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doi:10.2141/ jpsa.0130162
Copyright Ⓒ 2014, Japan Poultry Science Association.
≪Research Note≫
Influence of Valine Analogues on Protein Synthesis of
Chicken Embryo Myoblasts
Kazumi Kita1, 2 and Ryosuke Makino2
1
Laboratory of Animal Nutrition, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan
The United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate 020-8550, Japan
2
The influence of various valine analogues on protein synthesis of chicken embryo myoblasts was examined.
Valine and its analogues (D-valine, methylvaline, valinol) were supplemented into Medium 199 containing 20 ng/ml
of chicken insulin-like growth factor-I (IGF-I) instead of fetal calf serum (FCS). Influence of branched chain amino
acids (isoleucine, leucine, valine) on myoblast protein synthesis was also examined. The levels of valine analogue
concentration were based on the valine concentration (213 μM) in Medium 199, and the concentrations of supplements were set at 213 μM (×1 of valine in Medium 199) and 2,130 μM (×10 of valine in Medium 199). Protein
synthesis was measured by incorporation of 3 H-phenylalanine. Methylvaline, which is one of valine derivatives
having a methylated amino group, and D-valine, which is an optical isomer of L-valine, had no influence on myoblast
protein synthesis although the concentrations of these compounds were 10-times higher (2,130 μM) than that of valine
in Medium 199. Leucine and valine had the potency to increase protein synthesis of chicken embryo myoblasts. The
supplementation of valinol, which is one of valine derivatives having a hydrated carboxyl group, with 10-times higher
concentration than that of valine in Medium 199, decreased myoblast protein synthesis. These results suggested that
valinol could be used as an inhibitor of myoblast protein synthesis.
Key words: analogues, branched chain amino acids, myoblast, protein synthesis, valine, valinol
J. Poult. Sci., 51: 191-194, 2014
Introduction
Branched-chain amino acids, particularly leucine, have an
anabolic effect on protein metabolism by increasing the rate
of protein synthesis (Alvestrand et al., 1990; Nair et al.,
1992; Blomstrand et al., 1997). Leucine serves as a substrate to directly activate mammalian target of rapamycin
complex 1 (mTORC1) signaling pathway (Dodd and Tee,
2012). The mTORC1 increases translational initiation processes (Anthony et al., 2000) including the rapid phosphorylation of the eukaryotic translation initiation factor 4E‒binding protein 1 and the serine/threonine protein kinase p70S6
kinase 1 (Kimball and Jefferson, 2006).
In chickens, we have already reported that leucine could
stimulate protein synthesis of chicken embryo myoblasts
(Oki et al., 2007). Compared to leucine, the information
about the influence of valine, which is also one of branched
amino acids, on protein synthesis of chicken embryo myoblasts has been limited. Especially, it seems to be scarcely to
Received: August 27, 2013, Accepted: September 30, 2013
Released Online Advance Publication: November 25, 2013
Correspondence: K. Kita. Laboratory of Animal Nutrition, Faculty of
Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan.
(E-mail: [email protected])
examine the influence of valine deficiency on muscle protein
synthesis in vivo and in ovo because valine can be provided
by protein degradation from body proteins and egg proteins.
In this case, the use of valine analogues would be available to
examine the effect of valine on muscle protein synthesis
because these chemical compounds would have the potency
to block valine incorporation into cytoplasm of cells. The
aim of this study, therefore, was to investigate the influence
of various valine analogues on protein synthesis of chicken
embryo myoblasts.
Materials and Methods
Two experiments were conducted. In Experiment 1, the
influence of valine and various valine analogues (D-valine,
methylvaline or valinol) on protein synthesis of chicken embryo myoblasts was examined. Ten fertilized eggs of Single
Comb White Leghorn chickens were purchased from local
hatchery (Koiwai Farm Co., Ltd, Shizukuishi, Iwate, Japan)
and incubated for 19 days. At this time, embryos were taken
from eggs, rinsed in Dulbecco’s phosphate buffered saline
(DPBS). After decapitated, large breast muscles (M. pectoralis major) were removed and minced finely with scissors.
The minced tissues were mixed with 50 ml of DPBS including 0.25% (w/v) trypsin and then incubated at 37℃ for
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Journal of Poultry Science, 51 (2)
45 min. After centrifugation (5,000×g, 3 min, 4℃), the supernatant was discarded and the pellet was mixed with 15 ml of
Medium 199 including 2.5 μg/ml Fungizone, 100 units penicillin, 100 μg/ml streptomycin, 50 μg/ml gentamycin and
10% FCS. Minced muscles were pipetted several times and
filtrated by passing through a piece of gauze to remove undigested tissues. The cells were settled down by centrifuge
for 5 min at 5,000×g at 4℃, and then the supernatant was
removed. After rinsing cells pellet, the cells were resuspended in 20 ml of Medium 199 with 10% FCS, and then
seeded in a Type-1 collagen-coated 48-well plate (Becton
Dickinson Laboware, Belford, MA, USA) at the cell density
2.0×106 cells/cm3 and incubated at 37℃ in 5%CO2/95%air
(v/v). All reagents except for penicillin-streptomycin to prepare chicken embryo myoblasts were purchased from Gibco
Laboratories Life Technologies Inc. (NY, USA). Penicillinstreptomycin reagent was purchased from Biological Industries Ltd. (Kibbutz Beit Haemek, Israel).
To investigate the influence of valine and its analogues (Dvaline, methylvaline, valinol) on myoblast protein synthesis,
these compounds were supplemented into Medium199 containing 20 ng/ml of chicken IGF-I instead of FCS. The levels
of valine analogue concentration were based on the valine
concentration (213 μM) in Medium 199, and the concentrations of supplements were set at 2,130 μM (×10 of valine in
Medium 199). And then, L-[2,6-3H] phenylalanine (Amersham
Life Science, Ltd., Tokyo, Japan) was added into the medium, in which the radioactivity was 37 kBq/ml. After further 1 day of incubation, the medium was drawn away and
cells were rinsed with ice-cold Medium 199. The intracellular free amino acids were removed by rinsing with ice-cold
5% (w/v) trichloroacetic acid (TCA). After discarding TCA,
cells were rinsed with ice-cold DPBS, and the supernatant
was removed. Then, 400 μl of 0.5 M NaOH/0.1% (v/v) Triton
X-100 was added into the well and incubated at room temperature for 30 min. After dissolving protein by pipetting,
the radioactivity in NaOH/Triton X-100 solution was measured using a scintillation counter as an index of protein synthesis.
In Experiment 2, the influence of various valine analogues
and branched chain amino acids (isoleucine, leucine, valine)
was also examined. In this experiment, the influence of various valine analogues and branched chain amino acids (isoleucine, leucine, valine) was examined. The concentrations
of valine analogues were set at 213 μM (×1 of valine in
Medium 199) and 2,130 μM (×10 of valine in Medium 199).
The concentrations of branched amino acids were based on
the concentration of each amino acid in Medium 199. The
concentrations of isoleucine were set at 305 μM (×1 of
isoleucine in Medium 199) and 3,050 μM (×10 of isoleucine
in Medium 199). The concentrations of leucine were set at
457 μM (×1 of leucine in Medium 199) and 4,570 μM (×10
of leucine in Medium 199). The concentrations of valine
were set at 213 μM (×1 of valine in Medium 199) and 2,130
μM (×10 of valine in Medium 199). Preparation of chicken
embryo myoblasts and the measurement of protein synthesis
were described in Experiment 1.
Influence of various valine analogues supplemented into culture medium on protein synthesis of
chicken embryo myoblasts. The number of wells in each
treatment was twelve. Bars presented means±SEM. Means
with asterisks are significantly different from the control
(None) at P＜0.05. MeVal: methylvaline.
Fig. 1.
Animal care was in compliance with applicable guidelines
from the Iwate University Policy on Animal Care and Use.
Statistical analysis of data was performed by one-way
ANOVA using the General Linear Model Procedures of SAS
(SAS/STAT version 6) (SAS Institute, 1999). After ANOVA,
a multiple comparison procedure (Dunnett’s test) was performed to compare each treatment and control. Differences
between means were considered to be significant at P＜0.05.
Results
In Experiment 1, methylvaline, which is one of valine
derivatives having a methylated amino group, and D-valine,
which is an optical isomer of L-valine, had no influence on
myoblast protein synthesis although the concentrations of
these compounds were 10-times higher (2,130 μM) than that
of valine in Medium 199 (Fig. 1). The supplementation of
valinol, which is one of valine derivatives having a hydrated
carboxyl group, decreased myoblast protein synthesis (Fig.
1).
In Experiment 2, methylvaline and D-valine had no influence on myoblast protein synthesis like Experiment 1.
Leucine and valine had the potency to increase protein synthesis of chicken embryo myoblasts. The supplementation of
valinol with 10-times higher concentration than that of valine
in Medium 199 dramatically decreased myoblast protein synthesis similarly to Experiment 1 (Fig. 2).
Discussion
Since the establishment of primary cell culture system, it
has been well recognized that FSC was required to grow and
maintain animal cells. In avian species, FCS was also used
for normal growth of fibroblasts and myoblasts derived from
chicken embryos (Kita et al., 1996; Kita and Okumura,
2001). As we have previously reported that chicken IGF-I
Kita and Makino: Valine Analogues and Myoblast Protein Synthesis
193
Influence of various valine analogues and branched chain
amino acids supplemented into culture medium on protein synthesis
of chicken embryo myoblasts. The number of wells in each treatment
was six. One missing value in the ×1 D-valine treatment. Bars presented means±SEM. Means with asterisks are significantly different
from the control (None) at P＜0.05. MeVal: methylvaline.
Fig. 2.
could be used for healthy growth of chicken embryo myoblasts (Kita and Okumura, 2001), in the present study,
chicken IGF-I was supplemented into Medium 199 instead of
FCS to avoid the influence of valine in FCS.
As shown in Figs. 1 and 2, valinol with 10-times higher
concentration than that of valine in Medium 199 successfully
suppressed protein synthesis of chicken embryo myoblasts.
It has been widely established that free amino acids in body
fluid and gastrointestinal trust are incorporated into cytoplasm via various amino acid transporters called by another
term, System. Recently, Bröer (2008) summarized the physiological characteristics of amino acid transporters. Phenylalanine is incorporated by System L (LAT1, SLC3A2/SLC
7A5; LAT2, SLC3A2/SLC7A8; LAT3, SLC43A1; LAT4,
SLC43A2), System T (TAT1, SLC16A10), System B0 (B0
AT1, SLCC6A19) and System B0,+ (ATB0,+, SLC6A14).
Valine is incorporated by System L (LAT1, LAT2), System
B0 (B0AT1; B0AT2, SLC6A15) and System B0,+. In this experiment, radioactive phenylalanine was used to determine
protein synthesis, which suggested that the suppression of
myoblast protein synthesis by valinol would be resulted by
the decrease in phenylalanine and/or valine incorporated into
cytoplasm of myoblasts. Although Babu et al. (2003) reported that valinol could inhibit the amino acid incorporation
by LAT3, which is one of System L amino acid transporters,
the inhibitory effect of valinol on amino acid transporters
except for LAT3 has not been clarified. Therefore, we need
to investigate the influence of valinol on amino acid incorporation by System L amino acid transporters in the future.
In conclusion, valinol, which is one of valine analogue
having a hydrated carboxy group, could be used as an inhibitor of myoblast protein synthesis associated with an inhibition of amino acid incorporation into cytoplasm.
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