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Studies on hepatic protein synthesis in rats force-fed a threonine-devoid diet
EXPERIMENTAL
AND
Studies
MOLECULAR
on
PATHOLOGY
Hepatic
13, 12-24
Protein
Synthesis
a Threonine-Devoid HERSCHEL Department
(1970)
SIDRANSKY
in Rats
Force-Fed
Diet’
AND ETHEL
VERNEY
of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 16213 Received February 11, 19YO
Young Sprague-Dawley rats were force-fed a threonine-devoid or complete diet for one, two, or three feedings in 1 day. Rats force-fed the threonine-devoid diet for three feedings and killed the following morning (16 hours later) developed increased hepatic protein synthesis and decreased gastrocnemius muscle protein into proteins. At the synthesis, as measured by in vivo “C-leucine incorporation same time, sucrose density-gradient patterns of hepatic polyribosomes of experimental animals demonstrated a shift toward heavier aggregates, and in VitTO hepatic protein synthesis of experimental animals w&s increased when compared to control animals. Similar changes were not observed in experimental animals tube-fed one or two feedings and killed up to 6 hours later. Increases in hepatic protein synthesis were observed in animals killed 16 hours after two feedings of the threonine-devoid diet. Thus, it appears that the earliest increase in hepatic protein synthesis due to force-feeding a threonine-devoid diet occurs after two or three feedings in 1 day followed by an interval of 16 hours.
In earlier studies from this laboratory (Sidransky and Farber, 1958a; Sidransky and Clark, 1961) we reported that young rats force-fed a threonine-devoid diet for 3 to 7 days developed many o’f the pathologic changes resembling those found in children with kwashiorkor. Among the biochemical changes in the livers of these experimental animals were increasesin lipid, in glycogen, in total RNA, in the incorporation in vivo of 32Por 14C-orotate into ribosomal RNA, and in the incorporation in vivo and in vitro of 14C-amino acid into proteins (Sidransky and Farber, 19588; Sidransky et al., 1964; Sidransky et al., 1969; Staehelin et al., 1967). Along with the increased hepatic protein synthesis, the hepatic ribosomes revealed a shift from lighter toward heavier polyribosomes with a decrease in monosomes(Sidransky et al., 1964; Staehelin et al., 1967). The present investigation was undertaken to learn more about the pathogenesis of the enhanced hepatic protein synthesis in rats force-fed a threonine-devoid diet. In an attempt to gain someinsight into the mechanism responsible for the alteration in protein metabolism in the liver, we studied how early the changes could be detected after beginning the threonine-devoid diet. This was especially relevant since earlier studies by others (Fleck et al., 1965; Sox and Hoagland, 1966; Webb et al., 1966; Wilson and Hoagland, 1967; Wunner et al., 1966) and from our own labora1 This investigation was supported by United States Public Health Service Grant AM05908 from the National Institute of Arthritis and Metabolic Diseases and by Grant GM-10269 from the National Institute of General Medical Sciences, 12
HEPATIC
PROTEIN
SYNTHESIS
IN THREONINE
DEFICIENCY
13
tory (Staehelin et al., 1967) have indicated that the livers of fasted animals respond rapidly to a single feeding of proteins or of a complete amino acid mixture with a shift in polyribosomes from lighter to heavier aggregates and with enhanced protein synthesis. Therefore, in this study rats were force-fed a threonine-devoid diet for one, two, or three feedings in 1 day, and in viva and in vitro measurements of hepatic protein synthesis were conducted. Our present results indicate that it was necessary to wait 16 hours after the force-feeding of the deficient diet for two or three feedings before increases in hapatic protein synthesis were detectable. METHODS Male and female rats of the Sprague-Dawley strain,2 1 month old, weighing on the average 70 gm, were used in most experiments. In some experiments in which animals were fasted for 1 to 5 days, animals weighing 120 to 180 gm were used. The animals were maintained on a commercial diet3 before the experiments were begun. In all experiments groups of rats, each of the same sex, age, and weight, were used. The basal diet was similar to that used in earlier experiments (Sidransky and Clark, 1961; Sidransky and Farber, 1958a, 1958b; Sidransky et al., 1964; Sidransky et al., 1969; Staehelin et al., 1967). The percentage of the components was as follows: essential amino acids, 9.2; nonessential amino acid, 8.1; vitamin-sucrose mixture, 5; salt mixture, 4; corn oiJ4 5; cod liver oiJ5 1.5; and dextrin, 67.2. Dextrin was substituted for the threonine in the threonine-devoid diet and for phenylalanine in the phenylalanine-devoid diet. In a few experiments, instead of the preceding diets, rats were fed a complete amino acid mixture or a threonine-devoid amino acid mixture. The amino acid mixtures (essential and nonessential amino acids) were the same as those used in the basal diet. The diets or amino acid mixtures were blended with distilled water so that a milliliter of diet mixture contained 0.5 gm of diet. Rats were force-fed using plastic tubes at 7:30 AM, 1:00 PM, and 7: 30 PM. The amount of diet administered in the one or two feeding experiments was 1.2 to 3 gm per feeding. When animals were force-fed for 3 feedings during 1 day, they received an average daily feeding of 1 gm diet/l0 gm initial body weight unless indicated otherwise as in Table III. Except in the case where animals were fasted, animals were tube-fed the complete diet for 1 to 5 days before the experiments were begun. Rats had free access to water. They were housed in individual wire cages with raised bottoms and kept in an air-conditioned room which was without light from 8 PM to 8 AM. Rats were weighed at the beginning and end of each experiment. They were anesthetized with ether and exsanguinated at appropriate intervals after the last feeding. In animals receiving three feedings during 1 day, the animals were killed 16 hours after the last evening feeding (Tables III and V). In in vivo incorporation experiments in which rats were tube-fed once or twice, rats received 2.5 to 5 &Ji (10 &.?i/~ mole) of 14C-L-leucine, uniformly labeled, in aqueous solution either intraperitoneally or orally 0.5 to 5 hours before killing (Table I). In experiments in which rats were tube-fed three feedings in 1 day, rats received 2.5 e Sprague-Dawley, Inc., Madison, Wisconsin. 3 Wayne Lab-Blox, Allied Mills, Inc., Chicago. 4 Mazola, Corn Products Company, New York. 6 Cod liver oil liquid, Mead Johnson Laboratories,
Evansville,
Indiana.
14
SIDRANSKY
AND
VERNEY
PC1 14C-L-leucine intraperitoneally 1 hour before killing (Tables HI-Y). In in vitro incorporation experiments, either postmitochondrial supernatant or ribosomes and supernatant (cell sap) of liver homogenates were used. The postmitochondrial supernatant was prepared in 0.1 M sucrose in medium B [0.03 M Tris (pH 7.5), 0.15 M NH&l, and 0.0035 M MgC&] and used for protein synthesis in vitro or for size distribution analysis of polyribosomes after addition of deoxycholate (0.7%, final concentration) (Sidransky et al., 1968; Staehelin et al., 1967). 14C-L-Leucine, 0.125 to 0.50 PCi, was added to each incubation tube. Radioactivity in protein was measured using a liquid scintillation spectrometer.‘j In a number of experiments the levels of free amino acids of pooled livers or plasma of control and experimental animals were determined in an amino acid analyzer.’ In some cases the specific activity of the free leucine in the liver was determined after measuring the radioactivity in a liquid scintillation spectrometer.* The methods used for chemical analyses for protein, glycogen, lipid, RNA, and DNA have been described in detail in earlier studies (Sidransky and Farber, 1958b; Sidransky et al., 1964). RESULTS One feeding of complete (C) or threonine-devoid (TD) diet Young rats were force-fed the complete diet for 1 to 8 days before they were begun on the experimental diet. Sixteen hours after the last evening feeding of complete diet, one group was given one tube-feeding of complete (C) diet and another group of threonine-devoid (TD) diet. In five experiments 14C-leucine was added to the two diets before they were fed, and all animals were killed 5 hours later. In three experiments 14C-leucine was administered intraperitoneally 44, 1, or 2 hours before killing which occurred 135 or 6 hours after the tube-feeding. Animals force-fed the TD diet showed no increase in 14C-leucine incorporation into hepatic proteins and into plasma proteins in comparison with control animals force-fed the C diet. On the contrary, there was an average 10 to 14% decrease in incorporation into total hepatic proteins and an 11 to 31% decrease in incorporation into plasma proteins of the experimental animals. The results are presented in Table I. Since all of the data of experiments presented in Table I showed no statistically significant differences between C and TD groups, the results are summarized in a simple tabular form. In four experiments in which the 14C-leucine was given orally, the radioactivity remaining in the gastrointestinal tract at time of killing was measured. The results revealed that from 11 to 15 % (average 13 %) more counts remained in the gastrointestinal tract of the TD rats than in that of the C rats. However, the acid-soluble counts in the livers of the TD rats ranged from 17 to 34 % (average 27 %) greater than that in the C rats. In some experiments in which rats were killed 5 or 6 hours after one tube-feeding of C or TD diet, hepatic protein, glycogen, RNA, and DNA were determined. The results revealed no differences in protein, RNA, and DNA, but for glycogen there 6 Packard Tri-Garb, Packard Instrument Company, Inc., Downers 7 Spinco, Model No. lZOB, Beckman Instruments, Inc., Fullerton, * Nuclear-Chicago Corporation, Des Plaines, Illinois.
Grove, Illinois. California.
HEPATIC
PROTEIN
SYNTHESIS TABLE
IN THREONINE
DEFICIENCY
15
I
ANALYSES OF LIVER, GASTROCNEMIUS MUSCLE, SPLEEN, AND KIDNEY OF RATS FORCE-FED A COMPLETE OR THREONINE-DEVOID DIET FOR ONE OR Two FEEDINGS
Analyses
Liver Weight, mg Protein, mg/liver Glycogen, mg/liver RNA, mg/liver DNA, mg/liver r4C-Leucine incorporation into protein In viva; CPM/liver In viva; CPM/mg plasma protein In vitro;b CPM/sample Gastrocnemius muscle Weight, mg Protein, mg/muscle “C-Leucine incorporation into protein In wivo; CPM/muscle Spleen Weight, mg Protein, mg/spleen 14C-Leucine incorporation into protein In vivo; CPM/spleen Kidney Weight, xng Protein, mg/kidney r4C-Leucine incorporation into protein In viva; CPM/kidney
Threonine-devoid diet group compared with complete diet groupa After one tube-feeding
After two tube-feedings
NC NC Sl. D NC NC
NC NC Sl. D NC NC
Sl. D Sl. D NC
Sl. D
NC NC Sl. D NC NC Sl. D
NC NC NC
0 NC, no change; Sl. D, slight decrease. b Rats weighed 1%180 gm after a l- to 5-day fast at start of experiments. periments rats weighed 65-75 gm at start of experiments.
In all other ex-
was a 22 % decreasein the TD group (Table I). 14C-Leucineincorporation into proteins of gastrocnemius muscle, spleen, and kidney was determined in six experiments, and the results indicated an 11% decrease in muscle, a 13 % decrease in spleen, and an 8 % decreasein kidney of TD group in comparison with C group. The results are summarized in Table I. In five experiments in vitro 14C-leucineincorporation into hepatic proteins was determined using cell-free preparations of liver from animals force-fed the C or TD diet for one feeding and killed 1 to 2 hours later. Larger animals, weighing 120 to 180 gm, after a l- to 5-day fast were used. The data indicate that in some cases there was diminished and in others increased incorporation in vitro in the TD groups in comparison with the C groups, In two experiments the specific activity of the leucine in the postmitochondrial supernatants of the C and TD groups was determined, and after correction for this the differences between C and TD groups were +15% and -11%.
16
SIDRANSKY
AND
VERNEY
8 .7(L t. .62 _ g3::w .a9 .2.I 07 0
I 2
I 4
, 1 , 6 8 IO EFFLUENT VOLUME (ml.) FIG. 1. Sucrose density-gradient patterns of hepatic polyribosomes of rats force-fed for three feedings in 1 day the complete diet (A, left pattern) or the threonine-devoid diet (B, right pattern).
In five experiments sucrose density gradients were performed on deoxycholatetreated postmitochondrial supernatants of liver of C and TD rats. In each case identical patterns were observed for the C and TD rats. The patterns for both groups of animals were similar to that of the pattern illustrated in Fig. 1B. Hepatic and plasma free amino acid levels were determined in five experiments, and these results are summarized in Table II. Of the 14 amino acids analyzed, the only major difference between the C and TD groups was in the threonine level which, after one feeding, showed a 69% decrease in the liver and an 89% decreasein the plasma of the TD group. In some experiments, instead of using a complete (C) or threonine-devoid (TD) diet, we used a complete (CAA) or a threonine-devoid (TDAA) amino acid mixture. The results of in vitro incorporation studies with hepatic postmitochondrial supernatants in four experiments in which rats were fasted overnight and then tube-fed the CAA or TDAA mixture in the morning 1 hour before killing revealed little or no difference between the two groups. Sucrose density gradient studies of hepatic polyribosomes also revealed no difference between the two groups. Two feedings of C or TD diet In two experiments the animals were force-fed the C or TD diets twice in 1 day, at 7: 30 AM and at 1: 00 PM, and killed 5 or 6 hours after the second feeding. In the first experiment the 14C-leucinewas added to the diets at the second feeding, and in the second experiment the 14C-leucine was given intraperitoneally 1 hour before killing. In both cases there was less (average -28%) incorporation into hepatic proteins of animals fed the TD diet in comparison with those fed the C diet (Table I). Hepatic and plasma free amino acid levels were determined in someexperiments (Table II). Hepatic level of threonine was decreased 61%, and plasma level was
HEPATIC
PROTEIN
SYNTHESIS
IN THREONINE
TABLE ANALYSES
OF FREE
AMINO
-.
OF LIVER AND OR THREONINE-DEVOID
Groupc No. of feedings.. No. of experiments.
Essential amino acids Isoleucine Leucine Methionine Phenylalanine Threonine Valine Nonessential amino acids Alanine Aspartic acid Half cystine Glutamic acid Glycine Proline Serine Tyrosine
Liver
- 1% ;? 5
--
5
.30 1.28 .27 .37 2.42 .96
.27 .99 .33 .31 .75 .74
10.81 7.90 .15 9.51 6.17 .93 5.61 .30
7 .86 7 .38 .08 8 .63 4 .38 .94 5 .39 .25
II
ACIDS
COMPLETE
free amino
; 2
.32 1.56 .60 .49 2.46 1.00
--
acids”
.28 1.21 .41 .43 .97 .95
PLASMA
OF RATS DIET
-
.05 .46 .13 .15 .46 .43
.45 .12 .15 .05 .65 .46 .55
.07 .42 .lO .lO .05 .49
.91 .30 .Ol .29 .41 .34 .52 -
.72 .21 .03 .26 .27 .23 .35 .15
25
y
3”, 1
2
.23 .40 .06 .16 .82 .31
.20 .31 .07 .13 .52 .33
.2.98 14.35 3 .86 .3.32 11.95 4 .12 .24 .30 .12 5.88 7.46 5 .64 6.88 6.59 4 .06 .84 1.02 .36 7.84 10.55 8 .07 .41 .42 .21
5.81 6.09 .15 8.93 4.33 .21 1.15 .19
.21 .34 .06 .ll .45 .30
-
A
FORCE-FED
-y -F z 1 _- --
Plasma
-
-
-
Y2
17
DEFICIENCY
free amino
5
1
acids* -7
32 ‘72 _-
--
.06
---
.04 .07 .02 .03 .19 .lO
.03 .05 .03 .03 .08 .08
.36 .04 .04 .20 .25 .19 .42 .06 - * Pooled specimens of livers from three to five animals of each group were used in each experiment. Values are pmoles per liver. * Pooled specimens of plasma from three to five animals of each group. Values are Nmoles/ 100 ml of plasma. e C, indicates complete diet; TD, indicates threonine-devoid diet. Rats weighed 65-75 gm. d Rats were killed 5 or 6 hours after 7:30 AM feeding. B Rats were killed 5 or 6 hours after the second feeding given at 1:OO PM. 1 Rats were killed 16 hours after the last (2nd or 3rd) feeding given at 7:30 PM. .45 .62 .13 .64 .64 .08 .19 .15
.68 -17 .Ol .33 .34 .29 .37 .16
.78 .21 .02 .38 .37 .31 .46 .18
.36 .03 .02 .23 .38 .25 .49 .ll
decreased 92% in the TD group in comparison with the C group. While hepatic protein, RNA, and DNA showed little or no changes, hepatic glycogen was decreased by 24% in the TD group (Table I). Three feedings of C or TD diet (1 day)
In contrast to the results obtained after one or two feedings of diet, rats forcefed three times in 1 day and then killed 16 hours later, the following morning, showed an increase in incorporation of l*C-leucine into hepatic and plasma proteins in the TD animals in comparison with those of the C animals. Table III summarized the results of four experiments in which the rats were fed on the average 0.95 gm diet/l0 gm initial body weight and six experiments in which the rats were fed on the average 1.16 gm diet/l0 gm initial body weight during the 1 day of three feedings. It is apparent that the group of animals fed the higher intake of TD diet had a larger increase (approximately double) in hepatic protein synthesis and plasma protein synthesis than did the animals fed the lower intake.
18
SIDRANSKY
AND TABLE
INCORPORATION in Vivo OF %-LEUCINE OF RATS FORCE-FED A COMPLETE
VERNEY III
INTO PROTEINS OR THREONINE-DEVOID
OF LIVER DIET
Incorporation EXP. No.
Groud
187
C TD C TD C TD C TD
190 193 334
Summary
198 249 272 329 340 355
Amount of No. of diet fed rats (gm,1O gm body wt)
3 4 4 4 2 2 5 5
*
Liver
AND PLASMA FOR 1 DAY
of W-leucine Liver
into proteins
of
PlaSma
Weight (9)
Protein @g/liver)
Specific activity (cpmh.8 protein)
0.97 0.97 0.97 0.97 0.99 0.99 0.95 0.95
2.16 2.54 2.40 2.89 2.92 3.11 4.61 5.09
441 471 458 470 527 584 930 998
850 1330 534 572 606 618 477 665
376.6 626.2 242.0 267.4 316.6 359.7 443.8 663.7
Total incorp. (cpm/liver X 10-q
Sy;;/;gity c protein) 1572 1771 816 903 702 797
C TD
14 15
AV. 0.95 0.95
% 100 137 zlz 5.2csd
% 100 106.9 f 1.6Cre
% loo 127 zk 12.7’
% 100 135 f 13.F
C TD C TD C TD C TD C TD C TD
4 4 5 3 4 5 3 4 4 4 2 4
1.10 1.10 1.17 1.17 1.29 1.29 1.10 1.10 1.13 1.13 1.17 1.17
2.52 3.35 2.30 3.24 2.70 2.89 2.63 3.15 2.67 3.27 2.21 2.68
496
467 732 465 693 547 866 857 1191 773 959 686 927
228.2 425.4 218.6 374.5 353.0 634.5 465.0 811.0 483.5 776.5 455.3 691.3
1043 1265 593 744 1107 1550 1186 1449 1126 1295 -
C TD
22 24
AV. 1.16 1.16
% 100 124 f 4.gd
% loo 144 + 4.9d
% 100 171 zt 4.6
% 106 125 f 3.8d
summary*
577 489 540 647 726 569 677 630 804 668 754 % 106 116 f 3.0d
% 100 112 f 2.1c*e
a C indicates complete diet; TD indicates threonine-devoid diet. In each experiment animals received 2.5 &i W-leutine intraperitoneally 1 hour before killing. In experiment 334 animals weighed 112 gm while in all other experiments snimals weighed 65 to 75 gm at the start of the experiments. b In each experiment, the experimental group W&B compared with its control group (10%). c Mean GEM. dp < 0.01. e 0.05 > P > 0.01.
In two experiments rats were force-fed the C or TD diet at high levels (1.1-1.2 gm diet/day/l0 gm initial weight divided into three feedings), and the livers were used for in vitro incorporation studies. In one experiment using a postmitochondrial supernatant incorporation system as described earlier (Sidransky et al., 1968; Staehelin et al., 1967), there was 112% greater incorporation of 14C-leucineinto hepatic proteins of the TD group than in the C group. These values were corrected for the specific activity of the leucine in each incubation system. In another experiment using hepatic microsomes of C and TD rats there was a 64% increase in incorporation of 14C-leucineinto hepatic proteins of the TD group over that in the C group. The results for hepatic protein, glycogen, lipid, RNA, and DNA for rats fed the higher level (1.16 gm diet for 10 gm body weight) of C and TD diets are summarized
HEPATIC
PROTEIN
SYNTHESIS TABLE
ANALYSES FORCE-FED
IN THREONINE
Liver Protein, mg/liver Glycogen, mg/liver Lipid, mg/liver RNA, mg/liver DNA, mg/liver Gestrocnemius muscle Weight, mg Protein, mg/muscle i4C-leucine incorporation CPM/muscle X 1OF Spleen Weight, mg Protein, mg/spleen 14C-leucine incorporation CPM/spleen X lO+ Kidney Weight, mg Protein, mg/kidney i4C-leucine incorporation CPM/kidney X 1OF
SPLEEN, AND KIDNEY OF RATS DIET FOR 1 DAY (3 FEEDINGS)
Complete diet
(22)
687 110 154 2.94 0.48
f f f zk f
26a. b 206 6b .17 .05
(13)”
333 f 59 f 51.1 f
25 3.0 3.3
(17)”
309 f 51 f 33.9 f
12 7.0 l.Ob
(13)c
213 f 43 f 222.0 f
20 4 32.4
(17)”
177 f 35 f 187.7 f
10 4 27.8
(13)”
354 f 6 65 f 12 288.6 f 36.5
(17)”
334 f 16 57 xk 14 301.5 f 24.7
(4) (4)
into protein,
into protein,
(24)
diet
195 8 8 .14 .Ol
(21)
572 20 116 2.60 0.47
Threonine-devoid
f f f f f
(11)
into protein,
19
IV
OF LIVER, GASTROCNEMIUS MUSCLE, A COMPLETE OR THREONINE-DEVOID
Analyses
DEFICIENCY
(12) (24) (4) (4)
a Number of animals in parentheses; Mean f SEM. Rats weighed 65-75 gm at start of experiments and were force-fed 1.16 gm diet/l0 gm body weight. b P < 0.01. c Results are from four experiments. In three experiments, organs from two to four animals of each group were pooled for the determinations.
in Table IV. There were increasesin hepatic protein, glycogen, lipid, and RNA in the animals fed the TD diet. These increases, except for glycogen, were lessin animals fed the lower level (.97 gm diet per 10 gm body weight) than in those fed the higher level of TD diet. In one experiment in which rats were force-fed 1.1 gm diet/l0 gm body weight, sucrose density-gradient patterns were performed on livers of C and TD rats. The results are shown in Fig. 1. There was a shift in hepatic polyribosomes from lighter to heavier aggregates in the TD rats. In some experiments hepatic and plasma free amino acid levels of C and TD animals were determined (Table II). Since the results of experiments in which the animals were fed the lower diet intake were similar to those with higher diet intake, the results were combined and averaged together. Hepatic threonine levels were decreased 37%, and plasma levels were decreased 58% in the experimental group. Hepatic levels of alanine, aspartic acid, glutamic acid, and serine were somewhat elevated in the TD group over that in the C group. The results of 14C-leucineincorporation into proteins of skeletal muscle, spleen, and kidney of C and TD rats in four experiments in which rats received 1.16 gm
20
SIDRANSKY
AND VERNEY
HEPATIC FREE THREONINE
PLASMA
0
2
FREE THREONINE
4
6 6 Time -Hours
IO
12
I4
FIG. 2. Hepatic and plasma free threonine levels in rats force-fed one feeding of complete (C) or threonine-devoid (TD) diet and killed 1% to 13 hours later.
diet/l0 gm body weight are summarized in Table IV. The changesin incorporation into organ protein in the TD group in comparison to the C group were, respectively, as follows: muscle, - 34 % ; spleen, - 15 % ; and kidney, + 5 %. Additional experiments with diferent schedulesof one, two, and threefeedings In a further attempt to determine whether one feeding of TD diet under different experimental conditions than previously described would alter hepatic protein synthesis, another three experiments were conducted. After a preliminary tubefeeding period of 2 days using the C diet, the animals were divided into two groups. Each group received one morning tube-feeding of 3 gm of either C or TD diet and some of the animals of each group were killed 145, 2, 3, 5, 6, 10, 13, and 24 hours later. In all casesthe rats received 14C-leucineintraperitoneally 1 hour before killing. The results of incorporation into hepatic proteins indicated little or no differences between the C and TD groups except after 10 and 13 hours where there was a 10 to 19% increase (statistically insignificant) in the TD group. In two of these experiments hepatic and plasma free amino acids were determined in animals of the C and TD groups at the different time intervals. Figure 2 reveals the free threonine levels in the liver and plasma of C and TD rats. At 1% to 6 hours the threonine levels in the liver and plasma of the TD group were much lower than in the C group. However, after 13 hours the threonine levels of the liver and plasma of the C group had decreased so that these levels were only somewhat higher than those of the TD group. Analyses of isoleucine, leucine, methionine, phenylalanine, valine, alanine, aspartic acid, cystine, glutamic acid, glycine, proline, serine, and tyrosine in liver and plasma of C and TD animals were also conducted at each time interval, and the values for each of these free amino acids were similar in the C and TD groups. In general, the shapesof the curves for each amino acid were similar to that for threonine in the C group (Fig. 2). In light of the earlier results obtained after three feedings of C and TD diets and killing the animals 16 hours following the third feeding (Table III), we conducted additional experiments in which animals received one or two feedings of TD diet in a manner different from that used in earlier experiments (Table I). In these addi-
HEPATIC
PROTEIN
SYNTHESIS
IN THREONINE
TABLE
DEFICIENCY
21
V
INCORPORATION in Viuo OF i4C-LEUCINE INTO PROTEINS OF LIVER AND PLASMA OF RATS FORCE-FED A COMPLETE, THREONINE-DEVOID, PHENYLALANINEDEVOID DIET OR COMBINATION IN 1 DAY (THREE FEEDINGS)
Incornoration Group”
c-c-c C-C-TD C-TD-TD TD-TD-TD PD-PD-PD C-PD-PD C-TD-PD C-PD-TD TD-PD-PD
No. of rats
16 6 12 15 11 10 13 9 3
of i4C-leucine into
Liver proteinb Specii@a;tivity 0 100 106 115 134 135 115 116 116 122
f f f f f f i i
2.0 0.7d 3.5Cnd 1.6d 6.8 6.3 4.1 0.0
Plasma proteinb
Total radioactivity (%) loo 95 126 171 144 119 118 125 117
f f f i i i f f
1.2 8.1 4.3’,d 0.7d 12.0 5.50 8.3 0.0
Specific activity (%) loo 106 107 126 115 108 106 114 118
f 5.3 f 5.4 f 3.9”,d f 5.2 i 12.3 i 2.8 f 8.4 f 0.0
Q C, indicates complete diet; TD, indicates threonine-devoid diet; PD, indicates phenylalanine-devoid diet. All rats weighed 65-75 gm at start of experiments. b In each experiment, the experimental groups were compared with the control (C-C-C) group (100%). c Mean f SEM. d P < 0.01. c 0.05 > P > 0.01.
tional experiments one group (C-C-TD) of animals was tube-fed the C diet at 7:30 AM and 1:00 PM and the TD diet at 7: 30 PM and another group (C-TD-TD) was fed the C diet at 7:30 AM and the TD diet at 1:00 PM and 7: 30 PM, and then all groups were killed 16 hours later, the following morning. The results of four experiments are presented in Table V and indicate that after one TD feeding (C-C-TD group) there was essentially no difference in 14C-leucineincorporation into hepatic and plasma proteins, but after 2 TD feedings (C-TD-TD group) there was a 26% increase in ‘*C-leucine incorporation into total hepatic proteins. Thus, these results indicate that under the conditions described above, two feedings of TD diet can initiate an increase of hepatic protein synthesis if the animals were studied 16 hours after the second feeding. Four experiments were conducted to determine whether the intermixing of single essential amino acid devoid diets, threonine-devoid (TD) and phenylalanine-devoid (PD), during l-day feeding experiments would produce changes in hepatic protein synthesis. The dietary combinations used in three feedings during 1 day were as follows: (1) C-C-C, (2) C-C-TD, (3) C-TD-TD, (4) TD-TD-TD, (5) PD-PD-PD, (6) C-PD-PD, (7) C-TD-PD, (8) C-PD-TD, and (9) TD-PD-PD. The results of in vivo 14C-leucineincorporation into hepatic and plasma proteins in these experiments are summarized in Table V. The results for the C-C-TD and C-TD-TD groups were discussedin the preceding paragraph, The results for the TD-TD-TD group in comparison with the C-C-C group are similar to those obtained in other experiments (Table III) and indicate a 71% increase in hepatic protein synthesis and a 26 % increase in plasma protein synthesis. Rats of the PD-PD-PD group-showed an
22
SIDRANSKY
AND VERNEY
increase in 14C-leucineincorporation into liver (44 %) and plasma (15 %) proteins which was a somewhat smaller response than was observed in the TD-TD-TD group. These differences in the results with TD and PD diets were similar to those observed in earlier experiments of 3 days’ duration (Sidransky and Verney, 1964, 1967). Rats fed the C-PD-PD diet showed lessincrease than those fed the PD-PDPD diet but a similar increase to those fed the C-TD-PD, C-PD-TD, or TD-PDPD diet. Hepatic and plasma free amino acids were determined in several of these experiments. The overall results obtained in the animals fed the C-C-C and TD-TDTD diets were the same as those obtained in earlier experiments (Table III) and were therefore, combined, averaged, and presented in Table II. Also, the hepatic free amino acid levels of the C-TD-TD group in two experiments are presented in Table II, and these results are similar to those of the TD-TD-TD group except that there were little or no increasesin someof the nonessential amino acids over that in the C-C-C group. DISCUSSION The results of this study indicate that young rats force-fed a TD diet for 1 day (three feedings) develop enhanced hepatic protein synthesis as measured by in vivo 14C-leucineincorporation into hepatic proteins and plasma proteins. Sucrose densitygradient patterns of hepatic polyribosomes demonstrate a shift toward heavier aggregates in the TD rats in comparison to C rats, and in vitro hepatic protein synthesis is enhanced in livers of TD rats. Such changes are not observed if animals are tube-fed one or two feedings of TD diet and killed up to 6 hours later. Small increasesin hepatic protein synthesis were found in animals killed 16 hours after two feedings of TD diet. Thus, it appears that two or three feedings of TD diet and an interval of 16 hours are necessary to induce enhanced hepatic protein synthesis. Concomitant with the increase in hepatic protein synthesis in rats force-fed the TD diet for 1 day, there was a decreasein protein synthesis of skeletal muscle and spleen with little or no change in kidney. Similar results were reported in experiments of 3 days duration (Sidransky and Verney, 1967,1968). Thus, it is conceivable that the increase in hepatic protein synthesis may be related in some way to the concomitant decrease in muscle protein synthesis and increase in muscle protein catabolism. Results using 3H-leucine incorporation into proteins before beginning the control or experimental diet and then measuring loss due to protein catabolism indicated that skeletal muscle protein catabolism contributes amino acids which are then incorporated in greater amounts into hepatic proteins of the TD animals (Sidransky and Verney, 1970). Skeletal muscle degradation may be an important factor in controlling the levels of plasma free amino acids and possibly also the levels of hepatic free amino acids. Actually at 16 hours after the last (3rd) feeding, the plasma and hepatic free amino acid levels drop appreciably from peak levels at 6 hours after a feeding (Table II), and even the decreaseof plasma and hepatic free threonine levels in the TD in comparison with the C animals are now, respectively, only 58 % and 37 %. Whether these altered levels of amino acids in the plasma and liver play an influential role is at present not clear. Studies by others (Kumata and Harper, 1962; Peng et al., 1969; Sanahuja and Harper, 1963) using amino acid imbalanced diets have investigated the influence of dietary intake on plasma amino acid levels in relation to the altered nutritional state of the animals.
HEPATIC
PROTEIN
SYNTHESIS
IN THREONINE
DEFICIENCY
23
In evaluating the present results, it may be worthwhile to review the data of free amino acid levels in relation to hepatic and muscle protein synthesis after one, two, or three feedings of C or TD diet and compare the results in experimental animals with those in control animals. During the first 6 hours after one feeding of TD diet, the hepatic and plasma amino acid levels of all amino acids except threonine are high, yet hepatic and muscle protein synthesis are minimally decreased. After 10 to 13 hours following one feeding of TD diet, the hepatic and plasma amino acid levels of all amino acids, especially threonine, are low, and hepatic protein synthesis shows a questionable slight increase. At 6 hours following two feedings of TD diet, the hepatic and plasma amino acid levels of all amino acids except threonine are high, yet hepatic protein synthesis is minimally decreased. However, at 16 hours after two or three feedings of TD diet, the hepatic and plasma amino acid levels of all amino acids, including threonine, are low, yet hepatic protein synthesis is increasedwhile muscle protein synthesis is decreased. Since the free amino acid levels at lo-13 hours after one TD feeding and at 16 hours after two or three TD feedings are similar yet the hepatic protein synthesis is different, the alterations in free amino acid levels in the liver and plasma may not be the solefactor responsible. Since muscle protein synthesis seemsto decrease and muscle protein catabolism seemsto increase (Sidransky and Verney, 1970) with time after feeding the TD diet, it is possible that it is necessary for skeletal muscle protein metabolism to become affected, which may take 6-10 hours, before hepatic protein synthesis becomes stimulated. Decrease in skeletal muscle protein synthesis and decrease in hepatic and plasma free amino acids as induced by fasting 1 to 5 days do not induce a proper setting since one feeding of TD diet to such animals, which alters the free amino acid levels, will not enhance hepatic protein synthesis. Also in an earlier study (Sidransky et al., 1969) it was demonstrated that the acute administration of threonine to rats force-fed a TD diet for 3 days did not depressthe enhanced hepatic protein synthesis. Thus, once the proper setting has been induced by force-feeding the TD diet for 1 or 3 days, a sequenceof events has been set in motion which is no longer rapidly responsive to added threonine. The failure to observe enhanced hepatic protein synthesis following one feeding of TD diet or TDAA mixture in comparison to one feeding of C diet or CAA mixture is consistent with other studies by Fleck et al. (1965) with rats and with our own earlier studies (Sidransky et al., 1968) with mice. These studies indicated that animals fasted overnight and then given one tube-feeding of purified complete diet or amino acid mixture devoid of single essential amino acids excluding tryptophan (Fleck et al., 1965; Sidransky et al., 1968) showed a hepatic polyribosomal pattern and in vitro protein synthesis similar to that of animals receiving the complete control diet or amino acid mixture. Our present results stress the importance of the quantity of deficient diet in inducing alterations in hepatic protein synthesis. Rats force-fed for 1 day 1.16 gm diet/l0 gm body weight showed a greater increase (71%) than did rats fed 0.95 gm diet/l0 gm body weight (35%) in the TD groups in comparison to controls. These data are consistent with earlier results of experiments of 3 to 7 days duration where the amount of diet consumed played an mportant role (Sidransky and Farber, 1958b). Our findings in an earlier study (Sidransky and Clark, 1961) indicated that the amount of calories supplied by carbohydrate was important.
SIDRANSKY
24
AND
VERNEY
REFERENCES FLECK, A., SHEPHERD, J., and MUNRO, H. N. (1965). Protein synthesis in rat liver: Influence of amino acids in diet on microsomes and polysomes. Science 150,628-629. KUMATA, U. S., and HARPER, A. E. (1962). Amino acid balance and imbalance VII. Effects of amino acid imbalance on blood amino acid pattern. Proc. Sot. Exp. Biol. Med. 110,512-517. PENG, Y., BENEVENGA, N. J., and HARPER, A. E. (1969). Amino acid balance and food intake effect of previous diet on plasma amino acids. Amer. J. Physiol. 216, 1020-1025. SANAHUJA, J. C., and HARPER, A. E. (1963). Effect of dietary amino acid pattern on plasma amino acid pattern and food intake. Amer. J. Physiol. 204,686-690. SIDRANSKY, H., and CLARK, S. (1961). Chemical pathology of acute amino acid deficiencies. IV. Influence of carbohydrate intake on the morphologic and biochemical changes in young rats fed threonine- or valine-devoid diets. Arch. Pathol. 72,468-479. SIDRANSKY, H., and FARBER, E. (1958a). Ch emical pathology of acute amino acid deficiencies. I. Morphologic changes in immature rats fed threonine-, methionine-, or histidinedevoid diets. Arch. Pathol. 66, 119-134. SIDRANSKY, H., and FARBER, E. (1958b). Chemical pathology of acute amino acid deficiencies. II. Biochemical changes in rats fed threonine- or methionine-devoid diet. Arch. Pathol. 66, 135-149. SIDRANSKY, H., SARMA, D. S. R., BONGIORNO, M., and VERNEY, E. (1968). Effect of dietary tryptophan on hepatic polyribosomes and protein synthesis in fasted mice. J. Biol. Chem. 243, 1123-1132. SIDRANSKY, H., STAEHELIN, T., and VERNEY, E. (1964). Protein synthesis enhanced in the liver of rats force-fed a thronine-devoid diet. Science 146, 766-768. SIDRANSKY, H., and VERNEY, E. (1964). Chemical pathology of acute amino acid deficiencies. VII. Morphologic and biochemical changes in young rats force-fed arginine-, leucine-, isoleucine- or phenylalanine-devoid diets. Arch. Pathol. 78, 134-148. SIDRANSKY, H., and VERNEY, E. (1967). Decreased protein synthesis in the skeletal muscle of rats force-fed a threonine-devoid diet. Biochim. Biophys. Acta 138, 426-429. SIDRANSKY, H., and VERNEY, E. (1968). Effect of hypophysectomy on pathologic changes in rats force-fed a threonine-devoid diet. J. Nutr. 96, 28-36. SIDRANSKY, H., and VERNEY, E. (1970). Skeletal muscle metabolism in rats force-fed a threonine-devoid diet. Fed. Proc Fed Amer. Sot. Exp. Biol. 29, 365. SIDRANSKY, H., WAGLE, D. S., BONGIORNO, M., and VERNEY, E. (1969). Studies on blood glucose and hepatic glycogen in rats force-fed a threonine-devoid diet. J. Nutr. 98,477486. SIDRANSKY, H., WAGLE, D. S., and VERNEY, E. (1969). Hepatic protein synthesis in rats force-fed a threonine-devoid diet and treated with cortisone acetate or threonine. Lab. Invest.
20, 364-370.
Sox, H. C., JR., and HOAGLAND, M. B. (1966). Functional alterations in rat liver polysomes associated with starvation and refeeding. J. Mol. Biol. 20,113-121. STAEHELIN, T., VERNEY, E., and SIDRANSKY, H. (1967). The influence of nutritional change on polyribosomes of the liver. Biochim. Biophys. Acta 145.105-119. WEBB, T. E., BLOBEL, G., and POTTER, V. R. (1966). Polyribosomes in rat tissues III. The response of the polyribosome pattern of rat liver to physiologic stress. Cancer Res. 26, 253257. WILSON, S. H., and HOAGLAND, M. B. (1967). Physiology of rat-liver polysomes. The stability of messenger ribonucleic acid and ribosomes. Biochem. J. 103, 556-566. WUNNER, W. H., BELL, J., and MUNRO, H. N. (1966). The effect of feeding with a tryptophan-free amino acid mixture on rat liver polysomes and ribosomal ribonucleic acid. Biothem. J. 101,417-428.
AND
Studies
MOLECULAR
on
PATHOLOGY
Hepatic
13, 12-24
Protein
Synthesis
a Threonine-Devoid HERSCHEL Department
(1970)
SIDRANSKY
in Rats
Force-Fed
Diet’
AND ETHEL
VERNEY
of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 16213 Received February 11, 19YO
Young Sprague-Dawley rats were force-fed a threonine-devoid or complete diet for one, two, or three feedings in 1 day. Rats force-fed the threonine-devoid diet for three feedings and killed the following morning (16 hours later) developed increased hepatic protein synthesis and decreased gastrocnemius muscle protein into proteins. At the synthesis, as measured by in vivo “C-leucine incorporation same time, sucrose density-gradient patterns of hepatic polyribosomes of experimental animals demonstrated a shift toward heavier aggregates, and in VitTO hepatic protein synthesis of experimental animals w&s increased when compared to control animals. Similar changes were not observed in experimental animals tube-fed one or two feedings and killed up to 6 hours later. Increases in hepatic protein synthesis were observed in animals killed 16 hours after two feedings of the threonine-devoid diet. Thus, it appears that the earliest increase in hepatic protein synthesis due to force-feeding a threonine-devoid diet occurs after two or three feedings in 1 day followed by an interval of 16 hours.
In earlier studies from this laboratory (Sidransky and Farber, 1958a; Sidransky and Clark, 1961) we reported that young rats force-fed a threonine-devoid diet for 3 to 7 days developed many o’f the pathologic changes resembling those found in children with kwashiorkor. Among the biochemical changes in the livers of these experimental animals were increasesin lipid, in glycogen, in total RNA, in the incorporation in vivo of 32Por 14C-orotate into ribosomal RNA, and in the incorporation in vivo and in vitro of 14C-amino acid into proteins (Sidransky and Farber, 19588; Sidransky et al., 1964; Sidransky et al., 1969; Staehelin et al., 1967). Along with the increased hepatic protein synthesis, the hepatic ribosomes revealed a shift from lighter toward heavier polyribosomes with a decrease in monosomes(Sidransky et al., 1964; Staehelin et al., 1967). The present investigation was undertaken to learn more about the pathogenesis of the enhanced hepatic protein synthesis in rats force-fed a threonine-devoid diet. In an attempt to gain someinsight into the mechanism responsible for the alteration in protein metabolism in the liver, we studied how early the changes could be detected after beginning the threonine-devoid diet. This was especially relevant since earlier studies by others (Fleck et al., 1965; Sox and Hoagland, 1966; Webb et al., 1966; Wilson and Hoagland, 1967; Wunner et al., 1966) and from our own labora1 This investigation was supported by United States Public Health Service Grant AM05908 from the National Institute of Arthritis and Metabolic Diseases and by Grant GM-10269 from the National Institute of General Medical Sciences, 12
HEPATIC
PROTEIN
SYNTHESIS
IN THREONINE
DEFICIENCY
13
tory (Staehelin et al., 1967) have indicated that the livers of fasted animals respond rapidly to a single feeding of proteins or of a complete amino acid mixture with a shift in polyribosomes from lighter to heavier aggregates and with enhanced protein synthesis. Therefore, in this study rats were force-fed a threonine-devoid diet for one, two, or three feedings in 1 day, and in viva and in vitro measurements of hepatic protein synthesis were conducted. Our present results indicate that it was necessary to wait 16 hours after the force-feeding of the deficient diet for two or three feedings before increases in hapatic protein synthesis were detectable. METHODS Male and female rats of the Sprague-Dawley strain,2 1 month old, weighing on the average 70 gm, were used in most experiments. In some experiments in which animals were fasted for 1 to 5 days, animals weighing 120 to 180 gm were used. The animals were maintained on a commercial diet3 before the experiments were begun. In all experiments groups of rats, each of the same sex, age, and weight, were used. The basal diet was similar to that used in earlier experiments (Sidransky and Clark, 1961; Sidransky and Farber, 1958a, 1958b; Sidransky et al., 1964; Sidransky et al., 1969; Staehelin et al., 1967). The percentage of the components was as follows: essential amino acids, 9.2; nonessential amino acid, 8.1; vitamin-sucrose mixture, 5; salt mixture, 4; corn oiJ4 5; cod liver oiJ5 1.5; and dextrin, 67.2. Dextrin was substituted for the threonine in the threonine-devoid diet and for phenylalanine in the phenylalanine-devoid diet. In a few experiments, instead of the preceding diets, rats were fed a complete amino acid mixture or a threonine-devoid amino acid mixture. The amino acid mixtures (essential and nonessential amino acids) were the same as those used in the basal diet. The diets or amino acid mixtures were blended with distilled water so that a milliliter of diet mixture contained 0.5 gm of diet. Rats were force-fed using plastic tubes at 7:30 AM, 1:00 PM, and 7: 30 PM. The amount of diet administered in the one or two feeding experiments was 1.2 to 3 gm per feeding. When animals were force-fed for 3 feedings during 1 day, they received an average daily feeding of 1 gm diet/l0 gm initial body weight unless indicated otherwise as in Table III. Except in the case where animals were fasted, animals were tube-fed the complete diet for 1 to 5 days before the experiments were begun. Rats had free access to water. They were housed in individual wire cages with raised bottoms and kept in an air-conditioned room which was without light from 8 PM to 8 AM. Rats were weighed at the beginning and end of each experiment. They were anesthetized with ether and exsanguinated at appropriate intervals after the last feeding. In animals receiving three feedings during 1 day, the animals were killed 16 hours after the last evening feeding (Tables III and V). In in vivo incorporation experiments in which rats were tube-fed once or twice, rats received 2.5 to 5 &Ji (10 &.?i/~ mole) of 14C-L-leucine, uniformly labeled, in aqueous solution either intraperitoneally or orally 0.5 to 5 hours before killing (Table I). In experiments in which rats were tube-fed three feedings in 1 day, rats received 2.5 e Sprague-Dawley, Inc., Madison, Wisconsin. 3 Wayne Lab-Blox, Allied Mills, Inc., Chicago. 4 Mazola, Corn Products Company, New York. 6 Cod liver oil liquid, Mead Johnson Laboratories,
Evansville,
Indiana.
14
SIDRANSKY
AND
VERNEY
PC1 14C-L-leucine intraperitoneally 1 hour before killing (Tables HI-Y). In in vitro incorporation experiments, either postmitochondrial supernatant or ribosomes and supernatant (cell sap) of liver homogenates were used. The postmitochondrial supernatant was prepared in 0.1 M sucrose in medium B [0.03 M Tris (pH 7.5), 0.15 M NH&l, and 0.0035 M MgC&] and used for protein synthesis in vitro or for size distribution analysis of polyribosomes after addition of deoxycholate (0.7%, final concentration) (Sidransky et al., 1968; Staehelin et al., 1967). 14C-L-Leucine, 0.125 to 0.50 PCi, was added to each incubation tube. Radioactivity in protein was measured using a liquid scintillation spectrometer.‘j In a number of experiments the levels of free amino acids of pooled livers or plasma of control and experimental animals were determined in an amino acid analyzer.’ In some cases the specific activity of the free leucine in the liver was determined after measuring the radioactivity in a liquid scintillation spectrometer.* The methods used for chemical analyses for protein, glycogen, lipid, RNA, and DNA have been described in detail in earlier studies (Sidransky and Farber, 1958b; Sidransky et al., 1964). RESULTS One feeding of complete (C) or threonine-devoid (TD) diet Young rats were force-fed the complete diet for 1 to 8 days before they were begun on the experimental diet. Sixteen hours after the last evening feeding of complete diet, one group was given one tube-feeding of complete (C) diet and another group of threonine-devoid (TD) diet. In five experiments 14C-leucine was added to the two diets before they were fed, and all animals were killed 5 hours later. In three experiments 14C-leucine was administered intraperitoneally 44, 1, or 2 hours before killing which occurred 135 or 6 hours after the tube-feeding. Animals force-fed the TD diet showed no increase in 14C-leucine incorporation into hepatic proteins and into plasma proteins in comparison with control animals force-fed the C diet. On the contrary, there was an average 10 to 14% decrease in incorporation into total hepatic proteins and an 11 to 31% decrease in incorporation into plasma proteins of the experimental animals. The results are presented in Table I. Since all of the data of experiments presented in Table I showed no statistically significant differences between C and TD groups, the results are summarized in a simple tabular form. In four experiments in which the 14C-leucine was given orally, the radioactivity remaining in the gastrointestinal tract at time of killing was measured. The results revealed that from 11 to 15 % (average 13 %) more counts remained in the gastrointestinal tract of the TD rats than in that of the C rats. However, the acid-soluble counts in the livers of the TD rats ranged from 17 to 34 % (average 27 %) greater than that in the C rats. In some experiments in which rats were killed 5 or 6 hours after one tube-feeding of C or TD diet, hepatic protein, glycogen, RNA, and DNA were determined. The results revealed no differences in protein, RNA, and DNA, but for glycogen there 6 Packard Tri-Garb, Packard Instrument Company, Inc., Downers 7 Spinco, Model No. lZOB, Beckman Instruments, Inc., Fullerton, * Nuclear-Chicago Corporation, Des Plaines, Illinois.
Grove, Illinois. California.
HEPATIC
PROTEIN
SYNTHESIS TABLE
IN THREONINE
DEFICIENCY
15
I
ANALYSES OF LIVER, GASTROCNEMIUS MUSCLE, SPLEEN, AND KIDNEY OF RATS FORCE-FED A COMPLETE OR THREONINE-DEVOID DIET FOR ONE OR Two FEEDINGS
Analyses
Liver Weight, mg Protein, mg/liver Glycogen, mg/liver RNA, mg/liver DNA, mg/liver r4C-Leucine incorporation into protein In viva; CPM/liver In viva; CPM/mg plasma protein In vitro;b CPM/sample Gastrocnemius muscle Weight, mg Protein, mg/muscle “C-Leucine incorporation into protein In wivo; CPM/muscle Spleen Weight, mg Protein, mg/spleen 14C-Leucine incorporation into protein In vivo; CPM/spleen Kidney Weight, xng Protein, mg/kidney r4C-Leucine incorporation into protein In viva; CPM/kidney
Threonine-devoid diet group compared with complete diet groupa After one tube-feeding
After two tube-feedings
NC NC Sl. D NC NC
NC NC Sl. D NC NC
Sl. D Sl. D NC
Sl. D
NC NC Sl. D NC NC Sl. D
NC NC NC
0 NC, no change; Sl. D, slight decrease. b Rats weighed 1%180 gm after a l- to 5-day fast at start of experiments. periments rats weighed 65-75 gm at start of experiments.
In all other ex-
was a 22 % decreasein the TD group (Table I). 14C-Leucineincorporation into proteins of gastrocnemius muscle, spleen, and kidney was determined in six experiments, and the results indicated an 11% decrease in muscle, a 13 % decrease in spleen, and an 8 % decreasein kidney of TD group in comparison with C group. The results are summarized in Table I. In five experiments in vitro 14C-leucineincorporation into hepatic proteins was determined using cell-free preparations of liver from animals force-fed the C or TD diet for one feeding and killed 1 to 2 hours later. Larger animals, weighing 120 to 180 gm, after a l- to 5-day fast were used. The data indicate that in some cases there was diminished and in others increased incorporation in vitro in the TD groups in comparison with the C groups, In two experiments the specific activity of the leucine in the postmitochondrial supernatants of the C and TD groups was determined, and after correction for this the differences between C and TD groups were +15% and -11%.
16
SIDRANSKY
AND
VERNEY
8 .7(L t. .62 _ g3::w .a9 .2.I 07 0
I 2
I 4
, 1 , 6 8 IO EFFLUENT VOLUME (ml.) FIG. 1. Sucrose density-gradient patterns of hepatic polyribosomes of rats force-fed for three feedings in 1 day the complete diet (A, left pattern) or the threonine-devoid diet (B, right pattern).
In five experiments sucrose density gradients were performed on deoxycholatetreated postmitochondrial supernatants of liver of C and TD rats. In each case identical patterns were observed for the C and TD rats. The patterns for both groups of animals were similar to that of the pattern illustrated in Fig. 1B. Hepatic and plasma free amino acid levels were determined in five experiments, and these results are summarized in Table II. Of the 14 amino acids analyzed, the only major difference between the C and TD groups was in the threonine level which, after one feeding, showed a 69% decrease in the liver and an 89% decreasein the plasma of the TD group. In some experiments, instead of using a complete (C) or threonine-devoid (TD) diet, we used a complete (CAA) or a threonine-devoid (TDAA) amino acid mixture. The results of in vitro incorporation studies with hepatic postmitochondrial supernatants in four experiments in which rats were fasted overnight and then tube-fed the CAA or TDAA mixture in the morning 1 hour before killing revealed little or no difference between the two groups. Sucrose density gradient studies of hepatic polyribosomes also revealed no difference between the two groups. Two feedings of C or TD diet In two experiments the animals were force-fed the C or TD diets twice in 1 day, at 7: 30 AM and at 1: 00 PM, and killed 5 or 6 hours after the second feeding. In the first experiment the 14C-leucinewas added to the diets at the second feeding, and in the second experiment the 14C-leucine was given intraperitoneally 1 hour before killing. In both cases there was less (average -28%) incorporation into hepatic proteins of animals fed the TD diet in comparison with those fed the C diet (Table I). Hepatic and plasma free amino acid levels were determined in someexperiments (Table II). Hepatic level of threonine was decreased 61%, and plasma level was
HEPATIC
PROTEIN
SYNTHESIS
IN THREONINE
TABLE ANALYSES
OF FREE
AMINO
-.
OF LIVER AND OR THREONINE-DEVOID
Groupc No. of feedings.. No. of experiments.
Essential amino acids Isoleucine Leucine Methionine Phenylalanine Threonine Valine Nonessential amino acids Alanine Aspartic acid Half cystine Glutamic acid Glycine Proline Serine Tyrosine
Liver
- 1% ;? 5
--
5
.30 1.28 .27 .37 2.42 .96
.27 .99 .33 .31 .75 .74
10.81 7.90 .15 9.51 6.17 .93 5.61 .30
7 .86 7 .38 .08 8 .63 4 .38 .94 5 .39 .25
II
ACIDS
COMPLETE
free amino
; 2
.32 1.56 .60 .49 2.46 1.00
--
acids”
.28 1.21 .41 .43 .97 .95
PLASMA
OF RATS DIET
-
.05 .46 .13 .15 .46 .43
.45 .12 .15 .05 .65 .46 .55
.07 .42 .lO .lO .05 .49
.91 .30 .Ol .29 .41 .34 .52 -
.72 .21 .03 .26 .27 .23 .35 .15
25
y
3”, 1
2
.23 .40 .06 .16 .82 .31
.20 .31 .07 .13 .52 .33
.2.98 14.35 3 .86 .3.32 11.95 4 .12 .24 .30 .12 5.88 7.46 5 .64 6.88 6.59 4 .06 .84 1.02 .36 7.84 10.55 8 .07 .41 .42 .21
5.81 6.09 .15 8.93 4.33 .21 1.15 .19
.21 .34 .06 .ll .45 .30
-
A
FORCE-FED
-y -F z 1 _- --
Plasma
-
-
-
Y2
17
DEFICIENCY
free amino
5
1
acids* -7
32 ‘72 _-
--
.06
---
.04 .07 .02 .03 .19 .lO
.03 .05 .03 .03 .08 .08
.36 .04 .04 .20 .25 .19 .42 .06 - * Pooled specimens of livers from three to five animals of each group were used in each experiment. Values are pmoles per liver. * Pooled specimens of plasma from three to five animals of each group. Values are Nmoles/ 100 ml of plasma. e C, indicates complete diet; TD, indicates threonine-devoid diet. Rats weighed 65-75 gm. d Rats were killed 5 or 6 hours after 7:30 AM feeding. B Rats were killed 5 or 6 hours after the second feeding given at 1:OO PM. 1 Rats were killed 16 hours after the last (2nd or 3rd) feeding given at 7:30 PM. .45 .62 .13 .64 .64 .08 .19 .15
.68 -17 .Ol .33 .34 .29 .37 .16
.78 .21 .02 .38 .37 .31 .46 .18
.36 .03 .02 .23 .38 .25 .49 .ll
decreased 92% in the TD group in comparison with the C group. While hepatic protein, RNA, and DNA showed little or no changes, hepatic glycogen was decreased by 24% in the TD group (Table I). Three feedings of C or TD diet (1 day)
In contrast to the results obtained after one or two feedings of diet, rats forcefed three times in 1 day and then killed 16 hours later, the following morning, showed an increase in incorporation of l*C-leucine into hepatic and plasma proteins in the TD animals in comparison with those of the C animals. Table III summarized the results of four experiments in which the rats were fed on the average 0.95 gm diet/l0 gm initial body weight and six experiments in which the rats were fed on the average 1.16 gm diet/l0 gm initial body weight during the 1 day of three feedings. It is apparent that the group of animals fed the higher intake of TD diet had a larger increase (approximately double) in hepatic protein synthesis and plasma protein synthesis than did the animals fed the lower intake.
18
SIDRANSKY
AND TABLE
INCORPORATION in Vivo OF %-LEUCINE OF RATS FORCE-FED A COMPLETE
VERNEY III
INTO PROTEINS OR THREONINE-DEVOID
OF LIVER DIET
Incorporation EXP. No.
Groud
187
C TD C TD C TD C TD
190 193 334
Summary
198 249 272 329 340 355
Amount of No. of diet fed rats (gm,1O gm body wt)
3 4 4 4 2 2 5 5
*
Liver
AND PLASMA FOR 1 DAY
of W-leucine Liver
into proteins
of
PlaSma
Weight (9)
Protein @g/liver)
Specific activity (cpmh.8 protein)
0.97 0.97 0.97 0.97 0.99 0.99 0.95 0.95
2.16 2.54 2.40 2.89 2.92 3.11 4.61 5.09
441 471 458 470 527 584 930 998
850 1330 534 572 606 618 477 665
376.6 626.2 242.0 267.4 316.6 359.7 443.8 663.7
Total incorp. (cpm/liver X 10-q
Sy;;/;gity c protein) 1572 1771 816 903 702 797
C TD
14 15
AV. 0.95 0.95
% 100 137 zlz 5.2csd
% 100 106.9 f 1.6Cre
% loo 127 zk 12.7’
% 100 135 f 13.F
C TD C TD C TD C TD C TD C TD
4 4 5 3 4 5 3 4 4 4 2 4
1.10 1.10 1.17 1.17 1.29 1.29 1.10 1.10 1.13 1.13 1.17 1.17
2.52 3.35 2.30 3.24 2.70 2.89 2.63 3.15 2.67 3.27 2.21 2.68
496
467 732 465 693 547 866 857 1191 773 959 686 927
228.2 425.4 218.6 374.5 353.0 634.5 465.0 811.0 483.5 776.5 455.3 691.3
1043 1265 593 744 1107 1550 1186 1449 1126 1295 -
C TD
22 24
AV. 1.16 1.16
% 100 124 f 4.gd
% loo 144 + 4.9d
% 100 171 zt 4.6
% 106 125 f 3.8d
summary*
577 489 540 647 726 569 677 630 804 668 754 % 106 116 f 3.0d
% 100 112 f 2.1c*e
a C indicates complete diet; TD indicates threonine-devoid diet. In each experiment animals received 2.5 &i W-leutine intraperitoneally 1 hour before killing. In experiment 334 animals weighed 112 gm while in all other experiments snimals weighed 65 to 75 gm at the start of the experiments. b In each experiment, the experimental group W&B compared with its control group (10%). c Mean GEM. dp < 0.01. e 0.05 > P > 0.01.
In two experiments rats were force-fed the C or TD diet at high levels (1.1-1.2 gm diet/day/l0 gm initial weight divided into three feedings), and the livers were used for in vitro incorporation studies. In one experiment using a postmitochondrial supernatant incorporation system as described earlier (Sidransky et al., 1968; Staehelin et al., 1967), there was 112% greater incorporation of 14C-leucineinto hepatic proteins of the TD group than in the C group. These values were corrected for the specific activity of the leucine in each incubation system. In another experiment using hepatic microsomes of C and TD rats there was a 64% increase in incorporation of 14C-leucineinto hepatic proteins of the TD group over that in the C group. The results for hepatic protein, glycogen, lipid, RNA, and DNA for rats fed the higher level (1.16 gm diet for 10 gm body weight) of C and TD diets are summarized
HEPATIC
PROTEIN
SYNTHESIS TABLE
ANALYSES FORCE-FED
IN THREONINE
Liver Protein, mg/liver Glycogen, mg/liver Lipid, mg/liver RNA, mg/liver DNA, mg/liver Gestrocnemius muscle Weight, mg Protein, mg/muscle i4C-leucine incorporation CPM/muscle X 1OF Spleen Weight, mg Protein, mg/spleen 14C-leucine incorporation CPM/spleen X lO+ Kidney Weight, mg Protein, mg/kidney i4C-leucine incorporation CPM/kidney X 1OF
SPLEEN, AND KIDNEY OF RATS DIET FOR 1 DAY (3 FEEDINGS)
Complete diet
(22)
687 110 154 2.94 0.48
f f f zk f
26a. b 206 6b .17 .05
(13)”
333 f 59 f 51.1 f
25 3.0 3.3
(17)”
309 f 51 f 33.9 f
12 7.0 l.Ob
(13)c
213 f 43 f 222.0 f
20 4 32.4
(17)”
177 f 35 f 187.7 f
10 4 27.8
(13)”
354 f 6 65 f 12 288.6 f 36.5
(17)”
334 f 16 57 xk 14 301.5 f 24.7
(4) (4)
into protein,
into protein,
(24)
diet
195 8 8 .14 .Ol
(21)
572 20 116 2.60 0.47
Threonine-devoid
f f f f f
(11)
into protein,
19
IV
OF LIVER, GASTROCNEMIUS MUSCLE, A COMPLETE OR THREONINE-DEVOID
Analyses
DEFICIENCY
(12) (24) (4) (4)
a Number of animals in parentheses; Mean f SEM. Rats weighed 65-75 gm at start of experiments and were force-fed 1.16 gm diet/l0 gm body weight. b P < 0.01. c Results are from four experiments. In three experiments, organs from two to four animals of each group were pooled for the determinations.
in Table IV. There were increasesin hepatic protein, glycogen, lipid, and RNA in the animals fed the TD diet. These increases, except for glycogen, were lessin animals fed the lower level (.97 gm diet per 10 gm body weight) than in those fed the higher level of TD diet. In one experiment in which rats were force-fed 1.1 gm diet/l0 gm body weight, sucrose density-gradient patterns were performed on livers of C and TD rats. The results are shown in Fig. 1. There was a shift in hepatic polyribosomes from lighter to heavier aggregates in the TD rats. In some experiments hepatic and plasma free amino acid levels of C and TD animals were determined (Table II). Since the results of experiments in which the animals were fed the lower diet intake were similar to those with higher diet intake, the results were combined and averaged together. Hepatic threonine levels were decreased 37%, and plasma levels were decreased 58% in the experimental group. Hepatic levels of alanine, aspartic acid, glutamic acid, and serine were somewhat elevated in the TD group over that in the C group. The results of 14C-leucineincorporation into proteins of skeletal muscle, spleen, and kidney of C and TD rats in four experiments in which rats received 1.16 gm
20
SIDRANSKY
AND VERNEY
HEPATIC FREE THREONINE
PLASMA
0
2
FREE THREONINE
4
6 6 Time -Hours
IO
12
I4
FIG. 2. Hepatic and plasma free threonine levels in rats force-fed one feeding of complete (C) or threonine-devoid (TD) diet and killed 1% to 13 hours later.
diet/l0 gm body weight are summarized in Table IV. The changesin incorporation into organ protein in the TD group in comparison to the C group were, respectively, as follows: muscle, - 34 % ; spleen, - 15 % ; and kidney, + 5 %. Additional experiments with diferent schedulesof one, two, and threefeedings In a further attempt to determine whether one feeding of TD diet under different experimental conditions than previously described would alter hepatic protein synthesis, another three experiments were conducted. After a preliminary tubefeeding period of 2 days using the C diet, the animals were divided into two groups. Each group received one morning tube-feeding of 3 gm of either C or TD diet and some of the animals of each group were killed 145, 2, 3, 5, 6, 10, 13, and 24 hours later. In all casesthe rats received 14C-leucineintraperitoneally 1 hour before killing. The results of incorporation into hepatic proteins indicated little or no differences between the C and TD groups except after 10 and 13 hours where there was a 10 to 19% increase (statistically insignificant) in the TD group. In two of these experiments hepatic and plasma free amino acids were determined in animals of the C and TD groups at the different time intervals. Figure 2 reveals the free threonine levels in the liver and plasma of C and TD rats. At 1% to 6 hours the threonine levels in the liver and plasma of the TD group were much lower than in the C group. However, after 13 hours the threonine levels of the liver and plasma of the C group had decreased so that these levels were only somewhat higher than those of the TD group. Analyses of isoleucine, leucine, methionine, phenylalanine, valine, alanine, aspartic acid, cystine, glutamic acid, glycine, proline, serine, and tyrosine in liver and plasma of C and TD animals were also conducted at each time interval, and the values for each of these free amino acids were similar in the C and TD groups. In general, the shapesof the curves for each amino acid were similar to that for threonine in the C group (Fig. 2). In light of the earlier results obtained after three feedings of C and TD diets and killing the animals 16 hours following the third feeding (Table III), we conducted additional experiments in which animals received one or two feedings of TD diet in a manner different from that used in earlier experiments (Table I). In these addi-
HEPATIC
PROTEIN
SYNTHESIS
IN THREONINE
TABLE
DEFICIENCY
21
V
INCORPORATION in Viuo OF i4C-LEUCINE INTO PROTEINS OF LIVER AND PLASMA OF RATS FORCE-FED A COMPLETE, THREONINE-DEVOID, PHENYLALANINEDEVOID DIET OR COMBINATION IN 1 DAY (THREE FEEDINGS)
Incornoration Group”
c-c-c C-C-TD C-TD-TD TD-TD-TD PD-PD-PD C-PD-PD C-TD-PD C-PD-TD TD-PD-PD
No. of rats
16 6 12 15 11 10 13 9 3
of i4C-leucine into
Liver proteinb Specii@a;tivity 0 100 106 115 134 135 115 116 116 122
f f f f f f i i
2.0 0.7d 3.5Cnd 1.6d 6.8 6.3 4.1 0.0
Plasma proteinb
Total radioactivity (%) loo 95 126 171 144 119 118 125 117
f f f i i i f f
1.2 8.1 4.3’,d 0.7d 12.0 5.50 8.3 0.0
Specific activity (%) loo 106 107 126 115 108 106 114 118
f 5.3 f 5.4 f 3.9”,d f 5.2 i 12.3 i 2.8 f 8.4 f 0.0
Q C, indicates complete diet; TD, indicates threonine-devoid diet; PD, indicates phenylalanine-devoid diet. All rats weighed 65-75 gm at start of experiments. b In each experiment, the experimental groups were compared with the control (C-C-C) group (100%). c Mean f SEM. d P < 0.01. c 0.05 > P > 0.01.
tional experiments one group (C-C-TD) of animals was tube-fed the C diet at 7:30 AM and 1:00 PM and the TD diet at 7: 30 PM and another group (C-TD-TD) was fed the C diet at 7:30 AM and the TD diet at 1:00 PM and 7: 30 PM, and then all groups were killed 16 hours later, the following morning. The results of four experiments are presented in Table V and indicate that after one TD feeding (C-C-TD group) there was essentially no difference in 14C-leucineincorporation into hepatic and plasma proteins, but after 2 TD feedings (C-TD-TD group) there was a 26% increase in ‘*C-leucine incorporation into total hepatic proteins. Thus, these results indicate that under the conditions described above, two feedings of TD diet can initiate an increase of hepatic protein synthesis if the animals were studied 16 hours after the second feeding. Four experiments were conducted to determine whether the intermixing of single essential amino acid devoid diets, threonine-devoid (TD) and phenylalanine-devoid (PD), during l-day feeding experiments would produce changes in hepatic protein synthesis. The dietary combinations used in three feedings during 1 day were as follows: (1) C-C-C, (2) C-C-TD, (3) C-TD-TD, (4) TD-TD-TD, (5) PD-PD-PD, (6) C-PD-PD, (7) C-TD-PD, (8) C-PD-TD, and (9) TD-PD-PD. The results of in vivo 14C-leucineincorporation into hepatic and plasma proteins in these experiments are summarized in Table V. The results for the C-C-TD and C-TD-TD groups were discussedin the preceding paragraph, The results for the TD-TD-TD group in comparison with the C-C-C group are similar to those obtained in other experiments (Table III) and indicate a 71% increase in hepatic protein synthesis and a 26 % increase in plasma protein synthesis. Rats of the PD-PD-PD group-showed an
22
SIDRANSKY
AND VERNEY
increase in 14C-leucineincorporation into liver (44 %) and plasma (15 %) proteins which was a somewhat smaller response than was observed in the TD-TD-TD group. These differences in the results with TD and PD diets were similar to those observed in earlier experiments of 3 days’ duration (Sidransky and Verney, 1964, 1967). Rats fed the C-PD-PD diet showed lessincrease than those fed the PD-PDPD diet but a similar increase to those fed the C-TD-PD, C-PD-TD, or TD-PDPD diet. Hepatic and plasma free amino acids were determined in several of these experiments. The overall results obtained in the animals fed the C-C-C and TD-TDTD diets were the same as those obtained in earlier experiments (Table III) and were therefore, combined, averaged, and presented in Table II. Also, the hepatic free amino acid levels of the C-TD-TD group in two experiments are presented in Table II, and these results are similar to those of the TD-TD-TD group except that there were little or no increasesin someof the nonessential amino acids over that in the C-C-C group. DISCUSSION The results of this study indicate that young rats force-fed a TD diet for 1 day (three feedings) develop enhanced hepatic protein synthesis as measured by in vivo 14C-leucineincorporation into hepatic proteins and plasma proteins. Sucrose densitygradient patterns of hepatic polyribosomes demonstrate a shift toward heavier aggregates in the TD rats in comparison to C rats, and in vitro hepatic protein synthesis is enhanced in livers of TD rats. Such changes are not observed if animals are tube-fed one or two feedings of TD diet and killed up to 6 hours later. Small increasesin hepatic protein synthesis were found in animals killed 16 hours after two feedings of TD diet. Thus, it appears that two or three feedings of TD diet and an interval of 16 hours are necessary to induce enhanced hepatic protein synthesis. Concomitant with the increase in hepatic protein synthesis in rats force-fed the TD diet for 1 day, there was a decreasein protein synthesis of skeletal muscle and spleen with little or no change in kidney. Similar results were reported in experiments of 3 days duration (Sidransky and Verney, 1967,1968). Thus, it is conceivable that the increase in hepatic protein synthesis may be related in some way to the concomitant decrease in muscle protein synthesis and increase in muscle protein catabolism. Results using 3H-leucine incorporation into proteins before beginning the control or experimental diet and then measuring loss due to protein catabolism indicated that skeletal muscle protein catabolism contributes amino acids which are then incorporated in greater amounts into hepatic proteins of the TD animals (Sidransky and Verney, 1970). Skeletal muscle degradation may be an important factor in controlling the levels of plasma free amino acids and possibly also the levels of hepatic free amino acids. Actually at 16 hours after the last (3rd) feeding, the plasma and hepatic free amino acid levels drop appreciably from peak levels at 6 hours after a feeding (Table II), and even the decreaseof plasma and hepatic free threonine levels in the TD in comparison with the C animals are now, respectively, only 58 % and 37 %. Whether these altered levels of amino acids in the plasma and liver play an influential role is at present not clear. Studies by others (Kumata and Harper, 1962; Peng et al., 1969; Sanahuja and Harper, 1963) using amino acid imbalanced diets have investigated the influence of dietary intake on plasma amino acid levels in relation to the altered nutritional state of the animals.
HEPATIC
PROTEIN
SYNTHESIS
IN THREONINE
DEFICIENCY
23
In evaluating the present results, it may be worthwhile to review the data of free amino acid levels in relation to hepatic and muscle protein synthesis after one, two, or three feedings of C or TD diet and compare the results in experimental animals with those in control animals. During the first 6 hours after one feeding of TD diet, the hepatic and plasma amino acid levels of all amino acids except threonine are high, yet hepatic and muscle protein synthesis are minimally decreased. After 10 to 13 hours following one feeding of TD diet, the hepatic and plasma amino acid levels of all amino acids, especially threonine, are low, and hepatic protein synthesis shows a questionable slight increase. At 6 hours following two feedings of TD diet, the hepatic and plasma amino acid levels of all amino acids except threonine are high, yet hepatic protein synthesis is minimally decreased. However, at 16 hours after two or three feedings of TD diet, the hepatic and plasma amino acid levels of all amino acids, including threonine, are low, yet hepatic protein synthesis is increasedwhile muscle protein synthesis is decreased. Since the free amino acid levels at lo-13 hours after one TD feeding and at 16 hours after two or three TD feedings are similar yet the hepatic protein synthesis is different, the alterations in free amino acid levels in the liver and plasma may not be the solefactor responsible. Since muscle protein synthesis seemsto decrease and muscle protein catabolism seemsto increase (Sidransky and Verney, 1970) with time after feeding the TD diet, it is possible that it is necessary for skeletal muscle protein metabolism to become affected, which may take 6-10 hours, before hepatic protein synthesis becomes stimulated. Decrease in skeletal muscle protein synthesis and decrease in hepatic and plasma free amino acids as induced by fasting 1 to 5 days do not induce a proper setting since one feeding of TD diet to such animals, which alters the free amino acid levels, will not enhance hepatic protein synthesis. Also in an earlier study (Sidransky et al., 1969) it was demonstrated that the acute administration of threonine to rats force-fed a TD diet for 3 days did not depressthe enhanced hepatic protein synthesis. Thus, once the proper setting has been induced by force-feeding the TD diet for 1 or 3 days, a sequenceof events has been set in motion which is no longer rapidly responsive to added threonine. The failure to observe enhanced hepatic protein synthesis following one feeding of TD diet or TDAA mixture in comparison to one feeding of C diet or CAA mixture is consistent with other studies by Fleck et al. (1965) with rats and with our own earlier studies (Sidransky et al., 1968) with mice. These studies indicated that animals fasted overnight and then given one tube-feeding of purified complete diet or amino acid mixture devoid of single essential amino acids excluding tryptophan (Fleck et al., 1965; Sidransky et al., 1968) showed a hepatic polyribosomal pattern and in vitro protein synthesis similar to that of animals receiving the complete control diet or amino acid mixture. Our present results stress the importance of the quantity of deficient diet in inducing alterations in hepatic protein synthesis. Rats force-fed for 1 day 1.16 gm diet/l0 gm body weight showed a greater increase (71%) than did rats fed 0.95 gm diet/l0 gm body weight (35%) in the TD groups in comparison to controls. These data are consistent with earlier results of experiments of 3 to 7 days duration where the amount of diet consumed played an mportant role (Sidransky and Farber, 1958b). Our findings in an earlier study (Sidransky and Clark, 1961) indicated that the amount of calories supplied by carbohydrate was important.
SIDRANSKY
24
AND
VERNEY
REFERENCES FLECK, A., SHEPHERD, J., and MUNRO, H. N. (1965). Protein synthesis in rat liver: Influence of amino acids in diet on microsomes and polysomes. Science 150,628-629. KUMATA, U. S., and HARPER, A. E. (1962). Amino acid balance and imbalance VII. Effects of amino acid imbalance on blood amino acid pattern. Proc. Sot. Exp. Biol. Med. 110,512-517. PENG, Y., BENEVENGA, N. J., and HARPER, A. E. (1969). Amino acid balance and food intake effect of previous diet on plasma amino acids. Amer. J. Physiol. 216, 1020-1025. SANAHUJA, J. C., and HARPER, A. E. (1963). Effect of dietary amino acid pattern on plasma amino acid pattern and food intake. Amer. J. Physiol. 204,686-690. SIDRANSKY, H., and CLARK, S. (1961). Chemical pathology of acute amino acid deficiencies. IV. Influence of carbohydrate intake on the morphologic and biochemical changes in young rats fed threonine- or valine-devoid diets. Arch. Pathol. 72,468-479. SIDRANSKY, H., and FARBER, E. (1958a). Ch emical pathology of acute amino acid deficiencies. I. Morphologic changes in immature rats fed threonine-, methionine-, or histidinedevoid diets. Arch. Pathol. 66, 119-134. SIDRANSKY, H., and FARBER, E. (1958b). Chemical pathology of acute amino acid deficiencies. II. Biochemical changes in rats fed threonine- or methionine-devoid diet. Arch. Pathol. 66, 135-149. SIDRANSKY, H., SARMA, D. S. R., BONGIORNO, M., and VERNEY, E. (1968). Effect of dietary tryptophan on hepatic polyribosomes and protein synthesis in fasted mice. J. Biol. Chem. 243, 1123-1132. SIDRANSKY, H., STAEHELIN, T., and VERNEY, E. (1964). Protein synthesis enhanced in the liver of rats force-fed a thronine-devoid diet. Science 146, 766-768. SIDRANSKY, H., and VERNEY, E. (1964). Chemical pathology of acute amino acid deficiencies. VII. Morphologic and biochemical changes in young rats force-fed arginine-, leucine-, isoleucine- or phenylalanine-devoid diets. Arch. Pathol. 78, 134-148. SIDRANSKY, H., and VERNEY, E. (1967). Decreased protein synthesis in the skeletal muscle of rats force-fed a threonine-devoid diet. Biochim. Biophys. Acta 138, 426-429. SIDRANSKY, H., and VERNEY, E. (1968). Effect of hypophysectomy on pathologic changes in rats force-fed a threonine-devoid diet. J. Nutr. 96, 28-36. SIDRANSKY, H., and VERNEY, E. (1970). Skeletal muscle metabolism in rats force-fed a threonine-devoid diet. Fed. Proc Fed Amer. Sot. Exp. Biol. 29, 365. SIDRANSKY, H., WAGLE, D. S., BONGIORNO, M., and VERNEY, E. (1969). Studies on blood glucose and hepatic glycogen in rats force-fed a threonine-devoid diet. J. Nutr. 98,477486. SIDRANSKY, H., WAGLE, D. S., and VERNEY, E. (1969). Hepatic protein synthesis in rats force-fed a threonine-devoid diet and treated with cortisone acetate or threonine. Lab. Invest.
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Sox, H. C., JR., and HOAGLAND, M. B. (1966). Functional alterations in rat liver polysomes associated with starvation and refeeding. J. Mol. Biol. 20,113-121. STAEHELIN, T., VERNEY, E., and SIDRANSKY, H. (1967). The influence of nutritional change on polyribosomes of the liver. Biochim. Biophys. Acta 145.105-119. WEBB, T. E., BLOBEL, G., and POTTER, V. R. (1966). Polyribosomes in rat tissues III. The response of the polyribosome pattern of rat liver to physiologic stress. Cancer Res. 26, 253257. WILSON, S. H., and HOAGLAND, M. B. (1967). Physiology of rat-liver polysomes. The stability of messenger ribonucleic acid and ribosomes. Biochem. J. 103, 556-566. WUNNER, W. H., BELL, J., and MUNRO, H. N. (1966). The effect of feeding with a tryptophan-free amino acid mixture on rat liver polysomes and ribosomal ribonucleic acid. Biothem. J. 101,417-428.