression by administered L-tryptophan is discussed.
INTRODUCTION
The administration of L-tryptophan to the rat caused various biochemical changes in the liver, such as an induction of liver tryptophan pyrrolase (L-tryptophan : oxygen oxidoreductase, EC I.I~.I.Iz)~, depression of gluconeogenesis2 and others. During studies on the induction of liver tryptophan pyrrolase, an increment of fatty substances floating on the supernatant fraction after the centrifugation of liver homogenate was noticed with rats that had received L-tryptophan. The production of fatty Iiver by deficiency3 or imbalance4 of animo acids has been well recognized. Many other substances, ethionineb, erotic acid&, tetracycline’, aureomycinee, ethanols and CCIP1oplf can produce fatty liver. However, the production of fatty liver by a single interperitoneal injection of L-tr~toph~ has not been reported. The present studies showed that L-tryptophan rapidly induced a choline-resistant fatty liver in which the accuBiochim. Biophys.
Arta, 144 (1967)
Z&33--24X
Y. HIRATA,
234
T. KAWACHI,
T. SUGIMURA
mulated lipids were localized in the peripheral zones of the lobules and were all triglycerides. For a better understanding of the mechanism of the induction of the fatty liver by the administration of L-tryptophan, the fatty liver-inducing ability of various metabolites
of tryptophan
and the preventive
ability of adenine compounds
and lipo-
tropic factors will be described. METHODS
Male albino rats of the Wistar
strain
were maintained
on CLEA
CE-2
chow
pellets, and the animals weighing from 70-130 g were used. They were deprived of food overnight before treatment. Each chemical was injected intraperitoneally as a neutral aqueous solution.
The control animals received the same volume of distilled water. At
various intervals after the injection of chemicals, rats were decapitated and the liver was rapidly removed. A portion of liver was homogenized with 4 vol. of distilled water in a Waring blendor. Liver total lipids were extracted from the homogenate form-methanol (z : I), and were determined gravimetricallyl2. phosphorus,
neutral
lipids and total cholesterols
FISKE-SUBBAROW~~, by the modified method the method of HENLY’~, respectively. For the determination
of ATP
with IO vol. of chloroOn the extract, lipid
were determined
by the method
of
of HANDEL AND ZILVERSMIT~*, and by
concentration,
the liver was frozen as soon as
possible with light petroleum and solid CO, immediately after dissection, and was extracted with HClO,. The extract, neutralized with KOH, was subjected to the ATP assay according to the luciferin-luciferase means of the Farrand fluorometer. Fatty
method of STREHLER AND TOTTER’~ by acid composition was determined bygas-
liquid chromatography with a Shimazu GC-IB apparatus. Nitrogen was determined by the microKjeldahl-Nessler technique. Histological demonstration of fatty liver was carried out by Sudan III staining. ATPase in the microsomal fraction was assayed as follows. Microsomes prepared from I g of fresh liver were suspended in 0.25 M sucrose to a total of 4.0 ml. The reaction mixture contained 5 mM MgCl,, 130 mM choline chloride, 5 mM ATP-Tris salt, 0.2 M Tris buffer (pH 7.4) and 0.1 ml microsomal suspension in a total of 1.0 ml. This mixture was incubated at 37” for IO min, and the reaction was stopped by the addition of 1.0 ml of cold 10~)'~ thrichloroacetic acid. The trichloroacetic acid-soluble fraction was used for determination
of liberated
inorganic phosphate
by MARTIN AND DOTY’S”
method modified by TAKAHASHI~~. L-Tryptophan was purchased from Nippon Rikagaku Yakuhin Co. Ltd., Tokyo. ,4TP, ADP, AMP, adenosine, IMP and 3-hydroxyanthranilic acid were obtained from Sigma Chemical Co. U.S.A. L-Kynurenine sulfate was purchased from K and K Laboratories Inc., U.S.A. Anthranilic acid, kynurenic acid, picolinic acid, quinolinic acid, nicotinic acid, nicotinamide, inositol, vitamin Bg, folic acid and adenine sulfate were supplied from Tokyo Kasei Kogyo Co. Ltd., Tokyo. Tryptophan metabolites from Tokyo Kasei Kogyo Co. Ltd., were recrystallized before use.
Biochim.
Riophw.
Acta,
144
(1967)
233-241
TRYPTOPHAN
FATTY
LIVER
235
RESULTS
Effect of L-try$tophalz injection fihos$holiPids of rat liver
on the come&rations
of neutral lipids, cholesterols and
Table I shows the change in neutral lipids, cholesterols and phospholipids 4 h after the injection of 1.0 mg of L-tryptophan per g body weight. The level of neutral lipids increased, but the amount of total cholesterols and phospholipids did not change. This accumulation of neutral lipids was confirmed by histological examination. TABLE
I
NEUTRAL LIPIDS, CHOLESTEROLS
AND
PHOSPHOLIPIDS
IN
“TRYPTOPHAN
FATTY
LIVER”
Rats received an intraperitoneal injection of 1.0 mg of L-tryptaphan per g body weight, and were killed by decapitation 4 h later. Numbers of rats are shown in parentheses.
Control L-Tryptophan
(9) injected
(12)
Neutral lieids (mg/g of liver)
Total cholesterols (mgig 0-f liver)
12.2 + 1.2*
4.32 & o.35*
36.7 h 2.0~
4.20 *
35.0 f
20.0
+
1.3
0.13
Phospholipids (mg/g
of
liver)
1.6
* Standard error. TABLE
II
NEUTRAL
LIPIDS
IN
LIVER
AFTER
THE
INJECTION
OF L-TRYPTOPHAN
Rats received an intraperitoneal injection of 1.0 mg of L-tryptophan per g body weight, and were killed at the times indicated. Numbers of rats are shown in parentheses. Time
aftev injection
0 (3) 2.5 (2) 6.5 (4) 22
44
(3) (3)
(h)
Neutral 9.8, 15.5.
lipids 11.1,
(wag/g of 1iueF)
11.8
15.7
20.1,
16.1,
15.1,
‘4.3, 9.3,
14.5, 11.5,
12.2 14.5
11.2
Time course of the accumulation of the neutral lipids after the injection of various doses of L-tryptop han
Results on the accumulation of neutral lipids in liver after the administration of mg/g of body weight of r_-tryptophan are presented in Table II. The neutral lipid content showed a rapid increase, reaching its highest level within 2.5 h, and returned to the normal level 44 h after the administration. As shown in Table III, 0.5 mg of Ltryptophan per g body weight was most effective. I
Effect of other amino acids on the lipids in liver
Equimolar amounts of other amino acids were injected into rats intraperitoneally. r.-leucine, r_-threonine, L-tyrosine, L-lysine or L-methionine did not increase the total lipids of the liver. Among tested samples r,-tryptophan was the only amino acid that affected the lipid level of the liver. These results are summarized in Table IV. Fatty liver-inducing ability of tryptophan metabolites
various
To discover the mechanism metabolites of tryptophan
of the induction of the fatty liver by r.-tryptophan, were tested for their ability to induce fatty liver. Biochim.
Biophys.
Acta,
144 (1967)
233-241
Y. HIRATA, T. KAWACHI, T. SUGIMURA
236 TABLE
III
NEUTRALLIPIDS IN LIVER AFTER THE INJECTIoN OF L-TRYPTOPHAN Rats received an intraperitoneal injection of the indicated dose of L-tryptophan, and were killed 4.5 or 6 h later. Numbers of rats are shown in parentheses. The results are averages of two experiments. Half the rats were killed 4.5 h, and the other half 6 h, after the injection. i”l’eutval licids (nzgig 0.f liver)
Injected dose of r-tvyptophan (mglg of body weight)
-& 1.0* 12.6 - 1.6
(6)
r4.r
::: I.0
I:; (6)
2.0
(6)
2Z.I 15.2 _I_ ! I.0 1.8 19.1 i_ 1.8 ‘7.4 1. I.2
0
0.05 (5)
* Standard error.
TABLE
IV
EFFECT OF VARIOUS AMINO
ACIDS
ON
THE
LIPIDS
IN
LIVER
Rats were inject,ed intraperitoneally with 4.9 qmoles of each amino acid per g body weight, and were killed 6 h later. Numbers of rats are shown in parentheses. A&m
aced injected
None L-Tryptophan L-Leucine L-Threonine L-Lysine L-Methionine L-Tyrosine
(7) (9) (3) (3)
3.53 -’ 0.16 4.25 $ o. I I 3.34 3.59
\:I :I;‘, (3) 2.97
Total lipids (mg/N mg 01 liver) 2.g2-4.1g* 3.7g-4.8o* 2.78, 3.54, 3.32, 3.69, 3.52, 4.01, 3.89, 3.79. 3.29. 2.9%
3.70 3.75 3.62 3.82 2.64
* These represent the minimal and the maximal values.
The results are shown in Table V. 3-Hydroxyanthranilic acid and L-kynurenine proved to be as effective as L-tryptophan in increasing neutral lipids of the liver. These accumulations of neutral lipids were ascertained also by histological methods using Sudan III. Anthranilic acid, kynurenic acid, picolinic acid, quinolinic acid, nicotinic acid and nicotinamide did not cause an accumulation of neutral lipids. Fatty acid composition of the neutral lipids in the fatty liver The fatty acid composition of the neutral lipids that were increased by the administration of L-tryptophan or 3-hydroxyanthranilic acid was analyzed by gas-liquid chromatography. As shown in Table VI, there were slight decreases in saturated fatty acids and slight increases in unsaturated fatty acids in percentage of total fatty acids in the livers of the rats treated with L-tryptophan or 3-hydroxyanthranilic acid as compared with those in the livers of control rats. Prevention of the fatty liver To study the mode of action of L-tryptophan on the lipid metabolism in liver, the prevention of this fatty liver was studied. Lipotropic factors such as choline, methionine, inositol or folic acid, and other substances related to tryptophan metabolism such as vitamin B, or ATP, were injected intraperitoneally with L-tryptophan. The Biochim. Biophys. Acta. 144 (1967) 233-241
TRYPTOPHAN TABLE FATTY
FATTY
237
LIVER
V LIVER-INDUCING
ABILITY
OF VARIOUS
TRYPTOPHAN
METABOLITES
pmoles (0.5 mg as tryptophan) of various tryptophan metabolites per g of body weight were injected intraperitoneally. ‘The rats were killed 3 h after the injection, and the neutral lipids were measured. Numbers of rats are shown in parentheses. 2.45
None L-Tryptophan L-Kynurenine Anthranilic acid Kynurenic acid 3-Hydroxyanthranilic Picolinic acid Quinolinic acid Nicotinic acid Nicotinamide
9.3 * 0.6*
12)
12) 9)
17.0 &
1.4
IT.1
2.1
<
P < P <
0.001
0.00.5
0.005
<
P <
0.01
0.01
8.1 * 0.7 8.9 + 0.6
6)
acid
+
6) 9)
19.9
*
2.9
9.3 + I.5 TO.0 & 1.1 6.2 & 0.7 7.6 -_t 1.0
6)
15) 6)
6)
* Standard error.
TABLE FATTY
VI ACID
COMPOSITION
OF THE
NEUTRAL
LIPIDS
OF “TRYPTOPHAN
FATTY
LIVER”
2.45 @moles of L-tryptophan or 3-hydroxyanthranilic acid per g of body weight were injected. The rats were killed 3 h after the injection, and neutral lipids were extracted. Methyl esters of fatty acids from neutral lipids were analyzed by gas-liquid chromatography on a Shimazu GC-IB apparatus. A column of IO:/, polydiethyleneglycol succinate on Diasolid S was used at 195’. N, was the carrier gas. Each fatty acid is represented as a percentage of the total. Substance injected
Fatty acid Cl,
None r,-Tryptophan 3-Hydroxyanthranilic
acid
:0
4.0 2.9 3.9
__-
CM : 0
Cl, : 1
Cl, : 0
c,* : 1
Cl, : 2-
27.4 26.1 24.8
9.2 8.7 8.8
7.0 4.3 5.4
25.8 30.3 2j.6
26.7 27.8 30.4
results are presented inTable VII. Simultaneous injection of folic acid or ATPprevented the accumulation of neutral lipids in the liver that would have been caused by L-tryptophan administration alone. This prevention was histologically confirmed by Sudan III staining. The administration of choline, methionine or inositol failed to prevent the fatty liver. Vitamin B,, a cofactor of many enzymes concerned in the metabolism of L-tryptophan, had no effect. Adenine compounds other than ATP, such as adenine, adenosine, AMP or ADP and IMP, were examined for their ability to prevent fatty liver. Among them, ADP, AMP and adenosine were as effective as ATP. Adenine, reported to prevent the fatty liver induced by a diet containing erotic acidaB, showed no significant decrease in the lipid level. The slight preventive effect of fatty liver production by the injection of IMP was not statistically significant. Concentration of A
TP in fatty
liver
The levels of ATP in the liver after the injection of L-tryptophan or L-kynurenine are given in Table VIII. The ATP levels in the liver decreased to about 60% of the normal level while the lipid content increased to ISO~/~ of that after the administration of L-tryptophan or r.-kynurenine. Biochim. Biophys. Acta, 144 (1967)
233-241
Y. HIRATA, T. KAWACHI, T. SUGIMURA
238 TABLE
VII
PREVENTION
OF
“TRYPTOPHAN
FATTY
LIVER”
BY
VARIOUS
SUBSTANCES
(2.45 pmoles) of L-tryptophan and 2.45 pmoles of substances indicated per g of body weight were simultaneously injected into rats. The rats were killed 3 h after the injection. Numbers of rats are shown in parentheses.
0.5
mg
Sub&we
Neutral lipids (mg/g of liver)
znjected
None L-Tryptophan L-Tryptophan + choline L-Tryptophan+ L-methionine L-Tryptophan-tinositol L-Tryptophan + folic acid L-Tryptophan+vitamin I3, L-Tryptophan + ATP *
(12) (12)
( 6) ( 6)
9.3 z 0.6~ 17.0 *
1.4
20.1 * 2.5 ‘4.3 z 0.6
‘5.5 2: 1.5 ; 0”; ( 6) ( 6)
9.7 & 0.9 14.3 f 1.0
q.8 .k 0.g
0.1 < P < 0.2 0.2 < P < 0.3 O.OOI < P < 0.005 0.05 < P < 0.1 0.001
<
P
<
0.005
Standard error.
TABLE ATP
LEVEL
VII1 IN
LIVER
AFTER
THE
INJECTION
OF
L-TRYPTOPHAN
OR
L-KYNURENINE
2.45 /Lmoles of L-tryptophan or L-kynurenine per g body weight were intraperitoncally injected into rats. The rats were killed 3 h after the injection, and the concentrations of ATP in liver were measured according to the luciferin-luciferase method. Numbers of rats are given in parentheses. Substance injected
ATP
_~ ~____ None L-Tryptophan L-Kynureninc
(mpmoleslg
of liver)
570 :t 3S* 372 i 44 344 17 30
i:; (9)
_
* Standard error. A TPase activity in the fatty liver The marked decrease in the level of ATP in the liver might be due to the enhancement of ATPase activity in the liver. ATPase activity in the microsomal fraction of the fatty liver was therefore measured. Table IX shows that there was no difference in ATPase activity between the livers of control rats and L-tryptophan-administered rats. TABLE
IX
MICROSOMAL
ATPaX
ACTIVITY
IN
LIVER
AFTER
THE
ADMINISTRATION
OF
L-TRYPTOPHAN
A portion of the same liver that was used in the previous experiment (Table VIII), was assayed for ATPase activity in the microsomal fraction. Numbers of rats are given in parentheses. Substance injected
ATPase activity (,uatom P released per I0 min pev g of- liver)
None L-Tryptophan
37.9 4 3.79* 36.8 & 2.08
(5) (5)
* Standard error. DISCUSSION
Fatty liver can be induced in the rat by several agents. In fatty liver, the accumulating lipids are usually predominantly triglycerides. Abnormal accumulation of triglycerides in the liver is the result of an imbalance between the rate of synthesis and B&him.
Biophys. Acta,
144
(1967) 233-24’
TRYPTOPHAN
FATTY LIVER
239
the rate of utilization of hepatic triglycerides. Most factors that can produce fatty liver, for example administration of CC1410~19-26, ethioninea7-29, phosphorus or puromycinso, or with erotic acid36936, feeding of a choline-deficient diet31-34 or of a diet supplemented are known to block the secretion of hepatic triglycerides into the plasma. It is known that a major part of the hepatic triglycerides in the rat is secreted into the plasma as a moiety of plasma lipoprotein 37. It is suggested that the above agents disturb the metabolism of lipoprotein. In the fatty liver induced by CC1438-40,ethionine28y41p42, is an inhibition puromycin30 or phosphorus23, one of the basic metabolic disturbances of protein synthesis. It has been reported that administration of ethionine*“, CC1444*45, phosphorus**, ethanol45 or azaserine45, or feeding of a diet deficient in choline**, which produces fatty liver, leads to a rapid decrease in the concentration of ATP in the liver. The administration of ATP completely prevents the fatty liver induction by ethioninez*, CC1445, ethanol45 or azaserine45. The fatty liver induction by a diet containing erotic acid is also prevented by supplementation of the diet with adenine*8. In this paper we have shown that administration of L-tryptophan or L-kynurenine caused a marked decrease in the level of ATPin the liver and that the fatty liver induction by L-tryptophan was prevented by the administration of ATP, ADP, AMP or adenosine. Therefore, in tryptophan fatty liver, as in the majority of other types of fatty liver, it is suggested that a marked fall in the level of ATP depresses the protein synthesis leading to a failure of the liver to release triglycerides into the plasma as lipoproteins. It is also possible that a significant decrease in the cellular ATP level might disturb the completion of lipoprotein formation or alter the state of cellular membranes as to interfere with the transfer of preformed lipoprotein from the liver to the blood. To explain the striking decrease in the level of ATP in the liver, the enhancement of ATPase activity in liver might be considered. But we found no difference in hepatic microsomal ATPase activity between control rats and tryptophan-administrated rats. It has also been reported that ATPase activity does not change after the adminstration of Ccl, to induce fatty liver18. Recently QUAGLIARIELLO et aZ.47p48reported that, of all the tryptophan metabolites tested, only 3-hydroxyanthranilic acid uncoupled oxidative phosphorylation in vitro at the level of the first respiratory chain phosphorylation. It also inhibited the oxygen uptake of rat liver mitochondria respiring with NAD-dependent substrates. It is known that many substances that inhibit oxidative phosphorylation produce fatty infiltration of liver when administrated intraperitoneally to rats4a. 3,5-Dinitro-o-cresol, which inhibits oxidative phosporylation, depresses the concentration of ATP in many organsso. RAY, FOSTER AND LARDY~ found that administration of t-tryptophan to rats caused the rapid accumulation of citrate, malate, pyruvate, lactate and oxaloacetate to 5-35 times the normal concentration. They suggested that the administered tryptophan inhibited gluconeogenesis by blocking the conversion of oxaloacetate to phosphoenol pyruvate. Nevertheless, the level of phosphoenol pyruvate carboxykinase, when assayed in vitro, was enhanced. It seems reasonable to suppose that these effects of L-tryptophan may result from the inhibition of oxidation of a-oxoglutarate by 3-hydroxyanthranilic acid. Data presented in this paper, together with the reports from other investigators, suggest that the actual substance that produces the fatty liver is not L-tryptophan but may be its metabolite, 3-hydroxyanthranilic acid. The mechanism of depression of the ATPlevelintheliverafterasingleinjectionof~-tryptophanstillremainstobeelucidated. Biochinz.
Bio$hys.
Ada,
I++ (1967)
233-241
240
Y.
HIRATA, T. KAWACHI, T. SUGIMURA
Another factor in the ATP protective effect has been reported. HYAMS AND ISSELBACHER*~showed that Ccl,- or ethanol-treated animals injected with ATP become hypothermic, and they suggested that the hypothermia may be a factor in the protection. The preventive effect of adenine nucleotides on the tryptophan fatty liver may be due not only to stimulation of oxidative phosphorylation but to some pharmacological activity of tryptophan, or tryptophan in combination with adenine nucleotide. The possible role of ATP in relieving peripheral stress, increasing the transport of lipid, or elevating lipid synthesis de nova in the liver, remains to be clarified. Assays of ADP, AMP, adenosine and adenine in this fatty liver, and the effects of administered adenine compounds upon them, are under investigation. As to the effect of folic acid in preventing fatty liver, the dose of folic acid injected was well above the physiological level. As for the fatty acid composition of neutral lipids which accumulate in liver after administration of tryptophan, there was no striking change compared with that of the control. But a slight decrease in the percentage of saturated fatty acids and a slight increase in the percentage of unsaturated fatty acids suggest a metabolic disturbance of the synthesis of lipoprotein or its utilization by tryptophan. ACKNOWLEDGEMENTS We wish to thank Dr. T. BABA AND Mr. AOKI of the Pathology Division of this Institute for their help in histological diagnosis and Mrs. M. NAKAYASU for her help in the assay of ATPase activity. A part of this work was reported at the 38th General Meeting of The Japanese Biochemical Society at Fukuoka on October r7th, 1965. This work has been supported partly by a grant from the Ministry of Education to the research group on “Host Conditions and Growth of Cancer” headed by Dr. YUICHI YAMAMURA(No. 956107). REFERENCES I W. E. KNOX AND A. H. MEHLER, Science, 113 (1951) 237. 2 P. D. RAY, D. 0. FOSTER AND H. A. LARDY, J. Biol. Chem., 241 (1966) 3904. 3 A. E. HARPER, J. N&r., 50 (1953) 383; 56 (1955) 187. 4 A. E. HARPER, Am. J. Clin. Nutr., 6 (1958) 242. 5 E. FARBER, M. V. SIMPSON AND H. TARVER, J. Biol. Chem., 182 (1950) 91. 6 S. B. STANDERFER AND P. HANDLER, Proc. Sot. Exptl. Biol. Med., go (1955) 270. 7 G. Y. KIVMAN AND A. M. KHARITONOVA, Antibiotiki, 2 (1957) 49. 8 G. ZBINDEN AND A. STUDER, Schweiz. 2. Allgem. Pathol. Bakteriol., 20 (1957) IO. 9 C. T. ASHWORTH, Proc. Sot. Exptl. Biol. Med., 66 (1947) 382. IO A. S. AIYAR, P. FATTERPAKER AND A. SREENIVASAN, Biochem. J.. go (1964) 558. II M. C. SCHOTZ AND R. 0. RECKNAGEL, Biochim. Biophys. Acta, 41 (1960) 151. 12 J. FOLCH, M. LEES AND G. H. SLOANE-STANLEY, J. Biol. Chem., 226 (1957) 497. 13 C. H. FISKE AND Y. SUBBAROW, J. Biol. Chem., 66 (1925) 375. 14 E. V. HANDEL AND D. B. ZILVERSMIT, J. Lab. Clin. Med., 50 (1957) 152. 15 Z. HENLY, Am. J. Clin. Path., 27 (1957) 583. 16 B. L. STREHLER AND J. R. TOTTER, in D. GLICK, Methods of Biochemical Analysis, Vol .I, Interscience, New York, 195.4, I). 341. 17 J. B. MARTIN AND D. M. DOTY, Anal. Chem., PI (1949) 965. 18 T. TAKAHASHI, Seikagaku, 26 (1955) 690. 19 R. 0. RECKNAGEL AND B. LOMBARDI, J. Biol. Chem., 236 (1961) 564. 20 E. A. SMMUCKLER,0. A. ISERI AND E. P. BENDITT, Biochem. Biophys. Res. Commun., 5 (1961) 270. 21 M. HEIMBERG, I. WEINSTEIN, G. DISHMAPI’ AND A. DUNKERLEY, J. Biol. Chem., 237 (1962) 362322 H. M. MALING, A. FRANK AND M. G. HORNING, Biochim. Biophys. Acta, 64 (1962) 540.
Biochim. Biophys. Acta, 144 (1967) 233-241
TRYPTOPHAN
FATTY
LIVER
241
23 A. SEAKINS AND D. S. ROBINSON,Biochem. J., 86 (1963) 401. 24 M. POGGI AND R. PAOLETTX, Biociem. Pharmacol., 13 (1964) 949, 25 M. C. SCHOTZ,N. BAKER AND M. N.C~~v~z,J.Lipa'dHes., 5(x964)569.
26 B. L~MBARDI AND G. UB~zro,J.Li$idi&s.,6 (1965)498. 27 T. GI.IVECRONA, Acta P&&t. Seanad., 55 jIg62)2gI. 28 E. FARBER, B. LOMBARDI AND A. E. CASTILLO, Lab. Invest., IZ (x963)873. 2g G. UBAZI~ AND B. LOMBARDI,Lab. Invest., 14 (1965) 7x1. 30 D. S. ROBINSON AND A. SEAKINS,B&him. Rio$hys. Acta, 62 (xq62)163. 31 B. LOMBARDI AND R. 0. RECKNAGEL, Am.]. Pathal.,40(xg62) 571. 32 R. 0. RECKNAGEL AND A. K. GHOSHAL, ~~~~~a~~~~ BOG., 23 (1964) 243. 33 j.C. FORIIES, O.~.PETTERSONANDR.A.RT;T)O~PH,P~~~.Soc.~%~~~.Binl.NCed.,~rS frg65)59. 34 Il. S. i’d. HAINES AND S.MOOKERJEA, Canad. J. Biockem. Physiot., 43 (1965) 507. 35 H. G. WINDYUELLER, J,Bdol.Chsm, 239 (1964) 530. 36 P. S. ROHEINI, S. SWITZER,A. GIRARD AND H. A. EDER, Lab. Invest., 15 (rg66) 21. 3~ N. BAKER AND M. C. Scmrz, J. Lipid Res., 5 (1964) 188. 38 E. A. SN~UCKLER, 0. A. 1sg1+I AND E. P. BENDITT,.J.E~pt~,~e~, 116(rg6z)55. 39 E. A.SMUCKLER AND E, P. BENDITT,Science, 14o(Ig63)308. 40 E. A. SMLXKLER AND E. P. BENDITT,Biochenzistry, 4(rg65) 67x. 41 E. FARBER,K. H. SHIILL, S. VILLA-~R~VI~O,~.L~~EAR~IA~D~.THO~~S,~~~~~~~, 203(rg64) 34. 42 D. S. Rosmso~ ANYDP. M. HARRIS,BiocJzm. J., So (1g61f 361. 43 xf. H. SHULL,J.Biol.Chem., 237 (1962)1734. 44 M. U. DIANEANI,Biochem. ,I., 65 (1957) 116. 45 D. E. HYAMS AND K. J.XSSELBACHER, Nature, 204 (x964) 1196. 46 3%. E. HANDSCHUMACHER, W. A. CREASEY, J. J. JAFFE,C. A. PASTERNAK AND L. HANKIN,
Pmt. N&k. Acid SGi. U.S., $5 (rg60) 178. Q~IAGLIARIELLo, S. PAPA AND C. SACCONE,Baoclae~n. J.,84 (rg62)66P. 48 E. QUAGLIARIELLO, S. Pan, C. SACCONE ANU A. ALIFANS,B&hem.. J., gr (1964) 137.
47 E.
49 M. U, DIANZANI AND S. SCVRO, Biockent. J., 62 (x956) 205. 50 V. H. PARKER, Biochern. Jha 57 (1954) 381.
Biochinz. Biofihyys. Acta, 144 (1967) 233-241
ET BIOPHYSICA
233
ACTA
BBA 55365
FATTY
LIVER
YUKIKO
HIRATA,
INDUCED
TAKASHI
BY
INJECTION
KAWACHI
AND
OF L-TRYPTOPHAN
TAKASHI
SUGIMURA
Biochemistry Division, National Cancer Center Research Institute, Tsukiji, Chtso-ku, Tokyo (Japalz) {Received
March moth, rg67)
SUMMARY
r_-Tryptophan caused the accumulation of neutral lipids in liver within 2.5 h after its intraperitoneal injection into rats. This accumulation of neutral lipids continued for about 24 h. Peripheral fatty liver was diagnosed histologically by Sudan III staining. The minimal effective dose was 0.5 mg/g of body weight. The level of cholesterol and phospholipids in liver did not alter, 3-Hydroxyanthranilic acid and r.-kynurenine were as effective as r.-tryptophan in inducing the accumuIation of neutral lipids, but other metabolites of tryptophan, kynurenic acid, anthr~i~c acid, quinolinic acid, nicotinic acid and nicotinamide did not produce the lipid accumulation. The administration of other amino acids, such as L-leucine, L-lysine, L-tyrosine, L-threonine and L-methionine, did not increase the amount of total lipids in liver. Simultaneous administration of ATP, ADP, AMP, adenosine or folic acid with L-tryptophan prevented the fatty liver. A marked decrease in the concentration of ATP in the liver was shown by the administration of L-tryptophan or L-kynurenine. These observations support the concept that L-tryptophan-induced fattyliveris due to the decreased level of ATP. A possible mechanism of ATP depression by administered L-tryptophan is discussed.
INTRODUCTION
The administration of L-tryptophan to the rat caused various biochemical changes in the liver, such as an induction of liver tryptophan pyrrolase (L-tryptophan : oxygen oxidoreductase, EC I.I~.I.Iz)~, depression of gluconeogenesis2 and others. During studies on the induction of liver tryptophan pyrrolase, an increment of fatty substances floating on the supernatant fraction after the centrifugation of liver homogenate was noticed with rats that had received L-tryptophan. The production of fatty Iiver by deficiency3 or imbalance4 of animo acids has been well recognized. Many other substances, ethionineb, erotic acid&, tetracycline’, aureomycinee, ethanols and CCIP1oplf can produce fatty liver. However, the production of fatty liver by a single interperitoneal injection of L-tr~toph~ has not been reported. The present studies showed that L-tryptophan rapidly induced a choline-resistant fatty liver in which the accuBiochim. Biophys.
Arta, 144 (1967)
Z&33--24X
Y. HIRATA,
234
T. KAWACHI,
T. SUGIMURA
mulated lipids were localized in the peripheral zones of the lobules and were all triglycerides. For a better understanding of the mechanism of the induction of the fatty liver by the administration of L-tryptophan, the fatty liver-inducing ability of various metabolites
of tryptophan
and the preventive
ability of adenine compounds
and lipo-
tropic factors will be described. METHODS
Male albino rats of the Wistar
strain
were maintained
on CLEA
CE-2
chow
pellets, and the animals weighing from 70-130 g were used. They were deprived of food overnight before treatment. Each chemical was injected intraperitoneally as a neutral aqueous solution.
The control animals received the same volume of distilled water. At
various intervals after the injection of chemicals, rats were decapitated and the liver was rapidly removed. A portion of liver was homogenized with 4 vol. of distilled water in a Waring blendor. Liver total lipids were extracted from the homogenate form-methanol (z : I), and were determined gravimetricallyl2. phosphorus,
neutral
lipids and total cholesterols
FISKE-SUBBAROW~~, by the modified method the method of HENLY’~, respectively. For the determination
of ATP
with IO vol. of chloroOn the extract, lipid
were determined
by the method
of
of HANDEL AND ZILVERSMIT~*, and by
concentration,
the liver was frozen as soon as
possible with light petroleum and solid CO, immediately after dissection, and was extracted with HClO,. The extract, neutralized with KOH, was subjected to the ATP assay according to the luciferin-luciferase means of the Farrand fluorometer. Fatty
method of STREHLER AND TOTTER’~ by acid composition was determined bygas-
liquid chromatography with a Shimazu GC-IB apparatus. Nitrogen was determined by the microKjeldahl-Nessler technique. Histological demonstration of fatty liver was carried out by Sudan III staining. ATPase in the microsomal fraction was assayed as follows. Microsomes prepared from I g of fresh liver were suspended in 0.25 M sucrose to a total of 4.0 ml. The reaction mixture contained 5 mM MgCl,, 130 mM choline chloride, 5 mM ATP-Tris salt, 0.2 M Tris buffer (pH 7.4) and 0.1 ml microsomal suspension in a total of 1.0 ml. This mixture was incubated at 37” for IO min, and the reaction was stopped by the addition of 1.0 ml of cold 10~)'~ thrichloroacetic acid. The trichloroacetic acid-soluble fraction was used for determination
of liberated
inorganic phosphate
by MARTIN AND DOTY’S”
method modified by TAKAHASHI~~. L-Tryptophan was purchased from Nippon Rikagaku Yakuhin Co. Ltd., Tokyo. ,4TP, ADP, AMP, adenosine, IMP and 3-hydroxyanthranilic acid were obtained from Sigma Chemical Co. U.S.A. L-Kynurenine sulfate was purchased from K and K Laboratories Inc., U.S.A. Anthranilic acid, kynurenic acid, picolinic acid, quinolinic acid, nicotinic acid, nicotinamide, inositol, vitamin Bg, folic acid and adenine sulfate were supplied from Tokyo Kasei Kogyo Co. Ltd., Tokyo. Tryptophan metabolites from Tokyo Kasei Kogyo Co. Ltd., were recrystallized before use.
Biochim.
Riophw.
Acta,
144
(1967)
233-241
TRYPTOPHAN
FATTY
LIVER
235
RESULTS
Effect of L-try$tophalz injection fihos$holiPids of rat liver
on the come&rations
of neutral lipids, cholesterols and
Table I shows the change in neutral lipids, cholesterols and phospholipids 4 h after the injection of 1.0 mg of L-tryptophan per g body weight. The level of neutral lipids increased, but the amount of total cholesterols and phospholipids did not change. This accumulation of neutral lipids was confirmed by histological examination. TABLE
I
NEUTRAL LIPIDS, CHOLESTEROLS
AND
PHOSPHOLIPIDS
IN
“TRYPTOPHAN
FATTY
LIVER”
Rats received an intraperitoneal injection of 1.0 mg of L-tryptaphan per g body weight, and were killed by decapitation 4 h later. Numbers of rats are shown in parentheses.
Control L-Tryptophan
(9) injected
(12)
Neutral lieids (mg/g of liver)
Total cholesterols (mgig 0-f liver)
12.2 + 1.2*
4.32 & o.35*
36.7 h 2.0~
4.20 *
35.0 f
20.0
+
1.3
0.13
Phospholipids (mg/g
of
liver)
1.6
* Standard error. TABLE
II
NEUTRAL
LIPIDS
IN
LIVER
AFTER
THE
INJECTION
OF L-TRYPTOPHAN
Rats received an intraperitoneal injection of 1.0 mg of L-tryptophan per g body weight, and were killed at the times indicated. Numbers of rats are shown in parentheses. Time
aftev injection
0 (3) 2.5 (2) 6.5 (4) 22
44
(3) (3)
(h)
Neutral 9.8, 15.5.
lipids 11.1,
(wag/g of 1iueF)
11.8
15.7
20.1,
16.1,
15.1,
‘4.3, 9.3,
14.5, 11.5,
12.2 14.5
11.2
Time course of the accumulation of the neutral lipids after the injection of various doses of L-tryptop han
Results on the accumulation of neutral lipids in liver after the administration of mg/g of body weight of r_-tryptophan are presented in Table II. The neutral lipid content showed a rapid increase, reaching its highest level within 2.5 h, and returned to the normal level 44 h after the administration. As shown in Table III, 0.5 mg of Ltryptophan per g body weight was most effective. I
Effect of other amino acids on the lipids in liver
Equimolar amounts of other amino acids were injected into rats intraperitoneally. r.-leucine, r_-threonine, L-tyrosine, L-lysine or L-methionine did not increase the total lipids of the liver. Among tested samples r,-tryptophan was the only amino acid that affected the lipid level of the liver. These results are summarized in Table IV. Fatty liver-inducing ability of tryptophan metabolites
various
To discover the mechanism metabolites of tryptophan
of the induction of the fatty liver by r.-tryptophan, were tested for their ability to induce fatty liver. Biochim.
Biophys.
Acta,
144 (1967)
233-241
Y. HIRATA, T. KAWACHI, T. SUGIMURA
236 TABLE
III
NEUTRALLIPIDS IN LIVER AFTER THE INJECTIoN OF L-TRYPTOPHAN Rats received an intraperitoneal injection of the indicated dose of L-tryptophan, and were killed 4.5 or 6 h later. Numbers of rats are shown in parentheses. The results are averages of two experiments. Half the rats were killed 4.5 h, and the other half 6 h, after the injection. i”l’eutval licids (nzgig 0.f liver)
Injected dose of r-tvyptophan (mglg of body weight)
-& 1.0* 12.6 - 1.6
(6)
r4.r
::: I.0
I:; (6)
2.0
(6)
2Z.I 15.2 _I_ ! I.0 1.8 19.1 i_ 1.8 ‘7.4 1. I.2
0
0.05 (5)
* Standard error.
TABLE
IV
EFFECT OF VARIOUS AMINO
ACIDS
ON
THE
LIPIDS
IN
LIVER
Rats were inject,ed intraperitoneally with 4.9 qmoles of each amino acid per g body weight, and were killed 6 h later. Numbers of rats are shown in parentheses. A&m
aced injected
None L-Tryptophan L-Leucine L-Threonine L-Lysine L-Methionine L-Tyrosine
(7) (9) (3) (3)
3.53 -’ 0.16 4.25 $ o. I I 3.34 3.59
\:I :I;‘, (3) 2.97
Total lipids (mg/N mg 01 liver) 2.g2-4.1g* 3.7g-4.8o* 2.78, 3.54, 3.32, 3.69, 3.52, 4.01, 3.89, 3.79. 3.29. 2.9%
3.70 3.75 3.62 3.82 2.64
* These represent the minimal and the maximal values.
The results are shown in Table V. 3-Hydroxyanthranilic acid and L-kynurenine proved to be as effective as L-tryptophan in increasing neutral lipids of the liver. These accumulations of neutral lipids were ascertained also by histological methods using Sudan III. Anthranilic acid, kynurenic acid, picolinic acid, quinolinic acid, nicotinic acid and nicotinamide did not cause an accumulation of neutral lipids. Fatty acid composition of the neutral lipids in the fatty liver The fatty acid composition of the neutral lipids that were increased by the administration of L-tryptophan or 3-hydroxyanthranilic acid was analyzed by gas-liquid chromatography. As shown in Table VI, there were slight decreases in saturated fatty acids and slight increases in unsaturated fatty acids in percentage of total fatty acids in the livers of the rats treated with L-tryptophan or 3-hydroxyanthranilic acid as compared with those in the livers of control rats. Prevention of the fatty liver To study the mode of action of L-tryptophan on the lipid metabolism in liver, the prevention of this fatty liver was studied. Lipotropic factors such as choline, methionine, inositol or folic acid, and other substances related to tryptophan metabolism such as vitamin B, or ATP, were injected intraperitoneally with L-tryptophan. The Biochim. Biophys. Acta. 144 (1967) 233-241
TRYPTOPHAN TABLE FATTY
FATTY
237
LIVER
V LIVER-INDUCING
ABILITY
OF VARIOUS
TRYPTOPHAN
METABOLITES
pmoles (0.5 mg as tryptophan) of various tryptophan metabolites per g of body weight were injected intraperitoneally. ‘The rats were killed 3 h after the injection, and the neutral lipids were measured. Numbers of rats are shown in parentheses. 2.45
None L-Tryptophan L-Kynurenine Anthranilic acid Kynurenic acid 3-Hydroxyanthranilic Picolinic acid Quinolinic acid Nicotinic acid Nicotinamide
9.3 * 0.6*
12)
12) 9)
17.0 &
1.4
IT.1
2.1
<
P < P <
0.001
0.00.5
0.005
<
P <
0.01
0.01
8.1 * 0.7 8.9 + 0.6
6)
acid
+
6) 9)
19.9
*
2.9
9.3 + I.5 TO.0 & 1.1 6.2 & 0.7 7.6 -_t 1.0
6)
15) 6)
6)
* Standard error.
TABLE FATTY
VI ACID
COMPOSITION
OF THE
NEUTRAL
LIPIDS
OF “TRYPTOPHAN
FATTY
LIVER”
2.45 @moles of L-tryptophan or 3-hydroxyanthranilic acid per g of body weight were injected. The rats were killed 3 h after the injection, and neutral lipids were extracted. Methyl esters of fatty acids from neutral lipids were analyzed by gas-liquid chromatography on a Shimazu GC-IB apparatus. A column of IO:/, polydiethyleneglycol succinate on Diasolid S was used at 195’. N, was the carrier gas. Each fatty acid is represented as a percentage of the total. Substance injected
Fatty acid Cl,
None r,-Tryptophan 3-Hydroxyanthranilic
acid
:0
4.0 2.9 3.9
__-
CM : 0
Cl, : 1
Cl, : 0
c,* : 1
Cl, : 2-
27.4 26.1 24.8
9.2 8.7 8.8
7.0 4.3 5.4
25.8 30.3 2j.6
26.7 27.8 30.4
results are presented inTable VII. Simultaneous injection of folic acid or ATPprevented the accumulation of neutral lipids in the liver that would have been caused by L-tryptophan administration alone. This prevention was histologically confirmed by Sudan III staining. The administration of choline, methionine or inositol failed to prevent the fatty liver. Vitamin B,, a cofactor of many enzymes concerned in the metabolism of L-tryptophan, had no effect. Adenine compounds other than ATP, such as adenine, adenosine, AMP or ADP and IMP, were examined for their ability to prevent fatty liver. Among them, ADP, AMP and adenosine were as effective as ATP. Adenine, reported to prevent the fatty liver induced by a diet containing erotic acidaB, showed no significant decrease in the lipid level. The slight preventive effect of fatty liver production by the injection of IMP was not statistically significant. Concentration of A
TP in fatty
liver
The levels of ATP in the liver after the injection of L-tryptophan or L-kynurenine are given in Table VIII. The ATP levels in the liver decreased to about 60% of the normal level while the lipid content increased to ISO~/~ of that after the administration of L-tryptophan or r.-kynurenine. Biochim. Biophys. Acta, 144 (1967)
233-241
Y. HIRATA, T. KAWACHI, T. SUGIMURA
238 TABLE
VII
PREVENTION
OF
“TRYPTOPHAN
FATTY
LIVER”
BY
VARIOUS
SUBSTANCES
(2.45 pmoles) of L-tryptophan and 2.45 pmoles of substances indicated per g of body weight were simultaneously injected into rats. The rats were killed 3 h after the injection. Numbers of rats are shown in parentheses.
0.5
mg
Sub&we
Neutral lipids (mg/g of liver)
znjected
None L-Tryptophan L-Tryptophan + choline L-Tryptophan+ L-methionine L-Tryptophan-tinositol L-Tryptophan + folic acid L-Tryptophan+vitamin I3, L-Tryptophan + ATP *
(12) (12)
( 6) ( 6)
9.3 z 0.6~ 17.0 *
1.4
20.1 * 2.5 ‘4.3 z 0.6
‘5.5 2: 1.5 ; 0”; ( 6) ( 6)
9.7 & 0.9 14.3 f 1.0
q.8 .k 0.g
0.1 < P < 0.2 0.2 < P < 0.3 O.OOI < P < 0.005 0.05 < P < 0.1 0.001
<
P
<
0.005
Standard error.
TABLE ATP
LEVEL
VII1 IN
LIVER
AFTER
THE
INJECTION
OF
L-TRYPTOPHAN
OR
L-KYNURENINE
2.45 /Lmoles of L-tryptophan or L-kynurenine per g body weight were intraperitoncally injected into rats. The rats were killed 3 h after the injection, and the concentrations of ATP in liver were measured according to the luciferin-luciferase method. Numbers of rats are given in parentheses. Substance injected
ATP
_~ ~____ None L-Tryptophan L-Kynureninc
(mpmoleslg
of liver)
570 :t 3S* 372 i 44 344 17 30
i:; (9)
_
* Standard error. A TPase activity in the fatty liver The marked decrease in the level of ATP in the liver might be due to the enhancement of ATPase activity in the liver. ATPase activity in the microsomal fraction of the fatty liver was therefore measured. Table IX shows that there was no difference in ATPase activity between the livers of control rats and L-tryptophan-administered rats. TABLE
IX
MICROSOMAL
ATPaX
ACTIVITY
IN
LIVER
AFTER
THE
ADMINISTRATION
OF
L-TRYPTOPHAN
A portion of the same liver that was used in the previous experiment (Table VIII), was assayed for ATPase activity in the microsomal fraction. Numbers of rats are given in parentheses. Substance injected
ATPase activity (,uatom P released per I0 min pev g of- liver)
None L-Tryptophan
37.9 4 3.79* 36.8 & 2.08
(5) (5)
* Standard error. DISCUSSION
Fatty liver can be induced in the rat by several agents. In fatty liver, the accumulating lipids are usually predominantly triglycerides. Abnormal accumulation of triglycerides in the liver is the result of an imbalance between the rate of synthesis and B&him.
Biophys. Acta,
144
(1967) 233-24’
TRYPTOPHAN
FATTY LIVER
239
the rate of utilization of hepatic triglycerides. Most factors that can produce fatty liver, for example administration of CC1410~19-26, ethioninea7-29, phosphorus or puromycinso, or with erotic acid36936, feeding of a choline-deficient diet31-34 or of a diet supplemented are known to block the secretion of hepatic triglycerides into the plasma. It is known that a major part of the hepatic triglycerides in the rat is secreted into the plasma as a moiety of plasma lipoprotein 37. It is suggested that the above agents disturb the metabolism of lipoprotein. In the fatty liver induced by CC1438-40,ethionine28y41p42, is an inhibition puromycin30 or phosphorus23, one of the basic metabolic disturbances of protein synthesis. It has been reported that administration of ethionine*“, CC1444*45, phosphorus**, ethanol45 or azaserine45, or feeding of a diet deficient in choline**, which produces fatty liver, leads to a rapid decrease in the concentration of ATP in the liver. The administration of ATP completely prevents the fatty liver induction by ethioninez*, CC1445, ethanol45 or azaserine45. The fatty liver induction by a diet containing erotic acid is also prevented by supplementation of the diet with adenine*8. In this paper we have shown that administration of L-tryptophan or L-kynurenine caused a marked decrease in the level of ATPin the liver and that the fatty liver induction by L-tryptophan was prevented by the administration of ATP, ADP, AMP or adenosine. Therefore, in tryptophan fatty liver, as in the majority of other types of fatty liver, it is suggested that a marked fall in the level of ATP depresses the protein synthesis leading to a failure of the liver to release triglycerides into the plasma as lipoproteins. It is also possible that a significant decrease in the cellular ATP level might disturb the completion of lipoprotein formation or alter the state of cellular membranes as to interfere with the transfer of preformed lipoprotein from the liver to the blood. To explain the striking decrease in the level of ATP in the liver, the enhancement of ATPase activity in liver might be considered. But we found no difference in hepatic microsomal ATPase activity between control rats and tryptophan-administrated rats. It has also been reported that ATPase activity does not change after the adminstration of Ccl, to induce fatty liver18. Recently QUAGLIARIELLO et aZ.47p48reported that, of all the tryptophan metabolites tested, only 3-hydroxyanthranilic acid uncoupled oxidative phosphorylation in vitro at the level of the first respiratory chain phosphorylation. It also inhibited the oxygen uptake of rat liver mitochondria respiring with NAD-dependent substrates. It is known that many substances that inhibit oxidative phosphorylation produce fatty infiltration of liver when administrated intraperitoneally to rats4a. 3,5-Dinitro-o-cresol, which inhibits oxidative phosporylation, depresses the concentration of ATP in many organsso. RAY, FOSTER AND LARDY~ found that administration of t-tryptophan to rats caused the rapid accumulation of citrate, malate, pyruvate, lactate and oxaloacetate to 5-35 times the normal concentration. They suggested that the administered tryptophan inhibited gluconeogenesis by blocking the conversion of oxaloacetate to phosphoenol pyruvate. Nevertheless, the level of phosphoenol pyruvate carboxykinase, when assayed in vitro, was enhanced. It seems reasonable to suppose that these effects of L-tryptophan may result from the inhibition of oxidation of a-oxoglutarate by 3-hydroxyanthranilic acid. Data presented in this paper, together with the reports from other investigators, suggest that the actual substance that produces the fatty liver is not L-tryptophan but may be its metabolite, 3-hydroxyanthranilic acid. The mechanism of depression of the ATPlevelintheliverafterasingleinjectionof~-tryptophanstillremainstobeelucidated. Biochinz.
Bio$hys.
Ada,
I++ (1967)
233-241
240
Y.
HIRATA, T. KAWACHI, T. SUGIMURA
Another factor in the ATP protective effect has been reported. HYAMS AND ISSELBACHER*~showed that Ccl,- or ethanol-treated animals injected with ATP become hypothermic, and they suggested that the hypothermia may be a factor in the protection. The preventive effect of adenine nucleotides on the tryptophan fatty liver may be due not only to stimulation of oxidative phosphorylation but to some pharmacological activity of tryptophan, or tryptophan in combination with adenine nucleotide. The possible role of ATP in relieving peripheral stress, increasing the transport of lipid, or elevating lipid synthesis de nova in the liver, remains to be clarified. Assays of ADP, AMP, adenosine and adenine in this fatty liver, and the effects of administered adenine compounds upon them, are under investigation. As to the effect of folic acid in preventing fatty liver, the dose of folic acid injected was well above the physiological level. As for the fatty acid composition of neutral lipids which accumulate in liver after administration of tryptophan, there was no striking change compared with that of the control. But a slight decrease in the percentage of saturated fatty acids and a slight increase in the percentage of unsaturated fatty acids suggest a metabolic disturbance of the synthesis of lipoprotein or its utilization by tryptophan. ACKNOWLEDGEMENTS We wish to thank Dr. T. BABA AND Mr. AOKI of the Pathology Division of this Institute for their help in histological diagnosis and Mrs. M. NAKAYASU for her help in the assay of ATPase activity. A part of this work was reported at the 38th General Meeting of The Japanese Biochemical Society at Fukuoka on October r7th, 1965. This work has been supported partly by a grant from the Ministry of Education to the research group on “Host Conditions and Growth of Cancer” headed by Dr. YUICHI YAMAMURA(No. 956107). REFERENCES I W. E. KNOX AND A. H. MEHLER, Science, 113 (1951) 237. 2 P. D. RAY, D. 0. FOSTER AND H. A. LARDY, J. Biol. Chem., 241 (1966) 3904. 3 A. E. HARPER, J. N&r., 50 (1953) 383; 56 (1955) 187. 4 A. E. HARPER, Am. J. Clin. Nutr., 6 (1958) 242. 5 E. FARBER, M. V. SIMPSON AND H. TARVER, J. Biol. Chem., 182 (1950) 91. 6 S. B. STANDERFER AND P. HANDLER, Proc. Sot. Exptl. Biol. Med., go (1955) 270. 7 G. Y. KIVMAN AND A. M. KHARITONOVA, Antibiotiki, 2 (1957) 49. 8 G. ZBINDEN AND A. STUDER, Schweiz. 2. Allgem. Pathol. Bakteriol., 20 (1957) IO. 9 C. T. ASHWORTH, Proc. Sot. Exptl. Biol. Med., 66 (1947) 382. IO A. S. AIYAR, P. FATTERPAKER AND A. SREENIVASAN, Biochem. J.. go (1964) 558. II M. C. SCHOTZ AND R. 0. RECKNAGEL, Biochim. Biophys. Acta, 41 (1960) 151. 12 J. FOLCH, M. LEES AND G. H. SLOANE-STANLEY, J. Biol. Chem., 226 (1957) 497. 13 C. H. FISKE AND Y. SUBBAROW, J. Biol. Chem., 66 (1925) 375. 14 E. V. HANDEL AND D. B. ZILVERSMIT, J. Lab. Clin. Med., 50 (1957) 152. 15 Z. HENLY, Am. J. Clin. Path., 27 (1957) 583. 16 B. L. STREHLER AND J. R. TOTTER, in D. GLICK, Methods of Biochemical Analysis, Vol .I, Interscience, New York, 195.4, I). 341. 17 J. B. MARTIN AND D. M. DOTY, Anal. Chem., PI (1949) 965. 18 T. TAKAHASHI, Seikagaku, 26 (1955) 690. 19 R. 0. RECKNAGEL AND B. LOMBARDI, J. Biol. Chem., 236 (1961) 564. 20 E. A. SMMUCKLER,0. A. ISERI AND E. P. BENDITT, Biochem. Biophys. Res. Commun., 5 (1961) 270. 21 M. HEIMBERG, I. WEINSTEIN, G. DISHMAPI’ AND A. DUNKERLEY, J. Biol. Chem., 237 (1962) 362322 H. M. MALING, A. FRANK AND M. G. HORNING, Biochim. Biophys. Acta, 64 (1962) 540.
Biochim. Biophys. Acta, 144 (1967) 233-241
TRYPTOPHAN
FATTY
LIVER
241
23 A. SEAKINS AND D. S. ROBINSON,Biochem. J., 86 (1963) 401. 24 M. POGGI AND R. PAOLETTX, Biociem. Pharmacol., 13 (1964) 949, 25 M. C. SCHOTZ,N. BAKER AND M. N.C~~v~z,J.Lipa'dHes., 5(x964)569.
26 B. L~MBARDI AND G. UB~zro,J.Li$idi&s.,6 (1965)498. 27 T. GI.IVECRONA, Acta P&&t. Seanad., 55 jIg62)2gI. 28 E. FARBER, B. LOMBARDI AND A. E. CASTILLO, Lab. Invest., IZ (x963)873. 2g G. UBAZI~ AND B. LOMBARDI,Lab. Invest., 14 (1965) 7x1. 30 D. S. ROBINSON AND A. SEAKINS,B&him. Rio$hys. Acta, 62 (xq62)163. 31 B. LOMBARDI AND R. 0. RECKNAGEL, Am.]. Pathal.,40(xg62) 571. 32 R. 0. RECKNAGEL AND A. K. GHOSHAL, ~~~~~a~~~~ BOG., 23 (1964) 243. 33 j.C. FORIIES, O.~.PETTERSONANDR.A.RT;T)O~PH,P~~~.Soc.~%~~~.Binl.NCed.,~rS frg65)59. 34 Il. S. i’d. HAINES AND S.MOOKERJEA, Canad. J. Biockem. Physiot., 43 (1965) 507. 35 H. G. WINDYUELLER, J,Bdol.Chsm, 239 (1964) 530. 36 P. S. ROHEINI, S. SWITZER,A. GIRARD AND H. A. EDER, Lab. Invest., 15 (rg66) 21. 3~ N. BAKER AND M. C. Scmrz, J. Lipid Res., 5 (1964) 188. 38 E. A. SN~UCKLER, 0. A. 1sg1+I AND E. P. BENDITT,.J.E~pt~,~e~, 116(rg6z)55. 39 E. A.SMUCKLER AND E, P. BENDITT,Science, 14o(Ig63)308. 40 E. A. SMLXKLER AND E. P. BENDITT,Biochenzistry, 4(rg65) 67x. 41 E. FARBER,K. H. SHIILL, S. VILLA-~R~VI~O,~.L~~EAR~IA~D~.THO~~S,~~~~~~~, 203(rg64) 34. 42 D. S. Rosmso~ ANYDP. M. HARRIS,BiocJzm. J., So (1g61f 361. 43 xf. H. SHULL,J.Biol.Chem., 237 (1962)1734. 44 M. U. DIANEANI,Biochem. ,I., 65 (1957) 116. 45 D. E. HYAMS AND K. J.XSSELBACHER, Nature, 204 (x964) 1196. 46 3%. E. HANDSCHUMACHER, W. A. CREASEY, J. J. JAFFE,C. A. PASTERNAK AND L. HANKIN,
Pmt. N&k. Acid SGi. U.S., $5 (rg60) 178. Q~IAGLIARIELLo, S. PAPA AND C. SACCONE,Baoclae~n. J.,84 (rg62)66P. 48 E. QUAGLIARIELLO, S. Pan, C. SACCONE ANU A. ALIFANS,B&hem.. J., gr (1964) 137.
47 E.
49 M. U, DIANZANI AND S. SCVRO, Biockent. J., 62 (x956) 205. 50 V. H. PARKER, Biochern. Jha 57 (1954) 381.
Biochinz. Biofihyys. Acta, 144 (1967) 233-241