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 distributiondependent effects require a muscle clock. A higher skeletal muscle index and grip strength were observed in older women who habitually consume a highprotein 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 ll OPEN ACCESS 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 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 2Organization SUMMARY The meal distribution of proteins throughout the day is usually skewed. However, its physiological implications 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 overloading-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 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 Agriculture [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 humans 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 volume in older adults (Norton et al., 2016). Thus, it is hypothesized that not only the total amount of protein intake but also its distribution across meals is important for muscle growth. This can be attributed to the postprandial anabolic threshold of protein being reached across three meals by equal distribution (Paddon-Jones and Rasmussen, 2009). However, digestive and absorptive capacities 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 knockouts 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/). ll OPEN ACCESS 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 humans 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 bioavailability 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-mealsper-day feeding. We show here that BCAAs are involved in inducing distribution-dependent hypertrophic effects. Additionally, 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 muscle 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-protein-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 overloadinginduced 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 suggest 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 (mutation in a whole body) and MKO mice. The high-protein breakfast enhanced the overloading-induced muscle hypertrophy in wildtype (WT) mice, but not in ClockD19 mice (Figures 2A and 2B). Similar results were observed in mice that had breakfast supplemented 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-protein 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 timedependent 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 twomeals-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-protein meal, and the timing of high-protein meals did not affect the elevation of plasma BCAA levels (Figures 3A–3C). A timingdependent 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 muscles of mice fed a high-protein meal at dinner (Figure S4B). In contrast, the muscle-free serine level was higher in the ll OPEN ACCESS Article 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 transporter) 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 highprotein 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 phosphorylation 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, Cell Reports 36, 109336, July 6, 2021 3 ll OPEN ACCESS Article 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 sham and overloaded muscles (Figure 3L; Figures S3F–S3H). Similar to Igf1 expression, the expressions of Myf5 and Myog 4 Cell Reports 36, 109336, July 6, 2021 were increased by overloading. The day-night variations in the B protein group were higher at ZT21 than at other time points (Figures 3J and 3K). The myogenic factor 5 (MYF5), but not ll Article OPEN ACCESS Figure 3. Day-night variation of free branched-chain amino acid level and gene expression in plasma and muscle of time-restricted proteinfed 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. Cell Reports 36, 109336, July 6, 2021 5 ll OPEN ACCESS Article 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 6 Cell Reports 36, 109336, July 6, 2021 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 assessed 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’ ll OPEN ACCESS Article 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 protein 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 highprotein 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 highprotein 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 Reverba, 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 breakfast 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 hypertrophic 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 administration 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 (Figure 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). Therefore, 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. Participants in the B protein group habitually ingested higher protein at breakfast than at dinner (Figures 5B and 5D). An opposite distribution 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 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 necessary 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 influence 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 skeletal 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 distribution 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 effects 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 addition, the expression levels of Igf1, Myog, and Myf5 and the autophagy 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 consumption 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 distribution of protein consumption influences muscle synthesis (Mamerow et al., 2014). In our study, feeding a high-protein meal in the late active phase (defined as dinner) attenuated muscle hypertrophy in mice. In comparison, mice that were fed a high-protein 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 influence the anabolic processes of overloading-induced muscle hypertrophy because this process was drastically altered immediately after synergistic ablation (Schiaffino et al., 2013). Second, Cell Reports 36, 109336, July 6, 2021 7 ll OPEN ACCESS Article 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) reported 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 distribution. Therefore, either the anabolic process and/or the experimental 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 overloading-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 8 Cell Reports 36, 109336, July 6, 2021 assumed that absorption and uptake of BCAAs in the small intestine and skeletal muscles could play a small role in the distribution-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 effects of dietary protein. Igf1 expression and the activation of protein 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 especially 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 ll Article protein group. In contrast, the activation of protein synthesis, assessed using the phosphorylation of S6K and mTOR, was not altered by the distribution of dietary protein intake. It was previously 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 after synergist ablation is important for skeletal muscle hypertrophy (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 signals being involved immediately after the induction of muscle hypertrophy 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 ablation of satellite cells during growth phase suppressed overloading-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 distribution-dependent hypertrophic effects. However, further studies are needed to assess myogenesis using isolated satellite 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 transcription of Myod and Myf5. In fact, an earlier in vitro study reported 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) expression levels were not affected by the distribution of dietary protein 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 upregulation of myogenic processes. Therefore, there is a possibility OPEN ACCESS that the upregulation of MYFs during skeletal muscle hypertrophy 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 muscles. Overloading increased LC3B protein levels, and this elevation 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. Muscle-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 muscle hypertrophic process, our pharmacological approach showed that the inhibition of autophagy by 3-MA injection attenuated overloading-induced muscle hypertrophy by a high-protein meal at breakfast, suggesting that the upregulation of LC3B-II could be involved in overloading-induced muscle hypertrophy. 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 molecular clocks (Brooks and Dang, 2019; Ma et al., 2011, 2012; Pastore et al., 2019). Additionally, it has been reported that the activation 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 (Martinez-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 underlying 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 results 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 ll OPEN ACCESS step counts and moderate to vigorous physical activity (MVPA) did not differ between the two groups, the participants in this human 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 human study exhibited high physical activity levels. Therefore, physical activity level could influence the positive correlation between protein intake at breakfast and SMI. However, no significant association was found between physical activity (MVPA and step counts) and SMI/muscle mass in this study. Similar results were obtained in a previous study of elderly women (Barbat-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 between 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 participants 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 between muscle functions and distribution of protein intake, focusing on two eating patterns characterized by even distribution 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 breakfast. 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. However, 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 intervention could help address age-associated muscle loss. Further studies targeting other populations, such as those with sarcopenia, 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 (Greenlund and Nair, 2003; Rosenberg, 1997). In particular, it has been reported that the rate of muscle mass loss accelerates after 60 years of age (Lexell, 1995; Lexell et al., 1988). It was also shown that females have a higher rate of muscle protein synthesis and myofibril synthesis due to a meal than males. Therefore, females are more dependent on dietary proteins for muscle 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 (muscle 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 regulation of myogenesis by the circadian clock may be involved in the effects dependent on protein feeding patterns. In our human 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 muscle volume. In the future, more detailed studies on the controlling mechanisms via the circadian clock will be required to ll Article OPEN ACCESS STAR+METHODS Andrews, J.L., Zhang, X., McCarthy, J.J., McDearmon, E.L., Hornberger, T.A., 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, 19090–19095. Detailed methods are provided in the online version of this paper and include the following: Aoyama, S., Jia, H., Nakazawa, K., Yamamura, J., Saito, K., and Kato, H. (2016). Dietary Genistein Prevents Denervation-Induced Muscle Atrophy in Male Rodents via Effects on Estrogen Receptor-a. J. Nutr. 146, 1147–1154. explore the effects of dietary protein distribution on muscle volume. d d d d d KEY RESOURCES TABLE RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability EXPERIMENTAL MODEL AND SUBJECT DETAILS B Animal experiments B Human experiments METHOD DETAILS B Animal experiments B Human experiments QUANTIFICATION AND STATISTICAL ANALYSIS B Animal experiments B Human experiments SUPPLEMENTAL INFORMATION Supplemental information can be found online at https://doi.org/10.1016/j. celrep.2021.109336. 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 Science (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. Takahashi, Y.T., S. Shimba, and K. Shinohara; formal analysis, S.A., H.-K.K., M. Tanaka, 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. 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Cell Reports 36, 109336, July 6, 2021 13 ll OPEN ACCESS Article 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) antibody Cell Signaling Technology Cat# 9234, RRID: AB_2269803 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) e1 Cell Reports 36, 109336, July 6, 2021 ll OPEN ACCESS Article 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) ELISA Kit ASSAYPRO Cat# EMI1001-1 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 Mouse: Kwl:ICR Tokyo Laboratory Animals Science N/A 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 This paper N/A 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/uken/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/ 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 Experimental models: Organisms/Strains Oligonucleotides Primers for real-time RT-PCR, see Table S4 Software and algorithms Other 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. Cell Reports 36, 109336, July 6, 2021 e2 ll OPEN ACCESS Article 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 Institute 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 recruitment 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 Declaration 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 automated 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 (Bentzinger 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 ClockLab software (Actimetrics, Wilmette, IL, USA) as previously described (Aoyama et al., 2018). e3 Cell Reports 36, 109336, July 6, 2021 ll Article OPEN ACCESS 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 muscles 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 phosphatase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). After homogenization, samples were rotated for 1.5 h at 4 C, and then centrifuged 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, Buckinghamshire, 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 calculated 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 (legumes, 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, Omron, 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. Cell Reports 36, 109336, July 6, 2021 e4 ll OPEN ACCESS Article 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 measurements 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, athletes, elderly, and patients undergoing hemodialysis, and is closely correlated with the gold standard measurement using dual-en€rstenberg and Davenport, ergy X-ray absorptiometry (DEXA), underwater weight method, and air displacement plethysmography (Fu 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., Niigata, 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 measurements 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 evaluated 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 distribution 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 comparisons. 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 twoway repeated-measures analysis of variance was used to compare protein intake in each meal in both groups. When significant interaction 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 Clock19 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 (Clock19) 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 ttest, * 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 timerestricted 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. Daynight 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. Daynight 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. DPro at each time point). ZT; Zeitgeber time. Figure S5. MYF5 and LC3-II protein level in the muscle of Clock19 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 microtubuleassociated 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 (Clock19) 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. Ingredient (%) Casein Corn Starch a-Corn Starch Sucrose Soybean Oil Cellulose AIN*−93G Mineral MIX AIN−93M Mineral MIX AIN−93 Vitamin MIX L-Cystine Choline bitartrate Tert−Butylhydroquinone L-Isoleucine L-Leucine L-Valine L-Alanine L-Arginine L-Asparagine L-Aspartate L-Cystine L-Glutamine L-Glutamate L-Glycine L-Histidine L-Lysine HCL L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tryptophan L-Tyrosine Total 20C 20.0 39.7486 13.2 10.0 7.0 5.0 3.5 − 1.0 0.3 0.25 0.0014 − − − − − − − − − − − − − − − − − − − − 100 14C 3C 14.0 46.5692 15.5 10.0 4.0 5.0 − 3.5 1.0 0.18 0.25 0.0008 − − − − − − − − − − − − − − − − − − − − 100 *AIN; American Institute of Nutrition 6 3.0 57.5692 15.5 10.0 4.0 5.0 − 3.5 1.0 0.18 0.25 0.0008 − − − − − − − − − − − − − − − − − − − − 100 High B (High BCAA) 3.0 54.3060 15.5 10.0 4.0 5.0 − 3.5 1.0 0.18 0.25 0.0008 0.816 1.428 1.02 − − − − − − − − − − − − − − − − − 100 Low B (Low BCAA) 3.0 43.9126 15.5 10.0 4.0 5.0 − 3.5 1.0 0.18 0.25 0.0008 − − − 0.464 0.597 0.576 0.575 0.091 1.667 1.667 0.295 0.462 1.605 0.442 0.819 1.705 0.921 0.693 0.197 0.882 100 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 D-Pro (n = 6) B-Pro (n = 7) 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 0 min 30 min Time after 20C meal 60 min (g/L) 120 min B-Pro (n = 4) D-Pro (n = 4) 0.19 ± 0.07 0.54 ± 0.37 0.40 ± 0.08 0.74 ± 0.42 0.70 ± 0.26 0.83 ± 0.31 0.46 ± 0.13 0.93 ± 0.16 0 min 30 min 60 min 120 min 0.34 ± 0.09 2.82 ± 0.76 0.74 ± 0.24 1.61 ± 0.41 0.86 ± 0.35 4.66 ± 2.37 1.84 ± 0.72 3.07 ± 0.92 Time after 3C meal (g/L) 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 highprotein diet at dinner. Mean ± SE. 7 Table S3 Characteristics of participants in Figure 5. Related to Figure 5. Total Participants B-Pro D-Pro 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 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 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 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 Variables P value Physical function Dietary intake Physical activity 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 Igf1 Pax3 Pax7 Myf5 Myf6 Myog Myod Per1 Per2 Bmal1 Cry1 Cry2 Clock Reverb Ror Atrogin1 Murf1 Foxo1 Foxo3 Mstn Snat2 Ubc Tbp Forward 5’-agctggtggatgctgttcagtt-3’ 5’-gcccacgtctattccacaa-3’ 5’-gagttcgattagccgagtgc-3’ 5’-ctgctctgagcccaccag-3’ 5’-agggcctcgtgataactg-3’ 5’-ccttgctcagctccctca-3’ 5’-tacagtggcgactcagatgc-3’ 5’-caagtggcaatgagtccaacg-3’ 5’-tgtgtgcttacacgggtgtccta-3’ 5’-ccacctcagagccattgataca-3’ 5’-atccgctgcgtctatatcctc-3’ 5’-ttggcatctgtcccttcctg-3’ 5'-aagattctgggtctgacaat-3' 5’-cttccgtgacctttctcagc-3’ 5’-ggaagagctccagcagataacg-3’ 5’-cagcttcgtgagcgacctc-3’ 5’-accgagtgcagacgatcatctc-3’ 5’-cttcaaggataagggcgaca-3’ 5’-gctaagcaggcctcatctca-3’ 5’-ggatgacagcagtgatggc-3’ 5’-caatgagatccgtgcaaaag-3’ 5’-gaccagcagaggctgatctt-3’ 5’-cagcctcagtacagcaatcaac-3’ Reverse 5’-atccacaatgcctgtctgaggt-3’ 5’-gaatagtgctttggtgtacagtgc-3’ 5’-cgggttctgattccacatct-3’ 5’-gacagggctgttacattcagg-3’ 5’-ggaagaaaggcgctgaaga-3’ 5’-tgggagttgcattcactgg-3’ 5’-tagtaggcggtgtcgtagcc-3’ 5’-cgaagtttgagctcccgaagtc-3’ 5’-acgtttggtttgcgcatgaa-3’ 5’-gagcaggtttagttccactttgtct-3’ 5’-cccgaatcacaaacagacg-3’ 5’-ggctcttgggtaggcatctc-3’ 5'-ttgcagcttgagacatcgct-3' 5’-cagctcctcctcggtaagtg-3’ 5’-gctgacatcagtacgaatgcag-3’ 5’-ggcagtcgagaagtccagtc-3’ 5’-aaagtcaatggccctcaaggc-3’ 5’-gacagattgtggcgaattga-3’ 5’-ttccgtcagtttgagggtct-3’ 5’-gcttgccatccgcttgcattag-3’ 5’-tgcttccaatcatcaccact-3’ 5’-cctctgaggcgaaggactaa-3’ 5’-taggggtcataggagtcattgg-3’ 9