Reduction in dietary lysine increases muscle free amino acids

through changes in protein metabolism in chickens

Genya Watanabe,*,y Hiroyuki Kobayashi,* Masahiro Shibata,z Masatoshi Kubota,x Motoni Kadowaki,*,x
and Shinobu Fujimura*,x,1

*Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan; yNational Agriculture and
Food Research Organization for Institute of Livestock and Grassland Science, Ibaraki 305-0901, Japan; zFaculty of
Applied Life Science, Nippon Veterinary and Life Science University, Tokyo 180-8602, Japan; and xCenter for
Transdisciplinary Research, Niigata University, Niigata 950-2181, Japan

ABSTRACT Taste is crucial to meat quality, and compared with the Lys 100% group (P , 0.01). Free
free Glu is an important taste-active component in threonine, glutamine, glycine, valine, isoleucine, leucine,
meat. Our recent study showed that the short-term tyrosine, phenylalanine, histidine, and 3-methyl-histi-
feeding of a low-Lys diet increases the concentration dine concentrations in breast muscle were also increased
of free Glu and other free amino acids in chicken muscle in the Lys 90% group (P , 0.05). Metabolome analysis
and improves its taste. Here, we investigated the also showed that free amino acids were increased in the
mechanisms by which the feeding of a low-Lys diet in- Lys 90% group. The mRNA abundance of m-calpain,
creases free Glu in chicken muscle. Two groups (n 5 10 caspase-3, and 20S proteasome C2 subunit were
per group) of 28-day-old female Ross strain broiler increased in the Lys 90% group (P , 0.05). Moreover,
chickens were fed diets with a graded Lys content of the free Glu concentration in muscle was correlated
90% or 100% of the recommended Lys requirement with mRNA abundance of m-calpain (r 5 0.74,
(according to National Research Council [1994] guide- P , 0.01), caspase 3 (r 5 0.69, P , 0.01), 20S pro-
lines) for 10 D. Free amino acid concentrations and the teasome C2 subunit (r 5 0.65, P , 0.01), and cathepsin
mRNA abundance of protein metabolism–related genes B (r 5 0.52, P , 0.05). Our study suggests that the
were measured in breast muscle, and breast muscle feeding of a low-Lys diet to chickens increased the free
metabolome analysis was conducted. Free Glu in mus- Glu content of breast muscle by promoting protein
cle was increased by 51.8% in the Lys 90% group degradation.
Key words: broiler, lysine, glutamate, free amino acid, protein metabolism
2019 Poultry Science -:-–-
https://doi.org/10.1016/j.psj.2019.11.025

INTRODUCTION been shown to increase final pH and decrease drip loss

of broiler breast meat (Berri et al., 2008), and reducing
The essential amino acid Lys is often the limiting die- dietary Lys has been shown to increase intramuscular
tary amino acid in farm animals that consume predomi- fat in the longissimus dorsi muscles of finishing gilts
nantly cereal grain–based diets (National Research (Katsumata et al., 2005). Conversely, there have been
Council, 1989). Accordingly, most studies of the effects few reports on the relationship between dietary Lys
of dietary Lys on farm animals have focused on feed effi- and the meat taste.
ciency, growth performance, and meat yield (Kendall In-mouth perception of taste is related to consumer
et al., 2008; Dozier et al., 2008; Cemin et al., 2017). satisfaction of meat (Resano et al., 2011). Various com-
However, there are also some reports on the effect of pounds contribute to meat flavor, including Glu, an
dietary Lys on meat quality. Increasing dietary Lys has important taste-active component in meat (Kato and
Nishimura, 1987; Maga, 1994). Of the 5 basic tastes,
Glu is “umami”, which has also been described as
Ó 2019 Published by Elsevier Inc. on behalf of Poultry Science “savory,” “beefy,” and “brothy” (Maga, 1994). Halpern
Association Inc. This is an open access article under the CC BY-NC-ND (2000) reported that appropriate concentrations of Glu
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Received July 27, 2018.
increase food palatability. For instance, the addition of
Accepted November 21, 2019. Glu to chicken soup increased its intensity of umami,
1
Corresponding author: [email protected] sweetness, and saltiness (Fujimura et al., 1996); thus,

1
2 WATANABE ET AL.

an increase in the free Glu content of chicken may improve chicks were housed in a brooder from day 0 to day 14,
its taste. However, dietary Glu is metabolized by intesti- where they were kept warm, and in battery cages from
nal cells during absorption, with only 5% of the Glu day 15 to day 28. The room temperature was kept at
appearing in the portal blood (Reeds et al., 1996). There- 22 6 2 C, and lighting was kept on a regular schedule
fore, it is difficult to increase the free-Glu content of mus- (light for 15 h, 04:00 h to 19:00 h). All chicks were
cle by the feeding of Glu-rich feeds; instead, an indirect allowed free access to feed and water. Chicks were raised
method is required. We recently found that the free-Glu on a commercial diet containing 200 g/kg crude protein
concentration in chicken muscle can be increased by the (CP) and 3.2 kcal/g metabolizable energy (ME). On day
feeding of a high- or low-Lys diet (Watanabe et al., 28, chickens were allocated to 2 groups, (n 5 10 per
2015; 2017). These were the first reports on the group) ensuring that the average weight of animals
relationship between dietary Lys and meat taste. was the same between groups. During the experimental
In the feeding of a high-Lys diet, the Lys degradation period, chickens were individually housed in cages. The
pathway contributed to the free-Glu production. Lys is study was carried out in accordance with the recommen-
mainly degraded by the saccharopine pathway in chickens dations in the Guide for the Care and Use of Laboratory
(Grove and Roghair, 1971; Wang and Nesheim, 1972), and Animals of the Science Council of Japan. All the animal
lysine a-ketoglutarate reductase (EC 1.5.1.8) is the experiments were conducted in compliance with the pro-
primary enzyme of this pathway. Saccharopine and tocol, which was reviewed by the Institutional Animal
a-aminoadipic acid are the intermediate metabolites of Care and Use Committee and approved by the President
the saccharopine pathway, and 1 mol of Glu is released of Niigata University (permit number: #26 Niigata
when 1 mol of these intermediates is metabolized. Our Univ. Res. 80-5).
recent study showed a significant increase in lysine
a-ketoglutarate reductase mRNA abundance in chicken Experimental Diets
muscle from the feeding of a high-Lys diet, and muscular
saccharopine and a-aminoadipic acid were also increased The composition of the 2 experimental diets is shown
(Watanabe et al., 2015). These results suggest that the in Table 1. The diets contained 100 or 90% of the Lys
Lys degradation pathways in muscle were involved in requirement, where 100% of the Lys requirement is
the increase in free Glu in muscles. The feeding of a low- 10.0 g/kg of the diet according to NRC (1994) guide-
Lys diet to broilers also increased the free-Glu concentra- lines. Both diets contained 3.2 kcal/g ME and 170 g/
tion in muscles (Watanabe et al., 2017). Although the kg CP. Dietary amino acid, ME, vitamins, and mineral
mechanism is yet to be determined, this could be explained contents met the NRC requirements. Chickens in each
by a change in protein metabolism. Proteins are synthe- group were fed their allocated diet for 10 consecutive
sized using free amino acids, and free amino acids are sup- days.
plied by food and the degradation of preexisting proteins.
Thus, a change in protein metabolism might affect the free Sample Collection
amino acid concentrations in muscles. For instance,
insulin-like growth factor (IGF) 1, which is a stimulator At the end of the trial, feed intake was recorded for each
of protein synthesis, was significantly decreased in plasma individual, and the chickens were weighed. At 13:30 h,
of pigs fed a low-Lys diet (Katsumata et al., 2002). Also, blood samples were taken from the wing vein, and
we have previously shown that 3-methyl-histidine (3M- chickens were killed by cutting the carotid arteries. Blood
His), which is an indicator of skeletal muscle protein degra- samples were collected in syringes treated with heparin
dation, is significantly increased in chicken breast muscle solution (100 unit/mL), deproteinized with 3% sulfosali-
by the feeding of a low-Lys diet (Watanabe et al., 2017). cylic acid, and the plasma stored at 220 C until determi-
Therefore, a change in protein metabolism by low Lys nation of amino acid concentrations. Breast muscle was
feeding might induce an increase in muscle free amino dissected to remove the epimysium, fat, vessels, and con-
acids including Glu. nective tissues and cut into small pieces. Muscle samples
In the present study, the effect of a low-Lys diet on the were frozen using liquid nitrogen and kept at 280 C until
concentration of free Glu and other free amino acids in free amino acid measurements and real-time reverse tran-
muscles was examined using broiler chickens. We also scriptase quantitative PCR (RT-qPCR) were performed.
determined small-molecule metabolites. Finally, we inves-
tigated the effect of a low-Lys diet on the mRNA abun- Preparation of Muscle Extract
dance of protein metabolism–related genes and their
correlation with free Glu content to clarify mechanisms Muscles were homogenized separately in 10%
of Glu regulation. perchloric acid using a high-speed homogenizer (Ultra
Turrax T25 basic; IKA Werke, Staufen, Germany).
The homogenate was centrifuged at 600 ! g and kept
MATERIALS AND METHODS
at 4 C for 20 min, and then the supernatant was neutral-
Animals ized with 10% (w/v) potassium hydrate. After removal of
the potassium crystals by filtration, the filtrate volume
Ross strain female chicks were purchased at birth from was adjusted to 50 mL using double-distilled water, and
a commercial hatchery (Onuma, Shibata, Japan). All the samples were stored at 220 C until analysis.
 LOW-LYSINE DIET INCREASES MUSCLE AMINO ACIDS 3
Table 1. Composition of the experimental diets. (5-kDa cutoff filter; Ultrafree MC; Millipore, Darmstadt,
Dietary Lys level (% of requirement)1 Germany). Samples were then completely evaporated,
and the residue dissolved in 50 mL of double-distilled wa-
Ingredients (% of diet) 100 90
ter. The prepared samples were analyzed using an Agilent
Corn grain 74.00 73.50 CE-TOFMS system (Agilent Technologies, Waldbronn,
Soybean meal 15.46 16.01
Fish white 5.00 5.00
Germany) as described by Matsumoto et al., 2012. The
Soybean oil 2.59 2.73 extracted cationic and anionic metabolites were analyzed
CaCO3 0.43 0.43 using a fused silica capillary (50 mm in diameter ! 80 cm)
CaHPO4 1.36 1.35
NaCl 0.15 0.15
with a commercial electrophoresis buffer (cation buffer so-
Vitamin and mineral 0.50 0.50 lution, H3301-1001; anion buffer solution; H3302-1021;
premixture2 Human Metabolome Technologies Inc.). CE-TOFMS
Lys-HCl 0.13 0.01
Arg 0.11 0.09
data processing was conducted, and peak information
Met 0.12 0.12 extracted using the automatic integration software Mas-
Thr 0.12 0.11 terHands (Keio University, Tsuruoka, Japan), including
Trp 0.01
Ile 0.01
ion mass/charge number (m/z), migration time for CE-
Val 0.01 TOFMS measurement (MT), and peak area (Sugimoto
Phe et al., 2009). The mass spectrometry scan range was 50
Total 100.00 100.00
Calculate analysis
to 1,000 (m/z). Detected metabolites were identified based
CP (%) 17.00 17.00 on m/z and their MT using the metabolite library main-
ME (kcal/g) 3.20 3.20 tained by Human Metabolome Technologies Inc. The rela-
Essential amino acids (%)
Lys 1.00 0.90
tive area was calculated as follows: relative area 5 peak
Arg 1.10 1.10 area/(peak area of internal control ! sample volume).
His 0.41 0.42 To calculate the fold-change between groups, the relative
Ile 0.73 0.73
Leu 1.50 1.52
area of the Lys 90% group was divided by the relative area
Met 0.38 0.38 of the Lys 100% group.
Phe 0.75 0.76
Thr 0.74 0.74
Trp 0.18 0.18 RNA Isolation and Real-Time RT-qPCR
Val 0.82 0.82 Analysis
Abbreviations: CP, crude protein; ME, metabolizable energy.
1
100% Lys requirement is 10.0 g/kg of diet (NRC, 1994). Total RNA was isolated from muscles using TRIzol re-
2
Content per kg of vitamin and mineral premixture: vitamin A 300,000 agent (Invitrogen, Carlsbad, CA) according to the man-
IU (retinyl acetate), vitamin D3 40,000 IU, vitamin E 2,000 IU (DL-alpha- ufacturer’s protocol. RNA yield and quality were
tocopheryl acetate), vitamin K3 192 mg, thiamin nitrate 360 mg, riboflavin
720 mg, calcium d-pantothenate 2,175 mg, nicotinamide 6,943 mg, pyri- determined spectrophotometrically by the absorbance
doxine hydrochloride 700 mg, biotin 30 mg, folic acid 110 mg, cyanoco- at the wavelengths of 260 and 280 nm, using a Nanodrop
balamin 2 mg, calcium iodinate 108 mg, MgO 198,949 mg, MnSO4 spectrophotometer (Thermo Fisher Scientific, Waltham,
32,982 mg, ZnSO4 19,755 mg, FeSO4 43,521 mg, CuSO4 4,019 mg, and
choline chloride 299,816 mg. MA). cDNA synthesis was performed as described by
Shibata et al. (2006). Briefly, first-strand cDNA was syn-
Measurement of Free Amino Acids thesized from 3 mg of total RNA using SuperScript II
RNase H2 reverse transcriptase (Invitrogen) with
The concentrations of free amino acids in plasma and
oligo-dT primers (Invitrogen). After reverse transcrip-
muscles were measured using an amino acid analyzer
tion, analysis of cDNA abundance of IGF-1, IGF-2, myo-
(JLC-500/V; JEOL, Tokyo, Japan). A multisegment
statin, m-calpain, m-calpain large subunit, caspase 3,
tandem packed column (LC-500AC4016, Li type, 4 mm
cathepsin B, 20S proteasome C1 subunit, and 20S pro-
diameter ! 160 mm; JEOL) was used. The detection
teasome C2 subunit was performed by real-time RT-
wavelengths were 440 and 570 nm. Amino acids were
qPCR using LightCycler FastStart DNA MasterPLUS
detected using the ninhydrin method.
SYBR Green I (Roche Diagnostics, Basel, Switzerland)
and a LightCycler 1.5 instrument (Roche Diagnostics).
Metabolome Analysis Specific product amplification was checked by melting
curve analysis. The primers for each gene were designed
To screen the specific metabolism affected by reduced based on sequences reported in the study by Ibuki et al.
dietary Lys, the breast muscle metabolome was measured (2013; 2014) and Watanabe et al. (2015). The primer
using a combined capillary electrophoresis and time-of- sets used for PCR are shown in Table 2. The house-
flight mass spectrometry (CE-TOFMS) system (Human keeping gene Gapdh was used as a normalizing control.
Metabolome Technologies Inc., Tsuruoka, Japan). Sam- All data were analyzed using LightCycler Software
ples in each group were pooled (n 5 1), with each pooled version 3.5 (Roche Diagnostics).
sample weighing approximately 50 mg. The pooled sam-
ples were homogenized in 1,800 mL of 50% (v/v) acetoni- Statistical Analysis
trile solution (1,500 rpm ! 120 s ! 5 times),
centrifuged at 2,300 ! g for 5 min at 4 C, and the aqueous Data were analyzed using the SAS (version 9.4; SAS
phase was passed through an ultrafiltration membrane Institute, Cary, NC). Student t tests were used for growth
4 WATANABE ET AL.

Table 2. Primers used in this study.1

Gene Primer sequences Accession number
IGF-1 Sense 50 -GCTGCCGGCCCAGAA-30 NM_001004384
Antisense 50 -ACGAACTGAAGAGCATCAACCA-30
IGF-2 Sense 50 -CCTGGCTCTGCTGGAAACC-30 NM_001030342
Antisense 50 -GAGAGGTCACGCTCTGACTTGA-30
Myostatin Sense 50 -ATGCAGATCGCGGTTGATC-30 NM_001001461
Antisense 50 -GCGTTCTCTGTGGGCTGACT-30
m-calpain Sense 50 -CACACAAGGAGGCCGACTTC-30 NM_205303
Antisense 50 -TCCGCTGTGTCTGACTGCTT-30
20S proteasome C1 subunit Sense 50 -TGAGGAACAAGGAGCCCATCT-30 AB_001935
Antisense 50 -TGCCCTTGTACTGATACACCAT-
GT-30
20S proteasome C2 subunit Sense 50 -CCAGTATCTCGTTTGGTGTCAC- AF_027978
TAC-30
Antisense 50 -CATAGCGCTGCGTTGGTATC-30
m-Calpain large subunit Sense 50 -GTGGCTCGGTTTGCTGATG-30 D_38026
Antisense 50 -AATCAAGCACCGGACACAATT-30
Caspase 3 Sense 50 -GGAACACGCCAGGAAACTTG-30 AF_083029
Antisense 50 -TCTGCCACTCTGCGATTTACA-30
Cathepsin B Sense 50 -GCTACTCGCCTTCCTACAAGGA-30 U_18083
Antisense 50 -GCGAGGGACACCGTAGGAT-30
GAPDH Sense 50 -GTGGTGGCCATCAATGATCCC-30 M_11213
Antisense 50 -TGTTGCTGGGGTCACGCTCC-30

Abbreviations: GAPDH, glyceraldehydes-3-phospate dehydrogenase; IGF, insulin-like growth factor.

1
The primers were designed based on sequence given in the study by Ibuki et al. (2013; 2014). All primers are shown
5 to 30 .
0

performance, free amino acid concentrations, and mRNA compared with those fed the Lys 100% diet. Feed intake,
abundance. Data are presented as means 6 SEM. Student body weight gain, and feed efficiency were not signifi-
t tests were demonstrated by the TTEST procedures in cantly different between the groups.
SAS. The Pearson correlation coefficient was calculated
to determine the relationship between free-Glu concentra- Free Amino Acids in Plasma and Muscle
tion and the mRNA abundance of protein metabolism–
related genes in muscles. Pearson correlation coefficient The free amino acid concentrations in plasma are
was analyzed using the TTEST procedures in SAS. Differ- shown in Table 4. The concentration of plasma Lys
ences and correlations of P , 0.05 were considered (P , 0.01) and Met (P , 0.05) was decreased in the
significant. Lys 90% group, whereas that of plasma His (P , 0.01)
was increased in the Lys 90% group (P , 0.01) compared
with the Lys 100% group. There were no significant dif-
ferences between groups for other plasma free amino
RESULTS
acids.
Growth Performance The free amino acid concentrations in muscles are
shown in Table 5. The muscle free Lys concentration
The growth performance of chickens fed the Lys 90% was decreased in the Lys 90% group (P , 0.01)
diet for 10 D is shown in Table 3. Total Lys intake was compared with that in the Lys 100% group. The concen-
decreased in chickens fed the Lys 90% diet (P , 0.05) tration of muscle free Glu was increased in the Lys 90%

Table 3. Effect of dietary lysine level on the growth performance of broilers.1

Dietary Lys level (% of requirement)
Growth performance and tissue weight 100 90 Significance
Initial body weight (g) 1,109.0 631.9 1,107.6 630.2 NS
Body weight gain (g/10 D) 811.5 633.8 812.5 625.6 NS
Feed intake (g/10 D) 1,467.1 650.8 1,466.0 640.3 NS
Feed efficiency (g body weight gain/g 0.552 60.008 0.554 60.006 NS
intake)
Lys intake (g/10 D) 14.7 60.5 13.2 60.4 *
Breast muscle: body weight (g/kg BW) 143.5 63.3 140.2 62.1 NS
Abdominal fat: body weight (g/kg BW) 17.2 61.8 16.3 61.2 NS
Liver: body weight (g/kg BW) 25.5 61.1 25.9 60.9 NS

*P , 0.05.
Abbreviations: BW, body weight; NS, not significant.
1
Values of Lys 100% and Lys 90% groups are means 6 SEM for 10 chickens.
 LOW-LYSINE DIET INCREASES MUSCLE AMINO ACIDS 5
Table 4. Effect of dietary lysine level on free amino acid concen- Table 5. Effect of dietary lysine level on free amino acid concen-
trations in plasma of broilers (nmol/mL plasma).1 trations in muscle of broilers (mg/g muscle).1
Dietary Lys level (% of requirement) Dietary Lys level (% of requirement)
Amino acids 100 90 Significance Amino acids 100 90 Significance
Tau 599.7 6 86.4 584.4 6 61.8 NS Tau 259.8 6 64.2 193.1 6 24.9 NS
Asp 56.1 6 2.0 54.5 6 5.6 NS Asp 66.2 6 5.6 62.4 6 4.4 NS
Thr 1,076.8 6 51.8 1,076.9 6 48.8 NS Thr 132.0 6 6.0 198.0 6 17.5 **
Ser 1,131.3 6 69.1 981.1 6 70.0 NS Ser 162.9 6 10.7 189.0 6 16.1 NS
Glu 508.3 6 22.1 447.9 6 38.2 NS Glu 136.4 6 9.1 207.0 6 9.7 **
Gln 836.8 6 43.7 746.4 6 57.9 NS Gln 268.3 6 23.1 363.0 6 32.5 *
Gly 887.6 6 74.0 871.1 6 52.3 NS Gly 205.5 6 25.6 472.1 6 63.6 **
Ala 1,264.5 6 115.8 1,135.7 6 96.2 NS Ala 194.7 6 10.9 237.4 6 23.4 NS
Val 208.1 6 12.8 215.3 6 10.3 NS Val 11.2 6 0.8 23.9 6 1.9 **
Met 99.6 6 5.6 82.4 6 3.4 * Met 8.9 6 1.0 10.7 6 1.4 NS
Ile 103.8 6 6.9 103.4 6 5.7 NS Ile 6.3 6 0.9 11.7 6 0.7 **
Leu 302.9 6 12.2 303.0 6 16.2 NS Leu 20.2 6 1.2 31.7 6 2.1 **
Tyr 286.0 6 24.8 251.2 6 22.2 NS Tyr 55.4 6 5.2 75.4 6 5.1 **
Phe 145.3 6 7.2 150.5 6 7.2 NS Phe 18.3 6 1.6 29.7 6 2.6 **
His 146.8 6 14.3 204.2 6 15.2 ** His 2.2 6 0.4 12.0 6 2.5 **
Lys 186.6 6 13.8 102.7 6 4.9 ** Lys 10.1 6 0.5 6.2 6 0.9 **
Arg 639.2 6 42.2 549.2 6 37.7 NS Arg 92.3 6 9.0 113.2 6 9.1 NS
3M-His 22.6 6 1.7 26.1 6 1.2 NS 3M-His 4.1 6 0.5 6.0 6 0.5 *

**P , 0.01; *P , 0.05. **P , 0.01; *P , 0.05.

Abbreviation: NS, not significant. Abbreviation: NS, not significant.
1
Values of Lys 100% and Lys 90% groups are means 6 SEM for 10 1
Values of Lys 100% and Lys 90% groups are means 6 SEM for 10
chickens. chickens.

group (P , 0.01) by a factor of 51.8%. Muscle free Thr, Pearson Correlation Between Free Glu and
Gly, Val, Ile, Leu, Tyr, Phe, His, Gln, and 3M-His con-
centrations were also increased in chickens fed the Lys
mRNA Abundance
90% diet (P , 0.05). Other muscle free amino acids Results for the Pearson correlation between free Glu
showed no significant differences between groups. concentration and mRNA abundance of protein
metabolism–related genes in muscles are shown in
Figure 1. The muscle free Glu concentration was corre-
Metabolome Analysis lated with the mRNA abundance of m-calpain
(r 5 0.74, P , 0.01), caspase 3 (r 5 0.69, P , 0.01),
A total of 133 metabolites were identified in the breast 20S proteasome C2 subunit (r 5 0.65, P , 0.01), and
muscle. We divided these into 3 groups on the basis of the cathepsin B (r 5 0.52, P , 0.05). mRNA abundance of
fold-change in the Lys 90% group compared with the Lys other protein metabolism–related genes was not signifi-
100% group, as follows: upregulated (.1.5-fold), downre- cantly correlated with muscle free Glu concentration
gulated (,0.67-fold), and unchanged (0.67 to 1.5-fold). (data not shown).
Using these criteria, 22 metabolites were upregulated
and 24 were downregulated in the Lys 90% group, as
shown in Table 6. The upregulated metabolites included DISCUSSION
amino acids and metabolic intermediates of amino acids,
metabolites of glycolysis and the citric acid cycle, and Glu is a taste-active component of meat, contributing a
nucleoside triphosphate. The downregulated metabolites “umami” flavor (Kato and Nishimura, 1987; Maga, 1994).
included Lys-related metabolites, such as b-Ala-Lys, sac- An increase in the free Glu content of meat enhances taste
charopine, N6-acetyllysine, cadaverine, pipecolic acid, (Imanari et al., 2008; Watanabe et al., 2017) and may
and nucleoside monophosphate. thereby improve meat taste. However, dietary Glu does
not proportionally affect portal blood Glu because the
majority of dietary Glu is metabolized by intestinal
mRNA Abundance of Protein Metabolism– cells during absorption in pigs (Reeds et al., 1996). Glu
Related Genes in Muscles metabolism in chicken intestinal cells is slightly different
from mammals because of the deficiency in ornithine
Results for mRNA abundance of protein metabolism– aminotransferase (Wu et al., 1995). However, Glu is the
related genes are shown in Table 7. All data are preferred fuel for respiration in chicken intestinal cells
expressed as a percentage of the Lys 100% group. (Watford et al., 1979), and Glu supplementation in the
mRNA abundance of m-calpain, caspase 3, and 20S pro- diet did not increase the free Glu concentration in the
teasome C2 subunit was significantly increased in the plasma (Maruyama et al., 1976). Therefore, it is difficult
Lys 90% group compared with the Lys 100% group to increase the free Glu content of muscle by feeding with
(P , 0.01 except m-calpain, P , 0.05). mRNA abun- Glu-rich feeds. We have previously reported that the
dance of other protein metabolism–related genes showed feeding of a high Val, Ile, or CP diet increases the free
no significant differences between groups. Glu content in breast muscle of broilers (Imanari et al.,
6 WATANABE ET AL.

Table 6. Effect of dietary lysine level on metabolites in breast muscle in broilers.1

Relative area
2
Compound name m/z MT Lys 100% Lys 90% Fold-change (vs. Lys 100%)
Upregulated
UTP 482.96 12.48 ND3 2.4E-03 ,1
Diethanolamine 106.09 7.23 ND 4.5E-04 ,1
ATP 505.99 11.66 6.9E-03 4.6E-02 6.58
His 156.08 6.92 6.1E-03 2.0E-02 3.31
3-Methylhistidine 170.09 7.09 6.7E-03 2.1E-02 3.14
GTP 521.99 11.36 5.5E-04 1.2E-03 2.25
50 -Deoxy-50 -methylthioadenosine 298.09 9.36 6.6E-05 1.4E-04 2.05
S-Adenosylmethionine 399.14 6.75 2.3E-03 4.7E-03 2.00
Cystathionine 223.07 9.21 5.5E-04 1.1E-03 1.93
Imidazolelactic acid 157.06 8.20 1.8E-04 3.3E-04 1.83
Gly 76.04 7.83 3.0E-01 5.4E-01 1.82
ADP 426.02 10.86 1.2E-02 2.1E-02 1.76
Ile 132.10 9.47 1.9E-02 3.3E-02 1.74
Sarcosine 90.05 8.84 7.5E-03 1.3E-02 1.70
Succinic acid 117.02 20.97 4.3E-03 7.0E-03 1.60
Glucose 1-phosphate 259.02 10.05 1.7E-02 2.7E-02 1.60
Homoserinelactone 102.06 6.75 4.6E-04 7.2E-04 1.57
Glucose 6-phosphate 259.02 9.78 3.7E-01 5.7E-01 1.55
Val 118.09 9.31 3.5E-02 5.4E-02 1.55
Arg 175.12 6.75 8.7E-02 1.3E-01 1.53
Citric acid 191.02 26.40 2.9E-03 4.5E-03 1.53
Histamine 112.09 4.60 1.4E-03 2.1E-03 1.50
Downregulated
Urea 61.04 18.08 2.0E-02 ND 1,
Homoarginine 189.13 6.64 1.0E-03 ND 1,
b-Ala-Lys 218.15 6.34 5.8E-04 ND 1,
Saccharopine 277.14 9.91 1.5E-04 ND 1,
Gly-Leu 189.12 8.95 3.8E-04 ND 1,
Ala-Ala 161.09 8.65 4.4E-04 ND 1,
CMP 322.04 9.53 1.2E-03 1.9E-04 0.16
Guanidoacetic acid 118.06 7.76 4.0E-03 8.0E-04 0.20
b-Ala 90.05 6.94 1.2E-01 2.7E-02 0.23
UMP 323.03 9.72 3.7E-03 1.0E-03 0.28
N2-Succinylornithine 233.11 9.51 1.5E-03 4.5E-04 0.31
GMP 362.05 9.07 3.1E-03 1.1E-03 0.34
3-Guanidinopropionic acid 132.08 7.56 1.1E-02 4.1E-03 0.36
Adenosine 268.10 9.18 2.9E-04 1.4E-04 0.49
AMP 346.06 9.17 3.1E-02 1.6E-02 0.52
Ribulose 5-phosphate 229.01 10.91 2.4E-03 1.3E-03 0.53
N6-Acetyllysine 189.12 9.11 8.2E-04 4.4E-04 0.53
Carnosine 227.11 6.36 1.0E100 5.4E-01 0.54
Inosine 269.09 16.90 1.7E-02 9.7E-03 0.56
Cadaverine 103.12 4.79 9.6E-05 5.6E-05 0.58
Spermine 203.22 4.33 1.6E-03 9.5E-04 0.60
Pipecolic acid 130.09 9.53 2.9E-03 1.8E-03 0.62
Ascorbic acid 175.02 8.38 1.2E-03 7.6E-04 0.62
Isobutyrylcarnitine 232.15 8.85 3.4E-04 2.2E-04 0.65

Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; CMP, cytidine monophosphate; GMP,
guanosine monophosphate; GTP, guanosine triphosphate; UMP, uridine monophosphate; UTP, uridine triphosphate.
1
All samples in each group were pooled (n 5 1).
2
Compound name based on mass-to-charge ratio (m/z) and migration time (MT) by using the metabolite library
maintained by Human Metabolome Technologies (Tsuruoka, Japan).
3
ND, not detected in Lys 100% or Lys 90% group.

2008; Kobayashi et al., 2011), indicating that dietary show that the muscle concentration of free Glu, as well
amino acid and protein levels regulate the free Glu as that of various other amino acids, was significantly
concentration in muscles. In addition, we recently increased in the Lys 90% group compared with the Lys
showed that the feeding of a high- or low-Lys diet in- 100% group. However, the plasma concentration of free
creases the muscle free Glu concentration of broilers amino acids, with the exception of Met, Lys, and His,
(Watanabe et al., 2015; 2017). was no different between groups. Therefore, muscle free
The goal of the present study was to elucidate the amino acids that accumulated from the feeding of a
mechanisms by which the feeding of a low-Lys diet in- low-Lys diet were not derived from plasma.
creases free Glu in chicken muscles. This study provides A metabolome analysis was performed to further inves-
new insight into amino acid metabolism in skeletal mus- tigate the metabolic changes in muscle induced by the low-
cle and may lead to a more efficient method for increasing Lys diet. Results showed that saccharopine and pipecolic
muscular free Glu to improve meat taste. Our results acid, which are intermediates in the Lys degradation
 LOW-LYSINE DIET INCREASES MUSCLE AMINO ACIDS 7
Table 7. Effect of dietary lysine level on mRNA abundance of factors of
protein metabolism (% of Lys 100% group) in broiler muscles.
Dietary Lys level (% of requirement)
Factors1 100 90 Significance
Factors of protein synthesis
IGF-1 100.0 6 9.1 110.4 6 7.1 NS
IGF-2 100.0 6 17.1 87.1 6 9.1 NS
Myostatin 100.0 6 15.3 91.3 6 3.7 NS
Factors of protein degradation
m-Calpain 100.0 6 5.3 121.2 6 5.8 *
m-Calpain large subunit 100.0 6 10.3 105.2 6 15.0 NS
Caspase 3 100.0 6 9.5 186.3 6 19.4 **
Cathepsin B 100.0 6 7.6 111.6 6 5.3 NS
20S proteasome C1 subunit 100.0 6 18.2 106.7 6 16.0 NS
20S proteasome C2 subunit 100.0 6 5.5 123.3 6 4.0 **

**P , 0.01; *P , 0.05.

Abbreviations: IGF, insulin-like growth factor; NS, not significant.
1
Abundance of mRNA in the breast muscle of chicks fed the Lys 100% or Lys 90%
group expressed as % of Lys 100% group. Values of Lys 100% and Lys 90% groups are
means 6 SEM for 10 chickens.

pathway, were decreased in the Lys 90% group. We have that occurs during low-Lys feeding. Also, metabolites of
previously shown that saccharopine and pipecolic acid glycolysis and the citric acid cycle and nucleoside triphos-
are increased by the feeding of a high-Lys diet and sug- phate were increased in the Lys 90% group compared with
gested that this change plays a key role in the increase in the Lys 100% group. In contrast, nucleoside monophos-
muscle free Glu from high Lys feeding (Watanabe et al., phate was decreased in the Lys 90% group, suggesting
2015). The mechanisms underlying the increase in muscle that adenosine triphosphate synthesis by glycolysis, and
free Glu thus appear to differ between low-Lys and high- the citric acid cycle was stimulated by low Lys feeding.
Lys feeding conditions. Of the 22 metabolites upregulated In addition, carnosine and ascorbic acid were decreased
from low-Lys feeding, 9 were amino acids, suggesting in the Lys 90%. Carnosine is a naturally occurring skeletal
increased free amino acids to be a major metabolic change muscle dipeptide comprised of b-alanine and histidine

Figure 1. The Pearson correlation of mRNA abundance of (A) m-calpain, (B) caspase 3, (C) 20S proteasome C2 subunit, and (D) cathepsin B with
free glutamate (Glu) content in broiler muscles. (A) r 5 0.74, P , 0.01; (B) r 5 0.69, P , 0.01; (C) r 5 0.65, P , 0.01; (D) r 5 0.52, P , 0.05.
8 WATANABE ET AL.

(Crush, 1970), and it reduces oxidative stress by its per- In conclusion, we found that reducing dietary Lys
oxyl radical-trapping ability (Kohen et al., 1988). An in- significantly increased the content of free Glu and other
crease in carnosine in broiler meat improves its drip loss free amino acids in chicken muscle. The mRNA abun-
(Kralik et al., 2018), 2-thiobarbituric acid reactive sub- dance of protein degradation enzymes also significantly
stances values (Hu et al., 2009; Kralik et al., 2018), and increased with low-Lys diet feeding and correlated with
shear force value (Hu et al., 2009). Ascorbic acid is also muscle free Glu concentration. These results suggest
an antioxidant that partly improves growth performance that protein degradation was induced by the reduction
and meat quality, such as pH and color in broilers under of dietary Lys, which released free amino acids that
heat stress (Imik et al., 2012). Therefore, the decrease in were stored in muscles. In the present study, growth per-
these antioxidants due to the reduction in dietary Lys formance did not decrease as a result of this protein
may induce a negative effect on the meat quality. The degradation in muscle; however, productivity may be
metabolome analysis reveals some metabolic changes affected by Lys restriction. In future studies, we will
induced by the feeding of a low-Lys diet. However, this determine the optimal Lys restriction level and feeding
analysis could not address all the factors contributing to duration based on the enhancement of meat taste by
Glu production, and further study is required. the increase in Glu and the chickens’ growth perfor-
The increase in muscle free amino acids from the feeding mance. In addition, results of metabolome analysis could
of a low-Lys diet might be explained by a change in protein not clearly have stated because metabolome analysis was
metabolism in muscle because free amino acids are mainly performed on a pooled sample in this study. Thus, we will
derived from the degradation of preexisting proteins confirm the reproducibility of the metabolome analysis
(Ohsumi, 2006), and proteins are synthesized from free by individual samples. Especially, we will analyze the ef-
amino acids. Protein metabolism is affected by many nutri- fect of the low-Lys diet on the antioxidants’ concentra-
tional factors including energy status (Proud, 2007), type tion in the meat. Moreover, the effects of reduction in
of protein (Morifuji et al., 2005; Wilkinson et al., 2007), dietary Lys on the meat quality, such as pH, drip loss,
amino acids (Borsheim et al., 2002; Kadowaki and 2-thiobarbituric acid reactive substances values, and
Kanazawa, 2003), and Leu (Nagasawa et al., 2002; shear force value will be examined. Based on these results,
Norton and Layman, 2006). In a recent study, Sato et al. we will test antioxidant supplementation in the low-Lys
reported that Lys and saccharopine both affect protein treatment.
metabolism by suppressing autophagic-proteolysis
through Akt (Sato et al., 2013; 2015). Here, we show
that the muscle 3M-His content was significantly ACKNOWLEDGMENTS
increased in the Lys 90% group. 3M-His in excreta is This work was supported by a Grant-in-Aid for Scien-
indicative of the rate of myofibrillar protein degradation tific Research (C), JSPS KAKENHI grant number
in chicken skeletal muscle (Hayashi et al., 1985); therefore, JP26450375, and a Grant-in-Aid for JSPS Research
an increase in muscle free 3M-His content may suggest an Fellow grant number JP15J03133 from the Japan Soci-
increase in the rate of myofibrillar protein degradation. ety for the Promotion of Science (JSPS). The authors
Together, these results suggest that dietary Lys content thank the Edanz Group (www.edanzediting.com/ac)
is an important factor in protein metabolism. for editing a draft of this manuscript.
To investigate changes in muscle protein metabolism
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