Article

Distribution of dietary protein intake in daily meals

influences skeletal muscle hypertrophy via the
muscle clock
Graphical abstract Authors
Shinya Aoyama, Hyeon-Ki Kim,
Rina Hirooka, ..., Shigeki Shimba,
Kazuyuki Shinohara, Shigenobu Shibata

Correspondence
shibatas@waseda.jp

In brief
Aoyama et al. show that the distribution of
protein intake across meals affects
muscle hypertrophy. The distribution-
dependent effects require a muscle
clock. A higher skeletal muscle index and
grip strength were observed in older
women who habitually consume a high-
protein diet at breakfast.

Highlights
d Distribution of dietary protein across meals influences
muscle hypertrophy

d BCAAs are involved in hypertrophic effects of protein feeding

distribution

d Hypertrophic effects of protein feeding distribution require

the muscle clock

d Breakfast protein intake is correlated with skeletal muscle

functions in older women

Aoyama et al., 2021, Cell Reports 36, 109336

July 6, 2021 ª 2021 The Author(s).
https://doi.org/10.1016/j.celrep.2021.109336 ll
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Article
Distribution of dietary protein intake
in daily meals influences skeletal muscle
hypertrophy via the muscle clock
Shinya Aoyama,1,2,5,6 Hyeon-Ki Kim,1,2,6 Rina Hirooka,1 Mizuho Tanaka,1 Takeru Shimoda,1 Hanako Chijiki,1
Shuichi Kojima,1 Keisuke Sasaki,1 Kengo Takahashi,1 Saneyuki Makino,1 Miku Takizawa,1 Masaki Takahashi,3
Yu Tahara,1 Shigeki Shimba,4 Kazuyuki Shinohara,5 and Shigenobu Shibata1,7,*
1Laboratory of Physiology and Pharmacology, School of Advanced Science and Engineering, Waseda University, Tokyo 162-8480, Japan
2Organization for University Research Initiatives, Waseda University, Tokyo 162-8480, Japan
3Institute for Liberal Arts, Tokyo Institute of Technology, Tokyo 152-8550, Japan
4Department of Health Science, School of Pharmacy, Nihon University, Chiba 274-8555, Japan
5Department of Neurobiology & Behavior, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8523, Japan
6These authors contributed equally
7Lead contact

*Correspondence: shibatas@waseda.jp
https://doi.org/10.1016/j.celrep.2021.109336

SUMMARY

The meal distribution of proteins throughout the day is usually skewed. However, its physiological implica-
tions and the effects of better protein distribution on muscle volume are largely unknown. Here, using the
two-meals-per-day feeding model, we find that protein intake at the early active phase promotes overload-
ing-induced muscle hypertrophy, in a manner dependent on the local muscle clock. Mice fed branched-chain
amino acid (BCAA)-supplemented diets at the early active phase demonstrate skeletal muscle hypertrophy.
However, distribution-dependent effects are not observed in ClockD19 or muscle-specific Bmal1 knockout
mice. Additionally, we examined the relationship between the distribution of proteins in meals and muscle
functions, such as skeletal muscle index and grip strength in humans. Higher muscle functions were
observed in subjects who ingested dietary proteins mainly at breakfast than at dinner. These data suggest
that protein intake at breakfast may be better for the maintenance of skeletal muscle mass.

INTRODUCTION attributed to the postprandial anabolic threshold of protein being

Dietary protein intake is important for skeletal muscle growth and
maintenance (Paddon-Jones and Rasmussen, 2009). They are
not only a source of body protein but also activate skeletal
muscle synthesis. In particular, branched-chain amino acids
(BCAAs), such as leucine, isoleucine, and valine, are known to
activate skeletal muscle synthesis via the mammalian target of
rapamycin (mTOR) pathway and are important for muscle growth
(Reidy and Rasmussen, 2016).
Surveys of diets in Western and Asian countries revealed that
protein intake during breakfast is usually low, and the distribution
of proteins across various meals throughout the day is skewed
(Ishikawa-Takata and Takimoto, 2018; US Department of Agri-
culture [USDA], 2012; Tieland et al., 2015). The distribution of
protein ingestion has been related to muscle functions, such
as muscle synthesis, grip strength, and muscle volume, in hu-
mans and rodents (Mamerow et al., 2014; Mishra et al., 2018;
Norton et al., 2017). It has been reported that supplementation
of protein at breakfast and lunch increases skeletal muscle vol-
ume in older adults (Norton et al., 2016). Thus, it is hypothesized
that not only the total amount of protein intake but also its distri-
bution across meals is important for muscle growth. This can be
reached across three meals by equal distribution (Paddon-Jones
and Rasmussen, 2009). However, digestive and absorptive ca-
pacities and metabolic processes exhibit day-night variations
(Tahara and Shibata, 2013, 2014). It is unclear whether protein
intake distribution along with the day-night variation of its
bioavailability and/or threshold influences muscle growth.
Several physiological functions, including nutritional metabolic
processes, undergo day-night oscillations. Most of these are
driven by the negative feedback loop of circadian core clock
genes comprising Circadian locomotor output cycles kaput
(Clock), Brain and muscle arnt-like 1 (Bmal1), Period1/2 (Per1/
2), and Cryptcrome1/2 (Cry1/2). A heterodimer of CLOCK and
BMAL1 activates the transcription of Pers and Crys via binding
to the E-box site of Pers and Crys. Increasing levels of PERs
and CRYs inhibit their own transcription. Most clock gene knock-
outs or mutant mice lack the metabolic day-night variation and
show dysfunctions (Tahara and Shibata, 2016), suggesting that
clock genes drive day-night variation in nutrient utilization. For
example, the absorptive capacity of a specific peptide such as
b-alanyl-L-histidine was found to change throughout the day,
because the expression of the peptide transporter was regulated
by the circadian clock (Okamura et al., 2014; Saito et al., 2008). In

Cell Reports 36, 109336, July 6, 2021 ª 2021 The Author(s). 1

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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recent years, it has been found that the circadian clock controls
the day-night oscillation of amino acid metabolism in murine
skeletal muscle (Dyar et al., 2018). Amino acid metabolism in hu-
mans was also found to vary in response to the time of the day,
that is, whether the meal is breakfast or dinner (Sato et al., 2018;
Takahashi et al., 2018). These studies suggest that the bioavail-
ability of dietary amino acids and proteins is dependent on the
day-night oscillation modulated by the circadian clock system.
Thus, we hypothesized that an appropriate distribution of daily
protein intake across meals promotes muscle function. Here,
we report that the skeletal muscle hypertrophy is dependent
on the dietary protein distribution in mice having two-meals-
per-day feeding. We show here that BCAAs are involved in
inducing distribution-dependent hypertrophic effects. Addition-
ally, the hypertrophic effects of protein distribution were not
observed in clock-disrupted mice, such as Clock mutant
(ClockD19) mice and muscle-specific Bmal1 knockout (MKO)
mice. Finally, we showed that the distribution of protein intake
at breakfast was positively correlated with the skeletal muscle
volume in healthy older women.
RESULTS
Response to overloading-induced skeletal muscle
hypertrophy differed according to the daily protein
distribution in mice
To examine the effects of protein intake distribution, we fed ICR
(Institute of Cancer Research) mice a 2.0-g meal twice a day at
Zeitgeber time (ZT) 12 (the early active phase) and ZT20 (the
late active phase), which accounted for breakfast and dinner,
respectively (Figure 1A, upper panel). Mice were fed an average
of 11.5% or 8.5% protein diet in a day in three patterns of protein
distribution (high protein at breakfast, equal distribution or high
protein at dinner) (Figure 1A, lower panel). We confirmed that
ICR mice were able to eat a 2.0-g meal within 4 h. Plantaris mus-
cle hypertrophy was induced by unilateral synergist ablation,
hereafter called overloading. Initial and final body weight and
growth rate did not substantially change in any group (Figures
S1A–S1D). The plantaris muscle weight of all groups was found
to be increased by overloading, and the response of muscle
weight to overloading was higher in the mice having high protein
at breakfast, compared with those having high protein at dinner
(Figures 1B and 1C). In the sham leg, muscle weight was not
altered in any group (Figure 1B). The ratio of overloaded muscle
weight to sham muscle weight in the high-protein-breakfast-fed
mice in the 8.5% group was 17% higher than that in the high-pro-
tein-dinner-fed mice in the 11.5% group, even though the daily
total protein intake in the former was lower than that in the latter
(Figure 1C). Locomotor activity levels were not significantly
different either (Figures S1E–S1H).
BCAA intake at breakfast accelerates muscle
hypertrophy
We next determined whether breakfast, including high BCAAs,
activated overloading-induced muscle hypertrophy, because
BCAAs activate muscle growth (Reidy and Rasmussen, 2016).
BCAA supplementation at breakfast promoted overloading-
induced muscle hypertrophy as opposed to supplementation
2 Cell Reports 36, 109336, July 6, 2021
Article
at dinner (Figures 1D and 1E). No time-dependent effect was
observed following supplementation with amino acids contained
in casein other than BCAAs (Figures 1F and 1G). These data sug-
gest that BCAAs are involved in the time-dependent effects of
protein ingestion on overloading-induced muscle hypertrophy
in mice. However, the body weight, locomotor activity pattern,
and total activity level were not altered by the feeding time of
BCAAs or amino acids other than BCAAs (Figures S1I–S1P).
Time-dependent effects of dietary protein require the
muscle clock
To explore the contribution of the circadian clock to the effects
caused by protein intake distribution, we examined ClockD19 (mu-
tation in a whole body) and MKO mice. The high-protein breakfast
enhanced the overloading-induced muscle hypertrophy in wild-
type (WT) mice, but not in ClockD19 mice (Figures 2A and 2B).
Similar results were observed in mice that had breakfast supple-
mented with BCAAs (Figures S2A and S2B). Day-night variations
in locomotor activity were observed in WT mice, but not in
ClockD19 mice (Figures 2D and 2E; Figures S2C, S2D, and S2F).
In contrast, active peaks were apparent before each meal in
both genotypes (Figure 2D; Figure S2D). Total activity levels
were not significantly altered by the protein and BCAAs or their
distribution across meals (Figure 2C; Figure S2E). The results of
ClockD19 mice suggested that Clock’s genetic inactivity and
disturbance of rhythmic locomotor activity could be associated
with daily timing-dependent effects of protein ingestion on muscle
growth. Next, we used tissue-specific conditional knockout mice
to examine the effects of clock genes and locomotor activity
separately. The enhancement of muscle hypertrophy by high-pro-
tein breakfast was observed in Bmal1flox/flox mice, but not in MKO
mice (Figures 2F and 2G). Notably, the total locomotor activity
level and hourly activity pattern of both genotypes did not change
under the two-meals-per-day condition (Figures 2H–2J). These
results suggest that the muscle clock is involved in the time-
dependent hypertrophic effects of proteins, and that the effect
of locomotor activity could be minor.
Effects of dietary protein distribution on day-night
variation in BCAA levels and gene expression in the two-
meals-per-day feeding model mice
To examine the effect of protein feeding at breakfast and dinner
on blood and muscular amino acid levels, we determined the
day-night variation of free amino acids in the plasma and skeletal
muscle of the mice (Figures 3A–3F; Figure S4). Plasma leucine,
isoleucine, and valine (BCAA) levels increased after a high-pro-
tein meal, and the timing of high-protein meals did not affect
the elevation of plasma BCAA levels (Figures 3A–3C). A timing-
dependent response of BCAAs to a high-protein meal was not
observed in the sham and overloaded muscles (Figures 3D–
3F). Therefore, the day-night variation of BCAAs did not involve
a time-dependent hypertrophic effect on protein ingestion.
Most free amino acids other than glycine, histidine, and serine
did not show timing-dependent responses in the overloaded
muscles (Figure S4B). The muscular free glycine and histidine
levels at specific time points were higher in the overloaded mus-
cles of mice fed a high-protein meal at dinner (Figure S4B). In
contrast, the muscle-free serine level was higher in the
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Figure 1. Meal distribution of protein or BCAAs regulates overloading-induced skeletal muscle hypertrophy
(A) Experimental scheme of two-meals-per-day condition. During acclimation periods, ICR mice were fed a 14% casein diet twice a day (Zeitgeber time [ZT]:
ZT12 16 and ZT20 0). In the experimental periods, a 2.0-g meal was provided at ZT12 and ZT20, which was defined as breakfast and dinner, respectively. Mice
were kept under the six patterns of protein distribution for 2 weeks. One week after the experimental period, unilateral skeletal muscle hypertrophy was induced
by synergist ablation (overloading).
(B) Plantaris muscle weight in time-restricted protein-fed mice.
(C) Ratio of overloading muscle weight to contralateral sham-surgery muscle weight.
(D and E) Plantaris muscle weight (D) and ratio of overloaded muscle weight to sham-surgery muscle weight (E) in time-restricted BCAA (high B)-fed mice.
(F and G) Plantaris muscle weight (F) and ratio of overloaded muscle weight to sham-surgery muscle weight (G) in time-restricted low BCAA-diet (low B)-fed mice.
The experiments have been independently repeated. Mean ± SE (B and C: n = 5 6; D and E: n = 8; F and G: n = 7). One or two-way ANOVA with Tukey’s or
unpaired t test, *p < 0.05, **p < 0.01. Kruskal-Wallis test with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, ##q < 0.01. 3C, 3% casein
diet. See also Figure S1.

overloaded muscle of mice fed a high-protein meal at breakfast
(Figure S4B). The expression of Snat2 (neutral amino acid trans-
porter) was not affected by the protein feeding distribution
(Figure S3B).
Next, we examined the day-night variation in plasma insulin-like
growth factor 1 (IGF-1) and muscular Igf1 levels in mice fed a high-
protein meal at breakfast (B protein group) or at dinner (D protein
group). Plasma IGF-1 levels did not show any day-night variation
and were not altered between the two groups (Figure S3O).
Muscular Igf1 expression was increased by overloading and
was higher at ZT21 in the B protein group than in the D protein
group (Figure 3G). Although it was expected that muscle synthe-
sis would be increased in the overloaded muscle of the B protein
group, protein synthesis, which was evaluated by using phos-
phorylation of mTOR and S6 kinase (S6K), was not altered in a
day (Figures 4A, 4D, and 4E). Additionally, we examined the
day-night variation in muscle-atrophy-related genes (Figures
3H–3I; Figures S3A and S3C–S3E). The expression and day-night
variation in these genes were not affected by the protein feeding
distribution, although the expression of Atrogin1 and Mstn tended
to increase and decrease in the overloaded muscle in both
groups, respectively (Figures S3A and S3C).
We also examined the day-night variation in the expression of
myogenic genes. The expression levels of Pax3, Pax7, Myod,

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Figure 2. Meal-distribution-dependent effects of protein are not observed in ClockD19 mice and muscle-specific Bmal1 knockout (MKO) mice
(A–E) Sham surgery and overloaded plantaris muscle weight (A), ratio of overloading muscle weight to contralateral sham-surgery muscle weight (B), total activity
(C), hourly activity pattern (D), and activity ratio in the light and dark phase (E) in time-restricted protein-fed wild-type (WT) and Clock mutant (ClockD19) mice
(20C 3C: 20% casein diet at breakfast and 3% casein diet at dinner; 3C 20C: 3% casein diet at breakfast and 20% casein diet at dinner).
(F–J) Sham surgery and overloaded plantaris muscle weight (F), ratio of overloading muscle weight to contralateral sham-surgery muscle weight (G), total activity
(H), hourly activity pattern (I), and activity ratio in the light and dark phases (J) in time-restricted protein-fed Bmal1flox/flox and MKO mice.
The experiment has been independently repeated. Mean ± SE (A E: n = 4 5; F and G: n = 10 17; H J: n = 9 13). Two-way ANOVA with the Sidak test or
unpaired t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Kruskal-Wallis test with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli,
#
q < 0.05. See also Figure S2.

and Myf6 were not altered by protein intake distribution in the were increased by overloading. The day-night variations in the
sham and overloaded muscles (Figure 3L; Figures S3F–S3H). B protein group were higher at ZT21 than at other time points
Similar to Igf1 expression, the expressions of Myf5 and Myog (Figures 3J and 3K). The myogenic factor 5 (MYF5), but not

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Figure 3. Day-night variation of free branched-chain amino acid level and gene expression in plasma and muscle of time-restricted protein-
fed mice
The mice were maintained under two-meals-per-day conditions. Mice fed high protein at breakfast (B-Pro) were fed a high-protein diet (20% casein) at breakfast
and a low-protein diet (3% casein) at dinner. Mice fed high protein at dinner (D-Pro) were fed a low-protein diet at breakfast and a high-protein diet at dinner.
(A C) Day-night variations in plasma leucine (Leu), isoleucine (Ile), and valine (Val) levels in time-restricted protein-fed mice.
(D–O) Day-night oscillations of (D F) muscular-free Leu, Ile, and Val concentrations and gene expression levels of (G) insulin-like growth factor 1 (Igf1), (H) muscle
ring finger protein 1 (Mufr1), (I) Ubiquitin C (Ubc), (J) Myogenic factor 5 (Myf5), (K) Myogenin (Myog), (L) Myogenic differentiation 1 (Myod), (M) Period2 (Per2), (N)
Brain and muscle arnt-like 1 (Bmal1), and (O) nuclear receptor subfamily 1, group D, member 1 (Nr1d1, known as Reverba) normalized by TATA-box binding
protein (Tbp) in the sham-surgery and overloaded muscle of time-restricted protein-fed mice. The p value of JTK_CYCLE for rhythmicity is shown above the white
and black bars. White and black bars indicate the light and dark conditions, respectively. Each arrow indicates the meal time of breakfast (B) and dinner (D).
Mean ± SE (A F: n = 4; G O: n = 4 6). Kruskal-Wallis test with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, *q < 0.05, **q < 0.01,
***q < 0.001 (B-Pro versus D-Pro at each time point). See also Figures S3 and S4 and Table S2.

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Figure 4. Effects of dietary protein distribution on myogenic factors, mTOR signaling pathway, and autophagy in overloading-induced
muscle hypertrophy and the role of autophagy in muscle hypertrophy due to dietary protein distribution
Mice fed high protein at breakfast (B-Pro) were fed a high-protein diet (20% casein) at breakfast and a low-protein diet (3% casein) at dinner. Mice fed high protein
at dinner (D-Pro) were fed a low-protein diet at breakfast and a high-protein diet at dinner.
(A) Representative blots in sham and overloaded muscle pre-and post-meal.
(B–F) Quantitative signal intensity of (B) myogenic factor 5 (MYF5), (C) myogenin (MYOG), (D) phosphorylated ribosomal protein S6 kinase (p-S6K)/total S6K, (E)
phosphorylated mechanistic target of rapamycin kinase (p-mTOR)/total mTOR, and (F) microtubule-associated protein 1 light chain 3 beta II (MAP1LC3B-II,
known as LC3-II) protein levels in sham and overloaded muscle before and after meals. MYF5, MYOG, and LC3B-II were normalized using Coomassie brilliant
blue (CBB) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
(G) Experimental scheme of the two-meals-per-day condition and injection. One week after the experimental period, unilateral skeletal muscle hypertrophy was
induced by synergist ablation (overloading). Vehicle (Veh) or 3-methyladenine (3-MA) was intraperitoneally injected once a day at ZT20 from the second week.
(H) Relative weight of sham- and overloading-induced muscles in Veh- and 3-MA-treated mice.
(I) Ratio of overloading muscle weight to contralateral sham-surgery muscle weight.
Mean ± SE (B F: n = 4, H and I: n = 6 7). #q < 0.05; B protein versus D protein using Kruskal-Wallis test with a two-stage linear step-up procedure of the
Benjamini, Krieger, and Yekutieli test. Three-way ANOVA with Sidak test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bre, breakfast; Din, dinner. See also
Figure S5.

myogenin (MYOG), protein levels showed a tendency similar to
its gene expression level in the overloaded muscles of mice
fed with a high-protein meal at breakfast or dinner (Figures 4A–
4C). Interestingly, the tendency of the temporal variation of
MYF5 protein level was observed in the overloaded muscle
collected from WT and Bmal1flox/flox mice, which was not
observed in Clock D19 mutant and MKO mice (Figure S5). These
data suggest that the effect of the distribution of protein intake
across meals was influenced by the day-night variation in
expression of Igf1 and myogenic genes.
Finally, to evaluate the expression of clock genes, we as-
sessed the expression levels of several clock genes in the
skeletal muscles of mice fed a high-protein meal at breakfast
or dinner (Figures 3M–3O; Figures S3I–S3M). In both groups’

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sham and overloaded muscles, Per2 and Bmal1 oscillation
showed a regular pattern (Figures 3M and 3N). The expression
of Cry1 showed a typical rhythmic pattern in the sham muscle,
but not in the overloaded muscle (Figure S3K). The effects of pro-
tein feeding distribution were also observed in the expression of
Reverba, Clock, and Per1. Rhythmic expression of Reverba and
Clock was observed in the overloaded muscle of mice fed a high-
protein meal at breakfast and dinner, respectively (Figure 3O;
Figure S3J). The high expression level of Per1 in the inactive
phase in the sham and overloaded muscles of mice fed a high-
protein meal at dinner was observed. Thus, the distribution of
protein intake across meals partially affected the oscillation or
expression of a part of muscular clock genes, such as Rev-
erba, Clock, and Per1, while the day-night variations of Per2,
Bmal1, Cry1/2, Rora, and the serum corticosterone levels were
not altered by the protein feeding distribution (Figures 3M–3O;
Figures S3I–S3N).
Effects of dietary protein distribution on autophagy and
its role in the overloading-induced skeletal muscle
hypertrophy
We tested the response of autophagy to protein intake at break-
fast and dinner. Our western blotting analyses at four time points
across the active phase (before and after each meal) revealed that
the LC3B-II protein level after dinner was maintained at a higher
level in the overloaded muscles of the B protein group (Figures
4A and 4F). Activation of autophagy is observed in the hypertro-
phic muscles by overloading and resistance exercise (Riedl
et al., 2016; Sanchez et al., 2014). In our study, overloading
increased the LC3B-II levels after dinner (Figure 4F). However,
the LC3B-II level was decreased across each meal in the sham
muscles of both groups and the overloaded muscles of the D
protein group. To examine the role of autophagy activation in
overloading-induced skeletal muscle hypertrophy, we tested the
effect of the autophagy inhibitor 3-methyladenine (3-MA) in
time-restricted protein-fed mice. Daily intraperitoneal administra-
tion of 3-MA attenuated overloading-induced muscle hypertrophy
in the B protein group, but not in the D protein group (Figures 4G–
4I). The decrease in muscle weight by 3-MA was seen only in the
overloaded muscle, but not in the sham-surgery muscle (Fig-
ure 4H). In addition, the LC3B-II level tended to increase at ZT0
(after dinner) in WT and Bmal1flox/flox mice, whereas this tendency
was not observed in ClockD19 and MKO mice (Figure S5). There-
fore, it can be suggested that intake of high protein at breakfast
and low protein at dinner could enhance overloading-induced
muscle hypertrophy via the activation of autophagy.
Association between distribution of protein across
meals and muscle functions in humans
Sixty older women were recruited and divided into two groups
according to the balance of protein intake between breakfast
and dinner, obtained from the results of a diet survey. Partici-
pants in the B protein group habitually ingested higher protein
at breakfast than at dinner (Figures 5B and 5D). An opposite dis-
tribution of proteins across meals existed in the D protein group
participants. The total daily protein intake was not significantly
different between the groups (Figures 5C and 5E). It should be
noted that the protein intake of participants in this study was
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higher than Japan’s recommended dietary allowance for elderly
women (NIHN, 2015). Also, their daily protein intake fulfilled the
requirement of 1.0–1.2 g/kg body weight/day, which was neces-
sary for maintaining and increasing muscle mass as reported in a
previous study (Bauer et al., 2013). Participants’ characteristics
are presented in Table S3. There was no significant difference
between the two groups in height, body mass, fat mass, body
mass index (BMI), physical function, dietary intake, and physical
activity. The physical activity level affecting skeletal muscle was
not different between the two groups, suggesting that the influ-
ence of physical activity on skeletal muscle mass could be
considered equivalent. Muscle mass tended to be higher in the
B protein group than in the D protein group (Figure 5F). The skel-
etal muscle index (SMI) and grip strength were significantly
higher in the B protein group than in the D protein group (Figures
5G and 5H). Furthermore, a significant positive correlation was
observed between SMI and the percentage of breakfast protein
intake relative to total protein intake (Figure 5I).
DISCUSSION
In this study, we examined the effects of dietary protein distribu-
tion across meals on skeletal muscle mass. In the animal
experiments, we found that the response of skeletal muscle
hypertrophy to overloading differed with the daily protein
intake pattern. A similar distribution-dependent response was
observed in the mice fed BCAA-supplemented diets, but not in
those that were fed diets supplemented with amino acids other
than BCAAs. We expected the circadian rhythm to influence
the distribution-dependent effects. In fact, the hypertrophic ef-
fects of protein or BCAA distribution were not observed in the
ClockD19 mice (mutation in a whole body) and muscle-specific
Bmal1 KO mice, suggesting that the local muscle clock is
involved in distribution-dependent hypertrophic effects. In addi-
tion, the expression levels of Igf1, Myog, and Myf5 and the auto-
phagy marker (LC3B-II levels) were higher in the overloaded
muscles of mice fed a high-protein diet in the early active phase.
The SMI and grip strength were higher in subjects who habitually
consumed a high-protein breakfast than in those who had a
high-protein dinner.
We found that overloading-induced muscle hypertrophy was
influenced by the distribution of proteins or BCAAs across
meals. Several diet surveys have reported that protein consump-
tion in humans is skewed during a day with people opting for a
high-protein meal at dinner (Ishikawa-Takata and Takimoto,
2018; USDA, 2012; Tieland et al., 2015), and this skewed distri-
bution of protein consumption influences muscle synthesis (Ma-
merow et al., 2014). In our study, feeding a high-protein meal in
the late active phase (defined as dinner) attenuated muscle hy-
pertrophy in mice. In comparison, mice that were fed a high-pro-
tein meal at the early active phase (defined as breakfast) showed
the highest response of muscle hypertrophy to overloading. The
effect of protein intake distribution was not observed in the
sham-surgery muscles. There are two possible explanations
for this finding. First, the distribution of protein intake could influ-
ence the anabolic processes of overloading-induced muscle
hypertrophy because this process was drastically altered imme-
diately after synergistic ablation (Schiaffino et al., 2013). Second,
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Figure 5. Association between meal distribution of protein intake and muscle functions in healthy older adults
(A) The graphical flow diagram describing the human study design. Older adults were divided into two groups based on the meal distribution of protein intake into
two groups. Participants in the breakfast protein group (B-Pro) habitually consumed more protein at breakfast than at dinner. The dinner protein group (D-Pro)
showed the opposite distribution of protein intake.
(B and C) Average protein intake per meal (B) and total protein intake (C) in both groups.
(D and E) Average protein intake per kilogram of body weight per meal (D) and total protein intake per kilogram of body weight (E) in both groups.
(F–H) Muscle mass (F), skeletal muscle index (SMI) (G), and hand grip strength (H) in both groups. Data are expressed as the mean ± standard error (B-Pro: n = 18,
D-Pro: n = 42). Two-way ANOVA, Bonferroni, *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired t test, #p < 0.05.
(I) Correlation between SMI and the percentage of breakfast protein intake relative to total protein intake. Pearson’s product-moment correlation coefficient. See
also Table S3.

the study period was too short, because Norton et al. (2017) re-
ported an equal distribution of protein intake in daily meals for a
long time, 11 weeks, maintained and/or increased volume of
intact muscle in rats compared with the skewed protein distribu-
tion. Therefore, either the anabolic process and/or the experi-
mental period could be the reasons for protein distribution not
affecting sham muscles.
Distribution-dependent hypertrophic effects of dietary protein
were observed in mice that were fed the BCAA-supplemented
diet at the early active phase, although supplementation with
other amino acids, including a casein diet, did not affect over-
loading-induced muscle hypertrophy. This indicates that BCAAs
play an important role in the distribution-dependent hypertrophic
effects of dietary proteins. The responses of plasma and
muscular-free BCAA concentration to the high-protein diet
were not altered by the time of feeding. Therefore, it can be
assumed that absorption and uptake of BCAAs in the small intes-
tine and skeletal muscles could play a small role in the distribu-
tion-dependent effects of dietary protein.
BCAAs, especially leucine, are amino acids with strong
anabolic activity (Duan et al., 2016). Because of this and the
fact that BCAAs were involved in timing-dependent hypertrophic
effects, in our study, it was speculated that the muscle protein
synthesis pathway might be involved in the time-dependent ef-
fects of dietary protein. Igf1 expression and the activation of pro-
tein translation-related pathways showed day-night variation,
and their oscillations were found to be controlled by circadian
clocks (Chaudhari et al., 2017; Lipton et al., 2015). Our results
also revealed overloading-induced Igf1 expression and espe-
cially high expression levels at the middle active phase in the
overloaded muscle of the B protein group. Such an expression
pattern was not observed in the overloaded muscle of the D

8 Cell Reports 36, 109336, July 6, 2021


Article
protein group. In contrast, the activation of protein synthesis, as-
sessed using the phosphorylation of S6K and mTOR, was not
altered by the distribution of dietary protein intake. It was previ-
ously observed that translation-related pathways, including the
mTOR pathway, are involved in skeletal muscle hypertrophy in
a model of synergist ablation (Bentzinger et al., 2013). Another
study demonstrated that the activation of mTOR immediately af-
ter synergist ablation is important for skeletal muscle hypertro-
phy (Ito et al., 2013). In our study, muscle protein synthesis
was assessed using muscle collected 1 week after synergist
ablation. Therefore, there could be a possibility of anabolic sig-
nals being involved immediately after the induction of muscle hy-
pertrophy or not being involved in the distribution-dependent
muscle hypertrophic effects. Further experiments and detailed
analyses are necessary to determine the correct explanation.
The expression of myogenic genes, such as Myog and Myf5,
was found to increase in the middle of the active phase in the
overloaded muscle of the B protein group, but not the D protein
group. These results were not observed in the sham-surgery
muscles. Previous studies reported that functional overloading
upregulated the expression of myogenic and activated satellite
cells (Adams et al., 1999; Bruusgaard et al., 2010; Serrano et al.,
2008), suggesting a role for myogenesis in overloading-induced
muscle hypertrophy. Murach et al. (2017) reported that the abla-
tion of satellite cells during growth phase suppressed overload-
ing-induced muscle hypertrophy using tamoxifen-inducible
genetically satellite cell ablated mice. These reports suggest
that the increased expression of Myog and Myf5 in mice fed a
high-protein diet at an early active phase is associated with dis-
tribution-dependent hypertrophic effects. However, further
studies are needed to assess myogenesis using isolated satel-
lite cells and myoblasts. In addition, ClockD19 and MKO mice
did not show acceleration of muscle hypertrophy and a slight
temporal increase in MYF5 levels following increased protein
intake at the early active phase, suggesting the important role
of the circadian clock in the effects of protein feeding time. In
global Bmal1 knockout mice and ClockD19 mice, the day-night
oscillation of Myod was not observed (Andrews et al., 2010).
In addition, a recent study revealed that the DNA binding motif
of Myog and Myf5 is enriched by cistrome analysis using the
BMAL1 antibody (Dyar et al., 2018), suggesting that BMAL1
regulates the day-night oscillation of myogenesis via the tran-
scription of Myod and Myf5. In fact, an earlier in vitro study re-
ported that Bmal1 / myoblast cells demonstrated a low ability
to differentiate and suppress MYFs such as MYOD and MYF5
(Chatterjee et al., 2013). Considering these reports, it can be
suggested that dietary protein intake influences myogenesis
via the circadian clock system. Although Myf5 expression did
not oscillate in our study, the slight temporal increase in the
middle active phase could be involved in the maintenance of
high MYF5 levels after dinner. Further studies are needed to
reveal the molecular mechanism of MYF5 regulation via the
muscle clock. Myf6 and Pax3 (Figures S3G and S3H) expres-
sion levels were not affected by the distribution of dietary pro-
tein intake. Overloading upregulated the expression of Myog
and Myf5, but not Myf6 and Pax3. The distribution of dietary
protein intake could influence the overloading-induced upregu-
lation of myogenic processes. Therefore, there is a possibility
ll
OPEN ACCESS
that the upregulation of MYFs during skeletal muscle hypertro-
phy could be controlled by circadian clocks, which could be
influenced by the protein feeding pattern.
We found that in the overloaded muscle of the B protein group,
a higher LC3B-II level was maintained, especially after dinner. In
contrast, such high levels of LC3B-II were not observed in the D
protein group. This difference was not observed in sham mus-
cles. Overloading increased LC3B protein levels, and this eleva-
tion was attenuated by protein feeding at dinner. It is speculated
that activation of autophagy in muscles accelerates muscle
degradation because it is a protein degradation system. Mus-
cle-specific Atg7 knockout mice have been reported to exhibit
muscle loss and dysfunction (Masiero et al., 2009). In addition,
the activation of autophagy and the subsequent muscle loss
were observed in constitutively active Foxo3 mice (Mammucari
et al., 2007). Thus, the role of autophagy is dependent on the
state of skeletal muscle health (Neel et al., 2013). Although it is
not yet clear whether autophagy plays a role in the skeletal mus-
cle hypertrophic process, our pharmacological approach
showed that the inhibition of autophagy by 3-MA injection atten-
uated overloading-induced muscle hypertrophy by a high-pro-
tein meal at breakfast, suggesting that the upregulation of
LC3B-II could be involved in overloading-induced muscle hyper-
trophy. In fact, chronic exercise is known to activate autophagy,
and its activation is required for exercise-induced improvement
of endurance capacity and glucose tolerance (He et al., 2012;
Lira et al., 2013). In this study, we evaluated only the LC3B-II
levels before and after meals. In the future, the assessment of
autophagy by flux assays will be required to investigate the
detailed mechanism of the protein feeding pattern. Additionally,
the tendency to increase LC3-II levels in overloaded muscle of
the B protein group was observed in the WT and Bmal1flox/flox
mice, but not in ClockD19 and MKO mice. Several studies have
shown the diurnal regulation of the autophagy system by molec-
ular clocks (Brooks and Dang, 2019; Ma et al., 2011, 2012; Pas-
tore et al., 2019). Additionally, it has been reported that the acti-
vation of autophagy occurs in a time-of-day-dependent manner
under two meals per day. This suggests that the autophagic
response to a meal differs depending on the eating time (Marti-
nez-Lopez et al., 2017). This activation of autophagy due to
two meals per day promotes muscle formation via the activation
of myogenesis (Martinez-Lopez et al., 2017). This suggests that
the muscle clock regulates the temporal autophagic response to
a meal. Further investigation of the detailed mechanism underly-
ing the regulation of autophagy by muscle clocks is required.
In our study with humans, we compared muscle functions,
such as volume and strength, between the two groups showing
opposite distribution of protein intake across meals. The results
showed that higher SMI and grip strength were observed in older
women who ingested high protein at breakfast than in those with
a high-protein meal at dinner. These data were similar to the re-
sults of our animal study, suggesting that protein intake at the
early active phase or breakfast could be effective for muscle
growth and/or maintenance in humans. Considering that protein
intake during the early active phase influences only overloaded
muscles, but not sham muscles, in our animal study, it is thought
that protein distribution-dependent effects on muscle mass are
susceptible to physical activity level and exercise. Although
Cell Reports 36, 109336, July 6, 2021 9
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OPEN ACCESS
step counts and moderate to vigorous physical activity (MVPA)
did not differ between the two groups, the participants in this hu-
man study showed approximately 38% higher step counts than
general older Japanese women (Ministry of Health Labor and
Welfare [MHLW], 2017), suggesting that participants in our hu-
man study exhibited high physical activity levels. Therefore,
physical activity level could influence the positive correlation be-
tween protein intake at breakfast and SMI. However, no signifi-
cant association was found between physical activity (MVPA
and step counts) and SMI/muscle mass in this study. Similar re-
sults were obtained in a previous study of elderly women (Bar-
bat-Artigas et al., 2016). Therefore, in our study participants,
the possibility that the differences in MVPA and step counts
are involved in the association between protein intake patterns
and muscle mass may be minor. However, the effects of
combining exercise training, such as resistance training, have
not been investigated. Therefore, further studies are required.
Another limitation of this study is that there is no recording of
the participants’ socioeconomic status (education, income,
etc.). Because previous studies have shown a relationship be-
tween socioeconomic status and sarcopenia (da Silva Alexandre
et al., 2014; Dorosty et al., 2016), a detailed examination of these
issues is required in the future. However, because the partici-
pants of this study live in the same area (Tokyo), it is considered
that the difference in socioeconomic status has little effect on the
results.
Most previous studies aimed at exploring the association be-
tween muscle functions and distribution of protein intake,
focusing on two eating patterns characterized by even distribu-
tion or skewed distribution of low protein at breakfast (Tessier
and Chevalier, 2018). In this study, we assessed muscle function
by comparing them with the opposite-skewed pattern. Although
previous studies have shown that higher muscle mass and grip
strength were observed in older adults who followed an even
pattern of protein intake as opposed to those who followed a
skewed pattern of low protein at breakfast (Farsijani et al.,
2017; Granic et al., 2016), there are no human studies that
compare the two types of eating patterns characterized by
even distribution or skewed distribution of high protein at break-
fast. Considering that the skewed distribution of high protein at
breakfast promoted overloading-induced muscle hypertrophy
compared with the even pattern in our animal study, it could
be possible to observe a similar relationship in humans. Howev-
er, we did not include the pattern of even distribution of protein
intake in our human study.
For the present human study, we included healthy older
women without sarcopenia. In a previous study targeting Korean
older adults, women were shown to have a higher prevalence of
sarcopenia and obesity than men (Oh et al., 2015). This suggests
that the development of preventive interventions is important for
elderly women. Our results indicate that early nutritional inter-
vention could help address age-associated muscle loss. Further
studies targeting other populations, such as those with sarcope-
nia, are needed.
In addition, this was a cross-sectional study. A previous study
reported that long-term intake of protein for breakfast and lunch
is effective in increasing lean tissue mass (Norton et al., 2016).
However, there has been no study on the effect of long-term pro-
10 Cell Reports 36, 109336, July 6, 2021
Article
tein ingestion at breakfast or dinner on muscle mass. Therefore,
it is necessary to consider the influence of protein intake for
breakfast or dinner on muscle function.
Limitations of study
Our results from animal studies suggest that the autophagic and
myogenic responses to dietary protein distribution could be
involved in overloading-induced skeletal muscle hypertrophy.
However, the molecular implications of CLOCK and BMAL1 for
the time-specific effect of protein intake on muscle hypertrophy
are still unknown. Considering that the tendency for temporal
variation in LC3B-II and MYF5 levels was not observed in the
overloaded muscle collected from ClockD19 and MKO mice, it
was suggested that the muscle clock was involved in the
autophagic and myogenic response to the protein feeding
distribution. Future research is required to reveal the molecular
mechanisms underlying the hypertrophic effects of protein
feeding distribution.
Sex and age were different between our animal and human
studies. In particular, considering the age of participants, the
data may imply that protein intake at breakfast is beneficial
for preventing muscle atrophy, rather than the promotion of
muscle hypertrophy, which is inferred from animal experiments.
Skeletal muscle mass and strength decrease with age (Green-
lund and Nair, 2003; Rosenberg, 1997). In particular, it has
been reported that the rate of muscle mass loss accelerates af-
ter 60 years of age (Lexell, 1995; Lexell et al., 1988). It was also
shown that females have a higher rate of muscle protein syn-
thesis and myofibril synthesis due to a meal than males. There-
fore, females are more dependent on dietary proteins for mus-
cle synthesis than males (Horstman et al., 2019; Smith et al.,
2008). Hence we investigated the effects of differences in the
distribution of dietary protein intake on physical function (mus-
cle mass and strength) in elderly women. However, older adults
are less sensitive to amino acid stimulation of muscle protein
synthesis than younger adults (Katsanos et al., 2005; Wall
et al., 2015). Differences in the distribution of dietary protein
intake may have different effects on muscle function in young
adults. This is because the activation of the mTOR signaling
pathway due to leucine is reduced (Cuthbertson et al., 2005;
Katsanos et al., 2006). Therefore, it is necessary to perform
further human studies that take into account these issues
(gender and age) to confirm the results obtained in the animal
study.
In summary, our study on mice showed that the distribution
of protein intake throughout the day influenced skeletal muscle
hypertrophy, and high protein intake at breakfast accelerated
overloading-induced skeletal muscle hypertrophy. BCAAs
played a central role in the hypertrophic effects of the protein
feeding pattern. In addition, there is a possibility that the regu-
lation of myogenesis by the circadian clock may be involved in
the effects dependent on protein feeding patterns. In our hu-
man study, a higher SMI and grip strength were observed in
older adult women who consumed a high-protein diet at
breakfast compared with at dinner, suggesting a relationship
between dietary protein distribution in a day and skeletal mus-
cle volume. In the future, more detailed studies on the control-
ling mechanisms via the circadian clock will be required to
 ll
Article OPEN ACCESS

explore the effects of dietary protein distribution on muscle Andrews, J.L., Zhang, X., McCarthy, J.J., McDearmon, E.L., Hornberger, T.A.,
volume. Russell, B., Campbell, K.S., Arbogast, S., Reid, M.B., Walker, J.R., et al.
(2010). CLOCK and BMAL1 regulate MyoD and are necessary for maintenance
of skeletal muscle phenotype and function. Proc. Natl. Acad. Sci. USA 107,
STAR+METHODS 19090–19095.
Aoyama, S., Jia, H., Nakazawa, K., Yamamura, J., Saito, K., and Kato, H.
Detailed methods are provided in the online version of this paper (2016). Dietary Genistein Prevents Denervation-Induced Muscle Atrophy in
and include the following: Male Rodents via Effects on Estrogen Receptor-a. J. Nutr. 146, 1147–1154.
Aoyama, S., Kojima, S., Sasaki, K., Ishikawa, R., Tanaka, M., Shimoda, T., Hat-
d KEY RESOURCES TABLE tori, Y., Aoki, N., Takahashi, K., Hirooka, R., et al. (2018). Day-Night Oscillation
d RESOURCE AVAILABILITY of Atrogin1 and Timing-Dependent Preventive Effect of Weight-Bearing on
B Lead contact Muscle Atrophy. EBioMedicine 37, 499–508.
B Materials availability Barbat-Artigas, S., Pinheiro Carvalho, L., Rolland, Y., Vellas, B., and Aubertin-
B Data and code availability Leheudre, M. (2016). Muscle Strength and Body Weight Mediate the Relation-
ship Between Physical Activity and Usual Gait Speed. J. Am. Med. Dir. Assoc.
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
17, 1031–1036.
B Animal experiments
Bauer, J., Biolo, G., Cederholm, T., Cesari, M., Cruz-Jentoft, A.J., Morley, J.E.,
B Human experiments
Phillips, S., Sieber, C., Stehle, P., Teta, D., et al. (2013). Evidence-based rec-
d METHOD DETAILS ommendations for optimal dietary protein intake in older people: a position pa-
B Animal experiments per from the PROT-AGE Study Group. J. Am. Med. Dir. Assoc. 14, 542–559.
B Human experiments Bentzinger, C.F., Lin, S., Romanino, K., Castets, P., Guridi, M., Summermatter,
d QUANTIFICATION AND STATISTICAL ANALYSIS S., Handschin, C., Tintignac, L.A., Hall, M.N., and Ru €egg, M.A. (2013). Differ-
B Animal experiments ential response of skeletal muscles to mTORC1 signaling during atrophy and
B Human experiments hypertrophy. Skelet. Muscle 3, 6.
Bollwein, J., Diekmann, R., Kaiser, M.J., Bauer, J.M., Uter, W., Sieber, C.C.,
and Volkert, D. (2013). Distribution but not amount of protein intake is associ-
SUPPLEMENTAL INFORMATION
€rnberg. Nutr.
ated with frailty: a cross-sectional investigation in the region of Nu
J. 12, 109.
Supplemental information can be found online at https://doi.org/10.1016/j.
celrep.2021.109336. Brooks, R.C., and Dang, C.V. (2019). Autophagy: clocking in for the night shift.
EMBO J. 38, e102434.

ACKNOWLEDGMENTS
This study was partially supported by the Council for Science, Technology,
and Innovation, SIP, ‘‘Technologies for creating next-generation agriculture,
forestry, and fisheries’’ (funding agency: Bio-oriented Technology Research
Advancement Institution, NARO) (S. Shibata), Japan Society for the Promotion
of Science (JSPS) KAKENHI Grant Numbers 17K18176 and 20K19710 (to
S.A.), Grant for Young Scientists of Japan Society of Nutrition and Food Sci-
ence (S.A.), and JST-Mirai Program Grant Number JMPJM120D5 (Japan; S.
Shibata).
AUTHOR CONTRIBUTIONS
Conceptualization, S.A., H.-K.K., and S. Shibata; methodology, S.A., H.K.-K.,
R.H., M. Tanaka, T.S., H.C., S.K., K. Sasaki, K.T., S.M., M. Takizawa, M. Taka-
hashi, Y.T., S. Shimba, and K. Shinohara; formal analysis, S.A., H.-K.K., M. Ta-
naka, H.C., and T.S.; writing – original draft, A.S. and H.-K.K.; writing – review &
editing, S.A., H.-K.K., and S. Shibata; visualization, S.A.; supervision, S. Shi-
bata; funding acquisition, S.A. and S. Shibata.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: February 8, 2019
Revised: March 20, 2021
Accepted: June 11, 2021
Published: July 6, 2021
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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Anti-LC3B antibody Cell Signaling Technology Cat# 2775, RRID: AB_915950
Anti-Myf5 antibody abcam Cat# ab125301
Anti-myogenin (F5D) antibody Santa Cruz Biotechnology Cat#sc-12732, RRID: AB_627980
Anti-p70 S6 Kinase (49D7) antibody Cell Signaling Technology Cat# 2708, RRID: AB_390722
Anti-Phospho-p70 S6 Kinase (Thr389) (108D2) Cell Signaling Technology Cat# 9234, RRID: AB_2269803
antibody
Anti-mTOR (7C10) antibody Cell Signaling Technology Cat# 2983, RRID: AB_2105622
Anti-Phospho-mTOR (Ser2448) (D9C2) antibody Cell Signaling Technology Cat# 5536, RRID: AB_10691552
Goat polyclonal GAPDH (V-18) antibody Santa Cruz Biotechnology Cat# sc-20357, RRID: AB_641107
Donkey anti-goat IgG-HRP antibody Santa Cruz Biotechnology Cat# sc-2020, RRID: AB_631728
Anti-rabbit IgG, HRP-linked antibody Cell Signaling Technology Cat# 7074, RRID: AB_2099233
m-IgGKAPPA BP-HRP Santa Cruz Biotechnology Cat# sc-516102, RRID: AB_2687626
Chemicals, peptides, and recombinant proteins
3-Methyladenine AdipoGen LIFE SCINECES AG-CR1-3597-M100; CAS: 5142-23-4
Casein ORIENTAL YEAST N/A
Corn Starch RIENTAL YEAST N/A
a-Corn Starch RIENTAL YEAST N/A
Sucrose RIENTAL YEAST N/A
Soybean Oil Wako Chemicals 190-03776; CAS: 8001-22-7
Cellulose RIENTAL YEAST N/A
AIN* 93G Mineral MIX RIENTAL YEAST N/A
AIN 93M Mineral MIX RIENTAL YEAST N/A
AIN 93 Vitamin MIX RIENTAL YEAST N/A
Choline bitartrate SIGMA-ALDRICH C1629-100G, CAS: 87-67-2
L-Isoleucine Wako Chemicals 121-00862; CAS: 73-32-5
L-Leucine Wako Chemicals 128-00855; CAS: 61-90-5
L-Valine Wako Chemicals 224-00084; CAS: 72-18-4
L-Alanine Wako Chemicals 010-01042; CAS: 56-41-7
L-Arginine Wako Chemicals 017-04612; CAS: 74-79-3
L-Asparagine SIGMA-ALDRICH A0884-25G; CAS: 70-47-3
L-Aspartate SIGMA-ALDRICH A9506-25G; CAS: 2068-80-6
L-Cystine Wako Chemicals 037-05292; CAS: 56-89-3
L-Glutamine Wako Chemicals 074-00522; CAS: 56-85-9
L-Glutamate Wako Chemicals 070-00502; CAS: 56-86-0
L-Glycine Wako Chemicals 073-00732; CAS: 56-40-6
L-Histidine Wako Chemicals 084-00682; CAS: 71-00-1
L-Lysine HCL Wako Chemicals 121-01462; CAS: 657-27-2
L-Methionine Wako Chemicals 133-01602; CAS: 63-68-3
L-Phenylalanine Wako Chemicals 161-01302; CAS: 63-91-2
L-Proline Wako Chemicals 161-04602; CAS: 147-85-3
L-Serine Wako Chemicals 199-00402; CAS: 56-45-1
L-Threonine Wako Chemicals 204-01322; CAS: 72-19-5
L-Tryptophan Wako Chemicals 204-03382; CAS: 73-22-3
L-Tyrosine Wako Chemicals 202-03562; CAS: 60-18-4
(Continued on next page)

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Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Critical commercial assays
One-Step SYBR RT-PCR Kit Takara Bio Cat# RR086A
Mouse Insulin-like Growth Factor 1 (IGF-1) ASSAYPRO Cat# EMI1001-1
ELISA Kit
Corticosterone ELISA Kit ASSAYPRO Cat# EC3001-1
Mouse Insulin ELISA Kit Mercodia AB Cat# 10-1247-01
AccQ-Tag Ultra Derivatization Kit Waters Cat# 186003836
AccQ-Tag Ultra RP Column Waters Cat# 186003837
AccQ-Tag Ultra Eluent A Waters Cat# 186003838
AccQ-Tag Ultra Eluent B Waters Cat# 186003839
Experimental models: Organisms/Strains
Mouse: Kwl:ICR Tokyo Laboratory N/A
Animals Science
Mouse: C57BL/6J-Clockm1Jt/J Jackson Laboratory IMSR Cat# JAX:002923, RRID:IMSR_JAX:002923
Mouse: C57BL/6J-Bmal1 (flox/flox) mice Shimba et al., 2011 N/A
Mouse: B6.FVB(129S4)-Tg(Ckmm-cre)5Khn/J Jackson Laboratory IMSR Cat# JAX: 006475, RRID:IMSR_JAX:006475
Oligonucleotides
Primers for real-time RT-PCR, see Table S4 This paper N/A
Software and algorithms
ClockLab Actimetrics RRID:SCR_014309, URL: https://actimetrics.com/
products/clocklab/
GraphPad Prism version 7 and 8 GraphPad RRID:SCR_002798, URL: https://www.graphpad.com
IBM SPSS Statistics version IBM RRID:SCR_002865, URL: https://www.ibm.com/uk-
en/products/software
JTK_cycle Hughes et al., 2010 URL: https://openwetware.org/wiki/HughesLab:
JTK_Cycle
ImageJ N/A URL: https://imagej.nih.gov/nih-image/
Other
Area sensor Omron Cat# F5B
Frozen Cell Crusher: Cryo-Press MICROTEC Cat# CP-100WP
Piko Real PCR system Thermo Fisher Scientific Cat# 12675885
Image Quant LAS-3000 system GE healthcare N/A
ChemiDoc Touch Gel Imaging System Bio-Rad Laboratories Cat#1708370
Tissuelyser II QIAGEN Cat# 85300
Body Composition Analyzer InBody Cat# InBody 230
Stadiometer (seca213L) As One Cat# 62-6185-09
Omron Activity Monitor (Active style Pro) Omron Cat# HJA-750C
Grip Strength Dynamometer (T.K.K. 5401) Takei Cat# T-2177
Gait speed measure instrument (YW) Yagami Cat# 3421000

RESOURCE AVAILABILITY

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Shigenobu
Shibata (shibatas@waseda.jp).

Materials availability
This study did not generate new unique reagents.

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Data and code availability

This study did not include dataset and code.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animal experiments
Male Kwl:ICR mice, aged 5 weeks, were obtained from Tokyo Laboratory Animal Science. C57BL/6J-Clockm1Jt/J (Clock mutant)
mice were obtained from Jackson Laboratory, USA (JAX:002923), and backcrossed with ICR mice as described in our previous
report (Kamagata et al., 2017). Five to six-week-old male wild-type (WT) and Clock mutant (ClockD19) mice were used in this study.
Conditional Bmal1flox/flox and muscle-specific Bmal1 knockout (MKO) mice were generated, as described previously (Shimba et al.,
2011). To obtain MKO mice, we used mice expressing a Cre transgene driven by the muscle creatine kinase (Mck) promoter (Jackson
Laboratory, USA). Five- to ten-week-old male littermates, Bmal1flox/flox and MKO C57BL/6J mice were used in this study. The mice
were maintained in a conventional room maintained at 22 ± 2  C and 60 ± 5% humidity under a 12-h light (08:00 20:00)/dark cycle.
Zeitgeber time 0 (ZT0) was designated as lights-on time and ZT12 as lights-off time. The mice were provided with an American Insti-
tute of Nutrition (AIN)-93M diet (Reeves et al., 1993) and water. Experiments were performed in a non-blinded condition. This study
was approved by the Committee for Animal Experimentation at Waseda University [approval number: 2017-A077, 2019-A113] and
the animals were treated in accordance with the committee’s guidelines.

Human experiments
For the present study, cross-sectional data were collected from October 2017 to February 2018 in Tokyo (Japan). Sixty women aged
65 years and above (mean age, 69.6 ± 0.5 y) were recruited for this study. Sixty (n = 60) elderly women aged 65 years and above were
recruited for this study. This study was conducted on healthy elderly women with no regular exercise habits. Therefore, it was
intended only for humans who were confirmed to be on no medication, no disease history, and no smoking habit at the initial recruit-
ment stage (Table S3). The women were assigned to two groups: breakfast protein group (B-Pro, n = 18) and dinner protein group
(D-Pro, n = 42). Grouping was performed using the protein intake calculated from the food frequency questionnaire (FFQ) completed
by the participants. We advised the subjects in the B-Pro group to take a high-protein meal at breakfast and those in the D-Pro group
to do so at dinner. Before the study, informed consent was obtained from each participant after a detailed description of the study
(i.e., purpose, methods, and risk) was delivered. This study was conducted in accordance with the guidelines laid down in the Decla-
ration of Helsinki and was approved by the ethics committee of Waseda University [approval number: 2017-231(1)]. Participants were
recruited only if they met the following criteria: non-smoker, no known history of cardiovascular disease, and body mass index (BMI) <
30 kg/m2.

METHOD DETAILS

Animal experiments
Animal diets
All diets including high-protein diet, low-protein diet, and specific amino acid-supplemented diet were prepared on the basis of the
composition of AIN-93G or AIN-93M diets (Reeves et al., 1993). The composition of the various diets is shown in Table S1.
Two-meals per day regime
Mice were maintained in a 2-meals-per-day regime. For acclimation to this time-restricted feeding condition, mice were housed in
groups (four mice per cage) and were fed the AIN-93M diet in two 4 h windows each day (ZT12–16 and ZT20–0) for a week using an
automated time-restricted feeding device (Tahara et al., 2017). In the experiment where samples were collected at a single time point,
mice were provided the same amount of food at ZT12 and ZT20. The amount of food depended on the mouse strain (ICR mice: 2.0 g,
C57BL/6J: 1.4 g). We confirmed that the mice were able to consume the diet within 4 h. A hand-made automated feeding device was
used to provide food at each time point. In the experiment where sample collection was performed at multiple time points, mice were
housed (four mice per cage) and fed in two 4 h windows (ZT12–16 and ZT20–0) because there was not enough number of the auto-
mated feeding device. Mice were subsequently housed individually for 3 days before sampling and were manually provided with the
same amount of food (2.0 g) at ZT12 and ZT20.
Synergist ablation
The mice were anesthetized with 3% isoflurane (Wako Chemical, Osaka, Japan). Functional overload of the plantaris muscle was
induced by unilateral surgical ablation of the distal tendons of the gastrocnemius and soleus muscles as described previously (Bent-
zinger et al., 2013; Terena et al., 2017). The incision in the contralateral leg was closed without surgical ablation of the distal tendons,
and the plantaris muscle served as a sham muscle.
Locomotor activity analysis
Locomotor activity of mice was continuously monitored using an area sensor (F5B; Omron, Kyoto, Japan) and analyzed with Clock-
Lab software (Actimetrics, Wilmette, IL, USA) as previously described (Aoyama et al., 2018).

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Injection of 3-methyladenine (3-MA)

Vehicle (PBS) or 3-MA (30 mg/kg BW) was intraperitoneally injected once a day at ZT20 from the 2nd week. One week after two meals
per day, unilateral synergist ablation was performed to induce muscle hypertrophy. One week after the synergist ablation, the mus-
cles were collected.
Plasma IGF1, plasma insulin and serum corticosterone concentration
Plasma IGF1, plasma insulin and serum corticosterone levels were measured using a commercial kit (ASSAYPRO, St Charles, MO,
USA and Mercodia AB, Uppsala, Sweden). The assays were performed according to the manufacturer’s instructions.
Measurement of plasma and muscular free BCAA concentration
The concentrations of plasma and muscular free amino acids were measured using high-performance liquid chromatography with
pre-column 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization as previously described (Kanda et al., 2016). The
EDTA-treated plasma and the supernatant of perchloric acid extracts of the plantaris muscle were used.
Total RNA extraction and real-time RT-PCR
Total RNA was extracted from skeletal muscle using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Real-time reverse
transcription PCR was performed using the One-Step SYBR RT-PCR Kit (Takara Bio Inc., Shiga, Japan) with specific primer pairs
(Table S4) on a Piko Real PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The relative expression levels of the target
genes were normalized to those of the TATA box binding protein (Tbp). Data were analyzed using the DDCt method as described
in our previous report (Aoyama et al., 2018).
Protein extraction and western blotting
Frozen gastrocnemius muscles were ground into a powder using a frozen cell crusher (Cryo-Press, MICROTEC, Tokyo, Japan) and
homogenized using Tissuelyser II (QIAGEN, Frederick, MD, USA) with a homogenizing buffer (40 mM Tris, pH 7.5; 1 mM EDTA; 5 mM
EGTA; 0.5% Triton X-100) in the presence of a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA), and phospha-
tase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). After homogenization, samples were rotated for 1.5 h at 4 C, and then centri-
fuged at 14,000 3 g for 30 min at 4 C. Protein concentrations were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific,
Waltham, MA, USA). Western blotting analysis was conducted as previously described (Aoyama et al., 2016). Briefly, SDS-PAGE was
performed and the proteins in the gel were transferred onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Bucking-
hamshire, UK). The membranes were then incubated overnight at 4 C with the primary antibodies listed in the Key resources table.
The immunoreactive protein bands were detected with an enhanced chemiluminescence kit (GE Healthcare, Buckinghamshire, UK)
and quantified using a LAS-3000 system (GE Healthcare, Buckinghamshire, UK), ChemiDoc Touch Gel Imaging System (Bio-Rad
Laboratories, USA) and ImageJ (https://imagej.nih.gov/nih-image/).

Human experiments
Anthropometry
Body mass was measured to the nearest 0.1 kg using a digital balance (Inbody 230, InBody Co., Ltd., Tokyo, Japan). Height was
measured to the nearest 0.1 cm using a wall-mounted stadiometer (YS-OA, As One Corp., Japan). Body mass index (BMI) was calcu-
lated as weight in kilograms divided by the square of height in meters.
Dietary assessment
The FFQ is one of the most commonly used evaluation methods for meals. Most FFQs for Japanese people are highly effective in
estimating nutrients (Wakai, 2009). It is based on evaluating the content of meals by simple questions involving 29 food groups
and 10 cooking methods divided according to the food group. Eleven FFQ items were identified as primary protein sources, that
is, all food derived from animal products (meat, egg, milk, fish), cereals (e.g., rice, breads, pasta), and protein-rich vegetables (le-
gumes, soybean). The frequency of consumption of the main protein sources at each meal (breakfast, lunch, dinner) was recorded
(Bollwein et al., 2013). FFQ is relatively inexpensive, less burdensome to respondents, and does not require a trained interviewer
(Collins et al., 2014; Marks et al., 2006). Furthermore, previous studies have demonstrated the validity of the FFQ (Steinemann
et al., 2017; Sunami et al., 2016). The validity is also shown in the protein intake, which is the parameter evaluated in this study. In
addition, our survey included an image of the portion size of the meal in the questionnaire to improve data accuracy.
Average daily protein intake is depicted as grams per day (g/d), and as a percentage of daily energy intake (E%). Energy intake is
expressed as kcal/d. The amount of protein intake in each meal was calculated by adding the amount of protein obtained from the
primary protein sources per meal. All participants were requested to provide their eating pattern for the past month.
Physical activity assessment
To evaluate daily physical activity levels, all participants were asked to wear a triaxial accelerometer (Active style Pro HJA-750C, Om-
ron, Kyoto, Japan) for a week. The participants were asked to wear the accelerometer each day at all times except during the shower.
By measuring the magnitude and frequency of the acceleration, the device determined the level of intensity (METs) generated by
activity every 10 s from 0-8 METs (where 0 is the lowest and 8 is the highest). Data from the participants, who wore the accelerometer
for at least a total of 10 h (600 min) daily for at least four weekdays and a day on the weekend were included. In addition, the duration
of moderate to vigorous physical activity (MVPA) was calculated on a daily basis and used to estimate weekly activity by calculating a
weighted average of daily weekday and weekend activities [that is weekly MVPA = (average daily weekday MVPA 3 5) + (average
daily weekend MVPA 3 2)]. All minute recordings that were R 3 METs were classified as MVPA.

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Muscle mass
Muscle mass was measured via direct segmental multi-frequency bioimpedance analysis (InBody230, InBody Co., Ltd., Tokyo,
Japan). InBody230 uses a multi-frequency, segmental measurement method and an 8-point tactile electrode. Multi-frequency mea-
surements were conducted using multiple frequencies at 20 and 100 kHz for each body segment (arms, trunk, and legs). The analyzer
automatically displays measurements of lean body mass and fat mass. This analyzer has been assessed in normal populations, ath-
letes, elderly, and patients undergoing hemodialysis, and is closely correlated with the gold standard measurement using dual-en-
ergy X-ray absorptiometry (DEXA), underwater weight method, and air displacement plethysmography (Fu €rstenberg and Davenport,
2011; Kim et al., 2015; Utter and Lambeth, 2010; So et al., 2012). In addition, this tool is not based on statistical data from any specific
population. Thus, it can accurately assess people with very different body types, whether obese, elderly, or athletic (Cha et al., 1997;
Cha et al., 1995). The skeletal muscle index was calculated as appendicular muscle mass in kilograms divided by the square of height
in meters (Cruz-Jentoft et al., 2010).
Muscle strength
In the present study, muscle strength was measured by handgrip strength of both hands in the standing position as described earlier
(Granic et al., 2016). This was measured using a digital hand dynamometer (T.K.K.5401, Takei Scientific Instruments Co., Ltd., Nii-
gata, Japan). The standardized measurement protocol involved a standing position with the elbow straight by the side. For each
participant, the dynamometer was adjusted for the hand size. Two measurements were taken using the dominant hand, and the
mean of these values was used for the analysis.
Balance test
The participants were made to stand on one leg on a flat surface for as long as possible. The arms were held by the body with the eyes
opened. Time was measured with a stopwatch beginning from the moment when one foot was lifted from the floor and stopped if the
participant was unable to maintain the test position or if the participant could retain the test position for more than 120 s. Two mea-
surements were taken and a good record was used for the analysis.
The ‘sit to stand’ test
A previous study described the five times sit-to-stand test (Csuka and McCarty, 1985). When measuring the time taken to change
from a sitting to a standing position and vice versa, participants were instructed to stand up from sitting five times as quickly as
possible without using their arms for support. The total duration was recorded in seconds.
Time up and go
The Time Up and Go was used to assess the mobility and balance of the participants. Each participant was asked to sit on a standard
chair with their back against the chair. The participants were instructed to get up from the chair, walk to a line on the floor 3 m away at
a fast walking pace, turn, walk back to the chair, and sit down. The time taken to complete the test was recorded in seconds.
Usual gait speed
The 3 m gait speed test was used to evaluate gait speed. The participants were asked to walk over a 4 m course. Time was measured
for the last 3 m. Participants walked at their usual pace from a standing start and continued walking to a point past the line indicating
the end of the course. Usual gait speed was calculated by dividing distance in meters by the time in seconds (m/s). This was eval-
uated using a digital gait speed measuring instrument (YW, Yagami Co., Ltd., Tokyo, Japan).

QUANTIFICATION AND STATISTICAL ANALYSIS

Animal experiments
Data are presented as mean ± standard error (SE) values. GraphPad Prism version 7 and 8 (GraphPad Software) was used for statistical
analysis of animal experiments. To test whether data (sample size: n > 4) showed normal or non-normal distribution and equal or biased
variation, we used the Kolmogorov-Smirnov test and an F-test or Brown-Forsythe’s test, respectively. If the data showed normal dis-
tribution and equal variation, statistical significance was determined by unpaired t test or one-way ANOVA with a Tukey test or two-way
ANOVA with a Tukey test (if the interaction was significant) or Sidak test (if the interaction was not significant but the main effect was
significant) for post hoc analysis. If the data showed non-normal distribution or biased variation, statistical significance was determined
by Mann-Whitney U test or Kruskal-Wallis test with a two-stage linear step-up procedure from the Benjamini, Krieger, and Yekutieli test
for multiple comparisons. For data with a small sample size (n < 5), the statistical significance was determined using the Mann-Whitney
U test or Kruskal-Wallis test with a two-stage linear step-up procedure from the Benjamini, Krieger, and Yekutieli test for multiple com-
parisons. The JTK_cycle algorithm were used to detect day-24-h rhythms and to analyze day-night variation (Hughes et al., 2010).

Human experiments
Data are presented as mean ± SE values. SPSS Statistics version 25 (SPSS Japan Inc. Tokyo, Japan) was used for statistical analysis
of human experiments. All parameters were tested for normal or non-normal distributions using the Kolmogorov–Smirnov test. A two-
way repeated-measures analysis of variance was used to compare protein intake in each meal in both groups. When significant inter-
action effects were detected, we used the Tukey test for post hoc comparisons. To investigate the characteristics of participants
between B-Pro and D-Pro groups, we used an unpaired Student’s t test. Pearson’s product-moment correlation coefficient was
used to examine the relationship between the rate of protein intake in breakfast and SMI. P-values < 0.05 were considered to indicate
statistical significance.

e5 Cell Reports 36, 109336, July 6, 2021

Cell Reports, Volume 36

Supplemental information

Distribution of dietary protein intake

in daily meals influences skeletal muscle
hypertrophy via the muscle clock
Shinya Aoyama, Hyeon-Ki Kim, Rina Hirooka, Mizuho Tanaka, Takeru Shimoda, Hanako
Chijiki, Shuichi Kojima, Keisuke Sasaki, Kengo Takahashi, Saneyuki Makino, Miku
Takizawa, Masaki Takahashi, Yu Tahara, Shigeki Shimba, Kazuyuki
Shinohara, and Shigenobu Shibata
Figure S1. Effects of time-restricted protein or BCAAs feeding on body weight and locomotor
activity. Related to Figure 1.
(Figure legend continued on next page)

1
Figure S1 legend. (A, C) Body weight, (B, D) growth rate of mice in time-restricted protein-fed-mice
(20C−3C: 20% casein diet at breakfast and 3% casein diet at dinner, 11.5C−11.5C: 11.5% casein diet at
breakfast and dinner, 3C−20C: 3% casein diet at breakfast and 20% casein diet at dinner, 14C−3C: 14%
casein diet at breakfast and 3% casein diet at dinner, 8.5C−8.5C: 8.5% casein diet at breakfast and dinner,
3C−14C: 3% casein diet at breakfast and 14% casein diet at dinner). (E) Representative double plotted
actograms, (F, G) activity pattern and (H) total activity in time-restricted protein-fed mice. (I) Body weight
in time-restricted BCAA-fed mice (High B−3C: BCAA-supplemented diet at breakfast and 3% casein diet
at dinner, 3C−High B: 3% casein diet at breakfast and BCAA-supplemented diet at dinner). (M) Body
weight in time-restricted low BCAA-diet-fed mice (Low B−3C: low BCAA-diet at breakfast and 3% casein
diet at dinner, 3C−Low B: 3% casein diet at breakfast and low BCAA-diet at dinner). Representative double
plotted actograms (J, N), activity patterns (K, O) and total activity (L, P) in time-restricted high or low
BCAA-diet-fed mice. The experiments have been independently repeated. Mean ± SE, (A−D, F−H; n =
5−6, I, K, L, M, O, P; n = 6−8). ** P < 0.01, *** P < 0.001. Two-way ANOVA with Sidak test. ZT; Zeitgeber
time. n.s.; no significant difference.

Figure S2. Timing-dependent effects of BCAA ingestion on muscle hypertrophy and locomotor
activity in WT and Clock19 mice. Related to Figure 2.
(A) Sham-surgery and overloaded plantaris muscle weight, (B) ratio of overloading muscle weight to
contralateral sham-surgery muscle weight, (C) representative double plotted actograms, (D) hourly activity
pattern, (E) total activity and (F) activity ratio in the light and dark phase in time-restricted BCAA-fed wild
type (WT) and Clock mutant (Clock19) mice (High B−3C: high BCAA-diet at breakfast and 3% casein diet
at dinner, 3C−High B: 3% casein diet at breakfast and high BCAA-diet at dinner). The experiments have
been independently repeated. Mean ± SE, (n = 7−8). Two-way ANOVA with the Sidak test or unpaired t-
test, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < .0.0001. ZT; Zeitgeber time. n.s.; no significant
difference.

2
Figure S3. Day-night variation of muscular gene expression and blood hormone level in time-
restricted protein-fed mice. Related to Figure 3.
Mice fed high protein at breakfast (B-Pro) were fed a high-protein diet at breakfast and a low-protein diet
at dinner. Mice fed high protein at dinner (D-Pro) were fed a low-protein diet at breakfast and a high-protein
diet at dinner. Day-night oscillations of gene expression levels of (A) F-box protein 32 (known as Atrogin1),
(B) Solute carrier family 38, member 2 (Snat2), (C) Myostatin (Mstn), (D) Forkhead box O (Foxo)1, (E)
Foxo3, (F) Paired box (Pax)7, (G) Pax3, (H) Myogenic factor 6 (Myf6), (I) Period1 (Per1), (J) Circadian
locomotor output cycles kaput (Clock), (K) Cryptochrome (Cry)1, (L) Cry2, (M) RAR-related orphan
receptor alpha (Ror) in the sham-surgery and overloaded muscle of time-restricted protein-fed mice. Day-
night variations in (N) serum corticosterone and (O) plasma Insulin-like growth factor 1 (IGF-1) level in
the time-restricted protein-fed mice. The P value of JTK_CYCLE for rhythmicity is shown above white
and black bars. White and black bars mean light and dark condition. Each arrow means the meal time of
breakfast; B and dinner; D. Mean ± SE, (n = 4−6). Kruskal-Wallis test with two-stage linear step-up
procedure of Benjamini, Krieger and Yekutieli, * q < 0.05 (B-Pro vs. D-Pro at each time point). ZT;
Zeitgeber time. See also Table S4.

3
Figure S4. Day-night variation of blood and muscular free amino acids level in time-restricted protein-fed mice.
Related to Figure 3.
(Figure legend continued on next page)
4
Mice were maintained under two-meals-per-day conditions. Mice fed high protein at breakfast (B-Pro) were
fed a high-protein diet (20% casein) at breakfast and a low-protein diet (3% casein) at dinner. Mice fed high
protein at dinner (D-Pro) were fed a low-protein diet at breakfast and a high-protein diet at dinner. Day-
night variations in (A) plasma and (B) muscular free amino acid levels in time-restricted protein-fed mice.
Total amino acids; TAA, essential amino acids; EAA, nonessential amino acids; NEAA. The P value of
JTK_CYCLE for rhythmicity is shown in each panel. White and black bars indicate light and dark
conditions, respectively. Mean ± SE (n = 4). Kruskal-Wallis test with a two-stage linear step-up procedure
of Benjamini, Krieger, and Yekutieli, * q < 0.05, ** q < 0.01, *** q < 0.001, **** q < 0.0001 (B-Pro vs. D-
Pro at each time point). ZT; Zeitgeber time.

Figure S5. MYF5 and LC3-II protein level in the muscle of Clock19 and muscle-specific Bmal1 KO
mice fed a high-protein meal at breakfast or dinner. Related to Figure 4.
Representative blots and quantitative signal intensity of myogenic factor 5 (MYF5) and microtubule-
associated protein 1 light chain 3 beta II (MAP1LC3B-II, known as LC3-II) in time-restricted protein-fed
(A) wild-type (WT) and Clock mutant (Clock19) mice and (B) Bmal1flox/flox and muscle-specific Bmal1
knockout (MKO) mice. Protein levels were normalized using Coomassie Brilliant Blue (CBB). Mice in the
B-P group were fed a high-protein diet (20% casein: 20C) at breakfast and a low-protein diet (3% casein:
3C) at dinner. Mice in the D-P group were fed a low-protein diet at breakfast and a high-protein diet at
dinner. Mean ± SE, (A; n = 4, B; n = 4−5). ZT; Zeitgeber time.

5
Table S1 Diet compositions of animal experiment. Related to Figure STAR Methods.
High B Low B
Ingredient (%) 20C 14C 3C
(High BCAA) (Low BCAA)
Casein 20.0 14.0 3.0 3.0 3.0
Corn Starch 39.7486 46.5692 57.5692 54.3060 43.9126
a-Corn Starch 13.2 15.5 15.5 15.5 15.5
Sucrose 10.0 10.0 10.0 10.0 10.0
Soybean Oil 7.0 4.0 4.0 4.0 4.0
Cellulose 5.0 5.0 5.0 5.0 5.0
AIN*−93G Mineral MIX 3.5 − − − −
AIN−93M Mineral MIX − 3.5 3.5 3.5 3.5
AIN−93 Vitamin MIX 1.0 1.0 1.0 1.0 1.0
L-Cystine 0.3 0.18 0.18 0.18 0.18
Choline bitartrate 0.25 0.25 0.25 0.25 0.25
Tert−Butylhydroquinone 0.0014 0.0008 0.0008 0.0008 0.0008
L-Isoleucine − − − 0.816 −
L-Leucine − − − 1.428 −
L-Valine − − − 1.02 −
L-Alanine − − − − 0.464
L-Arginine − − − − 0.597
L-Asparagine − − − − 0.576
L-Aspartate − − − − 0.575
L-Cystine − − − − 0.091
L-Glutamine − − − − 1.667
L-Glutamate − − − − 1.667
L-Glycine − − − − 0.295
L-Histidine − − − − 0.462
L-Lysine HCL − − − − 1.605
L-Methionine − − − − 0.442
L-Phenylalanine − − − − 0.819
L-Proline − − − − 1.705
L-Serine − − − − 0.921
L-Threonine − − − − 0.693
L-Tryptophan − − − − 0.197
L-Tyrosine − − − − 0.882
Total 100 100 100 100 100
*AIN; American Institute of Nutrition

6
Table S2 Effects of time-restricted protein feeding on body and organ weights and the postprandial
response of insulin level. Related to Figure 3.
Items B-Pro (n = 7) D-Pro (n = 6)
Body weight (g) 37.6 ± 1.3 38.4 ± 0.9
Liver weight (g) 2.82 ± 0.16 2.65 ± 0.15
Relative liver weight (mg/gBW) 74.7 ± 1.8 68.7 ± 2.6
Epididymal fat weight (g) 0.78 ± 0.08 0.91 ± 0.06
Relative epdidymal fat weight (mg/gBW) 20.5 ± 1.6 23.7 ± 1.5
Postprandial response of plasma insulin B-Pro (n = 4) D-Pro (n = 4)
0 min 0.19 ± 0.07 0.70 ± 0.26
Time after 20C meal 30 min 0.54 ± 0.37 0.83 ± 0.31
(g/L) 60 min 0.40 ± 0.08 0.46 ± 0.13
120 min 0.74 ± 0.42 0.93 ± 0.16
0 min 0.34 ± 0.09 0.86 ± 0.35
Time after 3C meal 30 min 2.82 ± 0.76 4.66 ± 2.37
(g/L) 60 min 0.74 ± 0.24 1.84 ± 0.72
120 min 1.61 ± 0.41 3.07 ± 0.92
Mice in the B-Pro group were fed a high-protein diet (20% casein: 20C) at breakfast and a low-protein diet
(3% casein: 3C) at dinner. Mice in the D-Pro group were fed a low-protein diet at breakfast and a high-
protein diet at dinner. Mean ± SE.

7
Table S3 Characteristics of participants in Figure 5. Related to Figure 5.
Total Participants B-Pro D-Pro
Variables P value
n = 60 n = 18 n =42
Age (year) 69.57 ± 0.52 69.89 ± 1.01 69.43 ± 0.61 0.687
Height (cm) 155.30 ± 0.62 154.98 ± 1.33 155.44 ± 0.70 0.735
Body mass (kg) 54.85 ± 0.74 56.33 ± 1.48 54.22 ± 0.84 0.195
Fat mass (kg) 18.91 ± 0.53 19.63 ± 1.06 18.60 ± 0.60 0.371
BMI (kg/m2) 22.77 ± 0.29 23.49 ± 0.57 22.46 ± 0.33 0.102
Physical function
Muscle mass (kg/kg body mass) 0.34 ± 0.00 0.35 ± 0.01 0.34 ± 0.00 0.368
Hand grip (kg/kg body mass) 0.36 ± 0.01 0.38 ± 0.01 0.36 ± 0.01 0.197
Gait speed (m/s) 1.42 ± 0.02 1.45 ± 0.04 1.41 ± 0.03 0.471
Time Up and Go (s) 5.57 ± 0.10 5.63 ± 0.20 5.55 ± 0.12 0.717
Time sit to stand (s) 10.83 ± 0.38 10.52 ± 0.68 10.95 ± 0.47 0.609
One leg standing balance (s) 48.15 ± 4.90 42.31 ± 8.31 50.66 ± 6.05 0.440
Dietary intake
Energy intake (kcal/day) 2013.06 ± 76.12 1948.67 ± 108.78 2040.66 ± 98.72 0.584
Protein intake (en%) 15.27 ± 0.39 15.74 ± 0.81 15.07 ± 0.45 0.440
Fat intake (en%) 32.59 ± 0.70 32.65 ± 1.49 32.56 ± 0.78 0.954
Carbohydrate intake (en%) 52.14 ± 0.92 51.61 ± 1.96 52.37 ± 1.03 0.709
Animal-based protein (%) 54.41 ± 1.30 54.08 ± 2.45 54.56 ± 1.55 0.869
Physical activity
Step counts (steps/day) 6552.70 ± 382.00 7146.56 ± 787.53 6278.19 ± 429.54 0.313
MVPA (min/day) 78.50 ± 4.23 89.75 ± 9.00 73.68 ± 4.53 0.082

BMI; body mass index, en%; energy%, MVPA; moderate vigorous physical activity. Statistical P value between B-Pro

and D-Pro groups were shown.

8
Table S4 Primer sequence of real-time RT-PCR. Related to STAR Methods.
Primer Forward Reverse
Igf1 5’-agctggtggatgctgttcagtt-3’ 5’-atccacaatgcctgtctgaggt-3’
Pax3 5’-gcccacgtctattccacaa-3’ 5’-gaatagtgctttggtgtacagtgc-3’
Pax7 5’-gagttcgattagccgagtgc-3’ 5’-cgggttctgattccacatct-3’
Myf5 5’-ctgctctgagcccaccag-3’ 5’-gacagggctgttacattcagg-3’
Myf6 5’-agggcctcgtgataactg-3’ 5’-ggaagaaaggcgctgaaga-3’
Myog 5’-ccttgctcagctccctca-3’ 5’-tgggagttgcattcactgg-3’
Myod 5’-tacagtggcgactcagatgc-3’ 5’-tagtaggcggtgtcgtagcc-3’
Per1 5’-caagtggcaatgagtccaacg-3’ 5’-cgaagtttgagctcccgaagtc-3’
Per2 5’-tgtgtgcttacacgggtgtccta-3’ 5’-acgtttggtttgcgcatgaa-3’
Bmal1 5’-ccacctcagagccattgataca-3’ 5’-gagcaggtttagttccactttgtct-3’
Cry1 5’-atccgctgcgtctatatcctc-3’ 5’-cccgaatcacaaacagacg-3’
Cry2 5’-ttggcatctgtcccttcctg-3’ 5’-ggctcttgggtaggcatctc-3’
Clock 5'-aagattctgggtctgacaat-3' 5'-ttgcagcttgagacatcgct-3'
Reverb 5’-cttccgtgacctttctcagc-3’ 5’-cagctcctcctcggtaagtg-3’
Ror 5’-ggaagagctccagcagataacg-3’ 5’-gctgacatcagtacgaatgcag-3’
Atrogin1 5’-cagcttcgtgagcgacctc-3’ 5’-ggcagtcgagaagtccagtc-3’
Murf1 5’-accgagtgcagacgatcatctc-3’ 5’-aaagtcaatggccctcaaggc-3’
Foxo1 5’-cttcaaggataagggcgaca-3’ 5’-gacagattgtggcgaattga-3’
Foxo3 5’-gctaagcaggcctcatctca-3’ 5’-ttccgtcagtttgagggtct-3’
Mstn 5’-ggatgacagcagtgatggc-3’ 5’-gcttgccatccgcttgcattag-3’
Snat2 5’-caatgagatccgtgcaaaag-3’ 5’-tgcttccaatcatcaccact-3’
Ubc 5’-gaccagcagaggctgatctt-3’ 5’-cctctgaggcgaaggactaa-3’
Tbp 5’-cagcctcagtacagcaatcaac-3’ 5’-taggggtcataggagtcattgg-3’