Egg White Protein Feeding Facilitates Skeletal Muscle Gain in Young Rats With/Without Clenbuterol Treatment

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

Keiichi Koshinaka 1,*, Asuka Honda 1, Rihei Iizumi 1, Yuto Miyazawa 1, Kentaro Kawanaka 2 and Akiko Sato 1

1 Department of Health and Sports, Niigata University of Health and Welfare, 1398 Shimami-cho, Kita-ku, Niigata 950-3198, Japan; [email protected] (A.H.); [email protected] (R.I.); [email protected] (Y.M.); [email protected] (A.S.) 2 Faculty of Sports and Health Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan; [email protected] * Correspondence: [email protected]

Abstract: 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 Citation: Koshinaka, K.; Honda, A.; Iizumi, R.; Miyazawa, Y.; Kawanaka, on the arginine-rich composition of the protein source. We also demonstrated that the plausible K.; Sato, A. Egg White Protein mechanism of the stronger muscle-gain effect with EGG is not detectable in the mechanistic target of Feeding Facilitates Skeletal Muscle rapamycin (mTOR) or insulin signaling under our experimental conditions. We conclude that EGG Gain in Young Rats with/without may have a superior efﬁciency in muscle gain compared to other common animal-based proteins. Clenbuterol Treatment. Nutrients 2021, 13, 2042. https://doi.org/ Keywords: egg white protein; casein protein; whey protein; muscle gain; clenbuterol; arginine; mTOR 10.3390/nu13062042

Academic Editors: Sara Salucci and Maria Luz Fernandez 1. Introduction Skeletal muscle mass is moderated by the balance between the rate of protein synthesis Received: 1 May 2021 and protein breakdown, both of which are highly influenced by dietary protein intake [1]. Accepted: 12 June 2021 Different types of protein sources cause the efficiency of muscle anabolism to vary, along Published: 15 June 2021 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 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in nutritional and training-induced anabolic effects on the skeletal muscle of athletes as published maps and institutional affil- well as older subjects experiencing sarcopenia [2,3]. Protein quality is mainly defined by iations. 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 Copyright: © 2021 by the authors. protein source used in supplements on the consumer market. In comparison to soy protein, Licensee MDPI, Basel, Switzerland. This article is an open access article previous studies have demonstrated that single-dose (acute) WHE ingestion produced distributed under the terms and more muscle protein synthesis [6,7], and chronic WHE ingestion with resistance training conditions of the Creative Commons led to more muscle gain [8]. This difference in anabolic effects among protein sources is Attribution (CC BY) license (https:// at least partially attributed to different amounts of branched-chain amino acids (BCAA), creativecommons.org/licenses/by/ particularly leucine, which is well characterized as a critical and powerful stimulant for 4.0/). inducing anabolic effects on skeletal muscle [9]. Leucine can augment muscle protein

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synthesis independently of all other amino acids through activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling which is thought to be a primary pathway for muscle protein synthesis [9–11]. In parallel, the activation of the protein kinase B (Akt) pathway in insulin signaling also results in the activation of the mTORC1 pathway with simultaneous inhibition of protein breakdown by Forkhead box-containing protein O1 (FOXO1) [9]. The importance of sufficient dietary leucine to facilitate muscle protein synthesis is supported by previous studies showing that suboptimal ingestion of low amounts of protein stimulated muscle protein synthesis compared to the levels achieved with high amounts of protein when leucine was added [12,13]. WHE is leucine-rich (8.6 g/100 g protein) compared to typical plant-derived soy protein (5 g/100 g protein). Interestingly another animal-derived protein, egg white protein (EGG), contains lower levels of leucine (3.6 g/100 g protein) [14] but has an excellent score of 1.45 according to the Digestible Indispensable Amino Acid Score (DIAAS) formula and is comparable to whey protein (1.45 in DIAAS) [15]. Indeed, a previous study provided evidence that EGG supplementation in young adult males for 5 weeks, along with exercise training, effectively facilitated muscle strength adaptation with a large gain in lean body mass and cross-sectional area of skeletal muscle [16]. This suggests EGG is a useful and effective protein source for obtaining more muscle, but this anabolic effect has not been compared with that of other sources of protein supplementation [16]. While several studies did compare CAS versus WHE, or WHE versus plant-derived protein sources, there is scant information regarding the effectiveness of EGG on muscle anabolism compared to other excellent animal-derived protein sources [2]. In comparison to casein (CAS), EGG feeding reduced visceral fat mass in insulin-resistant animals [17–19]. A recent study conducted by Matsuoka et al. [20] demonstrated in an animal study that EGG ingestion resulted in greater body weight gain, food efficiency (body weight gain/food consumption), and protein efficiency (body weight gain/protein intake) compared to WHE. Although these authors did not discuss skeletal muscle mass, this result has given rise to the hypothesis that EGG might have a superior anabolic effect on skeletal muscle compared to other animal- derived proteins such as CAS and WHE, nevertheless with a lower amount of leucine. Therefore, in the present study, we examined the effect of EGG feeding on rat skeletal muscle gain in comparison to that of the common animal-derived protein sources CAS and WHE. To emphasize the potential of EGG, the effect was examined in clenbuterol-treated young rats, which is considered as the condition of being highly anabolism-dominant. We hypothesize that EGG feeding would result in greater muscle gain. Furthermore, we focused on the activation level of leucine-associated anabolic signaling in response to EGG after single-dose ingestion and after chronic ingestion, along with the interaction of clenbuterol treatment. Because EGG is an arginine-rich protein source and arginine functions in an anabolic hormone secretion promoter [21,22], a portion of the experiment was repeated with diets containing equal amounts of arginine.

2. Materials and Methods 2.1. Animals This research was approved by the Animal Studies Committee of Niigata University of Health and Welfare (29006-02103, 29016-30005, 02009, 29016). Male Wistar rats (4 weeks old) were obtained from CLEA Japan (Tokyo, Japan). Rats were housed individually at constant room temperature (23 ± 1 ◦C) in a 12-h light (06:00−18:00 h) and 12-h dark cycle and were provided standard laboratory chow (MF: Oriental Yeast, Tokyo, Japan) and water ad libitum until the experiment commenced. After an acclimatization period of one week, all rats were used in the experiment.

2.2. Experimental Diets The exact composition of each protein source is shown in Table1. According to these amounts, the protein concentration in Diet 1 was set as 20 g in 100 g diet (original diet). All materials, with the exception of protein sources, were obtained from Oriental Yeast Nutrients 2021, 13, 2042 3 of 20

(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 Table2. 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 Table2.

Table 1. Composition of nutrients in protein sources and diets.

CAS EGG WHE In each protein source (g/100g) Protein 91 81 78 Fat 1 1 6 In each diet (Diet 1) (g/100g) Casein protein 22 - - Egg white protein - 25 - Whey protein - - 26 Sucrose 5 5 5 α-Corn starch 46 47 47 Corn oil 11 7 6 Vitamin mix (AIN-76) 2 2 2 Mineral mix (AIN-76) 6 6 6 Cellulose 8 8 8 Final protein content 20 20 20 Final fat content 12 12 12

Table 2. Composition of amino acid.

Original (Diet 1) Arginine-modiﬁed (Diet 2) (mg/100g) CAS EGG WHE CAS EGG WHE 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 4370 2720 3770 4343 2703 3746 acid Serine 1120 1500 1160 1113 1491 1153 Threonine 764 907 1380 759 901 1371 Aspartic 1410 2150 2290 1401 2136 2276 acid Tryptophan 228 296 367 227 294 365 Cysteine 87 598 508 86 594 505 Nutrients 2021, 13, 2042 4 of 20

2.3. 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 (Figure1 ). After 24 h of fasting, rats were allowed to feed with Diet 1 containing the assigned protein, Nutrients 2021, 13, x FOR whilePEER REVIEW rats in the fasting group were maintained in the fasting state. One hour after feeding, 5 of 20 blood was taken, and the plantaris muscles were clamp-frozen in liquid nitrogen for western blot analysis.

Figure 1. Experimental design.Figure 1. Experimental design. 2.4. Experiment II: Chronic (7-Day) Effect of EGG Feeding under Normal Conditions (without Clenbuterol2.4. Treatment) Experiment II: Chronic (7-Day) Effect of EGG Feeding under Normal Conditions (without Rats wereClenbuterol randomly Treatment) assigned to one of the CAS, EGG, or WHE groups, and were maintained on DietRats 1 andwere clenbuterol randomly treatments assigned for to 3 one or 7 days,of the as CAS, described EGG, above, or WHE except groups, and were for saline ormaintained clenbuterol on administration Diet 1 and clenbuterol (Figure1). On treatments day 7, after for 6 3 h or of 7 fasting, days, as fat described pads above, except and skeletalfor muscles saline or were clenbuterol dissected outadministration under anesthesia (Figure and weighed.1). On day On 7, day after 3, some6 h of fasting, fat pads rats were sacrificed after 6 h of fasting, and plantaris muscles were clamp-frozen in liquid and skeletal muscles were dissected out under anesthesia and weighed. On day 3, some nitrogen for western blot analysis. rats were sacrificed after 6 h of fasting, and plantaris muscles were clamp-frozen in liquid 2.5. Experimentnitrogen III: Chronic for western Effect of blot EGG analysis. Feeding on Arginine Modification (without Clenbuterol Treatment) Rats were2.5. Experiment randomly assigned III: Chronic to either Effect a CAS, of EG EGG,G Feeding or WHE on group,Arginine and Modification were fed the (without arginine-modifiedClenbuterol Diet Treatment) 2 containing the respective protein source (Figure1). They were fed ad libitum for 7 days. On day 7, after the 6-h fasting, fat pads and skeletal muscles were Rats were randomly assigned to either a CAS, EGG, or WHE group, and were fed the dissected out under anesthesia and weighed. 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.

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2.6. 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 (Figure1). 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.

2.7. 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 (Figure1). 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.

2.8. Western Blot Analysis Plantaris muscles were homogenized in ice-cold buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM Na3VO4, 2 mM PMSF, aprotinin (10 µg/mL), and leupeptin (10 µg/mL). The homogenates were rotated end-over-end at 4 ◦C for 60 min and then centrifuged at 10,000× g for 10 min at 4 ◦C. Aliquots of supernatants were used for immunoblot analysis. Brieﬂy, supernatants were electrophoretically separated by SDS- PAGE and transferred to PVDF membranes. The membranes were incubated overnight at 4 ◦C with primary antibodies, followed by incubation for 60 min with appropriate HRP-conjugated second antibodies. Primary antibodies against mTOR, phospho-mTOR (Ser2448), Akt, phospho-Akt (Thr308), S6, and phospho-S6 (Ser235/236), cAMP response element-binding protein (CREB), and phospho-CREB (Ser133) were obtained from Cell Sig- naling Technology (Beverly, MA). Immunoreactive bands were visualized using enhanced chemiluminescence reagent (GE Healthcare Japan, Hino, Japan), and quantiﬁed using NIH Image software.

2.9. Blood Parameters Blood glucose levels were measured using the product Glutest Every (Sanwa Kagaku, Nagoya, Japan). Enzyme immunoassay kits were used for measuring insulin (Morinaga Institute of Biological Science, Yokohama, Japan), growth hormone (SPI-BIO, Montigny le Bretonneux, France), and IGF-1 ((R&D Systems, Minneapolis, MN, USA).

2.10. 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.

2.11. Statistics Values are expressed as means ± SE. Differences among multiple groups were deter- mined 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. Nutrients 2021, 13, x FOR PEER REVIEW 7 of 20

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3. Results 3.1.3. Results Experiment I 3.1. Experiment I We first attempted to identify the effect of EGG itself on muscle signaling (Figure 2) and bloodWe first parameters attempted (Figure to identify 3) 1 theh after effect the of feeding. EGG itself In response on muscle to signaling each protein (Figure feeding,2) and blood parameters (Figure3) 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 phosphorylation levels of Akt (Figure2A), mTOR (Figure2B), and S6 (Figure2C) were amplified by several magnitudes compared to basal (being fasted) conditions. However, amplified by several magnitudes compared to basal (being fasted) conditions. However, EGG-inducedEGG-induced amplification amplification waswas significantlysignificantly lower lower for for mTOR mTOR when when compared compared to WHEto WHE ((pp << 0.05), 0.05), and and also also weaker weaker thanthan bothboth CAS CAS and and WHE WHE in in S6 S6 (p (

Figure 2. Acute effect of each protein feeding on muscle signal transduction (Day 0). Rats were assigned to either a casein, Figureegg white, 2. Acute or wheyeffect proteinof each dietprotein group. feeding After on 24-h muscle fasting, signal rats transd were alloweduction (Day a feeding 0). Rats diet were containing assigned assigned to either protein. a casein, eggOne white, hour or after whey the protein feeding, diet plantaris group. muscles After 24-h were fasting, dissected rats out were for westernallowed blota feeding analysis diet of containing Akt (A), mTOR assigned (B), orprotein. S6 One(C). hour The after dots besidethe feeding, bars indicate plantaris individual muscles value.were dissected Values are out means for western± SE (n =blot 7). analysis CAS (C): of casein Akt ( protein,A), mTOR EGG (B (E):), oregg S6 (C).

Thewhite dots protein, beside WHEbars indicate (W): whey individual protein. Differentvalue. Values superscript are means represents ± SE statistical(n = 7). CAS differences (C): casein among protein, groups. EGG #: (E):p < 0.05egg vs.white protein, WHE (W): whey protein. Different superscript represents statistical differences among groups. #: p < 0.05 vs. EGG EGG group. group. 3.2. 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 (Figure4A). Although total food intake was identical among the groups (Figure4B), EGG feeding resulted in a significant and non-significant reduction in epididymal fat pad weight and retroperitoneal fat pad weight, respectively (Figure5A,B; p < 0.05). Under these conditions, in plantaris muscle from rats fed EGG, we also found a greater muscle-gain effect (Figure5C,D ; p < 0.05). Regarding the soleus muscle, no difference was observed between groups. On day 3, when phosphorylation levels of Akt (Figure6A), mTOR (Figure6B), and S6 (Figure6C) were measured after the 6-h fasting, these levels were comparable regardless of protein source.

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Figure 3. Acute effect of each protein feeding on blood parameters (Day 0). Rats were assigned to either a casein, egg white, Figure 3. orAcute whey effect protein of diet each group. protein After 24-h feeding fasting, on rats blood were allowed parameters a feeding (Day diet containing0). Rats were assigned assigned protein. Oneto either hour after a casein, egg white, or thewhey feeding, protein bloods diet were group. taken, After and then 24-h were fasting, analyzed rats for were measuring allowed blood a feedin glucoseg (Adiet), insulin containing (B), growth assigned hormone protein. One hour after( Cthe), or feeding, insulin-like bloods growth were factor taken, 1 (D). The and dots th besideen were bars analyzed indicate individual for measuring value. Values blood are glucose means ± (SEA), (n insulin= 7). CAS: (B), growth hormone casein(C), or protein, insulin-like EGG: egg growth white protein,factor 1 WHE: (D). wheyThe dots protein. beside Different bars superscript indicate representsindividual statistical value. differencesValues are among means ± SE (n = 7). CAS:groups. casein #: protein,p < 0.05 vs.EGG: EGG egg group. white protein, WHE: whey protein. Different superscript represents statistical differences among groups. #: p < 0.05 vs. EGG group.

3.2. 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). Alt- hough 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.

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Figure 4. Chronic effect of each protein feeding on body weight and food intake (during 7 days). Rats were assigned Figure 4. toChronic either an effect egg white, of each or wheyprotein protein feeding diet group, on body and wereweight maintained and food under intake ad libitum (during feeding 7 days). for 7 Rats days withoutwere assigned to either an eggclenbuterol white, administration. or whey protein Bodyweight diet group, (A) and and food were intake maintained were measured under daily. ad Total libitum food f intakeeeding (B )for was 7 thedays sum without of clenbuterol administration.consumed food duringBodyweight 7 days. The(A) dots and beside food barsintake indicate were individual measured value. daily. Values Total are food means intake± SE (n (B=) 5). was EGG: the egg sum of consumed foodwhite during protein, 7 days. WHE: wheyThe dots protein. beside Different bars superscript indicate representsindividual statistical value. differencesValues are among means groups. ± SE (n = 5). EGG: egg white protein, WHE: whey protein. Different superscript represents statistical differences among groups. 3.3. Experiment III In this experiment, we analyzed amino acid compositions in each diet used thus far (left side of Table2 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 Table2). During the 7-day feeding period with Diet 2, body weights were not different at any point (Figure7A) and total food intake was identical among groups (Figure7B). After the period, we could not find a significant difference in fad pad weight among groups (Figure8A,B). In contrast, in skeletal muscle, the EGG-induced greater muscle-gain effect remained in plantaris muscle compared to other protein feedings (Figure8C,D). The soleus muscle of EGG-fed rats was also relatively bigger than others, but it was not statistically different (Figure8C,D).

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Figure 4. 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.Nutrients The dots2021, 13 beside, 2042 bars indicate individual value. Values are means ± SE (n = 5). EGG: egg white9 of 20 protein, WHE: whey protein. Different superscript represents statistical differences among groups.

Figure 5. Chronic effect of each protein feeding on fat pad and muscle weight (Day 7). 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. 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. EGG group. Nutrients 2021, 13, x FOR PEER REVIEW 10 of 20

Figure 5. Chronic effect of each protein feeding on fat pad and muscle weight (Day 7). 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. The dots beside bars indicate individual value. Values Nutrients 2021, 13, 2042 10 of 20 are means ± SE (n = 7). CAS: casein protein, EGG: egg white protein, WHE: whey protein. #: p < 0.05 vs. EGG group.

Figure 6. Chronic effect of each protein feeding on muscle signal transduction (Day 3). Rats were assigned to either a casein, Figure 6. Chronic effect of each protein feeding on muscle signal transduction (Day 3). Rats were assigned to either a casein,egg white,egg white, or whey or whey protein protein diet group, diet group, and were and maintained were maintained under adunder libitum ad libitum feeding feeding for 2 days for without 2 days clenbuterolwithout clen- Nutrients 2021, 13, xbuterol FORadministration. PEERadministration. REVIEW On day On 3, ratsday were3, rats fasted were for fasted 6 h, and for then6 h, plantarisand then musclesplantaris were mu dissectedscles were out dissected for western out blot for analysiswestern of blot 11 of 20 analysisAkt (A of), mTORAkt (A (),B ),mTOR or S6 ((CB).), Theor S6 dots (C beside). The dots bars indicatebeside bars individual indicate value. individual Values value. are means Values± SE are (n =means 8). CAS: ± SE casein (n = 8). CAS:protein, casein EGG: protein, egg whiteEGG: protein,egg wh WHE:ite protein, whey WHE: protein. whey protein.

3.3. 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).

Figure 7. Chronic effect of each protein feeding with arginine-modiﬁcation on body weight and food intake (during 7 days). Figure 7. ChronicRats were effect assigned of each to either protein a casein, feedin egg white,g with or whey arginine-modification protein diet group, and were on maintained body weight under ad and libitum food feeding intake (during 7 days). Rats werefor 7assigned days without to clenbuteroleither a casein, administration. egg white, Bodyweight or whey (A) protein and food intakediet group, were measured and were daily. maintained Total food intake under ad libitum feeding for 7 days(B) was without the sum of clenbute consumedrol food administration. during 7 days. The Bodyweight dots beside bars ( indicateA) and individual food intake value. were Values measured are means ± daily.SE Total food intake (B) was(n the= 7). sum CAS: of casein consumed protein, EGG: food egg during white protein, 7 days. WHE: The whey dots protein. beside bars indicate individual value. Values are means ± SE (n = 7). CAS: casein protein, EGG: egg white protein, WHE: whey protein.

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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 Nutrientsfeeding2021, for13, 20427 days without clenbuterol administration. Bodyweight (A) and food intake were measured daily. Total food11 of 20 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.

3.4. 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 (Figure9). 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. Nutrients 2021, 13, x FOR PEER REVIEW 12 of 20

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.

3.4. 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+clen- Nutrients 2021, 13, 2042 buterol groups (p < 0.05) but no significant difference was observed between12 EGG+clen- of 20 buterol 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.

3.5. 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). Nutrients 2021, 13, x FOR PEER REVIEW 13 of 20

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.

3.5. 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). Nutrients 2021, 13, 2042 The total food intake in the CAS+clenbuterol group was significantly lower than13 of in 20 other groups (Figure 10B; p < 0.05).

Figure 10. Chronic effect of each protein feeding on body weight and food intake (during 7 days). Rats were assigned Figureto 10. either Chronic a casein, effect egg white,of each or protein whey protein feeding diet on group, body andweight were an maintainedd food intake under (during ad libitum 7 days). feeding Rats for were 7 days assigned with to eitheror a withoutcasein, egg clenbuterol white, administration.or whey protein Bodyweight diet group, (A and) and were food maintained intake were measuredunder ad daily.libitum Total feeding food intake for 7 (daysB) was with or withoutthe sumclenbuterol of consumed administration. food during Bodyweight 7 days. The dots (A) besideand food bars intake indicate were individual measured value. daily. Values Total are meansfood intake± SE ((nB=) 7).was the sum ofCAS: consumed casein protein, food during EGG: egg 7 days. white The protein, dots WHE:beside whey bars protein.indicate *: individualp < 0.05 vs. value. CAS+clenbuterol Values are group.means #:± pSE< ( 0.05n = vs.7). CAS: caseinWHE+clenbuterol protein, EGG: group.egg white $: p < pr 0.05otein, vs. otherWHE: three whey groups. protein. *: p < 0.05 vs. CAS+clenbuterol group. #: p < 0.05 vs. WHE+clenbuterol group. $: p < 0.05 vs. other three groups. In general, clenbuterol treatment decreased both epididymal and retroperitoneal fat padIn weightsgeneral, (Figure clenbuterol 11). As treatment shown, thedecreased greater bodyboth epididymal weight gain inand the retroperitoneal EGG group fat padwas weights not due (Figure to the 11). facilitation As shown, of fat the accumulation. greater body Rather, weight fat gain pad in weight the EGG after group EGG was notfeeding due to was the the facilitation lowest compared of fat accumulati to other groups,on. withRather, a prominent fat pad weight decrease after in the EGG relative feeding value of retroperitoneal fat pad weight when it was compared to the CAS+saline and was the lowest compared to other groups, with a prominent decrease in the relative value WHE+clenbuterol groups (Figure 11B; p < 0.05). of retroperitonealIn contrast to fat the effectpad ofweight EGG feedingwhen onit fatwas pad compared weight, a markedto the increaseCAS+saline was and WHE+clenbuterolobserved in muscle groups gain (Figure(Figure 12 11B;). Clenbuterol p < 0.05). 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).

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Figure 11. Chronic effect of each protein feeding on fat pad weight (Day 7). Rats were assigned to either a casein, egg Figurewhite, or11. whey Chronic protein effect diet group, of each and were protein maintained feeding under on ad fat libitum pad feedingweight for (Day 7 days 7). with Rats or without were clenbuterolassigned to eitheradministration. a casein, On egg day white, 7, rats were or fastedwhey for protein 6 h, and thendiet fat group, pads were and dissected were out.maintained An absolute under value (A ad) and libitum a relative ± feedingvalue to for body 7 weightdays (withB) were or calculated. without Theclenbuterol dots beside admi bars indicatenistration. individual On value.day 7, Values rats arewere means fastedSE for (n = 6 7). h, CAS: casein protein, EGG: egg white protein, WHE: whey protein. Different superscript represents statistical difference andamong then groups fat pads in same were fad pad dissec (p < 0.05).ted 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 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).

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Figure 12. Chronic effect of each protein feeding on muscle weight (Day 7). Rats were assigned to either a casein, egg 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 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 administration. On day 7, rats were fasted for 6 h, and then muscles were dissected out. An absolute value (A) and a ± valuerelative to value body weightto body ( Bweight) were ( calculated.B) were calculated. The dots The beside dots bars beside indicate bars indica individualte individual value. Values value. are Values means are meansSE (n =± SE 7). CAS:(n = 7). casein CAS: protein, casein protein, EGG: egg EGG: white egg protein, white protein, WHE:whey WHE: protein. whey protein. Different Different superscript superscript represents represents statistical statistical difference dif- amongference groupsamong ingroups same musclein same (musclep < 0.05). (p < 0.05).

To determinedetermine thethe molecular molecular mechanism mechanism behind behind the the EGG-induced EGG-induced facilitation facilitation in in muscle mus- gain,cle gain, we we examined examined the the effect effect of of EGG EGG feeding feeding on on the the phosphorylation phosphorylation levels levelsof of AktAkt andand mTOR signaling moleculesmolecules (Figure(Figure 1313).). Although wewe expectedexpected toto see an increase following chronic (7-day) clenbuterol treatment inin thethe phosphorylationphosphorylation statestate ofof AktAkt (A)(A) andand mTORmTOR (B),(B), wewe observedobserved a a marked marked downregulation downregulation of of these these phosphorylation phosphorylation levels levels after after 6-h 6-h fasting fast- and 20 h post-clenbuterol administration, especially in the case of EGG feeding. The same ing and 20 h post-clenbuterol administration, especially in the case of EGG feeding. The tendency was also observed in phospho-S6 (C) which is a downstream target of mTOR. The same tendency was also observed in phospho-S6 (C) which is a downstream target of levels of mTOR and S6 in the EGG+clenbuterol group were signiﬁcantly lower than those mTOR. The levels of mTOR and S6 in the EGG+clenbuterol group were significantly lower in the CAS+saline and WHE+clenbuterol (p < 0.05) groups when these were measured 20 h than those in the CAS+saline and WHE+clenbuterol (p < 0.05) groups when these were after the last clenbuterol administration and the 6-h fasting. measured 20 h after the last clenbuterol administration and the 6-h fasting.

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Figure 13. Chronic effect of each protein feeding on muscle signal transduction (Day 7). Rats were assigned to either a 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 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 clenbuterol administration. On day 7, rats were fated for 6 h, and then plantaris muscles were dissected out for western ± analysisblot analysis of Akt of (A Akt), mTOR (A), mTOR (B), or S6(B), (C or). TheS6 (C dots). The beside dots bars beside indicate bars individualindicate individual value. Values value. are Values means areSE means (n = 7±). SE CAS: (n = casein7). CAS: protein, casein EGG: protein, egg whiteEGG: protein,egg white WHE: protein, whey WHE: protein. whey CLE: protein. clenbuterol. CLE: clenbuterol. Different superscript Different superscript represents statisticalrepresents differencesstatistical differences among groups. among groups.

4.4. Discussion Discussion AsideAside from from thethe negativenegative conceptconcept linking egg consumption consumption and and the the adverse adverse effects effects of ofcholesterol, cholesterol, there there is isaccumulating accumulating evidence evidence that that eggs eggs and and egg-derived egg-derived foods foods perform perform pos- positiveitive functions functions in inpromoting promoting health health [25]. [25 In]. Inregards regards to toconsumption consumption of EGG of EGG and and EGG EGG hy- hydrolysate,drolysate, several several previous previous stud studiesies conducted conducted by by Ochiai Ochiai et al. et al.have have provided provided evidence evidence that thatthese these protein protein feedings feedings inhibit inhibiteded the theaccumulation accumulation of intracellular of intracellular triglyceride triglyceride in the in liver the liverand andskeletal skeletal muscle muscle together together with with visceral visceral mass mass in ininsulin-resistant insulin-resistant animals animals compared compared to tothose those consuming consuming CAS CAS [17–19]. [17–19]. They They also also found found E EGGGG to have anan inhibitoryinhibitory effect effect on on food food intakeintake when when fed fed a a high-fat high-fat diet, diet, and and they they expanded expanded their their measurement measurement of of this this to to include include muscle gain. One study [18] demonstrated that EGG feeding induced greater muscle gain 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 despite lower food intake, and another study [19] showed that this effect of EGG on mus- was not observed when it was compared under pair-fed conditions. These results seem cle was not observed when it was compared under pair-fed conditions. These results difficult to interpret for us, and we did not observe the inhibitory effect on food intake; seem difficult to interpret for us, and we did not observe the inhibitory effect on food however, we identified the stronger visceral fat-reducing effect in the EGG group compared intake; however, we identified the stronger visceral fat-reducing effect in the EGG group to both the CAS and WHE groups. Note that in these studies muscle mass was expressed compared to both the CAS and WHE groups. Note that in these studies muscle mass was as a value relative to body weight [17–19], so bodyweight reduction accompanied by a expressed as a value relative to body weight [17–19], so bodyweight reduction accompa- visceral fat mass reduction in EGG feeding might influence the muscle mass calculation nied by a visceral fat mass reduction in EGG feeding might influence the muscle mass through a secondary effect [18]. In the present study, we clearly demonstrated that the calculation through a secondary effect [18]. In the present study, we clearly demonstrated main result of EGG feeding is muscle gain, in the calculation of both absolute and relative that the main result of EGG feeding is muscle gain, in the calculation of both absolute and value, under anabolism-dominant conditions. This was demonstrated in comparison to notrelative only CASvalue, but under also anabolism-dominant WHE, and regardless conditions. of a lower leucineThis was amount. demonstrated in compar- isonWe to not also only provided CAS but a result also WHE, showing and that regardless EGG-induced of a lower facilitation leucine amount. in muscle gain is apparentWe withoutalso provided clenbuterol a result treatment. showing Therefore, that EGG-induced we focused facilitation on the contribution in muscle of gain EGG is itself.apparent When without EGG was clenbuterol ingested, treatment. activation levelsTherefore, of mTOR we focused and S6 were on the statistically contribution lower of thanEGG those itself. of When other EGG protein was sources ingested, or WHEactivation 1 h after levels EGG of mTOR feeding. and Although S6 were we statistically did not measurelower than leucine those concentration of other protein in blood, sources the levelor WHE might 1 h be after lower EGG in EGGfeeding. than Although others, and we thisdid would not measure be a plausible leucine candidateconcentration for lower in blood, activation the level of mTOR might signaling.be lower in Also, EGG given than thatothers, insulin and is this capable would of be activating a plausible mTOR candidate [9], lower for insulinlower activation secretion responseof mTOR after signaling. EGG would affect mTOR activation levels observed 1 h after EGG feeding.

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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 [17–19] 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. Pre- viously, 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 [29–31]. 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 3- kinase (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 Nutrients 2021, 13, 2042 18 of 20

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], clenbuterol- induced 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. Nutrients 2021, 13, 2042 19 of 20

5. 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.

Author Contributions: K.K. (Keiichi Koshinaka) designed all of the experiments, summarized the data, and wrote the manuscript. K.K. (Keiichi Koshinaka), R.I., Y.M. and A.S. performed the animal experiments, and K.K. (Keiichi Koshinaka), A.H. and K.K. (Kentaro Kawanaka) performed the biochemical analyses. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by a research fund (grant-2018-01) from SCETI K.K. and Pulviver SPRL, and a Grant-in-Aid for Advanced Research (grant number R01B09, H30C11, H29B08) from Niigata University of Health and Welfare. Institutional Review Board Statement: This research was approved by the Animal Studies Commit- tee of Niigata University of Health and Welfare (29006-02103, 29016-30005, 02009, 29016). Informed Consent Statement: Not applicable. Data Availability Statement: Each plot is in the article. Conﬂicts of Interest: This work was supported by a research fund from SCETI K.K. and Pulviver SPRL. The funders had no control over the interpretation, writing, or publication of this work. The authors have no further relationships with SCETI K.K. and Pulviver SPRL and did not obtain additional ﬁnancial support for this study.

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