Molecular and Cellular Biochemistry 108: 105-112, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands.
Effect of alanine supply on hepatic protein synthesis in animals maintained on a protein free diet
Dolores P6rez-Sala, Teresa Rivas Calleja, Roberto Parrilla and Matilde S. Ayuso Endocrine Physiology Unit, Centro de Investigaciones Biol6gicas, C.S.I.C., Vel{~zquez 144, 28006 Madrid, Spain
Key words: liver, protein synthesis, protein-free diet, alanine action Abstract
In contrast to what it is observed during starvation, animals maintained on a protein-free isocaloric diet showed an increase in the rate of hepatic peptide chain elongation as determined by measuring the ribosomal transit time in vivo. The loss of body nitrogen per se is insufficient to generate the signal(s) which arrests hepatic peptide chain elongation. This observation suggests that it is an increase in gluconeogenic demand, and not the negative nitrogen balance, which is implicated in determining reciprocal changes in the rate of protein synthesis. The rate of protein synthesis, as expressed per mg of DNA, does not change in protein deprived animals, while the R N A to DNA ratio decreased. These data also agree with a higher ribosomal efficiency at the elongation step. The animals maintained on a protein-free diet have a decreased hepatic content of protein and an increased concentration of valine, indicating an increased proteolysis. The enhanced rate of polypeptide elongation observed in animals kept on a protein-free diet was accompanied by decreases in the state of aggregation of polyribosomes and in the ability of liver extracts to form eIF-2 catalyzed ternary complexes. These observations suggest that the activity of the hepatic initiation factor in vivo may not be rate limiting. The administration of alanine in vivo to animals maintained on a protein-free diet showed a preferential effect in reaggregating polyribosomes. This action was neither accompanied by detectable effects on the rate of eIF-2 catalyzed ternary complexes formation nor by significant changes in the rate of elongation. It is concluded that factors other than eIF-2 activity or the rate of polypeptide elongation must be controlling the hepatic polyribosomal state of aggregation.
Introduction
Protein synthesis and gluconeogenesis, two apparently unrelated pathways, do share in common the use of amino acids as their only or preferred substrates. Reciprocal changes in the flux through these pathways have been observed in either physiological or pathological conditions [1]. Acute stimulations of gluconeogenesis by glucagon or fatty acids have also been reported which lead to oppo-
site changes in the rate of hepatic protein synthesis [1, 2]. The nature of the signalling process involved in coordinating the activity of these pathways remains to be elucidated. Variations in the phosphorylation potential [1, 2] and/or changes in the concentration of certain key amino acids [3] have been proposed to be responsible for such a coordination. The increased rate of gluconeogenesis observed after acute hormonal stimulation is accompanied by a decrease in the phosphorylation potential [1,
106 2]. This eventually might perturb the rate of elongation of polypeptide chains [2]. On the other hand, the concentration of certain amino acids, like alanine, may play an important regulatory role as this amino acid has been reported to display a powerful effect in stimulating hepatic peptide chain elongation in starved animals either in vivo or in isolated liver cells [3]. Starvation in the rat leads to an increase in hepatic gluconeogenesis and a decrease in protein synthesis [3, 4]. The liver from starved animals is also characterized by a lower phosphorylation potential and a decreased alanine content [3, 4]. The higher rate of gluconeogenesis, by increasing alanine demand and energy consumption, has been postulated to be responsible for the above mentioned changes, which eventually could induce a decrease in the rate of protein synthesis. Starvation is accompanied by a progressive loss of body nitrogen; therefore, the possibility should also be considered that negative nitrogen balance could promote the change in the rate of peptide chain elongation. Based on the observation that low protein diets perturb hepatic protein synthesis [5-9], we found it interesting to study the effect of alanine on hepatic protein synthesis under conditions of negative nitrogen balance and where gluconeogenesis is not stimulated. For this purpose animals were maintained on a protein-free diet with a high carbohydrate content. Our results indicate that a negative nitrogen balance by itself is not sufficient to induce a decrease in peptide chain elongation, and that, under these conditions, alanine administration has no effect.
Chemicals [U-3H]Valine and pS]Methionine were purchased from Amersham International (Amersham, Bucks., U.K.). Most of the reagents were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.).
Collection of liver biopsies and plasma samples The animals were anaesthetized with Nembutal (40 mg/kg body wt.) about 10 min before they were surgically manipulated. Small liver biopsies were taken as previously described [3] and immediately frozen with aluminum clamps precooled in liquid nitrogen [11]. Blood was withdrawn with heparinized syringes from the aortic bifurcation and the plasma obtained immediately by centrifugation at 4 ° C.
Determination of the state of aggregation of hepatic polyribosomes Liver biopsies were immediately cooled by immersion in ice cold buffer A (50 mM triethanolamineHC1, pH7.3, 5mM MgC12, 25mM KC1) and homogenized in 3 vol of this buffer. The homogenates were processed as previously described [3] to obtain the polyribosomal profiles. The polyribosomal fraction was calculated as the ratio between the area of the profile corresponding to polysomes and the area corresponding to monomers and dimers of ribosomes.
Material and methods
Animals Male Wistar albino rats weighing 200-220 g were used. The animals were maintained on a semiliquid diet, as described by De Carli and Lieber [10]. When indicated, all the protein-containing ingredients were substituted by isocaloric amounts of glucose during 4 or 8 days.
Determination of the rates of elongation of hepatic polypeptide chains and protein synthesis This was done by measuring the distribution of radioactivity incorporated into nascent peptides on the polyribosomes (n) and into total peptides (t) [12]. For these determinations, 20/zCi of [3H]valine was injected into the portal vein of anaesthetized rats and two small biopsies (about 1 g) were taken from each liver, either after 0.5 and i min or after 1 and 2rain. To prevent hemorrhage, liver
107 lobules were ligated immediately before the biopsies were taken. Polypeptide chain completion time in each case was calculated as the time needed for the ratio of the radioactivity incorporated in nascent peptides into the polyribosomes to the radioactivity incorporated into total peptides (n/t) to be decreased by 50% [3, 12]. The rate of protein synthesis was calculated as the amount of protein synthesized during one elongation cycle, which is equivalent to the nmoles of active ribosomes divided by the duration of the elongation cycle, that is, the determined time of completion of polypeptide chains (in min). The concentration of active ribosomes was calculated from the determination of total R N A (Table 5), considering the ribosomal RNA to be 80% of the total RNA (4 nmoles/g wet wt), and taking into account the percentage of active ribosomes (incorporated in polyribosomes) given by the polyribosomal fraction. The results obtained from these calculations are the nmoles of protein synthetized x g of liver wet weight-Ix min-k They are converted to mg of protein assuming and average molecular weight of liver protein of 50000 daltons.
Determination of the concentration of amino acids in plasma and liver For the determination of the plasma amino acid concentration, portions of plasma samples were deproteinized with 10% (w/v) sulphosalycilic acid. For the determination of the hepatic amino acid content, freeze-clamped liver biopsies were lyophilized. Approximately 200 mg of dry tissue were homogenized with 2 ml of 8% (w/v) HC104. After removal of the insoluble material, 0.7 ml portions of the supernatant were adjusted to p H 2 with 1 M lithium citrate, pH 1.3. The concentration of the different amino acids was determined by automated ion-exchange liquid chromatography.
Analysis of elF-2 activity The activity of eIF-2 in liver extracts was determined by measuring the formation of a ternary
complex between elF-2, GTP and [35S]methionyltRNA. The procedure was that described by Gupta et al. [13], as modified by Clemens and Pain [14]. Livers from rats weighing 100 to 120 g were homogenized with 3 vol of 50 mM triethanolamine-HC1, pH7.6, 5 mM magnesium acetate, 2.3% (w/v) glycerol, 0.5mM dithiothreitol and 120 or 250raM KCI. After centrifuging at 10,000 g for 10rain at 4 ° C, the supernatant was collected and centrifuged again at 150,000 g for 3 h at 4 ° C. The postribosomal supernatant was stored in aliquots in liquid nitrogen until the eIF-2 activity was assayed as previously described [15]. The incubation mixture contained 5% (v/v) of postribosomal supernatant. Final Mg 2+ concentration was 2raM. The reaction was initiated by the addition of 7 x 106cpm of [35S]methionyl-tRNA, prepared as previously described [16]. Under these conditions the rate of incorporation of [3SS]methionyl-tRNA into ternary complexes was linear with respect to incubation time and protein concentration.
Results
Effect of feeding a protein-free diet on the state of aggregation of hepatic polyribosomes, elF-2 activity and amino acid content Figure 1 shows the state of aggregation of hepatic polyribosomes of rats maintained on an isocaloric protein-free diet. After 4 days, a decrease in the state of aggregation was observed, although it was not statistically significant. At least 8 days on the diet were needed to consistently provoke a significant polyribosomal breakdown (Fig. 1, lower left panel). After either four or eight days on the protein-free diet, the administration of alanine was followed by an acute reaggregation of polyribosomes. The quantitation of this response at eight days is shown in Table 1. The administration of inorganic nitrogen as ammonium chloride led also to an acute and significant increase in the polyribosomal fraction (results not shown). The polyribosoreal breakdown observed in animals on a proteinfree diet could be a consequence of a lower rate of initiation of protein synthesis; therefore, the ability
108
t_..,__l.__J 0
2
body nitrogen balance due to feeding a protein-free diet, the hepatic content of amino acids decreased only by less than 20% (Table 3). Variations in the content of only three amino acids, aspartate, glutamate and proline could account for the decrease in total a-amino nitrogen. The higher concentration of valine probably reflects an accelerated rate of protein catabolism (Table 3). No significant changes in the total plasma content of amino acids were observed after eight days on a protein free diet. Decreases in glutamate and glutamine, and increases in glycine and serine were observed in plasma as well as in liver (Tables 3 and 4).
.I 4
4 days
Alanine
I'CO
,-1 m a. O 2
4
2
8 days
____._--I 2
/ 4
4
Effect of feeding a protein-free diet on the rate of hepatic protein synthesis
, Alanine
1 2
__,_L 4
GRADIENT (ml)
Fig. 1. Effects of feeding a protein-free diet and alanine administration on the state of aggregation of hepatic polyribosomes. Animals were maintained on a protein free diet for none, four or eight days, as indicated. The experiment was performed as described in Methods. Alanine (1 mmol/kg body wt.) was administered 10 rain before the animals were surgically manipulated. Each experimental condition was studied at least six times and representative tracings are presented.
of hepatic extracts to form ternary complexes was studied. The results, which are depicted in Table 2, indicate that the polyribosomal disaggregation was accompanied by a decrease in the ability of liver extracts to form ternary complexes. Despite the acute action of alanine in reaggregating polyribosomes (Fig. 1) its effect was not accompanied, at least in liver extracts, by an increase in the formation of ternary complexes (Table 2). Tables 3 and 4 show, respectively, the hepatic and plasma contents of amino acids in control and in protein deprived animals. Despite the negative
Animals maintained on a protein-free diet showed an increase in the rate of decay of the hepatic 'n/t' value (Fig. 2). Intraperitoneal administration of alanine to these animals produced a slight delay in the rate of decay (Fig. 2), while the administration of NH4C1 caused no apparent effect (results not shown). The polypeptide chain completion time was calculated from these data. Table 1 shows that in control animals the termination time was 1.7 min. This time was reduced to only one minute in protein deprived animals (Table 1). The rate of protein synthesis increased by 54% in livers from rats maintained on a protein-free diet. This was parallel by a similar increase in the DNA content (Table 5). Thus, the protein synthesis rate expressed as mg of protein x mg D N A - ; × day -~ was not altered (Table 1). Table 5 also shows that the hepatic content of total R N A per D N A was decreased by 35% in protein-deprived animals. The lack of effect on the rate of protein synthesis was accompanied by a decreased hepatic content of total protein (Table 5), indicating that there is an increased rate of proteolysis in protein deprived animals. Alanine, administered in a single dose, was not capable of inducing a further increase in the rate of protein synthesis (Table 1).
109 Table 1. Effect of feeding a protein-free diet on the hepatic polyribosomal fraction and rate of elongation of polypeptide chains
Control
5.4+ 0.3 86
Polyribosomal fraction % of active polyribosomes Termination time (rain) Rate of protein synthesis: nmoles × g wet wt -1 × min -1 mg protein × mg DNA -~ × day-1
Protein-free diet - Alanine
+ Alanine
4.1+ 0.3* 79
6.7+ 0.9 87
1.7
1.0
1.3
2.0 63
3.1 60
2.7 46
Animals were fed a protein-free diet for eight days. Liver biopsies were processed as described in Methods for the determination of either the polyribosomal state of aggregation or the ribosomal transit time. Alanine treated animals received an i.p. injection of 1 mmol of alanine 10 rain before the biopsies were taken. Control animals were given a similar volume of saline solution. The results are average values of at least 8 determinations _ SEM. * By t-test P < 0.01. Discussion
T h e progressive loss of liver protein induced by feeding a protein-free diet resulted in hepatic polyribosomal b r e a k d o w n ; h o w e v e r , in contrast to what has b e e n o b s e r v e d in starvation [3], the rate of peptide chain elongation, as reflected by the ribosomal transit time, was e n h a n c e d (Table 1). This finding allows us to c o n c l u d e that a progressive loss of b o d y nitrogen is not sufficient to induce a decrease in the rate of peptide chain elongation; therefore, as previously suggested [1], the activity Table 2. Effect of a protein-free diet on the ternary complex formation activity of liver extracts
Ternary complex formation (cpm × 10-3 x /zg DNA -~) KC1 concentration in extracts
Control diet Protein-free diet: - Alanine + Alanine
120 mM
250 mM
5.6 + 0.2
6.3 _+ 0.2
1.6 -+ 0.2 0.7 + 0.3
2.8 _+ 0.2 1.5 + 0.3
The liver extracts were obtained from animals maintained during eight days on a protein-free diet according to the protocol described in Methods. Alanine treated animals received an i.p. injection of 1 mmol of alanine 10 min before the biopsies were taken. The values are mean of six determinations in duplicate + SEM.
of the gluconeogenic p a t h w a y might be an important factor in determining the differential effects on peptide chains elongation o b s e r v e d during starvation or feeding a protein-free diet. Based on the ribosomal transit time and the state of aggregation of hepatic p o l y r i b o s o m e s (Table 1), it can be concluded that there is an increase in the rate of hepatic protein synthesis in protein deprived animals. H o w e v e r , when the rate o f protein synthesis was expressed as mg o f protein synthetized by m g of D N A (Table 1), no significant changes were observed. This observation is in conflict with reports f r o m o t h e r laboratories that indicate a fall in the overall rate of protein synthesis in the liver of protein deprived animals [7-9], or an increase [6]. W e have, at present, no explanation for these conflicting reports, but a correlation seems to exist between the effects of the diet on the animals b o d y weight and changes in hepatic protein content and protein synthesis. A n increased protein synthesis rate per g of tissue seems to be acc o m p a n i e d by m o d e s t variations in loss of liver protein and b o d y weight (Tables 1 and 5 and ref. 6). A decreased protein synthesis rate is a c c o m p a n i e d by m o r e i m p o r t a n t losses of b o d y weight and liver protein [9]. S o m e authors [9] had explained these discrepancies as caused by variations in the specific activity of the amino acid precursor not taken into account in some of the m e t h o d o l o g i e s used for the determination of protein synthesis rates in vivo. T o minimize changes in specific activty, a large dose of
110 the precursor amino acid is often used for measuring the in vivo rate of protein synthesis [9]. However, we considered conflicting to use a large dose of amino acid when trying to determine the effects of a negative nitrogen balance. The method used in our experiments, measurement of the ribosomal transit time, is independent of the specific activity of the precursor amino acid, provided that it does not change during the two minutes experimental time [12]. The plasma content of valine (Table 4) does not change in protein deprived animals, ruling out any difference in the specific radioactivity of the amino acid supplied to the liver. The observation that a negative correlation seems to exist between protein and RNA content and rates of peptide chain elongation (Tables 1 and Table 3. Effect of a protein-free diet on the hepatic content of
5) suggests that a feedback regulatory mechanism might exist. This possibility is consistent with unpublished results from our laboratory that show an accumulation Of hepatic protein while the rate of peptide elongation decreases in animals kept on an alcohol-containing diet. The peptide initiation factor elF-2 does not seem to be rate limiting for hepatic protein synthesis under physiological conditions. This conclusion is based on two observations: First, the acute reaggregation of polyribosomes observed in protein deprived animals after alanine administration (Fig. i and Table 1) is not accompanied by an increase in elF-2 activity. Second, the rate of protein elongation increases in protein deprived animals despite a Table 4. Effect of a protein-free diet on the plasma content of amino acids
amino acids Control Control
Protein-free diet
Protein-free diet - Alanine - Alanine
+ Alanine
+ Alanine
Aspartate Treonine Serine Glutamate Proline Glycine Alanine Citruline Valine Cistine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Tryptophane Arginine
(/xmoles x g dry wt -1) 22.3 +__ 1.9 4.4 + 0.4 11.9 + 3.2 2.5 ___ 0.26 5.3 + 0.8 4.0 + 0.6 2.4 + 0.26 7.9 + 0.5 7.6 + 0.5 10.1 + 1.0 5.9 + 0.3 7.9 + 0.5 22.9 + 4.2 16.6 + 1.7 11.6 + 1.7 6 . 1 + 0.5 10.0+ 0.3 8 . 9 + 0.5 2.8 _+ 0.4 2.9 + 0.56 4.5 + 0.8 0.05 + 0.009 0.2 + 0.01 0.4 + 0.2 0.3 +_ 0.03 0,5 + 0.04 0.2 + 0.05 0.1 _+ 0.02 0.4_+ 0.03 0.2 + 0.05 0.1 _+ 0.01 0.4 + 0.02 0.2 + 0.04 0.2 _+ 0.02 0.5 + 0.04 0.2 + 0.05 0.3 + 0.04 0.4 + 0.03 0.3 + 0,03 0.3 _+ 0.03 0.4 _+ 0.02 0.3 + 0.04 0.2 _+ 0.02 0.3 _+ 0.01 0.2 + 0.01 0.6 + 0.07 0.4 + 0.02 6.4 _+ 2.8 2,1 + 0.26 1.0 + 0.03 1.3 _+ 0.06 1.6 +_ 0,14 2.1 + 0.04 2.3 + 0.07 0.04_+ 0,003 0,008+ 0.001 0.07 + 0.02 0.07 + 0.01 0.03 _+ 0.008 0.1 _+ 0.02
Aspartate Treonine Serine Glutamate Glutamine Gtycine Alanine Valine Cistine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Tryptophane Arginine
(t~moles × g dry wt -1) 0.02 + 0.004 0.03 + 0.003 0.02 + 0.006 0.6 _+ 0.04 0.4 + 0.03 0.5 _+ 0.1 0.2 _+ 0.01 0.5 + 0.06 0.6 + 0.03 0 . 0 6 + 0.008 0.01-+ 0,001 0.1 + 0.01 0.5 +_ 0.04 0.3 + 0.02 0,4 + 0.04 0.2 _+ 0.007 0.6 + 0.05 0.6 + 0.04 0 . 5 + 0.04 0 . 4 + 0,04 1.1_+ 0.25 0.1 + 0.01 0.08 _+ 0.01 0.06 _+ 0,005 0.03 _+ 0.003 0.02 + 0.002 0.02 _+ 0.007 0.04 + 0.008 0.03 + 0.004 0,03 + 0.004 0.05 + 0.006 0.04 + 0.006 0.03 + 0.006 0.07 + 0.009 0.06 _+ 0.005 0.04 + 0.007 0.12 + 0.007 0.05 + 0.006 0.03 + 0.006 0.05 + 0.004 0.06 + 0.006 0.06 + 0.006 0.04 + 0.004 0.03 + 0.003 0.03 + 0.003 0.5 + 0.02 0.35 + 0.014 0.32 + 0.02 0.09 + 0.004 ' 0.17 + 0.01 0.21 + 0.02 0 . 0 4 + 0.007 0.05 + 0.002 0.11 + 0.01 0.1 +-- 0.008 0.07 _+ 0.009
Total amino acids
3.3
Total amino acids
75
Plasma samples obtained from animals maintained for 8 days on a control or protein-free diet were treated as described in Methods. When indicated, animals received an i.p. injection of 1 mmol of alanine or a similar volume of saline solution 10 min before the samples were taken. The values are means of eight determinations + SEM.
62
69
The experimental protocol was as described under Methods. Liver biopsies were obtained from animals maintained for 8 days either on control or on a protein-free diet. The values are means of eight determinations + SEM.
3.3
4.2
111 significant decrease in eIF-2 activity, as measured by the ability of liver extracts to form ternary complexes (Table 2). Alanine is one of the most powerful effectors so far described, capable of acutely stimulate hepatic protein synthesis in vivo [3]. Its effect is observable only in livers from starved animals where it stimulates both the initiation and the elongation steps of protein synthesis. Our results (Fig. 1 and Table 1) add further evidence to the role of alanine in stimulating protein synthesis when this process is previously inhibited, as is also the case during starvation [3]. The finding that alanine can reaggregate polysomes without significantly perturbing the rate of peptide elongation indicates that more than one site of alanine action should be considered. On the other hand, the administration of ammonium chloride led to a similar effect on ribosomes aggregation, in the absence of detectable effects on the polypeptide termination time (results not shown). These observations indicate that the steps of initiation and elongation of polypeptide chains are not linked in a compulsory manner in vivo. Thus, animals maintained on a protein-free diet offer an interesting model for the study of the functional relationship between the ribosomal state of aggregation and the rate of peptide synthesis.
1.0
PRoTEIN-FREE
CONTROL
DIET
%
0.5
n/t
L L
0.2
0.1
I
0
1
PROTEIN-FREE DIE"I'+ALANINE
i
20
•
,
1
.
±
,
20
,
.
1
Time (min.) Fig. 2. Effects of feeding a protein-free diet and acute alanine administration on the rate of decay of the hepatic n/t value. The experiment was performed as described in Methods. The animals were maintained for eight days on either control or protein-free diets. Alanine (1 mmol/kg body wt.) was administered 10min before the animals were surgically manipulated. The points represent means of at least eight experiments and the vertical bars the standard error.
Acknowledgements The authors wish to express their gratitude to M.J. Arias-Salgado and A. Rodrfguez for their excellent technical assistance. This work was supported in part by grants from Direcci6n General de Ciencia y Tecnologfa (PB87-0280 and 87065) and Fondo de Investigaciones Sanitarias (89/0467 and 89/0468). D. P.-S. was recipient of a fellowship from the Ministerio de Educaci6n y Ciencia.
Table 5. Effect of feeding a protein-free diet on the body weight and hepatic contents of D N A , R N A and protein
Control
D N A ( m g x g wet wt. -~) Total RNA (mg × g wet wt. 1) Total protein (mg × g wet wt.-1) RNA/DNA Body weight (g)
2.3_+ 0.13 9.9 + 0.6 170 + 8 4.2 + 0.07 162 _+ 2.5
Liver biopsies, obtained from animals fed control or proteinfree diets for 8 days, were processed as described in Methods. Alanine treated animals received intraperitoneally 1 mmol of alanine before the biopsies were collected. The values are means of eight different determinations _+ SEM. By t-test: * P < 0.001; * * P < 0.01.
,
2
Protein-free diet - Alanine
+ Alanine
3 . 7 + 0.1" 10.3 + 0.6 130-+ 15"* 2.7-+ 0.1" 116-+ 2.4
4.2_+ 0.1" 12.1 + 0.3 2.9-+ 0.09*
112 References 1. Ayuso MS, Vega P, Manch6n CG, Parrilla R: Interrelation between gluconeogenesis and hepatic protein synthesis. Biochim Biophys Acta 883: 33-40, 1986 2. Martin-Requero A, P6rez-Diaz J, Ayuso MS, Parrilla R: The mechanism of the glucagon-induced inhibition of hepatic protein synthesis. Arch Biochem Biophys 195: 223234, 1979 3. P6rez-Sala D, Parrilla R, Ayuso MS: Key role of L-alanine in the control of hepatic protein synthesis. Biochem J 241: 491-498, 1987 4. Parrilla R: The effect of starvation in the rat on metabolite concentrations in blood, liver and skeletal muscle. Pflugers Archiv 374: %14, 1978 5. Sato A, Noda K, Natori Y: The effect of protein depletion on the rate of protein synthesis in rat liver. Biochim Biophys Acta 561: 475-483, 1979 6. Garlick PJ, Miltward DJ, James WPT, Waterlow JC: Effect of protein deprivation and starvation on the rate of protein synthesis in the tissues of the rat. Biophys Biochim Acta 414: 71-84, 1975 7. Morgan EH, Peters T: The biosynthesis of rat serum albumin. V. Effect of protein depletion and refeeding on albumin and transferrin synthesis. J Biol Chem 246: 3500-3507, 1973 8. Conde RD, Scornik OA: Role of protein degradation in the growth of livers after a nutritional shift. Biochem J 158: 385-390, 1976
9. McNurlan MA, Garlick PJ: Protein synthesis in liver and small intestine in protein deprivation and diabetes. Am J Physiol 241: E238-245, 1981 10. De Carli LM, Lieber CS: Fatty liver in the rat after prolongued intake of ethanol with a nutritionally adequate new liquid diet. J Nutr 91: 331-446, 1967 11. Wollenberger A, Ristau O, Schoffa G: Eine einfache Technik der extrem schellen Abkiihlung gr613erer Gewebestiicke. Pflugers Arch Gesamte Physiol Menschen Tiere 270: 339-412, 1960 12. Mathews RW, Oronsky A, Haschemeyer AEV: Effect of thyroid hormone on polypeptide chain assembly kinetics in liver protein synthesis in vivo. J Biol Chem 248: 132%1333, 1973 13. Gupta NK, Woodley CL, Chen YC, Bose KK: Protein synthesis in rabbit reticulocytes. Assays, purification, and properties of different ribosomal factors and their role in peptide chain initiation. J Biol Chem 248: 4500-4511, 1973 14. Clemens MJ, Pain VM: Association of initiation factor eIF-2 with a rapidly sedimenting fraction from Ehrlich ascites-tumor cells. Biochem J 194: 357-360, 1981 15. Menaya J, Parrilla R, Ayuso MS: Effect of vasopressin on the regulation of protein synthesis initiation in liver cells. Biochem J 254: 773-779, 1988 16. Takeishi K, Ukita T, Nishimura S: Characterization of two species of methionine transfer rihonucleic acid from baker's yeast. J Biol Chem 243: 5761-5769, 1968
Address for offprints: S. Ayuso, Centro de Investigaciones Bio16gicas, C.S.I.C., C/. Vel~izquez 144, 28006 Madrid, Spain
Effect of alanine supply on hepatic protein synthesis in animals maintained on a protein free diet
Dolores P6rez-Sala, Teresa Rivas Calleja, Roberto Parrilla and Matilde S. Ayuso Endocrine Physiology Unit, Centro de Investigaciones Biol6gicas, C.S.I.C., Vel{~zquez 144, 28006 Madrid, Spain
Key words: liver, protein synthesis, protein-free diet, alanine action Abstract
In contrast to what it is observed during starvation, animals maintained on a protein-free isocaloric diet showed an increase in the rate of hepatic peptide chain elongation as determined by measuring the ribosomal transit time in vivo. The loss of body nitrogen per se is insufficient to generate the signal(s) which arrests hepatic peptide chain elongation. This observation suggests that it is an increase in gluconeogenic demand, and not the negative nitrogen balance, which is implicated in determining reciprocal changes in the rate of protein synthesis. The rate of protein synthesis, as expressed per mg of DNA, does not change in protein deprived animals, while the R N A to DNA ratio decreased. These data also agree with a higher ribosomal efficiency at the elongation step. The animals maintained on a protein-free diet have a decreased hepatic content of protein and an increased concentration of valine, indicating an increased proteolysis. The enhanced rate of polypeptide elongation observed in animals kept on a protein-free diet was accompanied by decreases in the state of aggregation of polyribosomes and in the ability of liver extracts to form eIF-2 catalyzed ternary complexes. These observations suggest that the activity of the hepatic initiation factor in vivo may not be rate limiting. The administration of alanine in vivo to animals maintained on a protein-free diet showed a preferential effect in reaggregating polyribosomes. This action was neither accompanied by detectable effects on the rate of eIF-2 catalyzed ternary complexes formation nor by significant changes in the rate of elongation. It is concluded that factors other than eIF-2 activity or the rate of polypeptide elongation must be controlling the hepatic polyribosomal state of aggregation.
Introduction
Protein synthesis and gluconeogenesis, two apparently unrelated pathways, do share in common the use of amino acids as their only or preferred substrates. Reciprocal changes in the flux through these pathways have been observed in either physiological or pathological conditions [1]. Acute stimulations of gluconeogenesis by glucagon or fatty acids have also been reported which lead to oppo-
site changes in the rate of hepatic protein synthesis [1, 2]. The nature of the signalling process involved in coordinating the activity of these pathways remains to be elucidated. Variations in the phosphorylation potential [1, 2] and/or changes in the concentration of certain key amino acids [3] have been proposed to be responsible for such a coordination. The increased rate of gluconeogenesis observed after acute hormonal stimulation is accompanied by a decrease in the phosphorylation potential [1,
106 2]. This eventually might perturb the rate of elongation of polypeptide chains [2]. On the other hand, the concentration of certain amino acids, like alanine, may play an important regulatory role as this amino acid has been reported to display a powerful effect in stimulating hepatic peptide chain elongation in starved animals either in vivo or in isolated liver cells [3]. Starvation in the rat leads to an increase in hepatic gluconeogenesis and a decrease in protein synthesis [3, 4]. The liver from starved animals is also characterized by a lower phosphorylation potential and a decreased alanine content [3, 4]. The higher rate of gluconeogenesis, by increasing alanine demand and energy consumption, has been postulated to be responsible for the above mentioned changes, which eventually could induce a decrease in the rate of protein synthesis. Starvation is accompanied by a progressive loss of body nitrogen; therefore, the possibility should also be considered that negative nitrogen balance could promote the change in the rate of peptide chain elongation. Based on the observation that low protein diets perturb hepatic protein synthesis [5-9], we found it interesting to study the effect of alanine on hepatic protein synthesis under conditions of negative nitrogen balance and where gluconeogenesis is not stimulated. For this purpose animals were maintained on a protein-free diet with a high carbohydrate content. Our results indicate that a negative nitrogen balance by itself is not sufficient to induce a decrease in peptide chain elongation, and that, under these conditions, alanine administration has no effect.
Chemicals [U-3H]Valine and pS]Methionine were purchased from Amersham International (Amersham, Bucks., U.K.). Most of the reagents were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.).
Collection of liver biopsies and plasma samples The animals were anaesthetized with Nembutal (40 mg/kg body wt.) about 10 min before they were surgically manipulated. Small liver biopsies were taken as previously described [3] and immediately frozen with aluminum clamps precooled in liquid nitrogen [11]. Blood was withdrawn with heparinized syringes from the aortic bifurcation and the plasma obtained immediately by centrifugation at 4 ° C.
Determination of the state of aggregation of hepatic polyribosomes Liver biopsies were immediately cooled by immersion in ice cold buffer A (50 mM triethanolamineHC1, pH7.3, 5mM MgC12, 25mM KC1) and homogenized in 3 vol of this buffer. The homogenates were processed as previously described [3] to obtain the polyribosomal profiles. The polyribosomal fraction was calculated as the ratio between the area of the profile corresponding to polysomes and the area corresponding to monomers and dimers of ribosomes.
Material and methods
Animals Male Wistar albino rats weighing 200-220 g were used. The animals were maintained on a semiliquid diet, as described by De Carli and Lieber [10]. When indicated, all the protein-containing ingredients were substituted by isocaloric amounts of glucose during 4 or 8 days.
Determination of the rates of elongation of hepatic polypeptide chains and protein synthesis This was done by measuring the distribution of radioactivity incorporated into nascent peptides on the polyribosomes (n) and into total peptides (t) [12]. For these determinations, 20/zCi of [3H]valine was injected into the portal vein of anaesthetized rats and two small biopsies (about 1 g) were taken from each liver, either after 0.5 and i min or after 1 and 2rain. To prevent hemorrhage, liver
107 lobules were ligated immediately before the biopsies were taken. Polypeptide chain completion time in each case was calculated as the time needed for the ratio of the radioactivity incorporated in nascent peptides into the polyribosomes to the radioactivity incorporated into total peptides (n/t) to be decreased by 50% [3, 12]. The rate of protein synthesis was calculated as the amount of protein synthesized during one elongation cycle, which is equivalent to the nmoles of active ribosomes divided by the duration of the elongation cycle, that is, the determined time of completion of polypeptide chains (in min). The concentration of active ribosomes was calculated from the determination of total R N A (Table 5), considering the ribosomal RNA to be 80% of the total RNA (4 nmoles/g wet wt), and taking into account the percentage of active ribosomes (incorporated in polyribosomes) given by the polyribosomal fraction. The results obtained from these calculations are the nmoles of protein synthetized x g of liver wet weight-Ix min-k They are converted to mg of protein assuming and average molecular weight of liver protein of 50000 daltons.
Determination of the concentration of amino acids in plasma and liver For the determination of the plasma amino acid concentration, portions of plasma samples were deproteinized with 10% (w/v) sulphosalycilic acid. For the determination of the hepatic amino acid content, freeze-clamped liver biopsies were lyophilized. Approximately 200 mg of dry tissue were homogenized with 2 ml of 8% (w/v) HC104. After removal of the insoluble material, 0.7 ml portions of the supernatant were adjusted to p H 2 with 1 M lithium citrate, pH 1.3. The concentration of the different amino acids was determined by automated ion-exchange liquid chromatography.
Analysis of elF-2 activity The activity of eIF-2 in liver extracts was determined by measuring the formation of a ternary
complex between elF-2, GTP and [35S]methionyltRNA. The procedure was that described by Gupta et al. [13], as modified by Clemens and Pain [14]. Livers from rats weighing 100 to 120 g were homogenized with 3 vol of 50 mM triethanolamine-HC1, pH7.6, 5 mM magnesium acetate, 2.3% (w/v) glycerol, 0.5mM dithiothreitol and 120 or 250raM KCI. After centrifuging at 10,000 g for 10rain at 4 ° C, the supernatant was collected and centrifuged again at 150,000 g for 3 h at 4 ° C. The postribosomal supernatant was stored in aliquots in liquid nitrogen until the eIF-2 activity was assayed as previously described [15]. The incubation mixture contained 5% (v/v) of postribosomal supernatant. Final Mg 2+ concentration was 2raM. The reaction was initiated by the addition of 7 x 106cpm of [35S]methionyl-tRNA, prepared as previously described [16]. Under these conditions the rate of incorporation of [3SS]methionyl-tRNA into ternary complexes was linear with respect to incubation time and protein concentration.
Results
Effect of feeding a protein-free diet on the state of aggregation of hepatic polyribosomes, elF-2 activity and amino acid content Figure 1 shows the state of aggregation of hepatic polyribosomes of rats maintained on an isocaloric protein-free diet. After 4 days, a decrease in the state of aggregation was observed, although it was not statistically significant. At least 8 days on the diet were needed to consistently provoke a significant polyribosomal breakdown (Fig. 1, lower left panel). After either four or eight days on the protein-free diet, the administration of alanine was followed by an acute reaggregation of polyribosomes. The quantitation of this response at eight days is shown in Table 1. The administration of inorganic nitrogen as ammonium chloride led also to an acute and significant increase in the polyribosomal fraction (results not shown). The polyribosoreal breakdown observed in animals on a proteinfree diet could be a consequence of a lower rate of initiation of protein synthesis; therefore, the ability
108
t_..,__l.__J 0
2
body nitrogen balance due to feeding a protein-free diet, the hepatic content of amino acids decreased only by less than 20% (Table 3). Variations in the content of only three amino acids, aspartate, glutamate and proline could account for the decrease in total a-amino nitrogen. The higher concentration of valine probably reflects an accelerated rate of protein catabolism (Table 3). No significant changes in the total plasma content of amino acids were observed after eight days on a protein free diet. Decreases in glutamate and glutamine, and increases in glycine and serine were observed in plasma as well as in liver (Tables 3 and 4).
.I 4
4 days
Alanine
I'CO
,-1 m a. O 2
4
2
8 days
____._--I 2
/ 4
4
Effect of feeding a protein-free diet on the rate of hepatic protein synthesis
, Alanine
1 2
__,_L 4
GRADIENT (ml)
Fig. 1. Effects of feeding a protein-free diet and alanine administration on the state of aggregation of hepatic polyribosomes. Animals were maintained on a protein free diet for none, four or eight days, as indicated. The experiment was performed as described in Methods. Alanine (1 mmol/kg body wt.) was administered 10 rain before the animals were surgically manipulated. Each experimental condition was studied at least six times and representative tracings are presented.
of hepatic extracts to form ternary complexes was studied. The results, which are depicted in Table 2, indicate that the polyribosomal disaggregation was accompanied by a decrease in the ability of liver extracts to form ternary complexes. Despite the acute action of alanine in reaggregating polyribosomes (Fig. 1) its effect was not accompanied, at least in liver extracts, by an increase in the formation of ternary complexes (Table 2). Tables 3 and 4 show, respectively, the hepatic and plasma contents of amino acids in control and in protein deprived animals. Despite the negative
Animals maintained on a protein-free diet showed an increase in the rate of decay of the hepatic 'n/t' value (Fig. 2). Intraperitoneal administration of alanine to these animals produced a slight delay in the rate of decay (Fig. 2), while the administration of NH4C1 caused no apparent effect (results not shown). The polypeptide chain completion time was calculated from these data. Table 1 shows that in control animals the termination time was 1.7 min. This time was reduced to only one minute in protein deprived animals (Table 1). The rate of protein synthesis increased by 54% in livers from rats maintained on a protein-free diet. This was parallel by a similar increase in the DNA content (Table 5). Thus, the protein synthesis rate expressed as mg of protein x mg D N A - ; × day -~ was not altered (Table 1). Table 5 also shows that the hepatic content of total R N A per D N A was decreased by 35% in protein-deprived animals. The lack of effect on the rate of protein synthesis was accompanied by a decreased hepatic content of total protein (Table 5), indicating that there is an increased rate of proteolysis in protein deprived animals. Alanine, administered in a single dose, was not capable of inducing a further increase in the rate of protein synthesis (Table 1).
109 Table 1. Effect of feeding a protein-free diet on the hepatic polyribosomal fraction and rate of elongation of polypeptide chains
Control
5.4+ 0.3 86
Polyribosomal fraction % of active polyribosomes Termination time (rain) Rate of protein synthesis: nmoles × g wet wt -1 × min -1 mg protein × mg DNA -~ × day-1
Protein-free diet - Alanine
+ Alanine
4.1+ 0.3* 79
6.7+ 0.9 87
1.7
1.0
1.3
2.0 63
3.1 60
2.7 46
Animals were fed a protein-free diet for eight days. Liver biopsies were processed as described in Methods for the determination of either the polyribosomal state of aggregation or the ribosomal transit time. Alanine treated animals received an i.p. injection of 1 mmol of alanine 10 rain before the biopsies were taken. Control animals were given a similar volume of saline solution. The results are average values of at least 8 determinations _ SEM. * By t-test P < 0.01. Discussion
T h e progressive loss of liver protein induced by feeding a protein-free diet resulted in hepatic polyribosomal b r e a k d o w n ; h o w e v e r , in contrast to what has b e e n o b s e r v e d in starvation [3], the rate of peptide chain elongation, as reflected by the ribosomal transit time, was e n h a n c e d (Table 1). This finding allows us to c o n c l u d e that a progressive loss of b o d y nitrogen is not sufficient to induce a decrease in the rate of peptide chain elongation; therefore, as previously suggested [1], the activity Table 2. Effect of a protein-free diet on the ternary complex formation activity of liver extracts
Ternary complex formation (cpm × 10-3 x /zg DNA -~) KC1 concentration in extracts
Control diet Protein-free diet: - Alanine + Alanine
120 mM
250 mM
5.6 + 0.2
6.3 _+ 0.2
1.6 -+ 0.2 0.7 + 0.3
2.8 _+ 0.2 1.5 + 0.3
The liver extracts were obtained from animals maintained during eight days on a protein-free diet according to the protocol described in Methods. Alanine treated animals received an i.p. injection of 1 mmol of alanine 10 min before the biopsies were taken. The values are mean of six determinations in duplicate + SEM.
of the gluconeogenic p a t h w a y might be an important factor in determining the differential effects on peptide chains elongation o b s e r v e d during starvation or feeding a protein-free diet. Based on the ribosomal transit time and the state of aggregation of hepatic p o l y r i b o s o m e s (Table 1), it can be concluded that there is an increase in the rate of hepatic protein synthesis in protein deprived animals. H o w e v e r , when the rate o f protein synthesis was expressed as mg o f protein synthetized by m g of D N A (Table 1), no significant changes were observed. This observation is in conflict with reports f r o m o t h e r laboratories that indicate a fall in the overall rate of protein synthesis in the liver of protein deprived animals [7-9], or an increase [6]. W e have, at present, no explanation for these conflicting reports, but a correlation seems to exist between the effects of the diet on the animals b o d y weight and changes in hepatic protein content and protein synthesis. A n increased protein synthesis rate per g of tissue seems to be acc o m p a n i e d by m o d e s t variations in loss of liver protein and b o d y weight (Tables 1 and 5 and ref. 6). A decreased protein synthesis rate is a c c o m p a n i e d by m o r e i m p o r t a n t losses of b o d y weight and liver protein [9]. S o m e authors [9] had explained these discrepancies as caused by variations in the specific activity of the amino acid precursor not taken into account in some of the m e t h o d o l o g i e s used for the determination of protein synthesis rates in vivo. T o minimize changes in specific activty, a large dose of
110 the precursor amino acid is often used for measuring the in vivo rate of protein synthesis [9]. However, we considered conflicting to use a large dose of amino acid when trying to determine the effects of a negative nitrogen balance. The method used in our experiments, measurement of the ribosomal transit time, is independent of the specific activity of the precursor amino acid, provided that it does not change during the two minutes experimental time [12]. The plasma content of valine (Table 4) does not change in protein deprived animals, ruling out any difference in the specific radioactivity of the amino acid supplied to the liver. The observation that a negative correlation seems to exist between protein and RNA content and rates of peptide chain elongation (Tables 1 and Table 3. Effect of a protein-free diet on the hepatic content of
5) suggests that a feedback regulatory mechanism might exist. This possibility is consistent with unpublished results from our laboratory that show an accumulation Of hepatic protein while the rate of peptide elongation decreases in animals kept on an alcohol-containing diet. The peptide initiation factor elF-2 does not seem to be rate limiting for hepatic protein synthesis under physiological conditions. This conclusion is based on two observations: First, the acute reaggregation of polyribosomes observed in protein deprived animals after alanine administration (Fig. i and Table 1) is not accompanied by an increase in elF-2 activity. Second, the rate of protein elongation increases in protein deprived animals despite a Table 4. Effect of a protein-free diet on the plasma content of amino acids
amino acids Control Control
Protein-free diet
Protein-free diet - Alanine - Alanine
+ Alanine
+ Alanine
Aspartate Treonine Serine Glutamate Proline Glycine Alanine Citruline Valine Cistine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Tryptophane Arginine
(/xmoles x g dry wt -1) 22.3 +__ 1.9 4.4 + 0.4 11.9 + 3.2 2.5 ___ 0.26 5.3 + 0.8 4.0 + 0.6 2.4 + 0.26 7.9 + 0.5 7.6 + 0.5 10.1 + 1.0 5.9 + 0.3 7.9 + 0.5 22.9 + 4.2 16.6 + 1.7 11.6 + 1.7 6 . 1 + 0.5 10.0+ 0.3 8 . 9 + 0.5 2.8 _+ 0.4 2.9 + 0.56 4.5 + 0.8 0.05 + 0.009 0.2 + 0.01 0.4 + 0.2 0.3 +_ 0.03 0,5 + 0.04 0.2 + 0.05 0.1 _+ 0.02 0.4_+ 0.03 0.2 + 0.05 0.1 _+ 0.01 0.4 + 0.02 0.2 + 0.04 0.2 _+ 0.02 0.5 + 0.04 0.2 + 0.05 0.3 + 0.04 0.4 + 0.03 0.3 + 0,03 0.3 _+ 0.03 0.4 _+ 0.02 0.3 + 0.04 0.2 _+ 0.02 0.3 _+ 0.01 0.2 + 0.01 0.6 + 0.07 0.4 + 0.02 6.4 _+ 2.8 2,1 + 0.26 1.0 + 0.03 1.3 _+ 0.06 1.6 +_ 0,14 2.1 + 0.04 2.3 + 0.07 0.04_+ 0,003 0,008+ 0.001 0.07 + 0.02 0.07 + 0.01 0.03 _+ 0.008 0.1 _+ 0.02
Aspartate Treonine Serine Glutamate Glutamine Gtycine Alanine Valine Cistine Methionine Isoleucine Leucine Tyrosine Phenylalanine Ornithine Lysine Histidine Tryptophane Arginine
(t~moles × g dry wt -1) 0.02 + 0.004 0.03 + 0.003 0.02 + 0.006 0.6 _+ 0.04 0.4 + 0.03 0.5 _+ 0.1 0.2 _+ 0.01 0.5 + 0.06 0.6 + 0.03 0 . 0 6 + 0.008 0.01-+ 0,001 0.1 + 0.01 0.5 +_ 0.04 0.3 + 0.02 0,4 + 0.04 0.2 _+ 0.007 0.6 + 0.05 0.6 + 0.04 0 . 5 + 0.04 0 . 4 + 0,04 1.1_+ 0.25 0.1 + 0.01 0.08 _+ 0.01 0.06 _+ 0,005 0.03 _+ 0.003 0.02 + 0.002 0.02 _+ 0.007 0.04 + 0.008 0.03 + 0.004 0,03 + 0.004 0.05 + 0.006 0.04 + 0.006 0.03 + 0.006 0.07 + 0.009 0.06 _+ 0.005 0.04 + 0.007 0.12 + 0.007 0.05 + 0.006 0.03 + 0.006 0.05 + 0.004 0.06 + 0.006 0.06 + 0.006 0.04 + 0.004 0.03 + 0.003 0.03 + 0.003 0.5 + 0.02 0.35 + 0.014 0.32 + 0.02 0.09 + 0.004 ' 0.17 + 0.01 0.21 + 0.02 0 . 0 4 + 0.007 0.05 + 0.002 0.11 + 0.01 0.1 +-- 0.008 0.07 _+ 0.009
Total amino acids
3.3
Total amino acids
75
Plasma samples obtained from animals maintained for 8 days on a control or protein-free diet were treated as described in Methods. When indicated, animals received an i.p. injection of 1 mmol of alanine or a similar volume of saline solution 10 min before the samples were taken. The values are means of eight determinations + SEM.
62
69
The experimental protocol was as described under Methods. Liver biopsies were obtained from animals maintained for 8 days either on control or on a protein-free diet. The values are means of eight determinations + SEM.
3.3
4.2
111 significant decrease in eIF-2 activity, as measured by the ability of liver extracts to form ternary complexes (Table 2). Alanine is one of the most powerful effectors so far described, capable of acutely stimulate hepatic protein synthesis in vivo [3]. Its effect is observable only in livers from starved animals where it stimulates both the initiation and the elongation steps of protein synthesis. Our results (Fig. 1 and Table 1) add further evidence to the role of alanine in stimulating protein synthesis when this process is previously inhibited, as is also the case during starvation [3]. The finding that alanine can reaggregate polysomes without significantly perturbing the rate of peptide elongation indicates that more than one site of alanine action should be considered. On the other hand, the administration of ammonium chloride led to a similar effect on ribosomes aggregation, in the absence of detectable effects on the polypeptide termination time (results not shown). These observations indicate that the steps of initiation and elongation of polypeptide chains are not linked in a compulsory manner in vivo. Thus, animals maintained on a protein-free diet offer an interesting model for the study of the functional relationship between the ribosomal state of aggregation and the rate of peptide synthesis.
1.0
PRoTEIN-FREE
CONTROL
DIET
%
0.5
n/t
L L
0.2
0.1
I
0
1
PROTEIN-FREE DIE"I'+ALANINE
i
20
•
,
1
.
±
,
20
,
.
1
Time (min.) Fig. 2. Effects of feeding a protein-free diet and acute alanine administration on the rate of decay of the hepatic n/t value. The experiment was performed as described in Methods. The animals were maintained for eight days on either control or protein-free diets. Alanine (1 mmol/kg body wt.) was administered 10min before the animals were surgically manipulated. The points represent means of at least eight experiments and the vertical bars the standard error.
Acknowledgements The authors wish to express their gratitude to M.J. Arias-Salgado and A. Rodrfguez for their excellent technical assistance. This work was supported in part by grants from Direcci6n General de Ciencia y Tecnologfa (PB87-0280 and 87065) and Fondo de Investigaciones Sanitarias (89/0467 and 89/0468). D. P.-S. was recipient of a fellowship from the Ministerio de Educaci6n y Ciencia.
Table 5. Effect of feeding a protein-free diet on the body weight and hepatic contents of D N A , R N A and protein
Control
D N A ( m g x g wet wt. -~) Total RNA (mg × g wet wt. 1) Total protein (mg × g wet wt.-1) RNA/DNA Body weight (g)
2.3_+ 0.13 9.9 + 0.6 170 + 8 4.2 + 0.07 162 _+ 2.5
Liver biopsies, obtained from animals fed control or proteinfree diets for 8 days, were processed as described in Methods. Alanine treated animals received intraperitoneally 1 mmol of alanine before the biopsies were collected. The values are means of eight different determinations _+ SEM. By t-test: * P < 0.001; * * P < 0.01.
,
2
Protein-free diet - Alanine
+ Alanine
3 . 7 + 0.1" 10.3 + 0.6 130-+ 15"* 2.7-+ 0.1" 116-+ 2.4
4.2_+ 0.1" 12.1 + 0.3 2.9-+ 0.09*
112 References 1. Ayuso MS, Vega P, Manch6n CG, Parrilla R: Interrelation between gluconeogenesis and hepatic protein synthesis. Biochim Biophys Acta 883: 33-40, 1986 2. Martin-Requero A, P6rez-Diaz J, Ayuso MS, Parrilla R: The mechanism of the glucagon-induced inhibition of hepatic protein synthesis. Arch Biochem Biophys 195: 223234, 1979 3. P6rez-Sala D, Parrilla R, Ayuso MS: Key role of L-alanine in the control of hepatic protein synthesis. Biochem J 241: 491-498, 1987 4. Parrilla R: The effect of starvation in the rat on metabolite concentrations in blood, liver and skeletal muscle. Pflugers Archiv 374: %14, 1978 5. Sato A, Noda K, Natori Y: The effect of protein depletion on the rate of protein synthesis in rat liver. Biochim Biophys Acta 561: 475-483, 1979 6. Garlick PJ, Miltward DJ, James WPT, Waterlow JC: Effect of protein deprivation and starvation on the rate of protein synthesis in the tissues of the rat. Biophys Biochim Acta 414: 71-84, 1975 7. Morgan EH, Peters T: The biosynthesis of rat serum albumin. V. Effect of protein depletion and refeeding on albumin and transferrin synthesis. J Biol Chem 246: 3500-3507, 1973 8. Conde RD, Scornik OA: Role of protein degradation in the growth of livers after a nutritional shift. Biochem J 158: 385-390, 1976
9. McNurlan MA, Garlick PJ: Protein synthesis in liver and small intestine in protein deprivation and diabetes. Am J Physiol 241: E238-245, 1981 10. De Carli LM, Lieber CS: Fatty liver in the rat after prolongued intake of ethanol with a nutritionally adequate new liquid diet. J Nutr 91: 331-446, 1967 11. Wollenberger A, Ristau O, Schoffa G: Eine einfache Technik der extrem schellen Abkiihlung gr613erer Gewebestiicke. Pflugers Arch Gesamte Physiol Menschen Tiere 270: 339-412, 1960 12. Mathews RW, Oronsky A, Haschemeyer AEV: Effect of thyroid hormone on polypeptide chain assembly kinetics in liver protein synthesis in vivo. J Biol Chem 248: 132%1333, 1973 13. Gupta NK, Woodley CL, Chen YC, Bose KK: Protein synthesis in rabbit reticulocytes. Assays, purification, and properties of different ribosomal factors and their role in peptide chain initiation. J Biol Chem 248: 4500-4511, 1973 14. Clemens MJ, Pain VM: Association of initiation factor eIF-2 with a rapidly sedimenting fraction from Ehrlich ascites-tumor cells. Biochem J 194: 357-360, 1981 15. Menaya J, Parrilla R, Ayuso MS: Effect of vasopressin on the regulation of protein synthesis initiation in liver cells. Biochem J 254: 773-779, 1988 16. Takeishi K, Ukita T, Nishimura S: Characterization of two species of methionine transfer rihonucleic acid from baker's yeast. J Biol Chem 243: 5761-5769, 1968
Address for offprints: S. Ayuso, Centro de Investigaciones Bio16gicas, C.S.I.C., C/. Vel~izquez 144, 28006 Madrid, Spain
