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The Journal of Nutrition Vol. 128 No. 9 September 1998,
pp. 1533-1536
Department of Home Economics, Aichi University of Education, Kariya, Aichi 448-8542, Japan and * Laboratory of Nutritional Biochemistry, School of Food and Nutritional Sciences, The University of Shizuoka, Yada, Shizuoka 422-8526, Japan
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The purpose of this study was to determine whether the quantity of dietary protein affects the rate of brain protein synthesis in aged rats. Experiments were conducted on three groups of 30-wk-old rats fed diets containing 0, 5 or 20 g casein/100 g for 10 d. The fractional rates of protein synthesis in brain, liver and kidney declined with a decrease in quantity of dietary protein. In brain, liver and kidney, RNA activity [g protein synthesized/(g RNA·d)] was significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. The results suggest that the rate of protein synthesis in the brain declines with a decrease in quantity of dietary protein in aged rats, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.
KEY WORDS: dietary protein · age · protein synthesis
brain ·
rats
Concentrations of tissue proteins are affected by alterations in dietary proteins and by age. These changes in protein metabolism may be reflected in the rate of protein synthesis and the polyribosomal profile of the endoplasmic reticulum, especially in liver and muscle (Goldspink et al. 1984 Many investigators have reported that protein synthesis declines in specific tissues (e.g., liver or muscle) and in the whole body throughout development in mammals after weaning (Attaix et al. 1986 Chemicals.
L-Tyrosine decarboxylase, Animals and diet.
Male Wistar rats (30 wk old, Japan SLC, Hamamatsu, Japan) were housed at 24°C in a room with a 12-h light:dark cycle. The rats were transferred to the experimental diets containing 0, 5 or 20% casein (Table 1) after consuming a commercial nonpurified diet (MF, Oriental Yeast, Tokyo, Japan) for 2 d. All rats were individually housed and given free access to food and water. The approval of Aichi University of Education Animal Care and Use Committee was given for our animal experiments.
Experimental design.
The experiment was conducted on three groups of rats. All rats were fed the experimental diets for 10 d. After 10 d, the fractional rates of protein synthesis in liver, kidney and brain were measured by the method of Garlick et al. (1980) Fractional rate of protein synthesis in tissues.
Radioactive L-[2,6-3H]phenylalanine was combined with unlabeled phenylalanine to yield a dose of 1.85 MBq and a concentration of 150 mmol/L saline. Rats were injected with the radioisotope via the tail vein at a dose of 1 mL/100 g body weight. Ten minutes after the injection, the rats were quickly decapitated. Tissue samples were homogenized with 10 volumes of cold 20 g/L perchloric acid and then centrifuged at 2800 × g for 15 min at 4°C. The supernatant was used for the measurements of specific radioactivity after adjusting the pH to 6.0-7.0 with saturated potassium citrate. The precipitate containing protein was washed three times with 5 mL of 20 g/L perchloric acid, suspended in 10 mL of 0.3 mol/L NaOH and incubated at 37°C for 1 h. Protein-bound phenylalanine was obtained by reprecipitating the protein with 2 mL of 200 g/L perchloric acid, washing the pellet with 5 mL of 20 g/L perchloric acid twice and hydrolyzing the protein in 10 mL of 6 mol/L HCl for 24 h at 110°C. The HCl was completely evaporated and the amino acids were dissolved in citrate buffer (pH 6.3). The determination of the specific radioactivity of [3H]phenylalanine involved its enzymatic conversion into phenethylamine, followed by a count of radioactivity (LS 5000TD, Beckman Japan, Tokyo, Japan) and fluorometric determination (F-3000, Hitachi, Tokyo, Japan) (Suzuki and Yagi 1976
Statistical analysis.
The means and pooled SEM are reported. Duncan's multiple range test was used to compare means after one-way ANOVA (Duncan 1955 The rats fed the protein-free diet lost body weight and consumed less food than the other two groups, which did not differ (Table 3). The relative weights of liver and brain regions did not differ. Compared with rats fed the 20% casein diet, rats fed the protein-free or 5% casein diets had lower relative kidney weights.
More research concerning age-related changes in brain composition and function (e.g., nutrient metabolism), is necessary to understand the modulating effects of nutritional factors (Smiciklas-Wright 1990
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Lewis et al. 1984, Millward et al. 1975 and 1976, Symmons et al. 1972, Yokogoshi et al. 1980a and 1980b). Several investigations have indicated that brain protein synthesis is also sensitive to the perturbation of dietary amino acid composition (Beverly et al. 1991, Yokogoshi et al. 1992) and of the internal environment (Siegel et al. 1971) in young rats.
and 1988, Goldspink and Kelly 1984, Waterlow et al. 1978). We demonstrated that the rate of protein synthesis in the brain decreased with age in rats after weaning (Hayase and Yokogoshi 1994). In older rats, however, little documentation of the effect of dietary protein on brain protein synthesis is available. Therefore, the purpose of our study was to determine whether the quantity of dietary protein affects brain protein synthesis in aged rats. In our previous report (Yokogoshi et al. 1992), a positive correlation between the rate of protein synthesis and RNA activity was found in the brain when the quality or quantity of dietary protein was manipulated. However, the reduction with age in brain protein synthesis was related to a fall in RNA concentration (Hayase and Yokogoshi 1994). The following two questions were considered in this study: 1) whether the quantity of dietary protein might affect brain protein synthesis in aged rats, and 2) whether greater RNA concentration or RNA activity in rats fed the higher protein diet resulted in a greater protein synthesis rate in the brain than those in rats fed the lower protein diet. Therefore, we examined three indicators of protein synthesis in rat brains, i.e., its rate, RNA concentration and RNA activity. The effects of the quantity of dietary protein on protein synthesis in liver and kidney were also investigated in aged rats. In a previous report (Yokogoshi et al. 1992), 0, 5 and 20% casein levels and a 10-d feeding period were chosen to investigate the effect of dietary protein on brain protein synthesis rate in weaned rats. Thus, to compare with those results, we used the same experimental conditions in this experiment.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
-phenethylamine and leucylalanine were purchased from Sigma Chemical (St. Louis, MO). L-[2,6-3H]Phenylalanine (1.5 TBq/mmol) was obtained from Amersham (Tokyo, Japan). All other reagents were purchased from Wako Pure Chemical (Osaka, Japan).
View this table:
Table 1.
Composition of experimental diets
. The rats were decapitated during the period 1000-1200 h. Liver, kidney and brain regions (Reinstein et al. 1979) were quickly removed and frozen in liquid nitrogen. The concentrations of protein and RNA in liver, kidney and brain were measured according to the methods of Lowry et al. (1951) with bovine serum albumin as a standard, and Fleck and Munro (1962), respectively.
). In a preliminary experiment, we determined whether the method of Garlick et al. (1980) could be used to measure the rate of protein synthesis in the brain under these experimental conditions. Specific radioactivities of free phenylalanine in the plasma, cerebral cortex and cerebellum in rats of the three groups were constant in each tissue (Table 2). The values were also not significantly different among the plasma, cerebral cortex and cerebellum, indicating that the precursor pool of labeled phenylalanine was not altered. In our previous report (Yokogoshi et al. 1992), the decrease in labeling of free phenylalanine at 3, 5 and 10 min in the brain was not significant after an injection of a large dose of [3H]phenylalanine. Therefore, the protein synthesis rates for brain regions, liver and kidney were calculated for animals killed at a single time point of 10 min after intravenous administration of the radioisotope.
View this table:
Table 2.
Specific radioactivities of free phenylalanine in plasma, cerebral cortex and cerebellum in aged rats fed diets with different quantities of protein1
, Snedecor and Cochran 1967). Linear regression analysis was used to assess the relationship between the rate of protein synthesis and RNA activity (Snedecor and Cochran 1967). Differences were considered significant at P < 0.05. In the hippocampus and brain stem, the rates of protein synthesis were determined from a pool of each region.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 3.
Effect of quantity of dietary protein on body weight gain, and liver, kidney and brain region relative weights in aged rats1
View this table:
Table 4.
Effect of quantity of dietary protein on fractional and absolute protein synthesis rates in liver,
kidney and brain regions of aged rats1
View this table:
Table 5.
Effect of quantity of dietary protein on RNA concentrations and RNA activities in liver, kidney and brain regions of aged rats1
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). The amino acid supply has been shown to be important for neuronal protein synthesis in the brain (Parks et al. 1976). Beverly et al. (1991) reported an apparent elevation in protein synthesis in the prepyriform cortex in rats fed an imbalanced diet after application of the dietary limiting amino acid. In previous studies, we found that protein synthesis in the brain decreased with a decrease in dietary protein in weaned rats and declined with age (Hayase and Yokogoshi 1994, Yokogoshi et al. 1992). However, little information is available regarding the effect of dietary quantity of protein on brain protein synthesis in aged rats.
). In weaned rats, a reduction with age in protein synthesis in the brain and muscle was related to a fall in RNA concentration (Hayase and Yokogoshi 1994, Waterlow et al. 1978). However, a positive correlation between the rate of protein synthesis and RNA activity was found in the brain of weaned rats when the dietary quality and quantity of protein were manipulated (Yokogoshi et al. 1992). Hormonal treatment such as insulin also appeared to elevate the rate of protein synthesis and RNA activity in the brain (Hayase and Yokogoshi 1995a). In the brain regions of rats in this study, RNA activity, rather than RNA concentration decreased with the decrease in dietary protein (Table 5). The lower RNA activity in rats fed the protein-free or 5% casein diet may have reduced the rate of brain protein synthesis in these groups. Therefore, the changes in quantity of dietary protein may have controlled RNA activity and thereby affected brain protein synthesis in aged rats. Little information is available on the mechanism by which dietary protein affects RNA activity in the brain of aged rats. We also reported that the aggregation of polyribosomes in the brain of weaned rats decreased with a decrease in dietary protein after only 5 h of feeding, and that there was a correlation between the polysomal profile and RNA activity (Yokogoshi et al. 1992). To determine the effect of quantity of dietary protein on brain protein synthesis in older rats, measurement of the ribosomal aggregation in the brain should be included in future studies.
). This possibility should be considered in future examinations of the mechanism by which the dietary protein alters brain protein metabolism.
). These observations suggest that in vivo, protein synthesis in liver and kidney also depend on the quantity of dietary protein in aged rats, and that the reduction in RNA activity associated with protein ingestion was correlated with the decrease in protein synthesis rate in these organs.
). However, insufficient information is available concerning the environmental factors (e.g., nutrition) that moderate the molecular mechanism responsible for the changes. In particular, Rowe and Kahn (1987) argued that the modifying effects of diet have been underestimated in aging research. These results indicate that brain protein synthesis was affected by the quantity of dietary protein in aged rats as assessed by protein synthesis rates. These effects in aged rats are important in understanding the relationships among nutrition, aging and brain function in mammals.
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FOOTNOTES |
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Manuscript received 11 February 1998. Initial reviews completed 30 March 1998. Revision accepted 19 May 1998.
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ACKNOWLEDGMENTS |
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The authors are grateful to M. Obayashi and M. Suzuki for their valuable technical assistance.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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-phenylethylamine in rat brain.
Anal. Biochem.
1976;
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