Rat Liver Glutathione: Possible Role as a Reservoir of Cysteine1 NORIKO TATEISHI,2 TANEAKI HIGASHI, AKIKO NARUSE, KAYOKO NAKASHIMA, HIROSHI SHIOZAKI ANDYUKIYA SAKAMOTO Department of Biochemistry, Institute for Cancer Research, Osaka University Medical School, 1-Fukushima, Fukushima-ku, Osaka 553, Japan ABSTRACT Rat liver contains a high concentration (7-8 HIM) of reduced glutathione and its level changes rapidly when starving or feeding rats. We concluded that one of the functions of liver glutathione was to act as a reservoir of cysteine. When starved rats were fed a protein-free diet, the increase in liver glutathione was dependent on the amount of cysteine added to the diet. A cysteine-dependent increase of glutathione was also observed in rats fed a diet containing gelatin with cysteine, but the increase was relatively lowered compared with rats fed a protein-free diet contain ing the same amount of cysteine. This suppression of the increase in gluta thione was observed much more clearly when the gelatin diet was fortified with tryptophan in addition to cysteine. In the presence of tryptophan, L-[35S]-cysteine in the diet appeared to be incorporated primarily into liver and serum proteins, and degradation of liver glutathione must also have been enhanced. Addition of excess cysteine to the diet masked the effects of gelatin and tryptophan, and stimulated glutathione synthesis in the liver as well as incorporation of dietary cysteine into protein fractions. Prolonged starvation of rats or injection of dibutyryl-3',5'-cyclic AMP lowered the flutathione level, but the level did not decrease below 2 to 3 mM. These ndings suggest that there may be at least two pools of glutathione. A labile fraction, constituting one-third to one-half the total liver glutathione, prob ably serves as a reservoir of cysteine which can be released by y-glutamyltransferase when necessary. J. Nutr. 107: 51-60, 1977. INDEXING KEY WORDS liver glutathione •dietary cysteine • liver protein synthesis •polysome profiles Reduced glutathione (GSH) is known to have several important physiological roles: It acts as a coenzyme of methylglyoxalase I [EC 4.4.1.5] (1) and other enzymes (25); a cysteine donor for mercapturic acid formation (6); a redox buffer (7); a protector of SH-enzymes (7), of the integrity of erythrocyte membranes (8, 9) and of the
One is why the level of GSH in the liver is so high. The concentrations of all coenzymes in the liver except GSH are between 0.03 mM and 0.8 mM (14-16) and those of adenine nucleotides are between 0.6 and about 2 mM (17), whereas that of GSH is as high as 7 to 8 mM (17). Another ques-
transparency of the lens (10, 11); a y-glutamvl frrnim drmnr in iminn arirl fri n snort uunyi grouP uonor in ammo acia transport in the v-glutamyl cycle (12) and a pro-
Received for publication December 29, 1975. * Part of this work was presented orally at the Xth international Confess of Nutrition In August, 197B (Kyoto). The work was supported in part by grants
tector against radiation injury (13).
^^^"^^^^VIS^W
However, there are at least two unanswered questions about liver glutathione.
se^^s^°^^hoM Tateishi. 51
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*?-
besentto Dr.
TATEISHI ET AL.
tion is why the level of liver GSH changes so rapidly after fasting or after refeeding animals. After 1 or 2 days of starvation, the liver GSH content of rats is between two thirds and half the normal level, and after feeding these animals, it rapidly returns to normal ( 17 ). On the contrary, the levels of coenzymes and adenine nucleotides in the liver do not change markedly after fasting and refeeding rats (17), except for that of acetyl CoA, which increases during star vation (16). These findings on rat liver GSH can not be adequately explained by the known physiological roles or GSH de scribed above. This paper reports a possible further role of liver GSH as a reservoir of cysteine. MATERIALS
AND METHODS
Animais. Male albino rats,3 weighing 130 g to 160 g, were housed individually in cages in an air-conditioned room (25°) and given food and water ad libitum, unless otherwise indicated. The rats were fed a stock diet4 prior to experimentation. The control diet contained: (in %) com oil,5 6; salt mixture,6 1; vitamin mixture,7 0.5; choline chloride,8 0.5; bovine milk casein,9 18; cellulose,10 5; corn starch,9 60 and agar8 to 100%. When powdered gelatin " or agar were used to replace casein, various amounts of cysteine (and/or tryptophan) were added to the diet, as described in table 1, and figures 1 and 2. Each rat was given 10 g of diet/day. This amount of diet was usually consumed almost completely except for the protein-free diet (table 1). In experiments using radioactive cysteine, L-[35S]-cysteine hydrochloride 12was added to the powdered diet described above and the diet was solidified by adding an equal weight of 4% agar solution. Therefore, 20 g of diet, equivalent to 10 g of the pow dered diet, were given per day in these ex periments. These amounts of food were usually consumed completely within 12 hours by rats which had been fasted for 24 to 48 hours. Preparation of post-mitochondrial super natant and analysis of polysome profiles of rat liver. Rats of each dietary group were killed by decapitation and their liver was quickly removed by the method of Drysdale and Munro (18). The liver was ho mogenized in 4 volumes of 0.25 M sucrose
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containing TKM buffer (0.05 M Tris buffer, pH 7.6, 0.025 M KCl, 0.005 M MgCl2) by 4 strokes of a loosely fitting motor-driven Teflon1S glass homogenizer and the homogenate was centrifugea at 18,000 X g, for 15 minutes at 2°.The post-mitochon drial supernatant thus obtained was mixed with rabbit anti-horse ferritin ™serum in the proportion of 40:1 (v/v) to remove ferritin particles (19) and «-amylase was added to degrade glycogen (20), because both ferritin and glycogen interfere with the polysome pattern. Sodium deoxycholate and Triton X-100 15were added to the postmitochondrial supernatant to concentra tions of 0.5% and the preparations were di luted with two volumes of TKM buffer. Then samples of 0.6 to 0.65 ml ( A2eo= about 18) were applied to a linear sucrose gradient ( 10 to 25% in TKM buffer, total volume = 28 ml ) with 1 ml of 2 M sucrose at the bottom. A layer of 0.5 ml of 3.5% sucrose was applied on top to trap soluble macromolecules. The tubes were centrifuged in a swinging-bucket rotor at 25,000 rpm for 3 hours in an ultracentrifuge.1* The absorption of the gradient at 254 nm was recorded automatically using a 10 mm light path flow-cell in an ultraviolet ana lyzer.17 3Donryu strain : Kitnyama LABES Co.. Kyoto. 4Stock diet (type M) containing (In %) carbo hydrate, 51.8 ; crude Hpld, 5.0 ; crude protein, 24.6 ; minerals. 7.4 : cellulose. 4.2 ; (Oriental Yeast Ind. Co., Ltd.. Tokyo.) "Nisshin Oil Co., Tokyo. « Salt mixture ; McCollum's salt misture containing (In %) ; calcium phosphate, monobasic 13.3, sodium chloride 4.29, magnesium sulfate, dlhydrate 14.84, sodium phosphate, dibasic, anhydrous 8.59, calcium lactate 32.2, potassium phosphate, dibasic 23.6 and ferric citrate 2.92 (Nakaral Chemicals, Ltd., Kyoto.) 'Vitamin mixture containing (In %) ; thlamln hydrochloride 0.059. rlboflavln 0.059, nlcotlnlc acid 0.294. calcium pantothenate 0.235, pyrldoxlne hydrochloride 0.029, menaqulnone 0.006, blotln 0.001, folle acid 0.002, cyanocobalamln 0.0002. Inosltol 1.176, ascorbic acid 0.588 and lactose 97.551. (Tanabe Amino Acid Research Foundation, Osaka.) • Nakaral Chemicals, Ltd., Kyoto. »WakoPure Chemicals Industries, Ltd., Osaka. »»Kokusaku Pulp Co., Tokyo. 11E. Merck AG, Darmstadt. u Product of the Radiochemlcal Centre, Amersham. Purchased from Japan Isotope Association. "E. I. DuPont de Nemours & Co. " Pentex ; Miles Laboratories, Inc., Kankakee, "Rohm Sc Haas Co., Philadelphia, Pennsylvania, Simultaneous use of sodium deoxycholate and Triton X-100 Is recommended by Dr. Ken Higashl In our laboratory. «In the SW 25.1 rotor of a Beckman L2 ultracentrifuge (Beckman Instruments, Inc., Splnco Divi sion, Palo Alto, Calif.). «ISCO Model 640 density gradient fractionator (Instrumentation Specialties Co., Inc., Lincoln, Nebraska).
53
RAT LIVER GSH AS A RESERVOIR OF CYSTEINE TABLE 1 Effect of the amount of L-cysteine in the diet on the increase of liver GSH
DietFasted
consumptiong7.4±1.0 wt.g50. liver4.02±0.50*2.84
for 40 hours Refed for 12 hours on Protein-free diet (18% agar) + CySH 0.5% +CySH 1.0% 5.0%CySH +CySH
±0.35.4±0.2 ±0.47 5.12±0.66 6.6±1.8 7.05±0.43 6.7±0.8 7.44±0.563.40±0.47 5.8 ±1.88.1
5.8±0.2 5.8±0.3 5.7±0.66.4±0.5
free protein diet (18% gelatin) + CySH 0.5% +CySH 1.0% +CySH 5.0%GSHnmoles/g
4.62±0.36 6.10±0.27 8.02±0.85Food
±0.8 9.8±0.7 9.6±0.4 9.1±0.7Liver
7.3±0.5 7.4±0.8 8.5±0.7
Protein diet (18% casein)
8.64±0.11
9.7±0.3
6.7±0.3
Rats weighing 145 to 160 g were starved for 40 hours and then fed 10 g of the indicated diets. Added amino acids are expressed as percentage of protein or agar. Twelve hours later, liver GSH was estimated. For the protein-free diet, n = 8. For other diets, n = 4. * Mean ±SD.
Preparation of trichloroacetic acid extract of rat liver. Rats were killed by decapita tion and their livers were quickly removed. A 25% (w/v) liver homogenate was pre pared in 0.15 M KC1 by 10 strokes of a motor-driven, Potter-type Teflon13 glass homogenizer. Then the homogenate was mixed with one-quarter volume of cold 25% trichloroacetic acid containing 5 mM EDTA and the mixture was centrifuged to separate the "acid supernatant". Cysteine can be determined more accurately using this acid supernatant than using the super natant from the "MSE homogenate" (0.21 M mannitol, 0.07 M sucrose and 0.1 mM EDTA ) as in previous work ( 17), because it contains no materials interfering with the ninhydrin reaction. This acid super natant was used for determination of both cysteine and glutathione. Determination of glutathione, cysteine and protein. GSH was estimated by the method of Saville (21). Total glutathione was also determined enzymatically (22, 23 ). The values determined by the two pro cedures always coincided well. This indi cates that most of the acid soluble sulfhydryl material in rat liver is glutathione with negligible amounts of other sulfhydryl compounds and that glutathione is present mainly in the reduced form.18 Cysteine was determined by the method of Gaitonde (24). Protein was assayed by the procedure
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of Lowry et al. (25) using bovine serum albumin as a standard. Assay for y-glutamyltransferase [EC 2.3.2.2]. The y-glutamyltransferase activity of the liver homogenate was assayed as described previously (17) using y-glutamylp-nitroanilide as substrate and glycylglycine as a -y-glutamyl group acceptor ( 26, 27). Measurement of radioactivity. A sample (50 to 100 /xi) of 25% liver homogenate was loaded on a glass fiber filter ( d = 2.4 cm) 10and immersed in 5% trichloroacetic acid containing 1 mM EDTA. The glass fiber filter was then boiled in this mixture for 10 minutes, washed successively three times with the same concentration of trichloroacetic acid, twice with ethanolether (1:1 v/v) and once with ethanolether (1:3 v/v), and dried. The acidic ex tract of liver obtained by adding onequarter volume of 25% trichloroacetic acid containing 5 /¿M EDTA to the 25% homog enate was added to ten volumes of alkaline tissue solubilizer.20 GSH and cysteine in the acid extract of the liver were separated by paper electrophoresis as described by Mooz "Naruse, A., Tatelehl, N., Hlgashl, T. & Sakamoto, T. (1973) 46th Ann. Meeting Jap. Blochem. Soc. (Nagoya) (Abstract; Seikagaku 45, 430). «Whatman GF/F filter (W & R Baiston Ltd., Maidstone). »NCS tissue solubilizer (Amersham/Searle Cor poration, Arlington Heights, Illinois).
54
TATEISHI ET AL.
and Meister (28). The bands of the two ma terials were located with ninhydrin reagent and cut off. Then they were immersed in, or mixed with a toluene scintillation fluid (8 g of 2,5-diphenyloxazole and 0.1 g of ì,4-bis[2-(5-phenyloxazolyl)]-benzene in 1 liter of toluene ) and their radioactivity was counted in a scintillation spectrometer.21 RESULTS Dependency of the rise in glutathione level on the amount of cysteine in the diet. To establish a quantitative relationship be tween the content of cysteine in the diet and the increase of liver GSH after feed ing starved rats, a protein-free diet con taining agar in place of casein and supple mented with cysteine at concentrations of 0.5%, 1.0% or 5.0% of the agar was fed to rats which had been fasted for 40 hours (table 1). The results showed that the increase of liver GSH 12 hours later was proportional to the amount of cysteine added, when this was within a physiologi cal range. With an unphysiologically high concentration of cysteine ("5.0%") the liver GSH concentration was not propor tional to the amount of cysteine in the diet.
i
r O
10
20 30 40 50 60 70 AMOUNT OFINGESTED CYSTEINE (MO/RAT)
80
Fig. 1 Effect on liver glutathione of ingestion of protein-free (agar) or gelatin-containing diet containing various amounts of cysteine after star vation. After fasting for 40 hours, the rats were given 10 g of diet containing 18% gelatin or agar, supplemented with cysteine corresponding to 0, 0.5%, 1.0% or 5.0% of the agar or gelatin. Twelve hours later, their liver glutathione level was estimated. The amount of ingested cysteine per rat was calculated from the intake of diet and its cysteine content. Each point represents one rat. —X—; agar diet, —•—;gelatin diet.
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Similar results were obtained when gela tin was used as the sole protein constitu ent of the diet and the diet was fortified with various amounts of cysteine (0.5%, 1.0% or 5.0% with respect to gelatin). Under physiological nutritional conditions (0.5% to 1.0% cysteine), a higher GSH level was attained with the protein-free diet than with gelatin diet, on the basis of net uptake of cysteine. Rats seemed to pre fer the gelatin diet to the agar diet so that their intake of the former was often larger than that of the agar diet. Therefore, in figure 1 the cysteine intake by each rat is plotted against the GSH content of the liver. Addition of gelatin to the diet in stead of agar suppressed the increase of liver GSH when the same amount of cys teine was ingested. Effect of the amount of L-tryptophan in the diet on the increase of liver GSH. The increase of liver GSH was dependent on the amount of cysteine added, as described above. However, the difference between the increases of GSH in the groups of rats fed agar-diet and those fed gelatindiet containing the same amount of cys teine suggested that some factors other than the cysteine content itself affected the hepatic GSH level. To examine this, the effect of a more nutritionally adequate gelatin-diet was tested. Namely, the diet was fortified with tryptophan, which is the first limiting amino acid in gelatin, in addition to cysteine. It was found that the increase in GSH when the cysteine-containing diet was fed was strongly inhibited by the addition of tryptophan (fig. 2). The level of GSH was lowered by addition of tryptophan at levels of 0.25% to 1.0% of the gelatin when the level of cysteine in diet was 0.5% to 1.0% of that of gelatin. With the diet containing the larger amount of cysteine (5% of the gelatin), the level of liver GSH was not lowered by adding tryptophan. Incorporation of dietary [3iS]-cysteine into rat liver fractions and serum under different nutritional conditions. To exam ine the metabolic fate of cysteine derived from food, rats were fasted for 40 hours and then fed diets containing gelatin as 21Tri-Carb scintillation spectrometer Model 3320 (Packard Instrument Co., Inc., Downers Grove, Illinois).
55
RAT LIVER GSH AS A RESERVOIR OF CYSTEINE
the protein constituent fortified (1) with cysteine, (2) cysteine + tryptophan or (3) excess cysteine + tryptophan. L-[35S]-Cysteine was added to all these diets. As shown in the previous section and figure 3, addition of tryptophan to the diet sup pressed the increase of liver GSH induced by dietary cysteine, although it did not affect the total intake of food. Addition of excess cysteine masked this effect of tryp tophan. Examination of the distribution of [35S]-cysteine in the acid soluble fraction of the liver and in liver GSH indicated that the differences in hepatic GSH contents of the three groups resulted from differences in the incorporation of dietary cysteine into GSH and in the turnover of GSH. The results showed that tryptophan stimulated the incorporation of [35S]-cysteine into the acid-insoluble fraction of liver. If cysteine
JJMOLES/G LIVER
0.1 —i—
0.2
CySH J*WLES/a LIVER
GSH 103CPM/IO MGLlVtR
*
„6
ACIDSOL. CYSH GSH
ACIDppr. SERUM (10ill)
«8.03oì14.0|ADDED
:0
CïSHas
»of gelatinADDED
0_T||ht11S 1-r~
5^
s-°lTup
7i) i.rf as r. afa j selitln •*,(
_j-rt;É ^Õ0
Fig. 2 Effect of addition of L-tryptophan to a diet containing gelatin and various amounts of cysteine on the increase of liver glutathione after starvation. The experimental conditions were similar to those described in the legend of table 1. The vertical bars show SD. The open triangle shows the level of liver glutathione after fasting for 40 hours. When the columns were marked from left to right, a, b,
1, liver weights
(g) of
each group of rats were (a) 7.2 ±0.4 (4), (b) 7.3 ±0.5 (4), (c) 7.1 ±0.2 (4), (d) 7.1 ±1.0 (6), (e) 7.4 ±0.8 (4), (f) 7.1 ±0.2 (4), (g) 7.8 ±0.7 (4), (h) 7.2 ±0.1 (4). (i) 8.5 ±0.8 (4), (j) 7.2 ±1.0 (4), (k) 8.1 (2) and (1) 8.3 ±0.6 (4). The two bars marked by asterisks differ from each other at P < 0.01. The two bars marked by crosses differ each other at P < 0.001. Student's i-test wasfrom employed (39, 40).
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Fig. 3 Incorporation of ["S]-cysteine from diet containing gelatin with cysteine alone, cys teine and tryptophan, or tryptophan and excess cysteine into liver fractions and serum of rats after starvation. After fasting for 40 hours, the rats were given 20 g of gelatin-containing diet solidified with agar. Dietary treatments are shown as 1) n 1% cysteine, 2) •1% cysteine +1% tryptophan, 3) 0 3% cysteine + 1% tryptophan. Added amino acids are expressed as % of gelatin. In 1) and 2), 10 /¿Ci and in 3) 30 /¿Ci of L-[MS]cysteine were added to the diet to give the same specific radioactivity. The three groups of rats consumed more than 90% of the food within 2 to 3 hours. The bars show standard deviations. Other experimental conditions were similar to those described in the legend of table 1. Liver weights of the rats are 1) 8.5 ±0.8 g, 2) 8.8 ±0.3 g and 3) 8.3 ±0.6 g, respectively.
is derived from GSH, the differences in net incorporations of cysteine into liver proteins in the different groups are in fact larger than those shown in figure 3. The incorporation of [35S]-cysteine into the serum of rats was also stimulated by the addition of tryptophan to the diet. The in corporation of radioactivity into serum proteins was essentially the same as those into whole serum in the three groups. When excess cysteine (3% of the gelatin content) with \% tryptophan was fed to
r,6
TATEISHI ET AL.
IA2S4=0.1
Fig. 4 Hepatic polysome patterns of rats under various nutritional conditions. Rats were fasted for 40 hours. The polysemes were obtained from rats under the following conditions: (1) fasted, (2) 14 hours after receiving a protein-free (18% agar) diet supplemented with cysteine corre sponding to 1% of the agar, (3) 14 hours after receiving a gelatin-containing (18%) diet forti fied with cysteine corresponding to 1% of the gelatin and fortified (4) 14 with hourscysteine after receiving the gelatin-diet ("1%") and tryptophan ("\%"). Columns on the left show the content of liver glutathione (^moles/g liver). Liver weights of the rats are (1) 4.5, (2) 7.4, (3) 7.6 and (4) 7.5 g, respectively. Other condi tions were as described.
rats, the incorporations of [35S]-cysteine into all fractions were larger. This prob ably indicates that under these conditions both GSH synthesis and other systems utilizing cysteine were maximally active.
Influence of addition of tryptophan to the diet on the liver polysome patterns. The results described in the previous sec tion suggest that the activities for protein synthesis in the liver were different in the different groups. Accordingly, we exam ined the polysome patterns of the postmitochondrial supernatant fractions of the livers of rats under various nutritional con ditions by sucrose density gradient centrifugation. All rats were fed the stock diet4 ad libitum and then fasted for 40 hours before the experiment. Then, they were treated as follows. Group 1 were examined directly (fig. 4-1). Group 2 were given protein-free diet supplemented with cysteine alone and killed 14 hours after the start of feeding (fig. 4-2). Group 3 were given diet containing cysteine and gelatin as a protein source and examined 14 hours later (fig. 4-3). Group 4 were given diet containing cysteine and tryptophan in addition to gelatin and examined 14 hours later (fig. 4-4). Rats with similar food in takes were selected for examination. Al though the GSH levels were very different in group 1 and 2, the polysome patterns of the two groups were similar, both showing large number of monosomes and disomes with fewer polysomes. The glutathione contents of groups 3 and 4 were also dif ferent but their polysome patterns were both typical profiles of well-fed animals. However, the pattern of group 4 (fig. 4—4) indicated more active protein synthesis than that of group 3 (fig. 4-3) with a de crease in monosomes and an increase in larger polysomes. These results are in agreement with those in figure 3, indicat-
TABLE 2 Effect of dibutyryl-3',5'-cyclic AMP on liver GSH and cysteine contents and y-glutamyltransferase (y-GTP) activity
TimeafterinjectionGSHCysteineLiver7-GTP wt.Liverprotein limole»/gliver 0 90
3.83±0.39* 2.84±0.23
nmoles/g liver
149±21 385±21
mg/g liver
nmoles/hr/mg protein
82± 2 309±56
4.2±0.2 3.9±0.1
208±17.5 223±20.6
* MeaniSD. Eight rats (weighing 135 to 150 g) were fasted for 16 hours (from 1800 to 1000 hours) and then 5 mg of dibutyryl-3',5'-cyclic AMP and 2 mg of theophylline per 100 g body weight were injected intraperitonealry. Four rats were killed immediately and four were killed 90 minutes later. For estimation of the 7-GTP activity the KC1 homogenate was used instead of the MSB homogenate used previously (17), because its enzyme activity was higher.
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RAT LIVER GSH AS A RESERVOIR
ing that supplementation of the gelatin diet with tryptophan enhanced the incor poration of cysteine into hepatic proteins and probably also into serum proteins, most of which are synthesized in the liver (29). At the same time, liver glutathione appeared to be mobilized. The clear differ ence polysoine patterns of (fig. in 4-2)the ("fasting-pattern") andgroup group2 3 or 4 (fig. 4-3 or 4-4) ("refed-pattern") must be considered in relation with the re sults shown in figure 2 and table 1. Active protein synthesis was accompanied by sup pressed increase of glutathione, as ob served in group 4, although nearly the same amount of cysteine was consumed in groups 2 and 4. These findings correspond to the results shown in table 1, figures 1 and 2, where an increase in glutathione ob served in the rats fed a cysteine contain ing agar diet was suppressed by using gelatin instead of agar, and more markedly by using gelatin fortified with tryptophan. The results suggest that when other condi tions are adequate and cysteine can be utilized for protein synthesis, increase of liver GSH is suppressed, and the cysteine moiety of GSH is mobilized for protein synthesis. However, when the supply of cysteine is sufficient but other conditions for protein synthesis are not adequate, or when there is excess cysteine over that re quired for protein synthesis, the cysteine is stored in the form of glutathione. ICOr*
2» M
72
96
HOURS
Of
" 0
2*
72
»
STARVATION
Fig. 5 Effect of prolonged fasting on liver glutathione, body weight and liver weight. Rats weighing 130 to 150 g were starved but given free access to water. Fasting was started at 1000 hours. Four rats each were killed 24, 48, 72 or 96 hours later and their liver glutathione (-O-), body weight (-X-) and liver weight (-•-) were estimated. Values of 145 g body weight and 6.5 g liver weight were taken as 100%. Vertical bars show so.
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OF CYSTEINE
57
Liver glutathione pool as a reservoir of cysteine. Assuming that liver GSH is uti lized as a source of cysteine for protein synthesis, the next problem is how much liver GSH can be mobilized. On fasting, the liver GSH concentration decreased rapidly to two-thirds of the normal concen tration within 1 day. However, it is inter esting that the concentration did not de crease below about 4 to 5 mM. Moreover in most rats, the concentration did not con tinue to decrease during further fasting for 3 to 4 days, although the liver weight and body weight continued to decrease gradu ally (fig. 5). In some rats, the GSH con centration in the liver decreased to a very low concentration of about 2 m,M with simultaneous sharp decreases in body weight and liver weight when rats were dying after prolonged starvation. This low concentration of GSH appears to represent the minimal requirement for survival. These findings suggest that there may be at least two pools of liver glutathione; one for many reactions in which glutathione itself is involved and the other for supply of cysteine. DISCUSSION
As shown previously (17), turnover of liver GSH was rapid in fasted rats and its increase on feeding these rats depended upon food intake; about 10 g of diet was necessary to attain the maximal increase in rats weighing 150 g. An intake of about 10 g of diet/day was necessary to maintain the normal level of GSH thus attained, and a smaller amount of food intake resulted in a decrease in the GSH content. Circadian change in liver GSH (30, 31), which is apparently related to the habit of nocturnal feeding, has also been reported. Moreover, recent results in our laboratory (32) showed that rat liver GSH decreased temporarily to a level of 2 to 3 mM after injection of dibutyryl-3',5'-cyclic AMP. These results indicate that GSH in rat liver turns over fairly rapidly under nor mal physiological conditions. Studies by Leaf and Neuberger (33) clearly demon strated that administration of cystine or methionine to fasted rats resulted in a significant increase in liver GSH. Their results were confirmed by feeding fasted rats a diet containing various amounts of cysteine, and an increase in liver gluta-
58
TATEISHI ET AL.
thione was found to be dependent on the amount of cysteine intake (fig. 1 and table 1). As shown in figure 1, however, an in crease of glutathione appeared to be af fected by other constituents of the diet: Addition of protein, gelatin in this case, suppressed the increase of glutathione, un less excessive amount of cysteine was given. This effect of protein was more clearly demonstrated when gelatin was fortified with tryptophan to make the diet more adequate nutritionally. Tryptophan may exert its effect on intestinal absorp tion of cysteine, or on utilization of cys teine in the liver or other parts of the body. We did not examine the former possibility, and it is unlikely that physiological con centration of tryptophan severely inhibit absorption of cysteine. We studied the metabolic fate of di etary cysteine in the liver, especially in re lation to GSH, and the effect of trypto phan on it. When fasted rats are refed a nutritionally complete mixture of amino acids, an increase in protein synthesis is reported to be accompanied by a shift in the pattern of liver polysomes to the heavier side (34, 35). Moreover polysome aggregation and protein synthesis has been found to be influenced by the con centration of tryptophan when it was the most limiting amino acid in the diet (36, 37). In accordance with these results, ad dition of tryptophan stimulated aggrega tion of ribosomes (fig. 4-4) and incorpora tion of cysteine into liver and serum pro teins (fig. 3); liver glutathione did not increase much above the level observed in starved rats on the other hand (fig. 4-1 and 4-4). Under this condition liver gluta thione appeared to turn over fairly rapidly; specific radioactivity of liver glutathione became higher, when tryptophan was added to the diet. If cysteine derived from unlabeled liver glutathione had been in corporated into liver and serum proteins, stimulation of protein synthesis by addi tion of tryptophan must have been under estimated, since newly supplied radio active cysteine was diluted with cysteine derived from unlabeled glutathione. It is unknown what portions of the cysteine de rived from liver glutathione were used for protein synthesis, catabolized in the liver, excreted in the bile and exported from the liver in the blood stream. But rapid change
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in the level of liver glutathione under various nutritional conditions, clearly in dicated that there is a mobile pool of glu tathione for these requirements. There must be another pool of gluta thione for many reactions requiring sulfhydryl compounds in liver tissues (1-13). After fasting, the level of liver glutathione did not decrease below about 4 to 5 mM. Recently we reported (32) that injection of dibutyryl-3',5'-cyclic AMP caused tem porary decrease of glutathione to a mini mum of 2 to 3 HIM and increased y-glutamyltransferase activity. Injection of saline in place of dibutyryl-3',5'-cyclic AMP did not significantly affect the level of gluta thione or y-glutamyltransferase activity (32). Similar results on the effect of dibutyryl-3',5'-cyclic AMP are shown in table 2. This compound appears to induce y-glutamyltransferase, which degrades glu tathione releasing cysteine. This minimum level of glutathione, however, appears to be high enough for many reactions re quiring sulfhydryl compounds. In our re cent experiments done to estimate the ap parent naif-life of the cysteine moiety in glutathione, at least two pools of gluta thione with different turnover rates in rat liver were demonstrated. The possibility of the presence of two pools of glutathione in the liver was also suggested by Palekar et al. (38), although remains to sulfoxibe ex amined whether the it"methionine mine-sensitive pool" is the same as the "pool for cysteine." Storage of cysteine as glutathione seems to be better than storage of free cysteine in many respects. For instance glutathione is less auto-oxidizable than cysteine, and its oxidized and reduced forms are both more soluble than cysteine (cystine). Moreover almost all tissues contain a powerful reducing system for oxidized glutathione; that is, glutathione reducÃ-ase [EC 1.6.4.2] and NADPH generating sys tems, such as NADP-linked isocitrate dehydrogenase [EC 1.1.1.42] and two dehydrogenases in the hexose monophosphate shunt. LITERATURE CITED 1. Lohmann, K. (1932) Beitrag zur enzymatischen Umwandlung von synthetischem Methylglyoxal in Milchsäure.Biochem. Z. 254, 332-354.
RAT LIVER GSH AS A RESERVOIR OF CYSTEINE 2. Strittmatter, P. & Ball, E. G. (1955) For maldehyde dehydrogenase, a glutathionedependent enzyme system. J. Biol. Chem. 213, 445-461. 3. Edwards, S. W. & Knox, W. E. (1956) Homogentisate metabolism: The isomerization of maleylacetoacetate by an enzyme which requires glutathione. J. Biol. Chem. 220, 7991. 4. Suzuki, I. (1965) Oxidation of elemental sulfur by an enzyme system of Thiobacillus thiooxidans. Biochim. Biophys. Acta 104, 359-371. 5. Lipke, H. & Kearns, C. W. (1959) DDT dehydrochlorinase. I. Isolation, chemical prop erties, and spectrophotometric assay. J. Biol. Chem. 234, 2123-2128. 6. Booth, J., Boyland, E. & Sims, P. (1961) An enzyme from rat liver catalysing conjuga tions with glutathione. Biochem. J. 79, 516524. 7. Jocelyn, P. C. (1972) Biochemistry of the SH group, pp. xvi-xvii, Academic Press, London. 8. Kosower, N. S. & Kosower, E. M. (1974) Protection of membranes by glutathione. In: Glutathione (Flohe, L., Benöhr,H. Gh., Sies, H., Waller, H. D. & Wendel, A., eds.) pp. 216-227, Georg Thieme Publishers, Stuttgart. 9. Cohen, G. & Hochstein, P. (1963) Gluta thione peroxidase: The primary agent for the elimination of hydrogen peroxide in erythrocytes. Biochemistry 2, 1420-1428. 10. Kinoshita, J. H. & Masurat, T. (1957) Studies on the glutathione in bovine lens. Arch. Ophthalmol. 57, 266-274. 11. Bellows, J. G. & Shoch, D. E. (1950) Alloxan diabetes and the lens. Am. J. Ophthal mol. 33, 1555-1564. 12. Orlowski, M. & Meister, A. (1970) The •y-glutamyl cycle: A possible transport sys tem for amino acids. Proc. Nati. Acad. Sci., U.S. 67, 1248-1255. 13. Rink, H. (1974) Thiol compounds in radi ation biology. In: Glutathione (Flohe, L., Benöhr,H. Gh., Sies, H., Waller, H. D. & Wendel, A., eds.) pp. 206-216, Georg Thieme Publishers, Stuttgart. 14. Williamson, D. H. & Brosnan, J. T. (1974) Concentrations of metabolites in animal tis sues. In: Methods of Enzymatic Analysis, (Bergmeyer, H. U., éd.)pp. 2266-2302, Ver lag Chemie, Weinheim. 15. Williamson, D. H., Veloso, D., Ellington, E. V. & Krebs, H. A. (1969) Changes in the concentrations of hepatic metabolites on ad ministration of dihydroxyacetone or glycerol to starved rats and their relationship to the control of ketogenesis. Biochem. J. 114, 575584. 16. Pearson, D. J. & Tubbs, P. K. (1967) Carnitine and derivatives in rat tissues. Biochem. J. 105, 953-963. 17. Tateishi, N., Higashi, T., Shinya, S., Naruse, A. & Sakamoto, Y. (1974) Studies on the regulation of glutathione level in rat liver. J. Biochem. 75, 93-103. 18. Drysdale, J. W. & Munro, H. N. (1967)
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19.
20.
21. 22.
23.
24.
25.
26. 27.
28.
29.
30.
31. 32.
33. 34.
59
Polysome profiles obtained from mammalian tissues by an improved procedure. Biochim. Biophys. Acta 138, 616-618. Ip, C. C. Y. & Harper, A. E. (1974) Liver polysome profiles and protein synthesis in rats fed a threonine-imbalanced diet. J. Nutr. 104 252—263 Gamulin, S., Gray, C. H. & Norman, M. R. ( 1972) Comparison of methods for prepar ing polysomes free of glycogen. Biochim. Biophys. Acta 259, 239-242. Saville, B. ( 1958) A scheme for the colori metrie determination of microgram amounts of thiols. Analyst 83, 670-672. Owens, C. W. I. & Belcher, R. V. (1965) A colorimetrie micromethod for the determi nation of glutathione. Biochem. J. 94, 705711. Tietze, F. (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues. Anal. Biochem. 27, 502-522. Gaitonde, M. K. (1967) A spectrophoto metric method for the direct determination of cysteine in the presence of other naturally occurring amino acids. Biochem. J. 104, 627633. Lowry, O. H., Rosebrough, N. J., Fan, A. L. & Randall, R. J. (1951) Protein measure ment with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Jacobs, W. L. W. (1971) A colorimetrie assay for -y-glutamyltranspeptidase. Clin. Chim. Acta 31, 175-179. Persijn, J. P., van der Silk, W. & Zwart, W. A. (1971) Colorimetrie assay for -y-glutamyltranspeptidase. Clin. Chim. Acta 35, 239240. Mooz, E. D. & Meister, A. (1971) Gluta thione biosynthesis. In: Methods in Enzymol., (Tabor, H. & Tabor, C. W., eds.) Vol. XVII B, pp. 483-495, Academic Press, New York. Asofsky, R. (1974) Plasma protein synthe sis by liver. In: The liver; normal and ab normal functions, Part A (Becker, F. F. ed.) pp. 87-102, Marcel Dekker, Inc., New York. Beck, L. V., Rieck, V. D. & Duncan B. ( 1958) Diurnal variation in mouse and rat liver sulfhydryl. Proc. Soc. Exp. Biol. Med. 97, 229-231. Calcutt, G. & Ting, S. M. (1969) Diurnal variations in rat tissue disulphide levels. Naturwissenschaften 56, 419-420. Higashi, T., Tateishi, N., Naruse, A. & Saka moto, Y. (1976) Decrease of glutathione and induction of -y-glutamyltransferase by dibutyryl-3',5'-cyclic AMP in rat liver. Bio chem. Biophys. Res. Comm. 68, 1280-1286. Leaf, G. & Neuberger, A. (1947) The ef fect of diet on the glutathione content of the liver. Biochem. J. 41, 280-287. Fleck, A., Shepherd, J. & Munro, H. N. ( 1965) Protein synthesis in rat liver: In fluence of amino acids in diet on microsomes and polysomes. Science 150, 628-629.
60
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35. Webb, T. E., Blobel, G. & Potter, V. R. (1966) Polysomes in rat tissues. III. The response of the polyribosome pattern of rat liver to physiologic stress. Cancer Res. 26, 253-257. 36. Sidransky, H., Bongiorno, M., Sarma, D. S. R. ôtVerney, E. (1967) The influence of tryptophan on hepatic polyribosomes and protein synthesis in fasted mice. Biochem. Biophys. Res. Comm. 27, 242-248. 37. Wunner, W. H., Bell, J. & Munro, H. N. (1966) The effect of feeding with a tryptophan-free amino acid mixture on rat-liver
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38.
, 39'
40.
polysemes and ribosomal ribonucleic acid. Biochem. J. 101, 417-428. Palekar, A. G., Täte, S. S. & Meister, A. ( 1975 ) Decrease in glutathione levels of kidney and liver after injection of methionine sulfoximine into rats. Biochem. Biophys. Res. Comm. 62, 651-657. **• * *tes' F' ¿ London. Fisher, R. A. (1963) Statistical methods for research workers, 13th ed. Oliver and Boyd, London.
One is why the level of GSH in the liver is so high. The concentrations of all coenzymes in the liver except GSH are between 0.03 mM and 0.8 mM (14-16) and those of adenine nucleotides are between 0.6 and about 2 mM (17), whereas that of GSH is as high as 7 to 8 mM (17). Another ques-
transparency of the lens (10, 11); a y-glutamvl frrnim drmnr in iminn arirl fri n snort uunyi grouP uonor in ammo acia transport in the v-glutamyl cycle (12) and a pro-
Received for publication December 29, 1975. * Part of this work was presented orally at the Xth international Confess of Nutrition In August, 197B (Kyoto). The work was supported in part by grants
tector against radiation injury (13).
^^^"^^^^VIS^W
However, there are at least two unanswered questions about liver glutathione.
se^^s^°^^hoM Tateishi. 51
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*?-
besentto Dr.
TATEISHI ET AL.
tion is why the level of liver GSH changes so rapidly after fasting or after refeeding animals. After 1 or 2 days of starvation, the liver GSH content of rats is between two thirds and half the normal level, and after feeding these animals, it rapidly returns to normal ( 17 ). On the contrary, the levels of coenzymes and adenine nucleotides in the liver do not change markedly after fasting and refeeding rats (17), except for that of acetyl CoA, which increases during star vation (16). These findings on rat liver GSH can not be adequately explained by the known physiological roles or GSH de scribed above. This paper reports a possible further role of liver GSH as a reservoir of cysteine. MATERIALS
AND METHODS
Animais. Male albino rats,3 weighing 130 g to 160 g, were housed individually in cages in an air-conditioned room (25°) and given food and water ad libitum, unless otherwise indicated. The rats were fed a stock diet4 prior to experimentation. The control diet contained: (in %) com oil,5 6; salt mixture,6 1; vitamin mixture,7 0.5; choline chloride,8 0.5; bovine milk casein,9 18; cellulose,10 5; corn starch,9 60 and agar8 to 100%. When powdered gelatin " or agar were used to replace casein, various amounts of cysteine (and/or tryptophan) were added to the diet, as described in table 1, and figures 1 and 2. Each rat was given 10 g of diet/day. This amount of diet was usually consumed almost completely except for the protein-free diet (table 1). In experiments using radioactive cysteine, L-[35S]-cysteine hydrochloride 12was added to the powdered diet described above and the diet was solidified by adding an equal weight of 4% agar solution. Therefore, 20 g of diet, equivalent to 10 g of the pow dered diet, were given per day in these ex periments. These amounts of food were usually consumed completely within 12 hours by rats which had been fasted for 24 to 48 hours. Preparation of post-mitochondrial super natant and analysis of polysome profiles of rat liver. Rats of each dietary group were killed by decapitation and their liver was quickly removed by the method of Drysdale and Munro (18). The liver was ho mogenized in 4 volumes of 0.25 M sucrose
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containing TKM buffer (0.05 M Tris buffer, pH 7.6, 0.025 M KCl, 0.005 M MgCl2) by 4 strokes of a loosely fitting motor-driven Teflon1S glass homogenizer and the homogenate was centrifugea at 18,000 X g, for 15 minutes at 2°.The post-mitochon drial supernatant thus obtained was mixed with rabbit anti-horse ferritin ™serum in the proportion of 40:1 (v/v) to remove ferritin particles (19) and «-amylase was added to degrade glycogen (20), because both ferritin and glycogen interfere with the polysome pattern. Sodium deoxycholate and Triton X-100 15were added to the postmitochondrial supernatant to concentra tions of 0.5% and the preparations were di luted with two volumes of TKM buffer. Then samples of 0.6 to 0.65 ml ( A2eo= about 18) were applied to a linear sucrose gradient ( 10 to 25% in TKM buffer, total volume = 28 ml ) with 1 ml of 2 M sucrose at the bottom. A layer of 0.5 ml of 3.5% sucrose was applied on top to trap soluble macromolecules. The tubes were centrifuged in a swinging-bucket rotor at 25,000 rpm for 3 hours in an ultracentrifuge.1* The absorption of the gradient at 254 nm was recorded automatically using a 10 mm light path flow-cell in an ultraviolet ana lyzer.17 3Donryu strain : Kitnyama LABES Co.. Kyoto. 4Stock diet (type M) containing (In %) carbo hydrate, 51.8 ; crude Hpld, 5.0 ; crude protein, 24.6 ; minerals. 7.4 : cellulose. 4.2 ; (Oriental Yeast Ind. Co., Ltd.. Tokyo.) "Nisshin Oil Co., Tokyo. « Salt mixture ; McCollum's salt misture containing (In %) ; calcium phosphate, monobasic 13.3, sodium chloride 4.29, magnesium sulfate, dlhydrate 14.84, sodium phosphate, dibasic, anhydrous 8.59, calcium lactate 32.2, potassium phosphate, dibasic 23.6 and ferric citrate 2.92 (Nakaral Chemicals, Ltd., Kyoto.) 'Vitamin mixture containing (In %) ; thlamln hydrochloride 0.059. rlboflavln 0.059, nlcotlnlc acid 0.294. calcium pantothenate 0.235, pyrldoxlne hydrochloride 0.029, menaqulnone 0.006, blotln 0.001, folle acid 0.002, cyanocobalamln 0.0002. Inosltol 1.176, ascorbic acid 0.588 and lactose 97.551. (Tanabe Amino Acid Research Foundation, Osaka.) • Nakaral Chemicals, Ltd., Kyoto. »WakoPure Chemicals Industries, Ltd., Osaka. »»Kokusaku Pulp Co., Tokyo. 11E. Merck AG, Darmstadt. u Product of the Radiochemlcal Centre, Amersham. Purchased from Japan Isotope Association. "E. I. DuPont de Nemours & Co. " Pentex ; Miles Laboratories, Inc., Kankakee, "Rohm Sc Haas Co., Philadelphia, Pennsylvania, Simultaneous use of sodium deoxycholate and Triton X-100 Is recommended by Dr. Ken Higashl In our laboratory. «In the SW 25.1 rotor of a Beckman L2 ultracentrifuge (Beckman Instruments, Inc., Splnco Divi sion, Palo Alto, Calif.). «ISCO Model 640 density gradient fractionator (Instrumentation Specialties Co., Inc., Lincoln, Nebraska).
53
RAT LIVER GSH AS A RESERVOIR OF CYSTEINE TABLE 1 Effect of the amount of L-cysteine in the diet on the increase of liver GSH
DietFasted
consumptiong7.4±1.0 wt.g50. liver4.02±0.50*2.84
for 40 hours Refed for 12 hours on Protein-free diet (18% agar) + CySH 0.5% +CySH 1.0% 5.0%CySH +CySH
±0.35.4±0.2 ±0.47 5.12±0.66 6.6±1.8 7.05±0.43 6.7±0.8 7.44±0.563.40±0.47 5.8 ±1.88.1
5.8±0.2 5.8±0.3 5.7±0.66.4±0.5
free protein diet (18% gelatin) + CySH 0.5% +CySH 1.0% +CySH 5.0%GSHnmoles/g
4.62±0.36 6.10±0.27 8.02±0.85Food
±0.8 9.8±0.7 9.6±0.4 9.1±0.7Liver
7.3±0.5 7.4±0.8 8.5±0.7
Protein diet (18% casein)
8.64±0.11
9.7±0.3
6.7±0.3
Rats weighing 145 to 160 g were starved for 40 hours and then fed 10 g of the indicated diets. Added amino acids are expressed as percentage of protein or agar. Twelve hours later, liver GSH was estimated. For the protein-free diet, n = 8. For other diets, n = 4. * Mean ±SD.
Preparation of trichloroacetic acid extract of rat liver. Rats were killed by decapita tion and their livers were quickly removed. A 25% (w/v) liver homogenate was pre pared in 0.15 M KC1 by 10 strokes of a motor-driven, Potter-type Teflon13 glass homogenizer. Then the homogenate was mixed with one-quarter volume of cold 25% trichloroacetic acid containing 5 mM EDTA and the mixture was centrifuged to separate the "acid supernatant". Cysteine can be determined more accurately using this acid supernatant than using the super natant from the "MSE homogenate" (0.21 M mannitol, 0.07 M sucrose and 0.1 mM EDTA ) as in previous work ( 17), because it contains no materials interfering with the ninhydrin reaction. This acid super natant was used for determination of both cysteine and glutathione. Determination of glutathione, cysteine and protein. GSH was estimated by the method of Saville (21). Total glutathione was also determined enzymatically (22, 23 ). The values determined by the two pro cedures always coincided well. This indi cates that most of the acid soluble sulfhydryl material in rat liver is glutathione with negligible amounts of other sulfhydryl compounds and that glutathione is present mainly in the reduced form.18 Cysteine was determined by the method of Gaitonde (24). Protein was assayed by the procedure
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of Lowry et al. (25) using bovine serum albumin as a standard. Assay for y-glutamyltransferase [EC 2.3.2.2]. The y-glutamyltransferase activity of the liver homogenate was assayed as described previously (17) using y-glutamylp-nitroanilide as substrate and glycylglycine as a -y-glutamyl group acceptor ( 26, 27). Measurement of radioactivity. A sample (50 to 100 /xi) of 25% liver homogenate was loaded on a glass fiber filter ( d = 2.4 cm) 10and immersed in 5% trichloroacetic acid containing 1 mM EDTA. The glass fiber filter was then boiled in this mixture for 10 minutes, washed successively three times with the same concentration of trichloroacetic acid, twice with ethanolether (1:1 v/v) and once with ethanolether (1:3 v/v), and dried. The acidic ex tract of liver obtained by adding onequarter volume of 25% trichloroacetic acid containing 5 /¿M EDTA to the 25% homog enate was added to ten volumes of alkaline tissue solubilizer.20 GSH and cysteine in the acid extract of the liver were separated by paper electrophoresis as described by Mooz "Naruse, A., Tatelehl, N., Hlgashl, T. & Sakamoto, T. (1973) 46th Ann. Meeting Jap. Blochem. Soc. (Nagoya) (Abstract; Seikagaku 45, 430). «Whatman GF/F filter (W & R Baiston Ltd., Maidstone). »NCS tissue solubilizer (Amersham/Searle Cor poration, Arlington Heights, Illinois).
54
TATEISHI ET AL.
and Meister (28). The bands of the two ma terials were located with ninhydrin reagent and cut off. Then they were immersed in, or mixed with a toluene scintillation fluid (8 g of 2,5-diphenyloxazole and 0.1 g of ì,4-bis[2-(5-phenyloxazolyl)]-benzene in 1 liter of toluene ) and their radioactivity was counted in a scintillation spectrometer.21 RESULTS Dependency of the rise in glutathione level on the amount of cysteine in the diet. To establish a quantitative relationship be tween the content of cysteine in the diet and the increase of liver GSH after feed ing starved rats, a protein-free diet con taining agar in place of casein and supple mented with cysteine at concentrations of 0.5%, 1.0% or 5.0% of the agar was fed to rats which had been fasted for 40 hours (table 1). The results showed that the increase of liver GSH 12 hours later was proportional to the amount of cysteine added, when this was within a physiologi cal range. With an unphysiologically high concentration of cysteine ("5.0%") the liver GSH concentration was not propor tional to the amount of cysteine in the diet.
i
r O
10
20 30 40 50 60 70 AMOUNT OFINGESTED CYSTEINE (MO/RAT)
80
Fig. 1 Effect on liver glutathione of ingestion of protein-free (agar) or gelatin-containing diet containing various amounts of cysteine after star vation. After fasting for 40 hours, the rats were given 10 g of diet containing 18% gelatin or agar, supplemented with cysteine corresponding to 0, 0.5%, 1.0% or 5.0% of the agar or gelatin. Twelve hours later, their liver glutathione level was estimated. The amount of ingested cysteine per rat was calculated from the intake of diet and its cysteine content. Each point represents one rat. —X—; agar diet, —•—;gelatin diet.
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Similar results were obtained when gela tin was used as the sole protein constitu ent of the diet and the diet was fortified with various amounts of cysteine (0.5%, 1.0% or 5.0% with respect to gelatin). Under physiological nutritional conditions (0.5% to 1.0% cysteine), a higher GSH level was attained with the protein-free diet than with gelatin diet, on the basis of net uptake of cysteine. Rats seemed to pre fer the gelatin diet to the agar diet so that their intake of the former was often larger than that of the agar diet. Therefore, in figure 1 the cysteine intake by each rat is plotted against the GSH content of the liver. Addition of gelatin to the diet in stead of agar suppressed the increase of liver GSH when the same amount of cys teine was ingested. Effect of the amount of L-tryptophan in the diet on the increase of liver GSH. The increase of liver GSH was dependent on the amount of cysteine added, as described above. However, the difference between the increases of GSH in the groups of rats fed agar-diet and those fed gelatindiet containing the same amount of cys teine suggested that some factors other than the cysteine content itself affected the hepatic GSH level. To examine this, the effect of a more nutritionally adequate gelatin-diet was tested. Namely, the diet was fortified with tryptophan, which is the first limiting amino acid in gelatin, in addition to cysteine. It was found that the increase in GSH when the cysteine-containing diet was fed was strongly inhibited by the addition of tryptophan (fig. 2). The level of GSH was lowered by addition of tryptophan at levels of 0.25% to 1.0% of the gelatin when the level of cysteine in diet was 0.5% to 1.0% of that of gelatin. With the diet containing the larger amount of cysteine (5% of the gelatin), the level of liver GSH was not lowered by adding tryptophan. Incorporation of dietary [3iS]-cysteine into rat liver fractions and serum under different nutritional conditions. To exam ine the metabolic fate of cysteine derived from food, rats were fasted for 40 hours and then fed diets containing gelatin as 21Tri-Carb scintillation spectrometer Model 3320 (Packard Instrument Co., Inc., Downers Grove, Illinois).
55
RAT LIVER GSH AS A RESERVOIR OF CYSTEINE
the protein constituent fortified (1) with cysteine, (2) cysteine + tryptophan or (3) excess cysteine + tryptophan. L-[35S]-Cysteine was added to all these diets. As shown in the previous section and figure 3, addition of tryptophan to the diet sup pressed the increase of liver GSH induced by dietary cysteine, although it did not affect the total intake of food. Addition of excess cysteine masked this effect of tryp tophan. Examination of the distribution of [35S]-cysteine in the acid soluble fraction of the liver and in liver GSH indicated that the differences in hepatic GSH contents of the three groups resulted from differences in the incorporation of dietary cysteine into GSH and in the turnover of GSH. The results showed that tryptophan stimulated the incorporation of [35S]-cysteine into the acid-insoluble fraction of liver. If cysteine
JJMOLES/G LIVER
0.1 —i—
0.2
CySH J*WLES/a LIVER
GSH 103CPM/IO MGLlVtR
*
„6
ACIDSOL. CYSH GSH
ACIDppr. SERUM (10ill)
«8.03oì14.0|ADDED
:0
CïSHas
»of gelatinADDED
0_T||ht11S 1-r~
5^
s-°lTup
7i) i.rf as r. afa j selitln •*,(
_j-rt;É ^Õ0
Fig. 2 Effect of addition of L-tryptophan to a diet containing gelatin and various amounts of cysteine on the increase of liver glutathione after starvation. The experimental conditions were similar to those described in the legend of table 1. The vertical bars show SD. The open triangle shows the level of liver glutathione after fasting for 40 hours. When the columns were marked from left to right, a, b,
1, liver weights
(g) of
each group of rats were (a) 7.2 ±0.4 (4), (b) 7.3 ±0.5 (4), (c) 7.1 ±0.2 (4), (d) 7.1 ±1.0 (6), (e) 7.4 ±0.8 (4), (f) 7.1 ±0.2 (4), (g) 7.8 ±0.7 (4), (h) 7.2 ±0.1 (4). (i) 8.5 ±0.8 (4), (j) 7.2 ±1.0 (4), (k) 8.1 (2) and (1) 8.3 ±0.6 (4). The two bars marked by asterisks differ from each other at P < 0.01. The two bars marked by crosses differ each other at P < 0.001. Student's i-test wasfrom employed (39, 40).
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Fig. 3 Incorporation of ["S]-cysteine from diet containing gelatin with cysteine alone, cys teine and tryptophan, or tryptophan and excess cysteine into liver fractions and serum of rats after starvation. After fasting for 40 hours, the rats were given 20 g of gelatin-containing diet solidified with agar. Dietary treatments are shown as 1) n 1% cysteine, 2) •1% cysteine +1% tryptophan, 3) 0 3% cysteine + 1% tryptophan. Added amino acids are expressed as % of gelatin. In 1) and 2), 10 /¿Ci and in 3) 30 /¿Ci of L-[MS]cysteine were added to the diet to give the same specific radioactivity. The three groups of rats consumed more than 90% of the food within 2 to 3 hours. The bars show standard deviations. Other experimental conditions were similar to those described in the legend of table 1. Liver weights of the rats are 1) 8.5 ±0.8 g, 2) 8.8 ±0.3 g and 3) 8.3 ±0.6 g, respectively.
is derived from GSH, the differences in net incorporations of cysteine into liver proteins in the different groups are in fact larger than those shown in figure 3. The incorporation of [35S]-cysteine into the serum of rats was also stimulated by the addition of tryptophan to the diet. The in corporation of radioactivity into serum proteins was essentially the same as those into whole serum in the three groups. When excess cysteine (3% of the gelatin content) with \% tryptophan was fed to
r,6
TATEISHI ET AL.
IA2S4=0.1
Fig. 4 Hepatic polysome patterns of rats under various nutritional conditions. Rats were fasted for 40 hours. The polysemes were obtained from rats under the following conditions: (1) fasted, (2) 14 hours after receiving a protein-free (18% agar) diet supplemented with cysteine corre sponding to 1% of the agar, (3) 14 hours after receiving a gelatin-containing (18%) diet forti fied with cysteine corresponding to 1% of the gelatin and fortified (4) 14 with hourscysteine after receiving the gelatin-diet ("1%") and tryptophan ("\%"). Columns on the left show the content of liver glutathione (^moles/g liver). Liver weights of the rats are (1) 4.5, (2) 7.4, (3) 7.6 and (4) 7.5 g, respectively. Other condi tions were as described.
rats, the incorporations of [35S]-cysteine into all fractions were larger. This prob ably indicates that under these conditions both GSH synthesis and other systems utilizing cysteine were maximally active.
Influence of addition of tryptophan to the diet on the liver polysome patterns. The results described in the previous sec tion suggest that the activities for protein synthesis in the liver were different in the different groups. Accordingly, we exam ined the polysome patterns of the postmitochondrial supernatant fractions of the livers of rats under various nutritional con ditions by sucrose density gradient centrifugation. All rats were fed the stock diet4 ad libitum and then fasted for 40 hours before the experiment. Then, they were treated as follows. Group 1 were examined directly (fig. 4-1). Group 2 were given protein-free diet supplemented with cysteine alone and killed 14 hours after the start of feeding (fig. 4-2). Group 3 were given diet containing cysteine and gelatin as a protein source and examined 14 hours later (fig. 4-3). Group 4 were given diet containing cysteine and tryptophan in addition to gelatin and examined 14 hours later (fig. 4-4). Rats with similar food in takes were selected for examination. Al though the GSH levels were very different in group 1 and 2, the polysome patterns of the two groups were similar, both showing large number of monosomes and disomes with fewer polysomes. The glutathione contents of groups 3 and 4 were also dif ferent but their polysome patterns were both typical profiles of well-fed animals. However, the pattern of group 4 (fig. 4—4) indicated more active protein synthesis than that of group 3 (fig. 4-3) with a de crease in monosomes and an increase in larger polysomes. These results are in agreement with those in figure 3, indicat-
TABLE 2 Effect of dibutyryl-3',5'-cyclic AMP on liver GSH and cysteine contents and y-glutamyltransferase (y-GTP) activity
TimeafterinjectionGSHCysteineLiver7-GTP wt.Liverprotein limole»/gliver 0 90
3.83±0.39* 2.84±0.23
nmoles/g liver
149±21 385±21
mg/g liver
nmoles/hr/mg protein
82± 2 309±56
4.2±0.2 3.9±0.1
208±17.5 223±20.6
* MeaniSD. Eight rats (weighing 135 to 150 g) were fasted for 16 hours (from 1800 to 1000 hours) and then 5 mg of dibutyryl-3',5'-cyclic AMP and 2 mg of theophylline per 100 g body weight were injected intraperitonealry. Four rats were killed immediately and four were killed 90 minutes later. For estimation of the 7-GTP activity the KC1 homogenate was used instead of the MSB homogenate used previously (17), because its enzyme activity was higher.
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RAT LIVER GSH AS A RESERVOIR
ing that supplementation of the gelatin diet with tryptophan enhanced the incor poration of cysteine into hepatic proteins and probably also into serum proteins, most of which are synthesized in the liver (29). At the same time, liver glutathione appeared to be mobilized. The clear differ ence polysoine patterns of (fig. in 4-2)the ("fasting-pattern") andgroup group2 3 or 4 (fig. 4-3 or 4-4) ("refed-pattern") must be considered in relation with the re sults shown in figure 2 and table 1. Active protein synthesis was accompanied by sup pressed increase of glutathione, as ob served in group 4, although nearly the same amount of cysteine was consumed in groups 2 and 4. These findings correspond to the results shown in table 1, figures 1 and 2, where an increase in glutathione ob served in the rats fed a cysteine contain ing agar diet was suppressed by using gelatin instead of agar, and more markedly by using gelatin fortified with tryptophan. The results suggest that when other condi tions are adequate and cysteine can be utilized for protein synthesis, increase of liver GSH is suppressed, and the cysteine moiety of GSH is mobilized for protein synthesis. However, when the supply of cysteine is sufficient but other conditions for protein synthesis are not adequate, or when there is excess cysteine over that re quired for protein synthesis, the cysteine is stored in the form of glutathione. ICOr*
2» M
72
96
HOURS
Of
" 0
2*
72
»
STARVATION
Fig. 5 Effect of prolonged fasting on liver glutathione, body weight and liver weight. Rats weighing 130 to 150 g were starved but given free access to water. Fasting was started at 1000 hours. Four rats each were killed 24, 48, 72 or 96 hours later and their liver glutathione (-O-), body weight (-X-) and liver weight (-•-) were estimated. Values of 145 g body weight and 6.5 g liver weight were taken as 100%. Vertical bars show so.
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OF CYSTEINE
57
Liver glutathione pool as a reservoir of cysteine. Assuming that liver GSH is uti lized as a source of cysteine for protein synthesis, the next problem is how much liver GSH can be mobilized. On fasting, the liver GSH concentration decreased rapidly to two-thirds of the normal concen tration within 1 day. However, it is inter esting that the concentration did not de crease below about 4 to 5 mM. Moreover in most rats, the concentration did not con tinue to decrease during further fasting for 3 to 4 days, although the liver weight and body weight continued to decrease gradu ally (fig. 5). In some rats, the GSH con centration in the liver decreased to a very low concentration of about 2 m,M with simultaneous sharp decreases in body weight and liver weight when rats were dying after prolonged starvation. This low concentration of GSH appears to represent the minimal requirement for survival. These findings suggest that there may be at least two pools of liver glutathione; one for many reactions in which glutathione itself is involved and the other for supply of cysteine. DISCUSSION
As shown previously (17), turnover of liver GSH was rapid in fasted rats and its increase on feeding these rats depended upon food intake; about 10 g of diet was necessary to attain the maximal increase in rats weighing 150 g. An intake of about 10 g of diet/day was necessary to maintain the normal level of GSH thus attained, and a smaller amount of food intake resulted in a decrease in the GSH content. Circadian change in liver GSH (30, 31), which is apparently related to the habit of nocturnal feeding, has also been reported. Moreover, recent results in our laboratory (32) showed that rat liver GSH decreased temporarily to a level of 2 to 3 mM after injection of dibutyryl-3',5'-cyclic AMP. These results indicate that GSH in rat liver turns over fairly rapidly under nor mal physiological conditions. Studies by Leaf and Neuberger (33) clearly demon strated that administration of cystine or methionine to fasted rats resulted in a significant increase in liver GSH. Their results were confirmed by feeding fasted rats a diet containing various amounts of cysteine, and an increase in liver gluta-
58
TATEISHI ET AL.
thione was found to be dependent on the amount of cysteine intake (fig. 1 and table 1). As shown in figure 1, however, an in crease of glutathione appeared to be af fected by other constituents of the diet: Addition of protein, gelatin in this case, suppressed the increase of glutathione, un less excessive amount of cysteine was given. This effect of protein was more clearly demonstrated when gelatin was fortified with tryptophan to make the diet more adequate nutritionally. Tryptophan may exert its effect on intestinal absorp tion of cysteine, or on utilization of cys teine in the liver or other parts of the body. We did not examine the former possibility, and it is unlikely that physiological con centration of tryptophan severely inhibit absorption of cysteine. We studied the metabolic fate of di etary cysteine in the liver, especially in re lation to GSH, and the effect of trypto phan on it. When fasted rats are refed a nutritionally complete mixture of amino acids, an increase in protein synthesis is reported to be accompanied by a shift in the pattern of liver polysomes to the heavier side (34, 35). Moreover polysome aggregation and protein synthesis has been found to be influenced by the con centration of tryptophan when it was the most limiting amino acid in the diet (36, 37). In accordance with these results, ad dition of tryptophan stimulated aggrega tion of ribosomes (fig. 4-4) and incorpora tion of cysteine into liver and serum pro teins (fig. 3); liver glutathione did not increase much above the level observed in starved rats on the other hand (fig. 4-1 and 4-4). Under this condition liver gluta thione appeared to turn over fairly rapidly; specific radioactivity of liver glutathione became higher, when tryptophan was added to the diet. If cysteine derived from unlabeled liver glutathione had been in corporated into liver and serum proteins, stimulation of protein synthesis by addi tion of tryptophan must have been under estimated, since newly supplied radio active cysteine was diluted with cysteine derived from unlabeled glutathione. It is unknown what portions of the cysteine de rived from liver glutathione were used for protein synthesis, catabolized in the liver, excreted in the bile and exported from the liver in the blood stream. But rapid change
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in the level of liver glutathione under various nutritional conditions, clearly in dicated that there is a mobile pool of glu tathione for these requirements. There must be another pool of gluta thione for many reactions requiring sulfhydryl compounds in liver tissues (1-13). After fasting, the level of liver glutathione did not decrease below about 4 to 5 mM. Recently we reported (32) that injection of dibutyryl-3',5'-cyclic AMP caused tem porary decrease of glutathione to a mini mum of 2 to 3 HIM and increased y-glutamyltransferase activity. Injection of saline in place of dibutyryl-3',5'-cyclic AMP did not significantly affect the level of gluta thione or y-glutamyltransferase activity (32). Similar results on the effect of dibutyryl-3',5'-cyclic AMP are shown in table 2. This compound appears to induce y-glutamyltransferase, which degrades glu tathione releasing cysteine. This minimum level of glutathione, however, appears to be high enough for many reactions re quiring sulfhydryl compounds. In our re cent experiments done to estimate the ap parent naif-life of the cysteine moiety in glutathione, at least two pools of gluta thione with different turnover rates in rat liver were demonstrated. The possibility of the presence of two pools of glutathione in the liver was also suggested by Palekar et al. (38), although remains to sulfoxibe ex amined whether the it"methionine mine-sensitive pool" is the same as the "pool for cysteine." Storage of cysteine as glutathione seems to be better than storage of free cysteine in many respects. For instance glutathione is less auto-oxidizable than cysteine, and its oxidized and reduced forms are both more soluble than cysteine (cystine). Moreover almost all tissues contain a powerful reducing system for oxidized glutathione; that is, glutathione reducÃ-ase [EC 1.6.4.2] and NADPH generating sys tems, such as NADP-linked isocitrate dehydrogenase [EC 1.1.1.42] and two dehydrogenases in the hexose monophosphate shunt. LITERATURE CITED 1. Lohmann, K. (1932) Beitrag zur enzymatischen Umwandlung von synthetischem Methylglyoxal in Milchsäure.Biochem. Z. 254, 332-354.
RAT LIVER GSH AS A RESERVOIR OF CYSTEINE 2. Strittmatter, P. & Ball, E. G. (1955) For maldehyde dehydrogenase, a glutathionedependent enzyme system. J. Biol. Chem. 213, 445-461. 3. Edwards, S. W. & Knox, W. E. (1956) Homogentisate metabolism: The isomerization of maleylacetoacetate by an enzyme which requires glutathione. J. Biol. Chem. 220, 7991. 4. Suzuki, I. (1965) Oxidation of elemental sulfur by an enzyme system of Thiobacillus thiooxidans. Biochim. Biophys. Acta 104, 359-371. 5. Lipke, H. & Kearns, C. W. (1959) DDT dehydrochlorinase. I. Isolation, chemical prop erties, and spectrophotometric assay. J. Biol. Chem. 234, 2123-2128. 6. Booth, J., Boyland, E. & Sims, P. (1961) An enzyme from rat liver catalysing conjuga tions with glutathione. Biochem. J. 79, 516524. 7. Jocelyn, P. C. (1972) Biochemistry of the SH group, pp. xvi-xvii, Academic Press, London. 8. Kosower, N. S. & Kosower, E. M. (1974) Protection of membranes by glutathione. In: Glutathione (Flohe, L., Benöhr,H. Gh., Sies, H., Waller, H. D. & Wendel, A., eds.) pp. 216-227, Georg Thieme Publishers, Stuttgart. 9. Cohen, G. & Hochstein, P. (1963) Gluta thione peroxidase: The primary agent for the elimination of hydrogen peroxide in erythrocytes. Biochemistry 2, 1420-1428. 10. Kinoshita, J. H. & Masurat, T. (1957) Studies on the glutathione in bovine lens. Arch. Ophthalmol. 57, 266-274. 11. Bellows, J. G. & Shoch, D. E. (1950) Alloxan diabetes and the lens. Am. J. Ophthal mol. 33, 1555-1564. 12. Orlowski, M. & Meister, A. (1970) The •y-glutamyl cycle: A possible transport sys tem for amino acids. Proc. Nati. Acad. Sci., U.S. 67, 1248-1255. 13. Rink, H. (1974) Thiol compounds in radi ation biology. In: Glutathione (Flohe, L., Benöhr,H. Gh., Sies, H., Waller, H. D. & Wendel, A., eds.) pp. 206-216, Georg Thieme Publishers, Stuttgart. 14. Williamson, D. H. & Brosnan, J. T. (1974) Concentrations of metabolites in animal tis sues. In: Methods of Enzymatic Analysis, (Bergmeyer, H. U., éd.)pp. 2266-2302, Ver lag Chemie, Weinheim. 15. Williamson, D. H., Veloso, D., Ellington, E. V. & Krebs, H. A. (1969) Changes in the concentrations of hepatic metabolites on ad ministration of dihydroxyacetone or glycerol to starved rats and their relationship to the control of ketogenesis. Biochem. J. 114, 575584. 16. Pearson, D. J. & Tubbs, P. K. (1967) Carnitine and derivatives in rat tissues. Biochem. J. 105, 953-963. 17. Tateishi, N., Higashi, T., Shinya, S., Naruse, A. & Sakamoto, Y. (1974) Studies on the regulation of glutathione level in rat liver. J. Biochem. 75, 93-103. 18. Drysdale, J. W. & Munro, H. N. (1967)
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35. Webb, T. E., Blobel, G. & Potter, V. R. (1966) Polysomes in rat tissues. III. The response of the polyribosome pattern of rat liver to physiologic stress. Cancer Res. 26, 253-257. 36. Sidransky, H., Bongiorno, M., Sarma, D. S. R. ôtVerney, E. (1967) The influence of tryptophan on hepatic polyribosomes and protein synthesis in fasted mice. Biochem. Biophys. Res. Comm. 27, 242-248. 37. Wunner, W. H., Bell, J. & Munro, H. N. (1966) The effect of feeding with a tryptophan-free amino acid mixture on rat-liver
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