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Departments of Home Economics and
*
Life Science, 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
2To whom correspondence should be addressed. E-mail: khayase{at}auecc.aichi-edu.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • 17-ß-estradiol • ovariectomy • protein synthesis • brain • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Lewis et al. 1984
, Millward et al. 1975
and 1976
,
Symmons et al. 1972
, Yokogoshi et al. 1980a
and 1980b
). We and other investigators have shown that protein
synthesis in the brain is also sensitive to the perturbation of dietary
amino acid composition (Beverly et al. 1991
,
Yokogoshi et al. 1992
) in young rats.
Many investigators have reported that protein synthesis declines in
specific tissues (e.g., liver, muscle) and in the whole body throughout
development in mammals after weaning (Attaix et al. 1986
and 1988
, Goldspink and Kelly 1984
, Waterlow et al. 1978
). We have demonstrated that the rate of brain
protein synthesis decreased with age in rats after weaning
(Hayase and Yokogoshi 1994
). In many postmenopausal
women, not only age but also sex hormone deficiency affects body
composition and function (Sowers 1996
). However, no
studies are available on the rate of brain protein synthesis during
estrogen deficiency.
Estrogen increases tissue protein synthesis by stimulating
transcriptional activity (Gordon et al. 1991
,
Hofbauer et al. 1999
, Lazier et al. 1994
,
Villa et al. 1995
). Recently, several investigators
suggested that estrogen might have a direct affect on brain function
(e.g., gene expression in neurons) (Roy et al. 1999
).
Therefore, the possible direct effects of estrogen on brain protein
synthesis in female rats are of interest. The purpose of our study was
to determine whether estrogen treatment affects the rate of brain
protein synthesis in ovariectomized female rats. In our previous
reports (Hayase et al. 1998
, Yokogoshi et al. 1992
), a positive correlation was found between the rate of
protein synthesis and the RNA activity in the brain when the dietary
quality or quantity of protein was manipulated. However, the reduction
with age in brain protein synthesis was related to a fall in the RNA
concentration (Hayase and Yokogoshi 1994
). Two questions
were considered in the present study: 1) whether 17-ß-estradiol might
affect the rate of brain protein synthesis in female rats and 2)
whether greater RNA concentration or RNA activity in ovariectomized
female rats treated with 17-ß-estradiol resulted in a greater brain
protein synthesis rate compared with untreated ovariectomized rats.
Therefore, we examined three indicators of protein synthesis in rat
brains: its rate, RNA concentration and RNA activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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L-Tyrosine decarboxylase, ß-phenethylamine, 17-ß-estradiol and leucylalanine were purchased from Sigma Chemical Co. (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).
Animals and diet.
Female Wistar rats (12 wk old; Japan SLC, Hamamatsu, Japan) were housed
at 24°C in a room with a 12-h light/dark cycle. The rats were fed a
20 g casein/100 g diet (Hayase et al. 1998
) after
they had been fed 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. Approval was granted by Aichi University
of Education Animal Care and Use Committee.
Experimental design.
The experiment was conducted on three groups of rats. All rats were fed
the 20 g casein/100 g diet for 16 d. On d 1, two groups were
ovariectomized and injected subcutaneously with either 17-ß-estradiol
dissolved in sesame oil [2 µg/(100 g body · d)] or sesame
oil for the last 3 d of the 16-d experimental period. The
sham-operated control group was administered the sesame oil only
for the last 3 d. The dose of sesame oil injected was 0.05 mL/100
g body. After 16 d, the fractional rates of protein synthesis in
brain were measured by the method of Garlick et al. (1980)
. Rats were decapitated between 1000 and 1200 h.
Brain regions (Reinstein et al. 1979
) were quickly
removed and frozen in liquid nitrogen. The concentrations of protein
and RNA in brain were measured according to the methods of Lowry et al. (1951),
with bovine serum albumin as a standard, and of
Fleck and Munro (1962)
, respectively. 17-ß-Estradiol
concentration in plasma was determined by enzyme-linked
immunosorbent assay (Estradiol Kit; Neogen, Lexington, KY).
Fractional rates 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. At 10 min
after the injection, rats were quickly decapitated. Specific
radioactivities of [3H]phenylalanine in tissue samples
were determined according to the method described in our previous
report (Hayase et al. 1998
). 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 this experimental condition. Specific
radioactivities of free phenylalanine in the plasma, cerebral cortex
and cerebellum in rats of the three groups were constant in each tissue
(Table 1
). The values also were 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 were calculated for rats killed at
the single time point of 10 min after intravenous administration of the
radioisotope.
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The means and pooled SEM values are reported. Duncan’s
multiple range test was used to compare means after one-way ANOVA
(Duncan 1955
, 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The relative weights of the various brain regions did not differ
among the experimental groups. Compared with the untreated
ovariectomized rats, the plasma concentration of 17-ß-estradiol was
significantly higher in that of ovariectomized rats treated with
estradiol or of the control rats.
|
). In pooled samples of hippocampus and brain stem, these rates were
lower in the ovariectomized rats.
|
). Correlations between the fractional rate of protein synthesis and RNA
activity were significant in the cerebral cortex (r = 0.905, P < 0.001) and cerebellum (r = 0.872, P < 0.001). The RNA concentrations of all
organs did not differ among groups (Table 4)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovariectomized rats had reduced fractional rates of protein synthesis
in brain regions, whereas treatment with 17-ß-estradiol reversed the
effect of estrogen deficiency (Table 3)
. The changes in the brain
protein synthesis likely depend on the body estrogen concentration as
previously demonstrated in the other tissues (Fiorelli et al. 1999
, Hofbauer et al. 1999
, Lazier et al. 1994
). 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
). Steroid hormones, especially
estrogen, appeared to increase the transcription rate (Hofbauer et al. 1999
). However, a positive correlation between the rate
of protein synthesis and RNA activity was found in the brain of weaned
and aged rats when the dietary quality and quantity of protein were
manipulated (Hayase et al. 1998
, Koie et al. 1999
, Yokogoshi et al. 1992
). Hormonal
treatments such as with 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 the present
study, RNA activity, rather than RNA concentration, in the ovariectomy
plus 17-ß-estradiol group was higher than that in the ovariectomized
group. The greater
RNA activity in the estrogen-treated ovariectomized rats may
reflect an increase in the rate of brain protein synthesis. Therefore,
the changes in estrogen concentration may have controlled RNA activity
and been one of the factors affecting brain protein synthesis in female
rats.
Little information is available on the mechanism by which estrogen
affects RNA activity in the brain of female rats. We also reported that
the aggregation of polyribosomes in the brain of weaned rats after only
a 5-h feeding decreased with a decrease in dietary protein quality, and
that a correlation was found between the polysomal profile and RNA
activity (Yokogoshi et al. 1992
). To determine the
effect of estrogen on brain protein synthesis in ovariectomized female
rats, in future studies the ribosomal aggregation in the brain should
be measured. In the present study, we determined the total RNA
concentration in the brain regions. However, because estrogen may
affect mRNA in the brain, this is another possibility to consider in
further examination of the mechanism by which the estrogen alters brain
protein metabolism. A deficiency of sex hormones also affects brain
function (Roy et al. 1999
, Smiciklas-Wright 1990
), but relatively insufficient information is available on
hormonal factors (e.g., estrogen) that moderate the molecular mechanism
responsible for the changes. In particular, Rowe and Kahn (1987)
argued that the modifying effects of diet and hormonal
factors have been underestimated in aging research. The present results
indicate that brain protein synthesis was affected by 17-ß-estradiol
in ovariectomized female rats, as evaluated by the protein synthesis
rates, and suggest the effects of estrogen on brain protein synthesis
in female rats are also of importance in understanding the relationship
among aging, nutrition, sex hormones and brain function in mammals.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received June 19, 2000. Initial review completed July 28, 2000. Revision accepted September 22, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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