EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
22, 85-97
( 19%)
Protein Synthesis in Rats Force-Fed for One Day Purified Diets Containing Complete, Threonine-Devoid or No Amino Acids and Adequate or Low Carbohydratel** ETHEL Department
of
VERNEY AND HFBSCHEX
SIDRANSKY
Pathology, University of South Florida, College of Medicine, Tampa, Florida 33620
Received May 20, 1974, and in revised form July 8, 1974 Young female rats were force-fed purified diets containing complete (C), threonine-devoid (TD) or no (AAD) amino acids and adequate or low carbohydrates for 1 day (three feedings) and were killed the following morning. Protein synthesis (in viva UC-leucine incorporation into proteins) was measured in liver, plasma, heart, gastrocnemius muscle, and spleen. The results revealed that protein synthesis was increased in the liver, plasma, and heart of rats force-fed the TD diet and was increased, but less marked, in the liver and plasma of rats force-fed the AAD diet. Protein synthesis in the gastrocnemius muscle and spleen was decreased in both experimental groups. Hepatic polyribosomes revealed greater aggregation and there was increased in vitro incorporation of “C-leucine into proteins of livers of rats force-fed the TD and the MD (less marked than with TD) diets in comparison with those of rats force-fed the C diet. Rats force-fed the experimental diets containing low carbohydrates did not reveal similar changes to those of rats force-fed the experimental diets containing adequate carbohydrates.
INTRODUCTION In earlier studies from our laboratory, we described in separate reports (Sidransky and Verney, 1970a, 1971) the effects on hepatic protein synthesis of force-feeding a threonine-devoid (Sidransky and Verney, 1970a) or an amino acids-free (protein-free) diet (Sidransky and Verney, 1971) in comparison to force-feeding a complete diet to rats for 1 day. Enhanced hepatic protein synthesis, determined by measuring in uivo 14C-leucine incorporation into proteins, was observed in rats force-fed a threonine-devoid diet (Sidransky and Verney, 1970a) and in rats force-fed a protein-free diet ( Sidransky and Verney, 1971). In addition, in U&O hepatic protein synthesis was increased and sucrose density gradient patterns of hepatic polyribosomes were shifted toward heavier aggregates in the experimental animals when compared to control animals in both studies (Sidransky and Vemey, 197Oa, 1971). Also in an earlier study (Sidransky and Rechcigl, 1962) from our laboratory, we reported that young rats force-fed for 3 days a purified diet devoid of amino acids developed no 1 This investigation was supported by United States Public Health Service Grant AM-16530 from the National Institute of Arthritis and Metabolic Diseases. a A preliminary report was presented at the Meeting of the Federation of American Societies for Experimental Biology in Atlantic City, N. J., April 1974; Fed. PTOC. 33, 695 (abst.). 85
86
VERNEY
AND
SIDRANSKY
pathologic (morphologic) changes in the liver, whereas the rats force-fed for 3 days a threonine-devoid diet developed a fatty liver with a periportal distribution of lipid and increased liver glycogen as described earlier in our experimental kwashiorkorlike model ( Sidransky and Farber, 1958a, b). In the present investigation, we were concerned with protein synthesis in the liver, heart, gastrocnemius muscle, and sp1een of rats force-fed a complete, threonine-devoid or amino acids-devoid diet for three feedings during 1 day and killed the following morning. By conducting these experiments, it was hoped to obtain further information regarding the similarities or differences in the effects of force-feeding equal quantities of isocaloric amounts of threonine-devoid and amino acids-devoid diets. Also, since in an earlier study (Sidransky and Clark, 1961) we reported that the amount of carbohydrate intake in the threonine-devoid diet influenced the morphologic and biochemical changes in the animals force-fed for 3 days, the influence of reducing the carbohydrate content of the threonine-devoid and amino acids-devoid diets on protein synthesis in the animals was investigated in this study of shorter duration. The results of our present studies indicate that the response of protein synthesis in the organs of rats force-fed for one day the threonine-devoid diet is generally more marked than that of rats force-fed for 1 day the amino acids-devoid diet. Also, the reduction of carbohydrate content of each of the two experimental diets caused a marked diminution or almost eliminated the changes in protein synthesis observed with animals force-fed for 1 day the experimental diets containing a higher or adequate quantity of carbohydrates. The present experiments of short (1 day) duration stress the importance of the type of deficient diet as well as the carbohydrate content on the induction of acute alterations in protein synthesis of the liver, heart, gastrocnemius muscle, and spleen. MATERIALS
AND
METHODS
Female rats of the Sprague-Dawley strain, 3 1 mo old, weighing on the average 65 g, were used. The animals were fed a commercial diet 4 before the experiments were begun, In all experiments, groups of animals, each of the same age and weight, were used. The basal diet was identical to that used in earlier experiments (Sidransky and Clark, 1961; Sidransky and Verney, 1970a). The percentage of dietary components was as follows: essential amino acids,5 9.2; nonessential amino acids,5 8.1; salt mixture (Hegsted IV)5 (S’d1 ransky and Farber, 1958a), 4; vitaminsucrose mixture 6 (Sidransky and Farber, 1958a), 5; corn oil,’ 5; cod liver oi1,s 1.5; and dextrin6 67.2. Dextrin was substituted for the threonine in the threoninedevoid diet and for all amino acids in the amino acids-free diet. In experiments where rats were fed the low carb.ohydrate (LC) diets, the diets were the same as the previous diets except that the dextrin content was reduced from 67.2 g/ s Sprague-Dawley, Madison, Wise. 4 Wayne Lab-Blox, Allied Mills, Inc., Chicago, Ill. s General Biochemicals, Inc., Chagrin Falls, Ohio. s&Methyl-l, 4-naphthoquinine. Eastman Organic Chemicals, vitamins, Nutritional Biochemical Corp., Cleveland, Ohio. 7 Cod Liver Oil Liquid, Mead Johnson Laboratories, Evansville, s Amersham/Searle Corp., Arlington Heights, Ill.
Rochester, Ind.
N.
Y. All
other
PROTEIN
SYNTHESIS
WlTH
DEFICIENT
DIETS
87
100 g diet to 24.2 g/57 g (43 g/100 g) diet without replacement, while all other dietary components were kept constant (Sidransky and Clark, 1961). The diets were blended with distilled water so that each milliliter of diet mixture contained 0.2!3-0.50 g of diet. Rats were force-fed using plastic tubes at SO0 AM, noon and 600 PM and each animal received on the average 1.2 g (4.8 cal) of regular diet or 0.7 g (2.8 cal) of low carbohydrate diet per 10 g of initial body wt. The basal complete diet was force-fed to all rats for 2 days prior to beginning the experimental diets: the threonine-devoid (TD ), the amino acids-devoid (AAD), or the complete ( C ) diet with adequate or low carbohydrate (LC) for 1 day. Rats had free accessto water. They were kept in individual wire cages with raised bottoms and in an air-conditioned room. All animals were killed in the morning approximately 16 hr after the last tube-feeding. Rats were weighed at the beginning and end of each experiment. The animals were killed by exsanguination. In in I&O experiments, the rats were killed by removing blood from the aorta after ether anesthesia, while in in vitro experiments, they were killed by decapitation. The organs, liver, heart, gastrocnemius (right), and spleen, were rapidly removed and weighed fresh. Pieces of the organs were homogenized in appropriate solutions. In studies on incorporation in uiuo, rats received 2.5 ,&i (10 &i/mole) L-leucineJ4C, uniformly labeled,* intraperitoneally 1 hr before killing. In studies on incorporation in u&o, postmitochondrial supernatants, mic,somes or ribosomes of liver homogenates were used. The postmitocbondrial supernatant, microsomes and ribosomes were prepared and used for assays as described earlier (Staehelin et al., 1967; Sidransky et al., 1968). r.,-LeucineJ4C, 0.5 &i, was added to each incubation tube. Radioactivity in protein was measured using a liquid scintillation spectrometer.a For size distribution analysis of hepatic polyribosomes, postmitochondrial supernatants were prepared in 0.25 M sucrose containing TKM (0.05 M Tris (pH 7.6)) 0.025 M KCl, and 0.005 LM MgClz) and then treated with deoxycholate (0.7%, final concentration) Staehelin et al., 1967; Sidransky et al., 1968). Samples were layered on linear 12 ml sucrose gradients (0.3 to 1.1 M sucrose containing TKM) and centrifuged in an ultracentrifuge lo at 38,000 rpm for 1 hr at 2” and analyzed as described earlier (Sidransky et al., 1968). The methods used for chemical analyses of protein, glycogen, RNA and DNA have been described in detail in earlier studies (Sidransky and Farber, 1958b; Sidransky and Verney, 1970a). RESULTS In Table I, the changes in body weight and the weights of the liver, heart, gastrocnemius muscle, and spleen of animals force-fed the different diets for 1 day are summarized. While rats force-fed the complete (C) diet gained weight ( + 1.7 g), the rats force-fed the threonine-devoid (TD) diet showed little change in weight (+O.l g), and the rats force-fed the amino acids-devoid (AAD) diet Iost weight (-1.3 g). AI1 rats force-fed the low carbohydrate (LC) diets lost 9 Packard Tri-Carb, Packard Instrument Company, Inc., Downers Grove, Ill. 10 Spinco SW 41 rotor in a model L2-65 Spinco ultracentrifuge, Beckman Instruments, Fullerton, Calif.
Inc.,
(32) -2.1 (32) -2.9 (33) -4.6
c-LC TD-LC AAD-LC
f f f
f f f
TABLE
I
0.34 0.34 0.30
0.35” 9.34 0.37 lOO(94 104 f 99 f
f 5.0)” 1.8 2.0
lOO(95 102 f 97 f
lOO(96 zk 12.9)” 97 f 3.3 99 f 2.9
(6) 100” 94 f 2.7 95 f 2.4
Gastrocnemius Muscle mg
lOO(97 102 f 93 f
(5) 100” 81 f 87 i
10.2)” 5.5 11.0
i
4.7d l.gd
Spleen mg
; Values for groups C and C-LC were set at 100% in each for the C group were as follows: liver, 2.58 f 0.06 g ; heart,
f 5.5)” 0.5 2.4
(3)lOOb 100 f 2.1 96 f 1.2c
mg
g
(6)100b 112 3~ 3.2” 93 f 2.2
Heart
Liver
AND ORGAN WEIGHTS OF RATS FORCE-FED PURIFIED DIETS CONTAINING COMPLETE (C), (TD) OR No (AAD) AMINO ACIDS AND ADEQUATE OR Low (LC) CARBOHPDRATES
a Number of animals in parenthesis; mean f SE. Rats weighed 65 g at st.art of experiments. b Number of experiments in parenthesis (in each experiment, 3-5 rats were used in each group) experiment and the experimental groups were compared with its cont,rol group. The organ weights 313 i 7.3 mg; gastrocnemius muscle, 369 f 15.9 mg; and spleen, 208 f 10.2 mg. e 0.05 > p > 0.01. dp < 0.01. e Values in parenthesis were compared with C group.
(46) +1.7 (43) +0.1 (47) -1.3
g
Body weight changes
C TD AAD
Group
CHANGES IN BODY THREONINE-DEVOID
iz
t!
E u
ECM
00 00
PROTEIN
SYNTHESIS
WITH TABLE
DEFICIENT
89
DIETS
II
ANALYSES OF LIVER OR RATS FORCE-FED COMPLETE (C), THREONINE-DEVOID ACIDS AND ADEQUATE OR Low
PURIFIED DIETS CONTAINING (TD) OR No (AAD) AMINO (LC) CARBOHYDRATES Livep
Group Protein
Glycogen
RNA
DNA
bx/liver) C TD AAD
(20)680 (19)773 (23)617
f f f
23.1 38.2b 23.1
C-L@ TD-LC AAD-LC
(15)627 (14)625 (15)570
xt 38.2 f 40.0 f 32.2
0 Number of animals in parenthesis b0.05 > p > 0.01. c p < 0.01. d Compared with C group.
(24)13.1 (25)55.4 (27)lS.l
f f f
3.1 5.9” 3.6
(11)24.3 (12)25.6 (13)21.1
f f f
1.0 1.1 O.gb
(11)8.88 (12)8.98 (13)8.65
i i f
0.39 0.50 0.29
(28) 7.5 f (28)15.6 f (29)25.8 f
1.6 3.5b 5.4”
(15)21.4 (14)22.8 (15)20.5
zt 0.4b f 0.5 f 0.6
(15)7.73 (14)8.25 (15)8.56
i i i
0.21b 0.31 0.31
; mean
f
SE.
weight. Those fed the AAD-LC diet lost the most (-4.6 g) and those fed the C-LC diet lost the least ( -2.1 g). The liver weights increased in rats force-fed the TD diet and decreased in rats fsorce-fed the other (AAD, C-LC, TD-LC, and AAD-LC) diets. The heart weights were essentially unchanged in rats force-fed the C and TD diets and decreased in rats force-fed the AAD diet and the groups force-fed the LC diets. The gastrocnemius muscle and splenic weights were somewhat decreased in rats fed the TD and AAD diets and further and similarly decreased in rats fed the three LC diets. In general, the only significant changes were in the increased liver weights in rats force-fed the TD diet, TABLE ANALYSES OF GAS~OCNEMIUS PURIFIED DIETS CONTAININQ No (AAD) AMINO ACIDS Group”
Gastroenemius
III
MUSCLE, HEART, AND SPLEEN OF RATS FORCE-FED COMPLETE (C), THREONINE-DEVOID (TD) OR AND ADEQUATE OR Low (LC) CARBOHYDRATES muscle
Heart
Glycogen
Protein
bsld
(mg/muscle)
protein
Spleen
protein
GmdorwQ
C TD AAD
(25)5.89 (26)6.@4 (28)6.56
zk 0.43 f 0.57 f 0.52
(20)55.9 (19)53.4 (‘22)53.5
f f f
3.7 3.8 3.6
(11)35.9 (12)42.9 (13)36.6
f i f
2.5 3.5 2.6
(21)40.9 (22)32.9 (23)36.5
f 3.3 f 2.6 f 2.5
C-L@ TD-LC AAD-LC
(29)3.45 (28)3.64 (29)5.07
f i f
(12)49.2 (14)44.5 (15)42.9
f f f
6.2 6.6 5.3
(12)41.5 (14)37.8 (1.5)36.7
f 2.9 f 4.1 f 2.9
(15)36.8 (12)40.5 (15)32.8
f f f
0.17” 0.19 0.43c
0 Number of animals in parenthesis; * Compared with C group. ‘p < 0.01.
mean
f
SE.
6.5 8.3 8.9
VERNEY
90
AND TABLE
SIDRANSKY IV
INCORPORATION IN VIVO OF W-LEUCINE INTO PROTEINS OF LIVER AND PLASMA FORCE-FED PURIFIED DIETS CONTAINING COMPLETE (C), THREONINE-DEVOID OR No (AAD) AMINO ACIDS AND ADEQUATE OR Low (LC) CARBOHYDRATES
Group
No. of raw
Incorporation of W-leucine Liver Specific activity %
C TD AAD
19 18 20
100 125 f 129 f
c-LC TD-LC AAD-LC
12 13 15
100(110 f 9.2)” 100 f 10.2 110 rrtr 3.4”
4.7” 6.0”
OF RATS (TD)
into proteins of6 Plasma
Total incorporation %
Specific activity %
100 141 f 7.2~ 119 l 5.0d
100 120 f 106 f
lOO(112 i 11.4)6 94 f 9.5 90 f 3.0”
lOO(102 f 3.5)” 104 f 13.6 111 f 22.7
5.6d 7.7
QResults of six experiments of adequate carbohydrate groups and of three experiments of LC groups. “Values for groups C and C-LC were set at 1007, in each experiment and the values (mean f SE) of the experimental groups were compared with those of the control groups. The values for the C group were as follows: Specific activity (cpm/mg protein), liver, 908.7 f 49.3 and plasma, 1285.1 f 64.7; Total incorporation (cpm/liver protein), (631.2 f 47.3) X 103. c p < 0.01. do.05 > p > 0.01. a Values in parenthesis were compared with C group.
the decreased liver and heart weights in rats force-fed the AAD diet, and the decreased splenic weights in rats force-fed the TD and AAD diets. Tables II and III summarize the analyses conducted on the composition of the liver, gastrocnemius muscle, heart, and spleen of rats force-fed the different diets. The main significant differences were as follows. Regular carbohydrate groups: TD group, increase of hepatic protein and glycogen; AAD group, decrease of hepatic RNA; Low carbohydrate (LC) groups: C-LC group compared with C group, decrease of hepatic RNA and DNA, and decrease of gastrocnemius muscle glycogen; AAD-LC group, increase in hepatic and gastrocnemius muscle glycogen; TD-LC group, increase in hepatic glycogen. TabIes IV and V summarize the incorporation in viva of W-leucine into proteins of liver, plasma, heart, gastrocnemius muscle, and spleen of rats fed the control and experimental diets. The results whether expressed as specific activity or as total incorporation into organ protein indicated in the TD and AAD groups an increase for liver, plasma, and heart and a decrease for gastrocnemius muscle and spleen. In general, the differences were more marked in the TD group than in the AAD group. Rats force-fed the low carbohydrate diets revealed only less differences between groups but in genera1 there was a decreased total incorporation into proteins of liver, heart, and spleen of the AAD-LC groups. Table VI summarizes the incorporation in vitro of 14C-leucine into protein when using postmitochondrial supernatants, microsomes or ribosomes prepared from the livers of the control and experimental animals. In ‘general, the results
12 13 15
c-LC TD-LC AAD-LC
lOO(105 f 17.0)” 108 f 3.2d 89 i 4.6d
100 132 f 2.3 108 i 6.5
S.A.
Heart
8.8” 4.7
lOO(116 f 12.9)” 96 f 10.0 81 f 7.4d
loo 154 f 107 f
Total incorporation
GASTROCNEMIUS
S.A.
lOO(103 f 14.5)” 94 zt 4.6 97 f 7.8
% 6.2c 6.3d
lOO(89 f 4.7d)” 83 f 8.8 84 f 12.7
100 70 f 75 f
Total incorporation
into proteins of* muscle
100 72 f 3.0” 80 f 18.0 lOO(99 f 7.0)’ 80 f 6.1c 73 i 4.0”
lOO(94 f 3.9)” 80 z!z 15.9 80 f 1.7”
Total incorporation
DIETS
100 93 f 17.3 83 f 24.2
S.A.
Spleen
MUSCLE AND SPLEEN OF RATS FORCE-FED PURIFIED ACIDS AND ADEQUATE OR Low (LC) CARBOHMRATES
of W-leucine Gastrocnemius
100 80 f 3.0” 91 f 5.7
Incorporation
OR No (AAD) AMINO
OF HEART,
(TD)
V
a Results of six experiments of adequate carbohydrate group and of three experiments of LC groups. 6 Values for groups C and C-LC were set at 100% in each experiment and the values (mean f SE) of the experimental groups were compared with those of the control groups. The values for the C group were as follows : Sp act (specific activity), cpm/mg protein, and total incorporation (cpm/organ protein X loo), respectively: heart, 247.8 f 8.7, 89.1 f 5.65; muscle 94.2 f 7.7, 49.0 Z!Z5.36; and spleen, 705.5 i 22.5, 288.0 i 63.0. cp < 0.01. do.05 > p > 0.01. * Values in parenthesis were compared with C group.
19 18 20
No. of ratsa
C TD AAD
Group
INCORPORATION IN VIVO OF W-LEUCINE INTO PROTEINS CONTAINING COMPLETE (C), THREONINE-DEVOID
TABLE
Ei 2
g ?I fi I3 2
3 2 E E
z”
2
VJZRNEY
92
AND TABLE
SIDRANSKY VI
INCOHPORATION IN VITRO OF W-LW~IN~ INTO PROTEINS BY H~PATIC POLYRIBOSOMYS USING POSTM~TOCHONDRIAL (PMS), MICROSOMAL OR RIBO~OMAL PREPARATIONS OF RUTS FORCE-FED PURIFIED DIETS CONTAINING COMPLETE (C), THREONINE-DEVOID (TD) OR No (AAD) AMINO ACIDS .~ND ADEQUATE OR Low (LC) CARBOHYDRATES Group
Source
of homogenate
PMS
fractions
Microsomesa
RibosomesU
% C TD AAD
(4) 1006 (4)149.1 (4)124.2
C TD AAD
(5)100(93.7 (5)lOl.O f (4)110.7 f
f f
(5) 1OOb (5) 161.0 (5)119.4
5.8” 3.@
f f
(5) 1OOb (5)133.1 (5)116.5
18.0c 34.9
f f
5.86 11.6
& 6.0)d 15.5 7.4
a Microsomes and ribosomes of each group were assayed using supernatants of their own groups. b Number of experiments in parenthesis (in each experiment, livers of 3-5 rats were pooled in each group). Values for group C and C-LC were set at lOO’$& in each experiment and the values (mean i SE) of the experimental groups were compared with those of the control group. cp < 0.01. d Value in parenthesis was compared with C group.
here are similar to those obtained in the in vivo incorporation studies (Tables IV and V ). There was increased incorporation ( 33-61s ) when using the fractions of the TD group and less increases (164X% ) with that of the AAD group. Little or no changes were present in the LC groups. In some experiments (Table TABLE
VII
COMPARISON OF THE EFFECTS OF TUBE-FEEDING FOR 1 DAY A THREONINE-DEVOID AND A AMINO ACIDS DEVOID (AAD) DIET IN COMPARISON TO A COMPLETE Determination”
Organ
weight
Protein
synthesis
Glycogen RNA
content
Polyribosomal
a?, Increase;
content
1, decrease;
NC,
1 1
NC 1
TD AAD
I
i
r NC
TD AAD
t (4+) t (2+)
TD AAD
t (4+) t cl+)
TD AAD no change.
Heart
1
TD AAD aggregation
Gastrocnemius muscle
T
TD AAD
content
Protein
Liver
Group
(TD) DIET (C) DIET
NC
1 3+
2+
t NC
PROTEIN 1,COMPLETE
aI 1
DIET
SYNTHESIS 4
WITH
2. THREONINE-
DEFICIENT
DEVOID
DlET
DIETS J
93
3. AMINO t?LlDSDEVOID DIET
4. COMPLETE LOW CARl3CtlYDkATE DIET
07
FIG. 1. Sucrose density gradient patterns of hepatic polyribosomes of deoxycholate-treated postmitochondrial supernatants of rats force-fed purified diets containing complete (l), threonine-devoid (2) or no (3) amino acids with adequate carbohydrate or purified diets containing complete (4), threonine-devoid (5) or no (6) amino acids with low carbohydrate.
VI) microsomes and ribosomes of each group were mixed with supernatants of other groups and then assayed for W-leucine incorporation into protein. These assays revealed similar results to those indicated in Table VI where complete systems were compared and indicate that the differences are due predominantly to the microsomes or ribosomes and not due to the supematant fractions. Sucrose density gradient analyses of deoxycholate-treated postmitochondrial supernatants of livers of control and experimental animals were studied, Figure 1 summarizes the polyribosome patterns of the six groups of animals. It is apparent that the livers of rats force-fed the threonine-devoid diet or the amino atidsdevoid diet had more aggregated polyribosomes (more marked in the TD than in the AAD) than those of the control animals. The livers of rats force-fed the low carbohydrate diets had more ‘disaggregated patterns, particularly in the GLC and TD-LC groups, but less so in the AAD-LC group. These findings are consistent with the changes in hepatic protein synthesis described in Tables IV and VI. DISCUSSION Some of the results of the present study are summarized in Table VII. It is apparent that there are a number of similarities as well as differences in the liver, gastrocnemius muscle and heart of rats force-fed for one day the threoninedevoid (TD) or the amino acids-devoid (AAD) diet. The results indicate that although the effects on protein synthesis of the liver, induced by force-feeding a threonine-devoid diet or an amino acids-devoid diet for 1 day are similar, they are more marked after the former diet. Thus, the presence of dietary amino acids
94
VERNEY AND SIDRANSKY
in the threonine-devoid diet plays a role in enhancing hepatic and cardiac protein synthesis. Actually, based on earlier studies (Sidransky and Verney, 1970a, 1971), there is evidence to suggest that the mechanisms involved in the liver may be different under the two experimental dietary conditions, There is evidence to suggest a protein-free diet may have its effect on hepatic protein synthesis through insulin ( Sidransky and Verney, 1971) . On the other hand, the effect due to the threonine-devoid diet is not due to elevated levels of insulin (Sidransky et al., 1969a) and is considered to be more related to an imbalanced state of circulatory amino acid levels (Sidransky and Rechcigl, 1962; Sidransky, 1972). The enhanced hepatic protein synthesis in rats force-fed the threonine-devoid diet for one day could not be attributed directly to a decreased level of hepatic free threonine since the hepatic threonine levels were decreased by 37% due to the TD diet (Sidransky and Verney, 1970a) and by 43% due to the AAD diet (Sidransky and Verney, 1971) in comparison to rats force-fed the complete diet. Also, the acute administration of threonine to rats after they had been forcefed the TD diet for 3 days had no influence on in vivo hepatic protein synthesis ( Sidransky et al., 1969b). In further consideration of the increased hepatic protein synthesis in rats force-fed the TD or the AAD diet, we wondered whether the increases could be in any way related to alterations in precursor pool sizes. Although the levels of free amino acids were not determined in this study, they were measured in two earlier studies ( Sidransky and Verney, 197Oa, 1971). These earlier results revealed that the spe&c activities of the W-leucine in the livers of the C and TD groups ( Sidransky and Verney, 1970b) and of the C and AAD groups ( Sidransky and Verney, 1971) were similar in both cases.Thus, altered pool sizes were not responsible for the differences in the in vivo hepatic protein synthesis in the control and experimental groups. Also, the in vitro incorporation studies in which the leucine pool size could be measured or controlled by using a common cell sap revealed that the hepatic polyribosomes of the TD (Sidransky and Verney, 1970a) and AAD (Sidransky and Verney, 1971) groups were more active in terms of protein synthesis. In addition, the hepatic polyribosome patterns of the TD and AAD groups were consistent with the in vivo and in vitro incorporation data indicating enhanced hepatic protein synthesis in the experimental groups. Thus, our results reveal that the enhanced protein synthesis in the experimental animals is clearly attributable to changes in the activities of the hepatic polyribosomes of the experimental animals. In an earlier study from our laboratory (Verney and Sidransky, 1974) we reported that the hearts of rats force-fed for 3 days a threonine-devoid diet revealed increases in weight, protein, RNA, glycogen, and in cardiac protein synthesis in in vivo and in &tro studies. Now in our present investigation we find that cardiac protein synthesis, based on in viva incorporation of 14C-leucine into protein, is significantly increased ‘over that in control animals in experiments of only 1 day’s duration. The hearts of rats force-fed the amino acids-devoid diet do not demonstrate such changes. Further studies are in progress relating to the alterations of cardiac protein metabolism in rats force-fed the threonine-devoid diet. Earlier studies from our laboratory (Sidransky and Rechcigl, 1962) have also reported that the morphologic consequences of force-feeding a threonine-devoid
PROTEIN
SYNTHESIS
WITH
DEFICIENT
DIETS
95
or protein-free diet for 3 days are quite different. While rats force-fed the threonine-devoid diet developed a periportal fatty liver with increased hepatic glycogen, protein, and RNA, such changes were not found in rats force-fed the protein-free diet for 3 days. In experiments of longer duration, Platt et UI. (Platt et al., 1964) h ave reported a number of pathologic changes in rats forcefed a protein-free diet for 6 days. Thus, it appears that the consequences of forcefeeding a threonine-devoid Norother single essential amino acids-devoid diets ( Sidransky, 1972) are indeed different than that of a protein- or amino acids-free diet and suggest altered mechanisms, differing either qualitatively or possibly quantitatively in inducing pathologic effects, are involved even though alterations in hepatic protein metabolism may appear to be similar during the earlier stages of the deficiencies. The results of our experiments in which the carbohydrate intake was lowered in the control (C-LC) and deficient ( TD-LC and AAD-LC ) diets are of much interest. Consistent with earlier experiments in which high amounts of carbohydrate in the TD diet accentuated while low amounts of carbohydrate in the TD diet diminished the pathologic changes in the liver and other organs (Sidransky and Clark, 1961), we now find that a decrease of carbohydrate content in the TD and AAD diets diminishes or eliminates the changes in protein synthesis of the liver and heart of the animals fed the deficient diets. These results would suggest that the elevated levels of hepatic protein synthesis in rats forcefed the TD (regular carbohydrate) diet may be involved or related to the pathologic changes such as fatty liver, increased glycogen, and increased RNA of the liver observed in earlier studies (Sidransky, 1972). Studies from other laboratories (Barnes et al., 1959; Heard et al., 1958; Macdonald, 1966) have also indicated the importance of the caloric intake in experiments with protein deficiency. Indeed, there have been reviews of the importance of protein-energy relationships by a number of workers (Miller, 1973; Morrison and Narayana Rao, 1967; Munro, 1964; Swanson, 1959). Thus, it is now apparent that the amount of carbohydrate in a deficient diet is of great importance in eliciting biochemical as well as morphologic alterations in organs such as the liver. Likewise, it is conceivable that the marked differences in morphologic and biochemical reactions in animals fed by stomach tube and those fed ad Zibitum deficient diets (Sidransky, 1972) may be due in (part or in whole to the decreased intake of carbohydrate, since the ad Zibitum fed animals consume much less diet which is chiefly composed of carbohydrate. Indeed, in the human nutritional disease, kwashiorkor a prominent feature of the dietary history is the relatively high intake of carbohydrate coupled with inadequate protein (Brock and Autret, 1952; Davies, 1952; Housden, 1956; Waterlow, 1948). In contrast to the numerous pathologic lesions described in kwashiorkor, there are few or no specific lesions, particularly of the liver, in states of general undernutrition with severe restriction of protein and carbohydrate intake as occurs with maramus (Jelliffe and Welbourn, 1963; Latham, 1965). REFERENCES R. H., KWONC, E., POND, W., LOWRY, R., and LOOSLI, J. K. (1959). and protein and serum cholesterol. II. Young swine. 1. Nuts. 69, 269.
BARNES,
Dietary
fat
VERNEY
96 BRICK, J. F., Monograph DAVIES,
AND SIDRANSKY
and AUTRET, M. (1952). “Kwashiorkor in Africa.” Series No. 8. Columbia University Press, New York.
J. N. P. (1952).
Nutrition
and
nutritional
diseases.
Ann.
HEARD, C. R. C., PLATT, B. S., and STEWART, R. J. C. low-protein diet with and without additional carbohydrate. HOUSDEN, L. (1950). Infant feeding in North-East Africa. JELLIFFE, D. B., and WELBOURN, H. F. calorie malnutrition of early childhood.” Malnutrition,” p. 12. Almquist & W&ells, LATHAM, M. ( 1965). “Human tion of the United Nations, MACWNALD,
I.
( 1960).
Nutrition Rome.
Some
effects
World
of
dietary
Africa.” sucrose
Organization
Med. 3, 99.
Rev.
(1958). The effects on pigs Proc. Nutr. Sot. 17, xli. hit. Med. J. 2, 456.
(1963). ‘Clinical signs In Blix “Mild-moderate Uppsala. in Tropical
Health
of
Food in
of
a
mild-moderate proteinForms of Protein-Calorie
and
Agriculture
experimental
Organiza-
protein
deficiency.
PTOC. Nutr. Sot. 19, XXIX. MILLER, Human
D. S. (1973). Protein energy interrelationships. Nutrition,” p. 93. Academic Press, New York.
MORRISON, A. B., and NARAYANA RAO, M. calories. In Bourne (Ed.), “World Review Publishing Co., Inc., New York. M~JNRO, H. N. (1964). General aspects by hormones. In Munro and Allison p. 381. Academic Press, New York.
In
Porter
(1967). Some relationships of Nutrition and Dietetics.”
H.
( 1972).
Chemical
and
Rolls
“Protein
cellular
pathology
of
in
between proteins and Vol. 7, p. 204. Hafner
of the regulation of protein metabolism by (Eds. ), “Mammalian Protein Metabolism,”
PLATT, B. S., HEARD, C. R. C., and STEWART, R. J. C. (Ed.), calorie deficiency. In Munro and Allison (Ed.), “Mammalian p. 445. Academic Press, New York. SIDRANSKY, deficiency.
and
(1964). Protein
diet and Vol. 1,
Experimental Metabolism,”
experimental
acute
proteinVol. 2, amino
acid
Meth. Achievm. Exp. Path. 6, 1.
SIDRANSKY, H., and CLARK, S. ( 1961). Chemical pathology of acute amino IV. Iniluence of carbohydrate intake in the morphologic and biochemical rats fed threonineor valine-devoid diets. Arch. Path. 72, 468. SIDFIANSW, H., and FARBER, E. (1958a). I. Morphologic changes in immature diets. Arch. Path. 66, 119.
Chemical rats fed
pathology threonine-,
H., and FARBER, E. (195813). Chemical II. Biochemical changes in rats fed threonine135.
of acute amino acid deficiencies. methionine-, or histidine-devoid
pathology of acute or methionine-devoid
SIDRANSKY,
acid deficiencies. changes in young
amino acid deficiencies. diets. Arch. Path. 66,
SIDRANSK~, H., and RECHCIGL, M., Jr. (1962). Ch emical pathology of acute amino acid deficiencies. V. Comparison of morphologic and biochemical changes in young rats fed protein-free or threonine-free diets. .I. Nutr. 78, 269. SIDRANSW, H., SARMA, D. S. R., BONGIORNO, M., and VERNEY, E. (1968). Effect of dietary tryptophan 243, 1123.
on hepatic
polyribosomes
and
protein
synthesis
in fasted
mice.
Biol. Chem.
J.
H., and VERNEY, E. (1970a). Studies on hepatic protein synthesis in rats force-fed a threonine-devoid diet. Exp. Mol. PathoE. 13, 12. SIDRANSKY, H., and VERNEY, E. (1970b). Skeletal muscle protein metabolism changes in rats force-fed a diet inducing an experimental kwashiorkor-like model. Am. J. Clin Nutr. 23, 1154. SIDRANSKY,
SIDRANSKY, H., and VERNEY, E. ( 1971). Studies thesis in rats force-fed a protein-free diet. J. SIDRAN~KY, H., WAGLE, D. S., BONGIORNO, M., glucose and hepatic glycogen in rats force-fed
H., WAGLE, D. force-fed a threonine-devoid Invest. 20, 364.
SIDRANSKY,
S. and VERNEY, E. diet and treated
on hepatic
polyribosomes
and
protein
syn-
Nutr. 101, 1153. and VERNEY, E. a threonine-devoid
(1969a). diet. J.
Studies
on
blood
Nutr. 98, 477.
(1969b). Hepatic protein synthesis with cortisone acetate or threonine.
in
rats Lab.
PROTEIN
SYNTHESIS
WITH
DEFICIENT
DIETS
97
T., VERNEY, E. and SIDRANSKY, H. ( 1967). The influence of nutritional change on polyribosomes of the liver. Biochdm. Biophys. Acta 145, 105. SWANSON, P. (1959). Food energy and the metabolism of nitrogen. In Albanese (Ed.), “Protein and Ammo Acid Nutrition,” p. 195. Academic Press, New York. VERNEY, E., and S~RANSKY, H. (1974). Alterations in cardiac protein metabolism in rats force-fed a threonine-devoid diet. j. Nub. 104, 463. WATERLOW, J. C. ( 1948). Fatty liver disease in infants in the British West Indies. Medical Research Council Special Report Series No. 263, Her Majesty’s Stationery Office, London. STAEHELXN,
AND
MOLECULAR
PATHOLOGY
22, 85-97
( 19%)
Protein Synthesis in Rats Force-Fed for One Day Purified Diets Containing Complete, Threonine-Devoid or No Amino Acids and Adequate or Low Carbohydratel** ETHEL Department
of
VERNEY AND HFBSCHEX
SIDRANSKY
Pathology, University of South Florida, College of Medicine, Tampa, Florida 33620
Received May 20, 1974, and in revised form July 8, 1974 Young female rats were force-fed purified diets containing complete (C), threonine-devoid (TD) or no (AAD) amino acids and adequate or low carbohydrates for 1 day (three feedings) and were killed the following morning. Protein synthesis (in viva UC-leucine incorporation into proteins) was measured in liver, plasma, heart, gastrocnemius muscle, and spleen. The results revealed that protein synthesis was increased in the liver, plasma, and heart of rats force-fed the TD diet and was increased, but less marked, in the liver and plasma of rats force-fed the AAD diet. Protein synthesis in the gastrocnemius muscle and spleen was decreased in both experimental groups. Hepatic polyribosomes revealed greater aggregation and there was increased in vitro incorporation of “C-leucine into proteins of livers of rats force-fed the TD and the MD (less marked than with TD) diets in comparison with those of rats force-fed the C diet. Rats force-fed the experimental diets containing low carbohydrates did not reveal similar changes to those of rats force-fed the experimental diets containing adequate carbohydrates.
INTRODUCTION In earlier studies from our laboratory, we described in separate reports (Sidransky and Verney, 1970a, 1971) the effects on hepatic protein synthesis of force-feeding a threonine-devoid (Sidransky and Verney, 1970a) or an amino acids-free (protein-free) diet (Sidransky and Verney, 1971) in comparison to force-feeding a complete diet to rats for 1 day. Enhanced hepatic protein synthesis, determined by measuring in uivo 14C-leucine incorporation into proteins, was observed in rats force-fed a threonine-devoid diet (Sidransky and Verney, 1970a) and in rats force-fed a protein-free diet ( Sidransky and Verney, 1971). In addition, in U&O hepatic protein synthesis was increased and sucrose density gradient patterns of hepatic polyribosomes were shifted toward heavier aggregates in the experimental animals when compared to control animals in both studies (Sidransky and Vemey, 197Oa, 1971). Also in an earlier study (Sidransky and Rechcigl, 1962) from our laboratory, we reported that young rats force-fed for 3 days a purified diet devoid of amino acids developed no 1 This investigation was supported by United States Public Health Service Grant AM-16530 from the National Institute of Arthritis and Metabolic Diseases. a A preliminary report was presented at the Meeting of the Federation of American Societies for Experimental Biology in Atlantic City, N. J., April 1974; Fed. PTOC. 33, 695 (abst.). 85
86
VERNEY
AND
SIDRANSKY
pathologic (morphologic) changes in the liver, whereas the rats force-fed for 3 days a threonine-devoid diet developed a fatty liver with a periportal distribution of lipid and increased liver glycogen as described earlier in our experimental kwashiorkorlike model ( Sidransky and Farber, 1958a, b). In the present investigation, we were concerned with protein synthesis in the liver, heart, gastrocnemius muscle, and sp1een of rats force-fed a complete, threonine-devoid or amino acids-devoid diet for three feedings during 1 day and killed the following morning. By conducting these experiments, it was hoped to obtain further information regarding the similarities or differences in the effects of force-feeding equal quantities of isocaloric amounts of threonine-devoid and amino acids-devoid diets. Also, since in an earlier study (Sidransky and Clark, 1961) we reported that the amount of carbohydrate intake in the threonine-devoid diet influenced the morphologic and biochemical changes in the animals force-fed for 3 days, the influence of reducing the carbohydrate content of the threonine-devoid and amino acids-devoid diets on protein synthesis in the animals was investigated in this study of shorter duration. The results of our present studies indicate that the response of protein synthesis in the organs of rats force-fed for one day the threonine-devoid diet is generally more marked than that of rats force-fed for 1 day the amino acids-devoid diet. Also, the reduction of carbohydrate content of each of the two experimental diets caused a marked diminution or almost eliminated the changes in protein synthesis observed with animals force-fed for 1 day the experimental diets containing a higher or adequate quantity of carbohydrates. The present experiments of short (1 day) duration stress the importance of the type of deficient diet as well as the carbohydrate content on the induction of acute alterations in protein synthesis of the liver, heart, gastrocnemius muscle, and spleen. MATERIALS
AND
METHODS
Female rats of the Sprague-Dawley strain, 3 1 mo old, weighing on the average 65 g, were used. The animals were fed a commercial diet 4 before the experiments were begun, In all experiments, groups of animals, each of the same age and weight, were used. The basal diet was identical to that used in earlier experiments (Sidransky and Clark, 1961; Sidransky and Verney, 1970a). The percentage of dietary components was as follows: essential amino acids,5 9.2; nonessential amino acids,5 8.1; salt mixture (Hegsted IV)5 (S’d1 ransky and Farber, 1958a), 4; vitaminsucrose mixture 6 (Sidransky and Farber, 1958a), 5; corn oil,’ 5; cod liver oi1,s 1.5; and dextrin6 67.2. Dextrin was substituted for the threonine in the threoninedevoid diet and for all amino acids in the amino acids-free diet. In experiments where rats were fed the low carb.ohydrate (LC) diets, the diets were the same as the previous diets except that the dextrin content was reduced from 67.2 g/ s Sprague-Dawley, Madison, Wise. 4 Wayne Lab-Blox, Allied Mills, Inc., Chicago, Ill. s General Biochemicals, Inc., Chagrin Falls, Ohio. s&Methyl-l, 4-naphthoquinine. Eastman Organic Chemicals, vitamins, Nutritional Biochemical Corp., Cleveland, Ohio. 7 Cod Liver Oil Liquid, Mead Johnson Laboratories, Evansville, s Amersham/Searle Corp., Arlington Heights, Ill.
Rochester, Ind.
N.
Y. All
other
PROTEIN
SYNTHESIS
WlTH
DEFICIENT
DIETS
87
100 g diet to 24.2 g/57 g (43 g/100 g) diet without replacement, while all other dietary components were kept constant (Sidransky and Clark, 1961). The diets were blended with distilled water so that each milliliter of diet mixture contained 0.2!3-0.50 g of diet. Rats were force-fed using plastic tubes at SO0 AM, noon and 600 PM and each animal received on the average 1.2 g (4.8 cal) of regular diet or 0.7 g (2.8 cal) of low carbohydrate diet per 10 g of initial body wt. The basal complete diet was force-fed to all rats for 2 days prior to beginning the experimental diets: the threonine-devoid (TD ), the amino acids-devoid (AAD), or the complete ( C ) diet with adequate or low carbohydrate (LC) for 1 day. Rats had free accessto water. They were kept in individual wire cages with raised bottoms and in an air-conditioned room. All animals were killed in the morning approximately 16 hr after the last tube-feeding. Rats were weighed at the beginning and end of each experiment. The animals were killed by exsanguination. In in I&O experiments, the rats were killed by removing blood from the aorta after ether anesthesia, while in in vitro experiments, they were killed by decapitation. The organs, liver, heart, gastrocnemius (right), and spleen, were rapidly removed and weighed fresh. Pieces of the organs were homogenized in appropriate solutions. In studies on incorporation in uiuo, rats received 2.5 ,&i (10 &i/mole) L-leucineJ4C, uniformly labeled,* intraperitoneally 1 hr before killing. In studies on incorporation in u&o, postmitochondrial supernatants, mic,somes or ribosomes of liver homogenates were used. The postmitocbondrial supernatant, microsomes and ribosomes were prepared and used for assays as described earlier (Staehelin et al., 1967; Sidransky et al., 1968). r.,-LeucineJ4C, 0.5 &i, was added to each incubation tube. Radioactivity in protein was measured using a liquid scintillation spectrometer.a For size distribution analysis of hepatic polyribosomes, postmitochondrial supernatants were prepared in 0.25 M sucrose containing TKM (0.05 M Tris (pH 7.6)) 0.025 M KCl, and 0.005 LM MgClz) and then treated with deoxycholate (0.7%, final concentration) Staehelin et al., 1967; Sidransky et al., 1968). Samples were layered on linear 12 ml sucrose gradients (0.3 to 1.1 M sucrose containing TKM) and centrifuged in an ultracentrifuge lo at 38,000 rpm for 1 hr at 2” and analyzed as described earlier (Sidransky et al., 1968). The methods used for chemical analyses of protein, glycogen, RNA and DNA have been described in detail in earlier studies (Sidransky and Farber, 1958b; Sidransky and Verney, 1970a). RESULTS In Table I, the changes in body weight and the weights of the liver, heart, gastrocnemius muscle, and spleen of animals force-fed the different diets for 1 day are summarized. While rats force-fed the complete (C) diet gained weight ( + 1.7 g), the rats force-fed the threonine-devoid (TD) diet showed little change in weight (+O.l g), and the rats force-fed the amino acids-devoid (AAD) diet Iost weight (-1.3 g). AI1 rats force-fed the low carbohydrate (LC) diets lost 9 Packard Tri-Carb, Packard Instrument Company, Inc., Downers Grove, Ill. 10 Spinco SW 41 rotor in a model L2-65 Spinco ultracentrifuge, Beckman Instruments, Fullerton, Calif.
Inc.,
(32) -2.1 (32) -2.9 (33) -4.6
c-LC TD-LC AAD-LC
f f f
f f f
TABLE
I
0.34 0.34 0.30
0.35” 9.34 0.37 lOO(94 104 f 99 f
f 5.0)” 1.8 2.0
lOO(95 102 f 97 f
lOO(96 zk 12.9)” 97 f 3.3 99 f 2.9
(6) 100” 94 f 2.7 95 f 2.4
Gastrocnemius Muscle mg
lOO(97 102 f 93 f
(5) 100” 81 f 87 i
10.2)” 5.5 11.0
i
4.7d l.gd
Spleen mg
; Values for groups C and C-LC were set at 100% in each for the C group were as follows: liver, 2.58 f 0.06 g ; heart,
f 5.5)” 0.5 2.4
(3)lOOb 100 f 2.1 96 f 1.2c
mg
g
(6)100b 112 3~ 3.2” 93 f 2.2
Heart
Liver
AND ORGAN WEIGHTS OF RATS FORCE-FED PURIFIED DIETS CONTAINING COMPLETE (C), (TD) OR No (AAD) AMINO ACIDS AND ADEQUATE OR Low (LC) CARBOHPDRATES
a Number of animals in parenthesis; mean f SE. Rats weighed 65 g at st.art of experiments. b Number of experiments in parenthesis (in each experiment, 3-5 rats were used in each group) experiment and the experimental groups were compared with its cont,rol group. The organ weights 313 i 7.3 mg; gastrocnemius muscle, 369 f 15.9 mg; and spleen, 208 f 10.2 mg. e 0.05 > p > 0.01. dp < 0.01. e Values in parenthesis were compared with C group.
(46) +1.7 (43) +0.1 (47) -1.3
g
Body weight changes
C TD AAD
Group
CHANGES IN BODY THREONINE-DEVOID
iz
t!
E u
ECM
00 00
PROTEIN
SYNTHESIS
WITH TABLE
DEFICIENT
89
DIETS
II
ANALYSES OF LIVER OR RATS FORCE-FED COMPLETE (C), THREONINE-DEVOID ACIDS AND ADEQUATE OR Low
PURIFIED DIETS CONTAINING (TD) OR No (AAD) AMINO (LC) CARBOHYDRATES Livep
Group Protein
Glycogen
RNA
DNA
bx/liver) C TD AAD
(20)680 (19)773 (23)617
f f f
23.1 38.2b 23.1
C-L@ TD-LC AAD-LC
(15)627 (14)625 (15)570
xt 38.2 f 40.0 f 32.2
0 Number of animals in parenthesis b0.05 > p > 0.01. c p < 0.01. d Compared with C group.
(24)13.1 (25)55.4 (27)lS.l
f f f
3.1 5.9” 3.6
(11)24.3 (12)25.6 (13)21.1
f f f
1.0 1.1 O.gb
(11)8.88 (12)8.98 (13)8.65
i i f
0.39 0.50 0.29
(28) 7.5 f (28)15.6 f (29)25.8 f
1.6 3.5b 5.4”
(15)21.4 (14)22.8 (15)20.5
zt 0.4b f 0.5 f 0.6
(15)7.73 (14)8.25 (15)8.56
i i i
0.21b 0.31 0.31
; mean
f
SE.
weight. Those fed the AAD-LC diet lost the most (-4.6 g) and those fed the C-LC diet lost the least ( -2.1 g). The liver weights increased in rats force-fed the TD diet and decreased in rats fsorce-fed the other (AAD, C-LC, TD-LC, and AAD-LC) diets. The heart weights were essentially unchanged in rats force-fed the C and TD diets and decreased in rats force-fed the AAD diet and the groups force-fed the LC diets. The gastrocnemius muscle and splenic weights were somewhat decreased in rats fed the TD and AAD diets and further and similarly decreased in rats fed the three LC diets. In general, the only significant changes were in the increased liver weights in rats force-fed the TD diet, TABLE ANALYSES OF GAS~OCNEMIUS PURIFIED DIETS CONTAININQ No (AAD) AMINO ACIDS Group”
Gastroenemius
III
MUSCLE, HEART, AND SPLEEN OF RATS FORCE-FED COMPLETE (C), THREONINE-DEVOID (TD) OR AND ADEQUATE OR Low (LC) CARBOHYDRATES muscle
Heart
Glycogen
Protein
bsld
(mg/muscle)
protein
Spleen
protein
GmdorwQ
C TD AAD
(25)5.89 (26)6.@4 (28)6.56
zk 0.43 f 0.57 f 0.52
(20)55.9 (19)53.4 (‘22)53.5
f f f
3.7 3.8 3.6
(11)35.9 (12)42.9 (13)36.6
f i f
2.5 3.5 2.6
(21)40.9 (22)32.9 (23)36.5
f 3.3 f 2.6 f 2.5
C-L@ TD-LC AAD-LC
(29)3.45 (28)3.64 (29)5.07
f i f
(12)49.2 (14)44.5 (15)42.9
f f f
6.2 6.6 5.3
(12)41.5 (14)37.8 (1.5)36.7
f 2.9 f 4.1 f 2.9
(15)36.8 (12)40.5 (15)32.8
f f f
0.17” 0.19 0.43c
0 Number of animals in parenthesis; * Compared with C group. ‘p < 0.01.
mean
f
SE.
6.5 8.3 8.9
VERNEY
90
AND TABLE
SIDRANSKY IV
INCORPORATION IN VIVO OF W-LEUCINE INTO PROTEINS OF LIVER AND PLASMA FORCE-FED PURIFIED DIETS CONTAINING COMPLETE (C), THREONINE-DEVOID OR No (AAD) AMINO ACIDS AND ADEQUATE OR Low (LC) CARBOHYDRATES
Group
No. of raw
Incorporation of W-leucine Liver Specific activity %
C TD AAD
19 18 20
100 125 f 129 f
c-LC TD-LC AAD-LC
12 13 15
100(110 f 9.2)” 100 f 10.2 110 rrtr 3.4”
4.7” 6.0”
OF RATS (TD)
into proteins of6 Plasma
Total incorporation %
Specific activity %
100 141 f 7.2~ 119 l 5.0d
100 120 f 106 f
lOO(112 i 11.4)6 94 f 9.5 90 f 3.0”
lOO(102 f 3.5)” 104 f 13.6 111 f 22.7
5.6d 7.7
QResults of six experiments of adequate carbohydrate groups and of three experiments of LC groups. “Values for groups C and C-LC were set at 1007, in each experiment and the values (mean f SE) of the experimental groups were compared with those of the control groups. The values for the C group were as follows: Specific activity (cpm/mg protein), liver, 908.7 f 49.3 and plasma, 1285.1 f 64.7; Total incorporation (cpm/liver protein), (631.2 f 47.3) X 103. c p < 0.01. do.05 > p > 0.01. a Values in parenthesis were compared with C group.
the decreased liver and heart weights in rats force-fed the AAD diet, and the decreased splenic weights in rats force-fed the TD and AAD diets. Tables II and III summarize the analyses conducted on the composition of the liver, gastrocnemius muscle, heart, and spleen of rats force-fed the different diets. The main significant differences were as follows. Regular carbohydrate groups: TD group, increase of hepatic protein and glycogen; AAD group, decrease of hepatic RNA; Low carbohydrate (LC) groups: C-LC group compared with C group, decrease of hepatic RNA and DNA, and decrease of gastrocnemius muscle glycogen; AAD-LC group, increase in hepatic and gastrocnemius muscle glycogen; TD-LC group, increase in hepatic glycogen. TabIes IV and V summarize the incorporation in viva of W-leucine into proteins of liver, plasma, heart, gastrocnemius muscle, and spleen of rats fed the control and experimental diets. The results whether expressed as specific activity or as total incorporation into organ protein indicated in the TD and AAD groups an increase for liver, plasma, and heart and a decrease for gastrocnemius muscle and spleen. In general, the differences were more marked in the TD group than in the AAD group. Rats force-fed the low carbohydrate diets revealed only less differences between groups but in genera1 there was a decreased total incorporation into proteins of liver, heart, and spleen of the AAD-LC groups. Table VI summarizes the incorporation in vitro of 14C-leucine into protein when using postmitochondrial supernatants, microsomes or ribosomes prepared from the livers of the control and experimental animals. In ‘general, the results
12 13 15
c-LC TD-LC AAD-LC
lOO(105 f 17.0)” 108 f 3.2d 89 i 4.6d
100 132 f 2.3 108 i 6.5
S.A.
Heart
8.8” 4.7
lOO(116 f 12.9)” 96 f 10.0 81 f 7.4d
loo 154 f 107 f
Total incorporation
GASTROCNEMIUS
S.A.
lOO(103 f 14.5)” 94 zt 4.6 97 f 7.8
% 6.2c 6.3d
lOO(89 f 4.7d)” 83 f 8.8 84 f 12.7
100 70 f 75 f
Total incorporation
into proteins of* muscle
100 72 f 3.0” 80 f 18.0 lOO(99 f 7.0)’ 80 f 6.1c 73 i 4.0”
lOO(94 f 3.9)” 80 z!z 15.9 80 f 1.7”
Total incorporation
DIETS
100 93 f 17.3 83 f 24.2
S.A.
Spleen
MUSCLE AND SPLEEN OF RATS FORCE-FED PURIFIED ACIDS AND ADEQUATE OR Low (LC) CARBOHMRATES
of W-leucine Gastrocnemius
100 80 f 3.0” 91 f 5.7
Incorporation
OR No (AAD) AMINO
OF HEART,
(TD)
V
a Results of six experiments of adequate carbohydrate group and of three experiments of LC groups. 6 Values for groups C and C-LC were set at 100% in each experiment and the values (mean f SE) of the experimental groups were compared with those of the control groups. The values for the C group were as follows : Sp act (specific activity), cpm/mg protein, and total incorporation (cpm/organ protein X loo), respectively: heart, 247.8 f 8.7, 89.1 f 5.65; muscle 94.2 f 7.7, 49.0 Z!Z5.36; and spleen, 705.5 i 22.5, 288.0 i 63.0. cp < 0.01. do.05 > p > 0.01. * Values in parenthesis were compared with C group.
19 18 20
No. of ratsa
C TD AAD
Group
INCORPORATION IN VIVO OF W-LEUCINE INTO PROTEINS CONTAINING COMPLETE (C), THREONINE-DEVOID
TABLE
Ei 2
g ?I fi I3 2
3 2 E E
z”
2
VJZRNEY
92
AND TABLE
SIDRANSKY VI
INCOHPORATION IN VITRO OF W-LW~IN~ INTO PROTEINS BY H~PATIC POLYRIBOSOMYS USING POSTM~TOCHONDRIAL (PMS), MICROSOMAL OR RIBO~OMAL PREPARATIONS OF RUTS FORCE-FED PURIFIED DIETS CONTAINING COMPLETE (C), THREONINE-DEVOID (TD) OR No (AAD) AMINO ACIDS .~ND ADEQUATE OR Low (LC) CARBOHYDRATES Group
Source
of homogenate
PMS
fractions
Microsomesa
RibosomesU
% C TD AAD
(4) 1006 (4)149.1 (4)124.2
C TD AAD
(5)100(93.7 (5)lOl.O f (4)110.7 f
f f
(5) 1OOb (5) 161.0 (5)119.4
5.8” 3.@
f f
(5) 1OOb (5)133.1 (5)116.5
18.0c 34.9
f f
5.86 11.6
& 6.0)d 15.5 7.4
a Microsomes and ribosomes of each group were assayed using supernatants of their own groups. b Number of experiments in parenthesis (in each experiment, livers of 3-5 rats were pooled in each group). Values for group C and C-LC were set at lOO’$& in each experiment and the values (mean i SE) of the experimental groups were compared with those of the control group. cp < 0.01. d Value in parenthesis was compared with C group.
here are similar to those obtained in the in vivo incorporation studies (Tables IV and V ). There was increased incorporation ( 33-61s ) when using the fractions of the TD group and less increases (164X% ) with that of the AAD group. Little or no changes were present in the LC groups. In some experiments (Table TABLE
VII
COMPARISON OF THE EFFECTS OF TUBE-FEEDING FOR 1 DAY A THREONINE-DEVOID AND A AMINO ACIDS DEVOID (AAD) DIET IN COMPARISON TO A COMPLETE Determination”
Organ
weight
Protein
synthesis
Glycogen RNA
content
Polyribosomal
a?, Increase;
content
1, decrease;
NC,
1 1
NC 1
TD AAD
I
i
r NC
TD AAD
t (4+) t (2+)
TD AAD
t (4+) t cl+)
TD AAD no change.
Heart
1
TD AAD aggregation
Gastrocnemius muscle
T
TD AAD
content
Protein
Liver
Group
(TD) DIET (C) DIET
NC
1 3+
2+
t NC
PROTEIN 1,COMPLETE
aI 1
DIET
SYNTHESIS 4
WITH
2. THREONINE-
DEFICIENT
DEVOID
DlET
DIETS J
93
3. AMINO t?LlDSDEVOID DIET
4. COMPLETE LOW CARl3CtlYDkATE DIET
07
FIG. 1. Sucrose density gradient patterns of hepatic polyribosomes of deoxycholate-treated postmitochondrial supernatants of rats force-fed purified diets containing complete (l), threonine-devoid (2) or no (3) amino acids with adequate carbohydrate or purified diets containing complete (4), threonine-devoid (5) or no (6) amino acids with low carbohydrate.
VI) microsomes and ribosomes of each group were mixed with supernatants of other groups and then assayed for W-leucine incorporation into protein. These assays revealed similar results to those indicated in Table VI where complete systems were compared and indicate that the differences are due predominantly to the microsomes or ribosomes and not due to the supematant fractions. Sucrose density gradient analyses of deoxycholate-treated postmitochondrial supernatants of livers of control and experimental animals were studied, Figure 1 summarizes the polyribosome patterns of the six groups of animals. It is apparent that the livers of rats force-fed the threonine-devoid diet or the amino atidsdevoid diet had more aggregated polyribosomes (more marked in the TD than in the AAD) than those of the control animals. The livers of rats force-fed the low carbohydrate diets had more ‘disaggregated patterns, particularly in the GLC and TD-LC groups, but less so in the AAD-LC group. These findings are consistent with the changes in hepatic protein synthesis described in Tables IV and VI. DISCUSSION Some of the results of the present study are summarized in Table VII. It is apparent that there are a number of similarities as well as differences in the liver, gastrocnemius muscle and heart of rats force-fed for one day the threoninedevoid (TD) or the amino acids-devoid (AAD) diet. The results indicate that although the effects on protein synthesis of the liver, induced by force-feeding a threonine-devoid diet or an amino acids-devoid diet for 1 day are similar, they are more marked after the former diet. Thus, the presence of dietary amino acids
94
VERNEY AND SIDRANSKY
in the threonine-devoid diet plays a role in enhancing hepatic and cardiac protein synthesis. Actually, based on earlier studies (Sidransky and Verney, 1970a, 1971), there is evidence to suggest that the mechanisms involved in the liver may be different under the two experimental dietary conditions, There is evidence to suggest a protein-free diet may have its effect on hepatic protein synthesis through insulin ( Sidransky and Verney, 1971) . On the other hand, the effect due to the threonine-devoid diet is not due to elevated levels of insulin (Sidransky et al., 1969a) and is considered to be more related to an imbalanced state of circulatory amino acid levels (Sidransky and Rechcigl, 1962; Sidransky, 1972). The enhanced hepatic protein synthesis in rats force-fed the threonine-devoid diet for one day could not be attributed directly to a decreased level of hepatic free threonine since the hepatic threonine levels were decreased by 37% due to the TD diet (Sidransky and Verney, 1970a) and by 43% due to the AAD diet (Sidransky and Verney, 1971) in comparison to rats force-fed the complete diet. Also, the acute administration of threonine to rats after they had been forcefed the TD diet for 3 days had no influence on in vivo hepatic protein synthesis ( Sidransky et al., 1969b). In further consideration of the increased hepatic protein synthesis in rats force-fed the TD or the AAD diet, we wondered whether the increases could be in any way related to alterations in precursor pool sizes. Although the levels of free amino acids were not determined in this study, they were measured in two earlier studies ( Sidransky and Verney, 197Oa, 1971). These earlier results revealed that the spe&c activities of the W-leucine in the livers of the C and TD groups ( Sidransky and Verney, 1970b) and of the C and AAD groups ( Sidransky and Verney, 1971) were similar in both cases.Thus, altered pool sizes were not responsible for the differences in the in vivo hepatic protein synthesis in the control and experimental groups. Also, the in vitro incorporation studies in which the leucine pool size could be measured or controlled by using a common cell sap revealed that the hepatic polyribosomes of the TD (Sidransky and Verney, 1970a) and AAD (Sidransky and Verney, 1971) groups were more active in terms of protein synthesis. In addition, the hepatic polyribosome patterns of the TD and AAD groups were consistent with the in vivo and in vitro incorporation data indicating enhanced hepatic protein synthesis in the experimental groups. Thus, our results reveal that the enhanced protein synthesis in the experimental animals is clearly attributable to changes in the activities of the hepatic polyribosomes of the experimental animals. In an earlier study from our laboratory (Verney and Sidransky, 1974) we reported that the hearts of rats force-fed for 3 days a threonine-devoid diet revealed increases in weight, protein, RNA, glycogen, and in cardiac protein synthesis in in vivo and in &tro studies. Now in our present investigation we find that cardiac protein synthesis, based on in viva incorporation of 14C-leucine into protein, is significantly increased ‘over that in control animals in experiments of only 1 day’s duration. The hearts of rats force-fed the amino acids-devoid diet do not demonstrate such changes. Further studies are in progress relating to the alterations of cardiac protein metabolism in rats force-fed the threonine-devoid diet. Earlier studies from our laboratory (Sidransky and Rechcigl, 1962) have also reported that the morphologic consequences of force-feeding a threonine-devoid
PROTEIN
SYNTHESIS
WITH
DEFICIENT
DIETS
95
or protein-free diet for 3 days are quite different. While rats force-fed the threonine-devoid diet developed a periportal fatty liver with increased hepatic glycogen, protein, and RNA, such changes were not found in rats force-fed the protein-free diet for 3 days. In experiments of longer duration, Platt et UI. (Platt et al., 1964) h ave reported a number of pathologic changes in rats forcefed a protein-free diet for 6 days. Thus, it appears that the consequences of forcefeeding a threonine-devoid Norother single essential amino acids-devoid diets ( Sidransky, 1972) are indeed different than that of a protein- or amino acids-free diet and suggest altered mechanisms, differing either qualitatively or possibly quantitatively in inducing pathologic effects, are involved even though alterations in hepatic protein metabolism may appear to be similar during the earlier stages of the deficiencies. The results of our experiments in which the carbohydrate intake was lowered in the control (C-LC) and deficient ( TD-LC and AAD-LC ) diets are of much interest. Consistent with earlier experiments in which high amounts of carbohydrate in the TD diet accentuated while low amounts of carbohydrate in the TD diet diminished the pathologic changes in the liver and other organs (Sidransky and Clark, 1961), we now find that a decrease of carbohydrate content in the TD and AAD diets diminishes or eliminates the changes in protein synthesis of the liver and heart of the animals fed the deficient diets. These results would suggest that the elevated levels of hepatic protein synthesis in rats forcefed the TD (regular carbohydrate) diet may be involved or related to the pathologic changes such as fatty liver, increased glycogen, and increased RNA of the liver observed in earlier studies (Sidransky, 1972). Studies from other laboratories (Barnes et al., 1959; Heard et al., 1958; Macdonald, 1966) have also indicated the importance of the caloric intake in experiments with protein deficiency. Indeed, there have been reviews of the importance of protein-energy relationships by a number of workers (Miller, 1973; Morrison and Narayana Rao, 1967; Munro, 1964; Swanson, 1959). Thus, it is now apparent that the amount of carbohydrate in a deficient diet is of great importance in eliciting biochemical as well as morphologic alterations in organs such as the liver. Likewise, it is conceivable that the marked differences in morphologic and biochemical reactions in animals fed by stomach tube and those fed ad Zibitum deficient diets (Sidransky, 1972) may be due in (part or in whole to the decreased intake of carbohydrate, since the ad Zibitum fed animals consume much less diet which is chiefly composed of carbohydrate. Indeed, in the human nutritional disease, kwashiorkor a prominent feature of the dietary history is the relatively high intake of carbohydrate coupled with inadequate protein (Brock and Autret, 1952; Davies, 1952; Housden, 1956; Waterlow, 1948). In contrast to the numerous pathologic lesions described in kwashiorkor, there are few or no specific lesions, particularly of the liver, in states of general undernutrition with severe restriction of protein and carbohydrate intake as occurs with maramus (Jelliffe and Welbourn, 1963; Latham, 1965). REFERENCES R. H., KWONC, E., POND, W., LOWRY, R., and LOOSLI, J. K. (1959). and protein and serum cholesterol. II. Young swine. 1. Nuts. 69, 269.
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