Table 2 Compositions of the experimental diets (g/1000 g diet).

Effective dose of Alaska Pollack protein for increase of muscle
Kenji Uchida (D3) and, Mina Fujitani, Taro Kishida
Supervisor: Prof. Taro KishidaCo-supervisor: Assist. Prof. Mina Fujitani

Value are means ± SEM *: p<0.05, * *: p<0.01 t-test

Fig. 2 APP or A-APP for Cas in the AIN-93 diet significantly
increased rat GA in 3 days after administration

Administration with the AIN-93 diet which were substituted APP(Each group n = 10)**

Weight of GA(g)Total food intake(g)

**

**
**

In the APP all and one third administration group, mass of the GA muscle and Body weightincreased significantly, and in the APP one ninth administration group was no significantdifference. All group was no significant difference of food intake.
[Exp. 2] Orally administration of APP 333 mg/kg for 3 days significantly increased GA.

Fig. 4

Dose of APP orally administration

Fig. 5 Effect of APP 333mg / kg administration(Each group n = 5)*(Each group n = 10)***(Each group n = 6)

Weight of GA (g)****Repeated oral administration: 6 weeks old male rats were orallyadministered with Cas and APP at 1000 mg/kg and 333 mg/kg
for 3 days. After administration, the GA of rat were evaluated.

*In the APP 333 mg/kg group, mass of the gastrocnemius muscle increased significantly. Although
there was no significant difference, the mean of gastrocnemius muscle mass in the APP 1000 mg/kg
group showed a value close to APP 333 mg/kg. Fig.5 is shown that APP 333 mg/kg administration
were increased significantly GA compared to Cas 333 mg/kg and sham administration.
And APP 333 mg/kg administration is minimum dose for increased GA.

Table 1 Amino acid(AA) composition of APP and Cas[Human effective dose (HED)] APP 53 mg/kg/day

AACas

APPAA

Cas

APPAA

Cas

APPAA

Cas

APP

Asp

6.41

9.30

Ala

2.76

5.40

Leu

8.39

7.26

Arg

3.30

5.86

Thr

3.86

4.08

Val

5.77

4.40

Tyr

5.02

3.26

Trp

1.12

1.01

Ser

5.21

4.00

Cys

0.32

1.03

Phe

4.57

3.47

Pro

9.64

2.94

Glu 20.00 14.00

Met

2.52

2.66

Lys

7.14

8.41

GlyIls

4.53

4.01

His

2.69

2.21

1.70

Repeated oral administration: 6 weeks old male rats were orally
administered with Cas at 333 mg/kg and APP at 333 mg/kg,
sham for 3 days. After administration, the GA of rat wereevaluated.

4.14

0.33

0.33
Animal weight in kg
HED = animal dose in mg/kg
Human weight in kg

0.19= 333 mg/kg
50 (*)= 53 mg/kg

The human equivalent dose based on body surface area suggests that 53 mg/kg or more may beeffective. *: Average body weight of Japanese women (50 kg)

APP A-APP

SPI

APP1/3 APP1/9200-

--

133

APP-

182-

-

59

A-APP--

160-

Soy been protein-L-Cysteine

Dietary administration (free intake): 6 weeks old male rats were administrated with the AIN-93 diet which weresubstituted APP for all, one third or one ninth of casein(APP 1/3, APP 1/9), or Cas-based control diet for 3 days. Afteradministration, GA of rat were evaluated.

Weight of GA(g)Weight of GA (g)Fig. 1 APP for Cas in the AIN-93 diet significantly increased
GA in 3 days and 7days after administrations (Each group n = 8)
3 days7days****

Fig. 3Weight of GA(g)[Previous studies] An administration of APP increased rat skeletalmuscle(Fig.1). Some report suggested that an administration ofbranched chain amino acids (BCAA) increased the skeletal muscle.
However, administration of A-APP is not effect (Fig.2) and BCAA of
APP is lower than Cas (Table 1). Therefore, these data suggested
that the effect of APP was independent of BCAA.

[Exp. 1] APP for one third of Cas in the AIN-93 significantly increased GA.

Body weight (g)[Objectives] We have confirmed that the administration of Alaska
pollack protein (APP) diet, which was totally substituted APP forcasein (Cas) in the AIN-93 diet significantly increased rat
gastrocnemius muscle (GA) in 3 days after administration, compared
to Cas-based control diet. But the administration of amino acid mixture,
which of the equivalent amino acid composition to APP(A-APP), was noteffective in increasing skeletal muscle. It suggested that a protein or adegraded peptide of APP might be involved in the efficacy byadministration of APP, but not the amino acids. In further study, weplan to investigate the effect of the administration of APP in the clinical
trial. For estimation of the dosage for humans, we investigated theeffective dose of APP for the skeletal muscle increasing effect of rat.

Weight of GA (g)CasCasein

--

208

178
20---

333

3

3

3α-Cornstarch

532

550

572

524

539

534

Sucrose

100

100

100

100

100

100

Cellulose

50

50

50

50

50

50

Soybean oil

70

70

70

70

70

70

AIN-93 mineral mixture

35

35

35

35

35

35

AIN-93 vitamin mixture

101010101010Total

1000 1000

1000

1000

1000

1000

% of crude protein

87.5

100.0

84.3

90.7

88.6

97.2

In previous studies and all the experiments, diets are AIN-93G orprotein source modified AiN-93G. Food intake is about 18-23 g/day.
Alaska pollack fillets (Nippon Suisan Kaisha, Ltd., Tokyo, Japan) wereused as the fish protein source in this study. The fillets were freezedried and ground. The fat component was extracted using hot ethanol(65°C, 60 min × 2) and removed through centrifugation. The
remaining ethanol was dried in a vacuum dryer (60°C, ~24 h). Therats were housed in individual stainless wire mesh cages in a room
under a 12 h light–dark cycle (dark phase: 15:00–3:00) at a constanttemperature (22±1°C). The animals were housed separately for 6
days for acclimatization to the environment. This study wasconducted by March 2019 in accordance with the ethical guidelinesof the Ehime University Animal Experimentation Committee and incomplete compliance with the National Institutes of Health: Guide for
the Care and Use of Laboratory Animals. All efforts were madetoward minimizing the number of animals used and limiting
experimentation to what was necessary to produce reliable scientificinformation.

[Conclusions] The increasing effect of APP on skeletal muscle
was exerted in more than one-third APP substitution in
dietary administration. Moreover, the effective amount of APP
was 333 mg/kg or lower in repeated oral administration. It
suggested that a protein or peptide was involved for the skeletal
muscle increasing effect of APP, not amino acid balance. Inaddition, considering the application to humans, the humanequivalent dose based on body surface area suggests that 53mg/kg or more may be effective. So, APP dose is 2.65 g for
day for 50 kg body weight. In considering the variation in bodyweight, we decided to dose of APP 4.5 g daily in the clinical
trial. This dose corresponds to about 30 g of Alaska pollack
fillet daily.