ISSN 00062979, Biochemistry (Moscow), 2017, Vol. 82, No. 8, pp. 957961. © Pleiades Publishing, Ltd., 2017.

Original Russian Text © V. Y. Brodsky, L. A. Malchenko, N. N. Butorina, D. S. Lazarev (Konchenko), N. D. Zvezdina, T. K. Dubovaya, 2017, published in Biokhimiya, 2017,
Vol. 82, No. 8, pp. 12371242.

Glutamic Acid as Enhancer of Protein Synthesis Kinetics

in Hepatocytes from Old Rats
V. Y. Brodsky1*, L. A. Malchenko1, N. N. Butorina1,
D. S. Lazarev (Konchenko)2, N. D. Zvezdina1, and T. K. Dubovaya2

1
Koltsov Institute of Developmental Biology, Russian Academy of Sciences, 117808 Moscow, Russia; Email: brodsky.idb@bk.ru
2
Pirogov Russian State Medical University, 117513 Moscow, Russia
Received April 25, 2017
Revision received May 4, 2017

Abstract—Dense cultures of hepatocytes from old rats (∼2 years old, body weight 530610 g) are different from similar cul
tures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4,
or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the
protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was
observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α
methyl4carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the pro
tein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell–cell
communications. Hence, glutamic acid, acting as a receptordependent transmitter, enhanced direct cell–cell communi
cations of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gan
gliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the com
munications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

DOI: 10.1134/S0006297917080119

Keywords: aging, protein synthesis kinetics, protein synthesis rhythm, glutamic acid, biochemistry of direct cell–cell com
munications

We showed earlier [1] that glutamic acid added into
hepatocyte culture could organize the rhythm of protein
synthesis. In this case, glutamic acid acted as a neuro
transmitter: inhibition of glutamic acid receptors on
hepatocyte membranes abolished its organizing function.
We observed such function in cultures and in vivo after
feeding rats with glutamic acid. That study was performed
on young animals, and it was interesting to determine
whether the effect of glutamic acid could be seen in old
animals. As differentiated from other known membrane
signaling factors of protein synthesis kinetics, such as
gangliosides, norepinephrine, serotonin [2], glutamic
acid can penetrate across the blood–brain barrier into the
brain. It is known that various parameters of protein syn
thesis are disturbed with aging, especially in neurodegen
erative diseases [3]. In the present study, we found that
glutamic acid acts as an organizer of protein synthesis
rhythm in hepatocytes of old rats; therefore, it is reason
Abbreviations: MCPG, αmethyl4carboxyphenylglycine.
* To whom correspondence should be addressed.
able to expect that it will have a positive influence not
only on hepatocytes, but also on neurons.
MATERIALS AND METHODS
Isolation and cultivation of hepatocytes, as well as
determination of protein synthesis kinetics, were
described by us in detail earlier [1]. Hepatocytes were iso
lated from 2.02.5yearold Wistar rats with body weight
of 530610 g on perfusing the liver with calciumfree
Hanks’ solution containing HEPES (Sigma, USA),
0.5 mM EGTA and 0.05% collagenase. The hepatocytes
were cultured in serumfree medium 199, which was sup
plemented with 0.2 mg/ml albumin for cell cultures
(Sigma) and 0.5 μg/ml insulin (Sigma). The cells were
cultured at 37°C under 10% CO2 and 90% air.
Suspensions containing ∼106/ml hepatocytes were placed
into Petri dishes above collagencoated glasses, and as a
result dense cultures of hepatocytes were obtained. In
such cultures, the protein synthesis rhythm was estab

957
958
lished soon after the medium change, i.e. autosynchro
nization of the cells occurred [2]. After 2 h, the cultures
were washed from unattached cells and debris. After 24 h,
the medium was changed for fresh medium with or with
out glutamic acid (see “Results and Discussion”). For
2 h, samples of the three cultures were taken every
10 min.
Cultures of each sample were incubated concurrent
ly for 10 min at 37°C in leucinefree medium 199 supple
mented with 3Hlabeled leucine (IMG; 2530 μCi/ml,
specific molar activity 70100 Ci/mmol). Then the cul
tures were washed with cold saline and treated with 5%
perchloric acid for 60 min, washed with ethanol, and pro
teins were dissolved with hyamine (benzethonium chlo
ride; Sigma). Incorporation of 3Hlabeled leucine into
proteins and the free leucine activity (the acidsoluble
fraction) were measured using a Perkin Elmer 2810TR
scintillation counter. Leucine incorporation into proteins
(Icorr) was calculated for each culture relative to the acid
soluble fraction. Such relative values normalize the cul
ture by cell number and by low variability of temperature
during the experiment.
A drugstore tablet of glutamic acid was minced to
powder, which was placed into warm saline and cen
trifuged. The supernatant was introduced into the medi
BRODSKY et al.
um with hepatocyte cultures. The dose of glutamic acid
was chosen in accordance with the dose used in clinical
practice and on considering the mean weight of humans
and rats. For rats, the dose of glutamic acid was
0.2 mg/ml (1.4 mM). Effects of doses of 0.4 and of
0.6 mg/ml were also studied. In one experiment with cul
tures of old rat hepatocytes, MCPG (αmethyl4car
boxyphenylglycine), an antagonist of glutamic acid
metabotropic receptors, was used at the dose of
0.01 mg/ml (reported in more detail in [1]).
RESULTS AND DISCUSSION
Glutamic acid (0.2 mg/ml) introduced in the medi
um with dense cultures of hepatocytes of old rats did not
change the mean level of protein synthesis, but it signifi
cantly increased the amplitude of oscillations (Fig. 1).
The same result was obtained on using 0.4 and 0.6 mg/ml
glutamic acid. Thus, under the influence of glutamic acid
the mean amplitude of the protein synthesis rhythm in
hepatocytes of old rats was increased approximately
twofold (table).
In Fig. 2, the level of protein synthesis is expressed in
percent of the mean for the definite variant of the experi

a b
610
Icorr, cpm

430

250
0 60 120 0 60 120

Time, min

Fig. 1. Effect of glutamic acid on protein synthesis kinetics in cultures of hepatocytes from an old 530g rat. After isolation of hepatocytes,
dense cultures were obtained. After 24 h, the cultures were washed and the medium was refreshed. a) Control: after 60 min samples were taken
every 10 min of three cultures, and in each culture the pool of free 3Hlabeled leucine and the leucine incorporation in proteins were deter
mined. b) Cultures from the same hepatocyte suspension were washed, the medium was supplemented with glutamic acid (0.2 mg/ml), and
after 60 min the protein synthesis kinetics were studied. Ordinate axis: Icorr (cpm) represents incorporation of 3Hlabeled leucine into proteins
relative to the pool of free leucine. Here and in the other figures: the common mean of relative incorporation for the given variant of the exper
iment (36 cultures) is shown by the solid line, and ± error of this mean is shown by dotted lines above and below the mean.

BIOCHEMISTRY (Moscow) Vol. 82 No. 8 2017

 GLUTAMIC ACID AND AGING 959

Amplitudes of oscillations in protein synthesis intensity in hepatocytes from old rats (body weight 530610 g, age ∼2
years); maximal value minus the minimal value in percent of the mean level taken as 100%. Introduction of glutamic
acid into the culture or feeding the rat with glutamic acid

Experiment number Control, without glutamic acid Glutamic acid concentration, mg/ml Effect of glutamic acid

Culture 1 47, 43, 28, 31 0.2 80, 98, 97

2 35, 32, 38 0.2 50, 45, 49

0.4 59, 49, 65
0.6 54, 58, 71

3 34, 45, 21 0.4 53, 49

33, 16, 26, 43 the same 70, 89, 72, 67

Common mean 34 ± 2 65 ± 4

Rat 1 30, 30, 30, 40, 60 – –

(saline [2])

Mean 38 ± 5

2 – 62 mg acid with mixed fodder 60, 62, 74

3 – the same 37, 45, 102, 92

Mean – 68 ± 8

ment. It was confirmed that glutamic acid acted through
specific receptors. Inhibition of the receptors prevented
the increase in the amplitude after the addition of glu
tamic acid into the medium: the kinetics were not differ
ent from the control. In intact old rats, the rhythm was
observed, but its amplitudes were low (table and Fig. 1).
In young rats studied earlier for many years (references
are given in [2]) amplitudes of the rhythm were high.
After the treatment with glutamic acid, hepatocytes from
old rats were not different from the cells of young animals
in protein synthesis rhythm.
Feeding an old rat with glutamic acid (Fig. 3)
revealed that the transmitter normalized the protein syn
thesis kinetics also in vivo. The rat was fed with mixed
fodder moistened with glutamic acid. In cultures
obtained from this rat, amplitudes were as high as after
the direct treatment of cells in culture with glutamic acid.
The control for these experiments coincided with the
control for the cultures presented in the table: the rats
before obtaining the cultures were fed with the same
mixed fodder. In one of the previous works, cultures of
hepatocytes isolated from old rats injected with saline
were studied [2, 4].
The rhythm of protein synthesis that similarly to
other hourly rhythms deprived of an external pacemaker
is a fundamental feature of the cell [2]. Observations on
the living cell have indicated that hourly periods are per
sistently inherent in oscillations of different parameters
[2, 5]. However, the mean rhythm of a cell population is
another thing [2, 6]. Expression of such summarized
rhythm depends on synchronization of individual oscilla
tions. If the cells are fully asynchronous (are oscillating in
counterphase with equal probability), the mean rhythm is
not seen at all, and the kinetics are linear. In the case of
synchronization of cells, a rhythm can be observed: the
more coinciding are individual oscillations with each
other, the higher are amplitudes of the summarized
rhythm. In our case in old rats, small amplitudes of the
rhythm were observed, i.e. the cells had low synchroniza
tion and poor cell–cell communications.
Signaling factors of protein synthesis rhythm organ
ization are a persistent component of blood as of the
intercellular medium of mammals. Gangliosides are syn
thesized and secreted by any cell. Norepinephrine, sero
tonin, and dopamine of blood are also produced outside
the nervous system, in adrenals, enterochromaffin cells of
intestine, etc., and are released into the blood. Many
hepatocytes of a twoyearold rat can be halfyearold or
oneyearold, i.e. their age can be a half or a quarter of
the rat’s life. The synthesis and secretion by old cells of
gangliosides are low and their concentration in the serum
fell, as well as the concentration of other signaling factors,
such as norepinephrine, serotonin, and melatonin, which
are synthesized by other cells not belonging to the nerv
ous system [79]. However, such cells not belonging to the
nervous system, including hepatocytes, can perceive a
synchronizing signal, and just this has been shown for
glutamic acid in the present work and for gangliosides in

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960 BRODSKY et al.

a b
150

100

50
Icorr, %

c d
150

100

50
0 60 120 0 60 120

Time, min

Fig. 2. Comparison of protein synthesis kinetics in cultures of hepatocytes from young and old rats. a) Daily dense cultures from a young rat
prepared for the study of protein synthesis similarly to the experiment shown in Fig. 1a (many such examples are presented in works cited in
a review [2]); bd) similar cultures from an old rat: b) control similar to Fig. 1a; c) into the medium of such cultures, glutamic acid (0.2 mg/ml)
was added; d) into such cultures the inhibitor MCPG (0.01 mg/ml) of glutamic acid receptors was added, and after 180 min the cultures were
treated with glutamic acid (0.4 mg/ml) for 60 min. Ordinate axis, Icorr in percent of value for each variant of the experiment; the mean values
of Icorr for variants (a)(d) were taken for 100%, and the other values are considered as parts of this value. The absolute mean value of Icorr for
the young 180g rat was 415 ± 12 cpm (a); for control of the old 510g rat – 318 ± 14 cpm (b); for glutamic acid in the cultures from the same
rat – 299 ± 9 cpm (c); for MCPG together with glutamic acid for the 580g rat – 303 ± 7 cpm (d).

earlier works. Hepatocytes of old rats do not differ from
hepatocytes of young animals in the ability to interact
with receptors. As we have observed, for the real situation
in the body it is important that blood serum of an old rat
did not synchronize the rhythm, whereas blood serum of
a young rat synchronized it. Addition of gangliosides into
the serum of old rats compensated agerelated changes in
the intercellular medium and enhanced its synchronizing
function to the level of young rats [2].
Studies on effects of glutamic acid are especially
interesting because it is different from other known
organizers of proteins synthesis kinetics: glutamic acid is
an amino acid and a transmitter capable of penetrating
into the brain and influencing brain biochemistry, includ

BIOCHEMISTRY (Moscow) Vol. 82 No. 8 2017

 GLUTAMIC ACID AND AGING 961

a b
600
260
Icorr, cpm

400
160

60 200
0 60 120 0 60 120
Time, min

Fig. 3. Glutamic acid effect in vivo. Rats were fed with mixed fodder moistened with solution containing 62 mg glutamic acid. Hepatocytes
were isolated after 60 min, and dense cultures were obtained. After 24 h, the cultures were washed, the medium was refreshed, and the pro
tein synthesis kinetics were studied. a) 600g rat; b) 580g rat.

ing the agingaffected protein synthesis. Glutamic acid is
used in treatment of human nervous diseases, especially
in pediatrics: in Down’s syndrome or cerebral paralysis,
and sometimes in epilepsy and schizophrenia. Our data
on normalization of protein synthesis kinetics and direct
cell–cell communications on aging are leading to the rec
ommendation to prescribe glutamic acid also to improve
conditions of elderly persons.
Acknowledgments
The work was supported by the Russian Foundation
for Basic Research (project No. 170400460).
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