Egg White Protein Feeding Facilitates Skeletal Muscle Gain in Young Rats with/without Clenbuterol Treatment

Based on the Digestible Indispensable Amino Acid Score (DIAAS), egg white protein (EGG) has an excellent score, comparable to that of whey protein but with a lower amount of leucine. We examined the effect of EGG feeding on rat skeletal muscle gain in comparison to that of two common animal-derived protein sources: casein (CAS) and whey (WHE). To explore the full potential of EGG, this was examined in clenbuterol-treated young rats. Furthermore, we focused on leucine-associated anabolic signaling in response to EGG after single-dose ingestion and chronic ingestion, as well as clenbuterol treatment. Because EGG is an arginine-rich protein source, a portion of the experiment was repeated with diets containing equal amounts of arginine. We demonstrated that EGG feeding accelerates skeletal muscle gain under anabolism-dominant conditions more efficiently than CAS and WHE and this stronger effect with EGG is not dependent on the arginine-rich composition of the protein source. We also demonstrated that the plausible mechanism of the stronger muscle-gain effect with EGG is not detectable in the mechanistic target of rapamycin (mTOR) or insulin signaling under our experimental conditions. We conclude that EGG may have a superior efficiency in muscle gain compared to other common animal-based proteins.


Introduction
Skeletal muscle mass is moderated by the balance between the rate of protein synthesis and protein breakdown, both of which are highly influenced by dietary protein intake [1]. Different types of protein sources cause the efficiency of muscle anabolism to vary, along with the amount of protein consumed. Therefore, the daily intake of high-quality protein through diet or supplementation has been recognized as a possible strategy for maximizing nutritional and training-induced anabolic effects on the skeletal muscle of athletes as well as older subjects experiencing sarcopenia [2,3]. Protein quality is mainly defined by the amount of essential amino acids and their bioavailability and digestibility, which are scored by various calculations [4]. Based on the Digestible Indispensable Amino Acid Score (DIAAS), animal-derived protein sources generally have higher scores than those of plant-derived proteins [5]. In accordance with this concept, several researchers have focused on the anabolic effect of animal-derived whey protein (WHE), the most popular protein source used in supplements on the consumer market. In comparison to soy protein, previous studies have demonstrated that single-dose (acute) WHE ingestion produced more muscle protein synthesis [6,7], and chronic WHE ingestion with resistance training led to more muscle gain [8]. This difference in anabolic effects among protein sources is at least partially attributed to different amounts of branched-chain amino acids (BCAA), particularly leucine, which is well characterized as a critical and powerful stimulant for inducing anabolic effects on skeletal muscle [9]. Leucine can augment muscle protein (Tokyo, Japan). CAS (Calcium Caseinate A, DMV, Netherlands), EGG (EAP-sport PLUS: SCETI, K.K., Japan), and WHE (Wheyco W80, Nippon Shinyaku, Japan) were obtained from SCETI, K.K., Japan. The amino acid composition of Diet 1 is shown in Table 2. To make the arginine concentration in the CAS and WHE diets equal to that of the EGG diets, arginine was added to both the CAS diet (460 mg/100 g) and WHE diet (632 mg/100 g), with the counter-addition of proline to the CAS diet (172 mg/100 g) and EGG diet (632 mg/100 g). The final amino acid composition in these arginine-modified diets is shown as Diet 2 in Table 2.  Arginine  610  1070  438  1063  1063  1063  Lysine  1400  1290  1790  1391  1282  1749  Histidine  457  385  332  454  383  330  Phenylalanine  935  1130  605  929  1123  601  Tyrosine  924  722  596  918  717  592  Leucine  1760  1600  2210  1749  1590  2196  Isoleucine  611  643  957  607  639  951  Methionine  554  750  452  551  745  449  Valine  788  844  877  783  839  871  Alanine  616  1240  1120  612  1232  1113  Glycine  357  730  383  355  725  381  Proline  2080  714  1280  2238  1338  1272  Glutamic  acid  4370  2720  3770  4343  2703  3746   Serine  1120  1500  1160  1113  1491  1153  Threonine  764  907  1380  759  901  1371  Aspartic  acid  1410  2150  2290  1401  2136  2276   Tryptophan  228  296  367  227  294  365  Cysteine  87  598  508  86 594 505

Experiment I: Acute Effect of EGG Feeding on Muscle Signal Transduction (on Day 0) under Normal Conditions (without Clenbuterol Treatment)
Rats were randomly assigned to either a fasting, CAS, EGG, or WHE group ( Figure 1). After 24 h of fasting, rats were allowed to feed with Diet 1 containing the assigned protein, while rats in the fasting group were maintained in the fasting state. One hour after feeding, blood was taken, and the plantaris muscles were clamp-frozen in liquid nitrogen for western blot analysis.

Experiment II: Chronic (7-Day) Effect of EGG Feeding under Normal Conditions Clenbuterol Treatment)
Rats were randomly assigned to one of the CAS, EGG, or WHE groups, maintained on Diet 1 and clenbuterol treatments for 3 or 7 days, as described abo for saline or clenbuterol administration ( Figure 1). On day 7, after 6 h of fasting and skeletal muscles were dissected out under anesthesia and weighed. On da rats were sacrificed after 6 h of fasting, and plantaris muscles were clamp-frozen nitrogen for western blot analysis.

Experiment III: Chronic Effect of EGG Feeding on Arginine Modification (without Clenbuterol Treatment)
Rats were randomly assigned to either a CAS, EGG, or WHE group, and we arginine-modified Diet 2 containing the respective protein source (Figure 1). T fed ad libitum for 7 days. On day 7, after the 6-h fasting, fat pads and skeleta were dissected out under anesthesia and weighed.

Experiment II: Chronic (7-Day) Effect of EGG Feeding under Normal Conditions (without Clenbuterol Treatment)
Rats were randomly assigned to one of the CAS, EGG, or WHE groups, and were maintained on Diet 1 and clenbuterol treatments for 3 or 7 days, as described above, except for saline or clenbuterol administration ( Figure 1). On day 7, after 6 h of fasting, fat pads and skeletal muscles were dissected out under anesthesia and weighed. On day 3, some rats were sacrificed after 6 h of fasting, and plantaris muscles were clamp-frozen in liquid nitrogen for western blot analysis.

Experiment III: Chronic Effect of EGG Feeding on Arginine Modification (without Clenbuterol Treatment)
Rats were randomly assigned to either a CAS, EGG, or WHE group, and were fed the arginine-modified Diet 2 containing the respective protein source (Figure 1). They were fed ad libitum for 7 days. On day 7, after the 6-h fasting, fat pads and skeletal muscles were dissected out under anesthesia and weighed.

Experiment IV: Acute Effect of Clenbuterol on Muscle Signal Transduction on Day 3
Rats were randomly assigned to one of the CAS+saline, CAS+clenbuterol, EGG+ clenbuterol, or WHE+clenbuterol groups, and were maintained for 2 days on Diet 1 for feeding and clenbuterol treatments as above ( Figure 1). On day 3, after 6 h of fasting and 20 h after the last clenbuterol administration, the rats received intraperitoneal injections of clenbuterol (1 mg/kg body weight) or saline alone. The rats were maintained in a fasting state under resting conditions for 2 h. Following this, the plantaris muscles were dissected out under anesthesia and clamp-frozen in liquid nitrogen for western blot analysis.

Experiment V: Chronic (7-Day) Effect of EGG Feeding with Clenbuterol
Rats were randomly assigned to one of the CAS+saline, CAS+clenbuterol, EGG+ clenbuterol, or WHE+clenbuterol groups, and were fed Diet 1 of the respective protein source ad libitum for 7 days (Figure 1). During this period, rats received once-daily intraperitoneal administration of either clenbuterol (1 mg/kg body weight: Toronto Research Chemicals, North York, Canada) dissolved in saline (2 mL/kg body weight) or saline alone, with daily measurement of food intake. On day 6, the last intraperitoneal administration was at 16:00. The next morning (at 06:00), the food was removed, and rats were maintained in the fasting state for 6 h. Following this, fat pads (epididymal and retroperitoneal) and skeletal muscles (soleus and plantaris) were dissected out under anesthesia and weighed. The plantaris muscles were clamp-frozen in liquid nitrogen for western blot analysis.

Amino Acid and Protein Compositions
The measurements of protein content in each protein source and the composition of amino acids in each diet were conducted by Sunatec, food analysis technology center, Yokkaichi, Japan.

Statistics
Values are expressed as means ± SE. Differences among multiple groups were determined using a one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test. When two mean values were compared, analysis was performed using an unpaired t-test. A value of p < 0.05 was considered significant.

Experiment I
We first attempted to identify the effect of EGG itself on muscle signaling ( Figure 2) and blood parameters (Figure 3) 1 h after the feeding. In response to each protein feeding, phosphorylation levels of Akt (Figure 2A), mTOR ( Figure 2B), and S6 ( Figure 2C) were amplified by several magnitudes compared to basal (being fasted) conditions. However, EGG-induced amplification was significantly lower for mTOR when compared to WHE (p < 0.05), and also weaker than both CAS and WHE in S6 (p < 0.05). For blood parameters, no significant difference was observed among groups except insulin ( Figure 3B) where WHE feeding led to a higher increase compared to EGG feeding (p < 0.05).

Experiment I
We first attempted to identify the effect of EGG itself on muscle signaling ( Figure 2) and blood parameters (Figure 3) 1 h after the feeding. In response to each protein feeding, phosphorylation levels of Akt (Figure 2A), mTOR ( Figure 2B), and S6 ( Figure 2C) were amplified by several magnitudes compared to basal (being fasted) conditions. However, EGG-induced amplification was significantly lower for mTOR when compared to WHE (p < 0.05), and also weaker than both CAS and WHE in S6 (p < 0.05). For blood parameters, no significant difference was observed among groups except insulin ( Figure 3B) where WHE feeding led to a higher increase compared to EGG feeding (p < 0.05). Figure 2. Acute effect of each protein feeding on muscle signal transduction (Day 0). Rats were assigned to either a casein, egg white, or whey protein diet group. After 24-h fasting, rats were allowed a feeding diet containing assigned protein.
One hour after the feeding, plantaris muscles were dissected out for western blot analysis of Akt (A), mTOR (B), or S6 (C). The dots beside bars indicate individual value. Values are means ± SE (n = 7). CAS (C): casein protein, EGG (E): egg white protein, WHE (W): whey protein. Different superscript represents statistical differences among groups. #: p < 0.05 vs. EGG group. Figure 2. Acute effect of each protein feeding on muscle signal transduction (Day 0). Rats were assigned to either a casein, egg white, or whey protein diet group. After 24-h fasting, rats were allowed a feeding diet containing assigned protein.
One hour after the feeding, plantaris muscles were dissected out for western blot analysis of Akt (A), mTOR (B), or S6 (C). The dots beside bars indicate individual value. Values are means ± SE (n = 7). CAS (C): casein protein, EGG (E): egg white protein, WHE (W): whey protein. Different superscript represents statistical differences among groups. #: p < 0.05 vs. EGG group.

Experiment II
Next, we examined the chronic effect of EGG feeding without clenbuterol treatment. Bodyweight throughout the experimental period was slightly higher following EGG feeding compared to WHE feeding, but it was not statistically significant ( Figure 4A). Although total food intake was identical among the groups ( Figure 4B), EGG feeding resulted in a significant and non-significant reduction in epididymal fat pad weight and retroperitoneal fat pad weight, respectively ( Figure 5A,B; p < 0.05). Under these conditions, in plantaris muscle from rats fed EGG, we also found a greater muscle-gain effect ( Figure 5C,D; p < 0.05). Regarding the soleus muscle, no difference was observed between groups. On day 3, when phosphorylation levels of Akt ( Figure 6A), mTOR ( Figure 6B), and S6 ( Figure 6C) were measured after the 6-h fasting, these levels were comparable regardless of protein source. rients 2021, 13, x FOR PEER REVIEW 8 o Figure 3. Acute effect of each protein feeding on blood parameters (Day 0). Rats were assigned to either a casein, egg white, or whey protein diet group. After 24-h fasting, rats were allowed a feeding diet containing assigned protein. One hour after the feeding, bloods were taken, and then were analyzed for measuring blood glucose (A), insulin (B), growth hormone (C), or insulin-like growth factor 1 (D

Experiment II
Next, we examined the chronic effect of EGG feeding without clenbuterol treatme Bodyweight throughout the experimental period was slightly higher following EGG fe ing compared to WHE feeding, but it was not statistically significant ( Figure 4A). A hough total food intake was identical among the groups ( Figure 4B), EGG feeding resul in a significant and non-significant reduction in epididymal fat pad weight and retrop itoneal fat pad weight, respectively ( Figure 5A,B; p < 0.05). Under these conditions . Acute effect of each protein feeding on blood parameters (Day 0). Rats were assigned to either a casein, egg white, or whey protein diet group. After 24-h fasting, rats were allowed a feeding diet containing assigned protein. One hour after the feeding, bloods were taken, and then were analyzed for measuring blood glucose (A), insulin (B), growth hormone (C), or insulin-like growth factor 1 (D). The dots beside bars indicate individual value. Values are means ± SE (n = 7). CAS: casein protein, EGG: egg white protein, WHE: whey protein. Different superscript represents statistical differences among groups. #: p < 0.05 vs. EGG group.   . Chronic effect of each protein feeding on body weight and food intake (during 7 days). Rats were assigned to either an egg white, or whey protein diet group, and were maintained under ad libitum feeding for 7 days without clenbuterol administration. Bodyweight (A) and food intake were measured daily. Total food intake (B) was the sum of consumed food during 7 days. The dots beside bars indicate individual value. Values are means ± SE (n = 5). EGG: egg white protein, WHE: whey protein. Different superscript represents statistical differences among groups.

Experiment III
In this experiment, we analyzed amino acid compositions in each diet used thus far (left side of Table 2 as Diet 1). A typical characteristic of EGG was that the amount of branched-chain amino acids (leucine, isoleucine, and valine) was almost equivalent to that in CAS, and lower than in WHE. Whereas amounts of arginine were two-fold higher in EGG compared to other proteins. Arginine is known as amino acid functioning in anabolic response [21,22]. It has a visceral fat-lowering effect [23]. Therefore, to examine whether the stronger muscle-gain effect and the stronger visceral fat-lowering effect induced by EGG was due to the higher content of arginine in EGG, we prepared another diet named "arginine-modified" Diet 2 (right side of Table 2). During the 7-day feeding period with Diet 2, body weights were not different at any point ( Figure 7A) and total food intake was identical among groups ( Figure 7B). After the period, we could not find a significant difference in fad pad weight among groups ( Figure 8A,B). In contrast, in skeletal muscle, the EGG-induced greater muscle-gain effect remained in plantaris muscle compared to other protein feedings ( Figure 8C,D). The soleus muscle of EGG-fed rats was also relatively bigger than others, but it was not statistically different ( Figure 8C,D).
fect of each protein feeding on body weight and food intake (during 7 days). Rats were assigned to or whey protein diet group, and were maintained under ad libitum feeding for 7 days without clenon. Bodyweight (A) and food intake were measured daily. Total food intake (B) was the sum of condays. The dots beside bars indicate individual value. Values are means ± SE (n = 5). EGG: egg white protein. Different superscript represents statistical differences among groups.  . Rats were assigned to either an egg white, or whey protein diet group, and were maintained under ad libitum feeding for 7 days without clenbuterol administration. On day 7, rats were fasted for 6 h, and then fat pads (A,B) and muscles (C,D) were dissected out. An absolute value (A,C) and a relative value to body weight (B,D) were calculated.

Experiment III
In this experiment, we analyzed amino acid compositions in each diet used thus far (left side of Table 2 as Diet 1). A typical characteristic of EGG was that the amount of branched-chain amino acids (leucine, isoleucine, and valine) was almost equivalent to that in CAS, and lower than in WHE. Whereas amounts of arginine were two-fold higher in EGG compared to other proteins. Arginine is known as amino acid functioning in anabolic response [21,22]. It has a visceral fat-lowering effect [23]. Therefore, to examine whether the stronger muscle-gain effect and the stronger visceral fat-lowering effect induced by EGG was due to the higher content of arginine in EGG, we prepared another diet named "arginine-modified" Diet 2 (right side of Table 2). During the 7-day feeding period with Diet 2, body weights were not different at any point ( Figure 7A) and total food intake was identical among groups ( Figure 7B). After the period, we could not find a significant difference in fad pad weight among groups ( Figure 8A,B). In contrast, in skeletal muscle, the EGG-induced greater muscle-gain effect remained in plantaris muscle compared to other protein feedings ( Figure 8C,D). The soleus muscle of EGG-fed rats was also relatively bigger than others, but it was not statistically different ( Figure 8C,D).  . Chronic effect of each protein feeding with arginine-modification on body weight and food intake (during 7 days). Rats were assigned to either a casein, egg white, or whey protein diet group, and were maintained under ad libitum feeding for 7 days without clenbuterol administration. Bodyweight (A) and food intake were measured daily. Total food intake (B) was the sum of consumed food during 7 days. The dots beside bars indicate individual value. Values are means ± SE (n = 7). CAS: casein protein, EGG: egg white protein, WHE: whey protein. Figure 7. Chronic effect of each protein feeding with arginine-modification on body weight and food intake (during 7 days). Rats were assigned to either a casein, egg white, or whey protein diet group, and were maintained under ad libitum feeding for 7 days without clenbuterol administration. Bodyweight (A) and food intake were measured daily. Total food intake (B) was the sum of consumed food during 7 days. The dots beside bars indicate individual value. Values are means ± SE (n = 7). CAS: casein protein, EGG: egg white protein, WHE: whey protein. Figure 8. Chronic effect of each protein feeding with arginine-modification on fat pad and muscle weight (Day 7). Rats were assigned to either a casein, egg white, or whey protein diet group, and were maintained under ad libitum feeding for 7 days without clenbuterol administration. On day 7, rats were fasted for 6 h, and then fat pads (A,B) and muscles (C,D) were dissected out. An absolute value (A,C) and a relative value to body weight (B,D) were calculated. The dots beside bars indicate individual value. Values are means ± SE (n = 7). CAS: casein protein, EGG: egg white protein, WHE: whey protein. #: p < 0.05 vs. other two groups.

Experiment IV
To examine whether the stronger muscle-gain effect of EGG was also superior to CAS and WHE under clenbuterol-treated conditions, we tested the acute effect of clenbuterol administration on these signaling molecules (Figure 9). Clenbuterol can increase cAMP level through activation of adenylate cyclase, and the increase finally leads to a CREB activation [24]. At 2 h post-administration, the CREB phosphorylation level, an indicator of cAMP-dependent response, was significantly amplified in all groups (A; p < 0.05 vs. CAS+saline) but the magnitude was identical among different protein sources. In these muscles, we also found considerable activation of Akt (B), mTOR (C), and S6 (D) in response to clenbuterol administration, with the lowest response observed in the CAS+clenbuterol group. In Akt and mTOR, these phosphorylation levels were significantly higher with EGG+clenbuterol and WHE+clenbuterol than those in the CAS+saline and CAS+clenbuterol groups (p < 0.05) but no significant difference was observed between EGG+clenbuterol and WHE+clenbuterol. muscles, we also found considerable activation of Akt (B), mTOR (C), and S6 (D) in response to clenbuterol administration, with the lowest response observed in the CAS+clenbuterol group. In Akt and mTOR, these phosphorylation levels were significantly higher with EGG+clenbuterol and WHE+clenbuterol than those in the CAS+saline and CAS+clenbuterol groups (p < 0.05) but no significant difference was observed between EGG+clenbuterol and WHE+clenbuterol. Figure 9. Acute effect of clenbuterol on muscle signal transduction (Day 3). Rats were assigned to either a casein, egg white, or whey protein diet group, and were maintained under ad libitum feeding for 2 days with/without saline or clenbuterol administration. On day 3, rats were administrated saline or clenbuterol. Two hours after the administration, plantaris muscles were dissected out for western blot analysis of CREB (A), Akt (B), mTOR (C), or S6 (D). The dots beside bars indicate individual value. Values are means ± SE (n = 6-7). CAS: casein protein, EGG: egg white protein, WHE: whey protein. Different superscript represents statistical differences among groups.

Experiment V
We further examined the effect of chronic EGG feeding under clenbuterol treatment. During the 7-day period, body weights in all groups were gradually increased ( Figure 10A). A tendency for greater gains began to appear in the EGG+clenbuterol group on day 2, and then on day 4, it reached a statistically significant level compared to the CAS+clenbuterol group (p < 0.05). Between EGG and WHE, body weight was greater in the EGG group throughout, but the difference was statistically significant only on day 6 (p < 0.05). The total food intake in the CAS+clenbuterol group was significantly lower than in other groups ( Figure 10B; p < 0.05). 10A). A tendency for greater gains began to appear in the EGG+clenbuterol group on day 2, and then on day 4, it reached a statistically significant level compared to the CAS+clenbuterol group (p < 0.05). Between EGG and WHE, body weight was greater in the EGG group throughout, but the difference was statistically significant only on day 6 (p < 0.05). The total food intake in the CAS+clenbuterol group was significantly lower than in other groups ( Figure 10B; p < 0.05). In general, clenbuterol treatment decreased both epididymal and retroperitoneal fat pad weights ( Figure 11). As shown, the greater body weight gain in the EGG group was not due to the facilitation of fat accumulation. Rather, fat pad weight after EGG feeding was the lowest compared to other groups, with a prominent decrease in the relative value of retroperitoneal fat pad weight when it was compared to the CAS+saline and WHE+clenbuterol groups ( Figure 11B; p < 0.05). In general, clenbuterol treatment decreased both epididymal and retroperitoneal fat pad weights ( Figure 11). As shown, the greater body weight gain in the EGG group was not due to the facilitation of fat accumulation. Rather, fat pad weight after EGG feeding was the lowest compared to other groups, with a prominent decrease in the relative value of retroperitoneal fat pad weight when it was compared to the CAS+saline and WHE+clenbuterol groups ( Figure 11B; p < 0.05).
In contrast to the effect of EGG feeding on fat pad weight, a marked increase was observed in muscle gain ( Figure 12). Clenbuterol treatment increased plantaris muscle weight in all groups, but the increase was significantly greater in the EGG group compared to other all groups ( Figure 12A,B; p < 0.05). The same tendency was also observed in the soleus muscle ( Figure 12A,B). The magnitude of the clenbuterol effect on the soleus muscle was relatively lower than that of the plantaris muscle but only EGG feeding led to a higher muscle gain compared to CAS+saline treatment (p < 0.05). Figure 11. Chronic effect of each protein feeding on fat pad weight (Day 7). Rats were assigned to either a casein, egg white, or whey protein diet group, and were maintained under ad libitum feeding for 7 days with or without clenbuterol administration. On day 7, rats were fasted for 6 h, and then fat pads were dissected out. An absolute value (A) and a relative value to body weight (B) were calculated. The dots beside bars indicate individual value. Values are means ± SE (n = 7). CAS: casein protein, EGG: egg white protein, WHE: whey protein. Different superscript represents statistical difference among groups in same fad pad (p < 0.05).
In contrast to the effect of EGG feeding on fat pad weight, a marked increase was observed in muscle gain (Figure 12). Clenbuterol treatment increased plantaris muscle Figure 11. Chronic effect of each protein feeding on fat pad weight (Day 7). Rats were assigned to either a casein, egg white, or whey protein diet group, and were maintained under ad libitum feeding for 7 days with or without clenbuterol administration. On day 7, rats were fasted for 6 h, and then fat pads were dissected out. An absolute value (A) and a relative value to body weight (B) were calculated. The dots beside bars indicate individual value. Values are means ± SE (n = 7). CAS: casein protein, EGG: egg white protein, WHE: whey protein. Different superscript represents statistical difference among groups in same fad pad (p < 0.05). Nutrients 2021, 13, x FOR PEER REVIEW 15 of 20 Figure 12. Chronic effect of each protein feeding on muscle weight (Day 7). Rats were assigned to either a casein, egg white, or whey protein diet group, and were maintained under ad libitum feeding for 7 days with or without clenbuterol administration. On day 7, rats were fasted for 6 h, and then muscles were dissected out. An absolute value (A) and a relative value to body weight (B) were calculated. To determine the molecular mechanism behind the EGG-induced facilitation in muscle gain, we examined the effect of EGG feeding on the phosphorylation levels of Akt and mTOR signaling molecules ( Figure 13). Although we expected to see an increase following chronic (7-day) clenbuterol treatment in the phosphorylation state of Akt (A) and mTOR (B), we observed a marked downregulation of these phosphorylation levels after 6-h fasting and 20 h post-clenbuterol administration, especially in the case of EGG feeding. The same tendency was also observed in phospho-S6 (C) which is a downstream target of mTOR. The levels of mTOR and S6 in the EGG+clenbuterol group were significantly lower than those in the CAS+saline and WHE+clenbuterol (p < 0.05) groups when these were measured 20 h after the last clenbuterol administration and the 6-h fasting. Figure 12. Chronic effect of each protein feeding on muscle weight (Day 7). Rats were assigned to either a casein, egg white, or whey protein diet group, and were maintained under ad libitum feeding for 7 days with or without clenbuterol administration. On day 7, rats were fasted for 6 h, and then muscles were dissected out. An absolute value (A) and a relative value to body weight (B) were calculated. The dots beside bars indicate individual value. Values are means ± SE (n = 7). CAS: casein protein, EGG: egg white protein, WHE: whey protein. Different superscript represents statistical difference among groups in same muscle (p < 0.05).
To determine the molecular mechanism behind the EGG-induced facilitation in muscle gain, we examined the effect of EGG feeding on the phosphorylation levels of Akt and mTOR signaling molecules ( Figure 13). Although we expected to see an increase following chronic (7-day) clenbuterol treatment in the phosphorylation state of Akt (A) and mTOR (B), we observed a marked downregulation of these phosphorylation levels after 6-h fasting and 20 h post-clenbuterol administration, especially in the case of EGG feeding. The same tendency was also observed in phospho-S6 (C) which is a downstream target of mTOR. The levels of mTOR and S6 in the EGG+clenbuterol group were significantly lower than those in the CAS+saline and WHE+clenbuterol (p < 0.05) groups when these were measured 20 h after the last clenbuterol administration and the 6-h fasting. Figure 13. Chronic effect of each protein feeding on muscle signal transduction (Day 7). Rats were assigned to either a casein, egg white, or whey protein diet group, and were maintained under ad libitum feeding for 6 days with or without clenbuterol administration. On day 7, rats were fated for 6 h, and then plantaris muscles were dissected out for western blot analysis of Akt (A), mTOR (B), or S6 (C). The dots beside bars indicate individual value. Values are means ± SE (n = 7). CAS: casein protein, EGG: egg white protein, WHE: whey protein. CLE: clenbuterol. Different superscript represents statistical differences among groups.

Discussion
Aside from the negative concept linking egg consumption and the adverse effects of cholesterol, there is accumulating evidence that eggs and egg-derived foods perform positive functions in promoting health [25]. In regards to consumption of EGG and EGG hydrolysate, several previous studies conducted by Ochiai et al. have provided evidence that these protein feedings inhibited the accumulation of intracellular triglyceride in the liver and skeletal muscle together with visceral mass in insulin-resistant animals compared to those consuming CAS [17][18][19]. They also found EGG to have an inhibitory effect on food intake when fed a high-fat diet, and they expanded their measurement of this to include muscle gain. One study [18] demonstrated that EGG feeding induced greater muscle gain despite lower food intake, and another study [19] showed that this effect of EGG on muscle was not observed when it was compared under pair-fed conditions. These results seem difficult to interpret for us, and we did not observe the inhibitory effect on food intake; however, we identified the stronger visceral fat-reducing effect in the EGG group compared to both the CAS and WHE groups. Note that in these studies muscle mass was expressed as a value relative to body weight [17][18][19], so bodyweight reduction accompanied by a visceral fat mass reduction in EGG feeding might influence the muscle mass calculation through a secondary effect [18]. In the present study, we clearly demonstrated that the main result of EGG feeding is muscle gain, in the calculation of both absolute and relative value, under anabolism-dominant conditions. This was demonstrated in comparison to not only CAS but also WHE, and regardless of a lower leucine amount.
We also provided a result showing that EGG-induced facilitation in muscle gain is apparent without clenbuterol treatment. Therefore, we focused on the contribution of EGG itself. When EGG was ingested, activation levels of mTOR and S6 were statistically lower than those of other protein sources or WHE 1 h after EGG feeding. Although we did not measure leucine concentration in blood, the level might be lower in EGG than others, and this would be a plausible candidate for lower activation of mTOR signaling. Figure 13. Chronic effect of each protein feeding on muscle signal transduction (Day 7). Rats were assigned to either a casein, egg white, or whey protein diet group, and were maintained under ad libitum feeding for 6 days with or without clenbuterol administration. On day 7, rats were fated for 6 h, and then plantaris muscles were dissected out for western blot analysis of Akt (A), mTOR (B), or S6 (C). The dots beside bars indicate individual value. Values are means ± SE (n = 7). CAS: casein protein, EGG: egg white protein, WHE: whey protein. CLE: clenbuterol. Different superscript represents statistical differences among groups.

Discussion
Aside from the negative concept linking egg consumption and the adverse effects of cholesterol, there is accumulating evidence that eggs and egg-derived foods perform positive functions in promoting health [25]. In regards to consumption of EGG and EGG hydrolysate, several previous studies conducted by Ochiai et al. have provided evidence that these protein feedings inhibited the accumulation of intracellular triglyceride in the liver and skeletal muscle together with visceral mass in insulin-resistant animals compared to those consuming CAS [17][18][19]. They also found EGG to have an inhibitory effect on food intake when fed a high-fat diet, and they expanded their measurement of this to include muscle gain. One study [18] demonstrated that EGG feeding induced greater muscle gain despite lower food intake, and another study [19] showed that this effect of EGG on muscle was not observed when it was compared under pair-fed conditions. These results seem difficult to interpret for us, and we did not observe the inhibitory effect on food intake; however, we identified the stronger visceral fat-reducing effect in the EGG group compared to both the CAS and WHE groups. Note that in these studies muscle mass was expressed as a value relative to body weight [17][18][19], so bodyweight reduction accompanied by a visceral fat mass reduction in EGG feeding might influence the muscle mass calculation through a secondary effect [18]. In the present study, we clearly demonstrated that the main result of EGG feeding is muscle gain, in the calculation of both absolute and relative value, under anabolism-dominant conditions. This was demonstrated in comparison to not only CAS but also WHE, and regardless of a lower leucine amount.
We also provided a result showing that EGG-induced facilitation in muscle gain is apparent without clenbuterol treatment. Therefore, we focused on the contribution of EGG itself. When EGG was ingested, activation levels of mTOR and S6 were statistically lower than those of other protein sources or WHE 1 h after EGG feeding. Although we did not measure leucine concentration in blood, the level might be lower in EGG than others, and this would be a plausible candidate for lower activation of mTOR signaling. Also, given that insulin is capable of activating mTOR [9], lower insulin secretion response after EGG would affect mTOR activation levels observed 1 h after EGG feeding.
Although EGG has an almost equivalent or lower leucine amount than CAS and WHE, it is known to be an arginine-rich protein source [15]. This higher arginine amount in EGG was one of the plausible explanations for the mechanism of its stronger muscle-gain effect on skeletal muscle, as arginine stimulates the anabolic hormone secretion. For example, a single arginine administration stimulated insulin [21] and growth hormone [22] secretion immediately after administration. In the present study, blood parameter responses immediately after each protein feeding were similar among groups except for insulin (Figure 10), which was lower in rats fed EGG. We cannot exclude the possibility that an insufficient number of blood sampling points might confound the overall trend. However, under our limited experimental conditions, the acute response of IGF-1, GH, and insulin could not explain the stronger muscle-gain effect of EGG.
Whereas young mammals have been used as a good experimental model to estimate the effect of nutrients or food on the whole body or fractional anabolic effect, it should be noted that arginine is categorized as a semi-essential amino acid and is needed for optimal growth in young mammals, but not in adults [26]. This is also true in rats and is supported by a study by Milner et al. [27], which demonstrated that rats fed an arginine-deficient diet, from immediately post-lactation until 18 days later, showed lower body weights compared to those fed a standard diet. In contrast to the results in the comparison to definitive conditions like "deficient", Suzumura et al. [28] demonstrated that when young (5-weeks old) rats ate a 20% CAS-based diet with additional arginine supplementation for 10 days, body weights were not different from the 20% CAS-based diet without additional arginine supplementation. This result implies that even during a growing period, arginine demands in the body and arginine supply (arginine intake and its synthesis) might reach a balancing point for skeletal muscle when rats eat arginine presented in 20% CAS. Besides these surrounding evidence for our interpretation, we could not find direct evidence showing the arginine-independent effect of EGG feeding in the previous study. Definitely, by using an arginine-modified diet, we again confirmed the biggest muscle mass in rats fed EGG diet. This is the first evidence, to our knowledge, to demonstrate that EGG-induced facilitation in muscle gain is not dependent on the arginine content in the protein source.
We found that EGG feeding resulted in a marked reduction in visceral fat mass under growth and clenbuterol-treated conditions. This is in accordance with previous studies [17][18][19] but the mechanism has not been well documented. Regarding this question, we found that an arginine-modified diet eliminated the fat-reducing effect of the EGG. Previously, Fu et al. [23] demonstrated that additional arginine supplementation for 10 weeks reduced visceral fat mass with marked bodyweight reduction in Zucker diabetic fatty rats, through arginine-dependent nitric oxide production. In that study, the authors also found that skeletal muscle mass was increased after additional arginine supplementation when it was expressed as a relative value, whereas it was identical when it was expressed as an absolute value. Given these results, our present results indicate that in contrast to the case of skeletal muscle, the abundant arginine content of EGG plays a predominant role in facilitating visceral fat mass reduction after EGG feeding.
To emphasize the potential of EGG and to see whether the EGG-induced facilitation in muscle gain would appear under highly anabolism-dominant conditions, we used clenbuterol. Clenbuterol has been known to be a strong anabolic reagent and it can induce significant muscle hypertrophy within a few days [29][30][31]. In accordance with these results, we successfully produced muscle hypertrophy after a 7-day treatment with the 1 mg/kg clenbuterol. This was observed mainly in fast-twitch glycolytic plantaris muscle, regardless of protein source. Traditionally, it has been thought that clenbuterol induces hypertrophy through a pathway related to adenylate cyclase-mediated cAMP production and the subsequent protein kinase A-CREB activation pathway [24]. More recently, in a manner similar to the leucine-induced anabolic effect, activation of the phosphatidylinositol 3kinase (PI3K)-Akt-mTOR pathway has been recognized to regulate the clenbuterol-induced anabolic effect [32]. When we measured phosphorylation levels of Akt and mTOR at day 7, following a 6-h fasting and 20 h post-clenbuterol administration, phospho-Akt and phospho-mTOR levels were downregulated in all groups treated with clenbuterol ( Figure 13), and the lowest these levels were observed in the most hypertrophied muscle in rats fed EGG. Because we did not measure blood parameters in this situation, we cannot mention whether the changes in blood would affect muscle signaling. However, our signaling results are not surprising because generally most, but not all [32], clenbuterolinduced anabolic responses (e.g., activated anabolic signaling and muscle protein synthesis) were more pronounced 2 to 3 days after starting clenbuterol treatment, and returned to basal or lower than basal levels after 7 days or more [31], although the increased muscle mass persisted [30]. We interpret these downregulations as proof of the most powerful effect of EGG in terms of negative feedback for preventing excess hypertrophy. Concurrently, these results also could suggest that the stronger muscle-gain effect of EGG is unlikely to be due to the absence of adequate negative feedback regulation. To further investigate, we also examined the acute effect of clenbuterol on these phosphorylations as well as CREB levels 2 h after injection. Although those in the CAS group were relatively lower compared to other protein sources, the highest increase in these activations was not observed in rats fed EGG. Taken together, these results indicate that sensitivity against clenbuterol to mTOR and insulin signaling is unlikely to explain the mechanism of EGG-induced facilitation in muscle gain.
We have also provided insight into clenbuterol-induced anabolic effects on skeletal muscle. Clenbuterol has a powerful anabolic effect and appears to cause physiological skeletal mass to achieve maximum efficiency. However, few animal studies have shown clenbuterol treatment to result in an additive anabolic response when combined with muscle activity-associated stimuli/conditions (i.e., tenotomy-induced compensatory hypertrophy) [29] or recovery conditions from immobilization-induced atrophy [33]. Similarly, GH administration was also reported to amplify the anabolic effect of clenbuterol [34]. To the best of our knowledge, this is the first evidence that the protein source, especially EGG, is an additional factor for accelerating the anabolic efficiency of clenbuterol. It is known that clenbuterol is a beta-adrenergic agonist and the potential contribution of the beta-adrenergic pathway for exercise-induced hypertrophy has been documented [35]. EGG might be a beneficial protein source for athletes with a desire to increase their muscle mass.
In the present study, we had only single sampling points for blood and muscle signal transductions, therefore this is a limitation and a point for further discussion. Nevertheless, our effort to identify the mechanism for the stronger muscle-gain effect induced by EGG on mTOR and insulin signaling might not be a good strategy considering our experimental conditions. Insufficient activation of mTOR signaling after single-dose EGG ingestion has also been reported previously [36]. Additional examination of relevant signaling pathways including muscle protein breakdown pathways would provide a clearer interpretation. Alternatively, whole egg, egg-derived foods including EGG, and egg-derived peptides are gaining attention because of their reported functions in countering type 2 diabetes via their antioxidant and anti-inflammatory properties [37]. The mechanism which is attributed to one or more common factors related to these positive effects might have some role in the present stronger muscle-gain effect in EGG. Further study is needed to resolve this molecular mechanism.
As another limitation of our experiments, we did not measure the effect of EGG feeding on muscle power output, muscle fiber-type compositions, or cross-sectional area. These surrounding results would be convincing proof. In addition, we cannot conclude from the present study whether EGG can induce its stronger muscle-gain effect under conditions that are catabolism-dominant, such as cachexia, malnutrition-associated muscle atrophy, muscle disuse, or sarcopenia associated with these conditions. We hope that these questions can be answered by future research.

Conclusions
In the present study, we demonstrated that EGG facilitates skeletal muscle gain under highly anabolism-dominant conditions compared to CAS and WHE and this stronger muscle-gain effect of EGG is not dependent on an arginine-rich protein source. The plausible mechanism of the stronger muscle-gain effect induced by EGG was not detectable in mTOR and insulin signaling under our experimental conditions. We conclude that EGG may have a superior efficiency in muscle gain compared to other common animal-based proteins.