Effect of Globin Digests on Physical Fatigue in Mice

Abstract

Globin digest (GD) inhibits dietary hypertriglyceridemia; however, its effects on physical fatigue remain unknown. Therefore, this study aimed to investigate the potential anti-fatigue effects of GD. Repeated administration of GD and valine (Val)-Val-tyrosine (Tyr)-proline (Pro), a component of GD, for five days prevented the forced walking-induced decrease in locomotion. Furthermore, GD treatment reversed the forced walking-induced increase in blood lactate levels in mice and increased phosphorylated AMP-activated protein kinase (p-AMPK) in the soleus muscle, suggesting that the anti-fatigue effect of GD involves AMPK activation in the soleus muscle through reduced blood lactate.

INTRODUCTION

Fatigue is classified into mental fatigue, which stems from stress caused by work and relationships, and physical fatigue, which is the physiological fatigue that arises from physical activities, such as work and exercise. Furthermore, severe exercise has been suggested to cause the accumulation of lactic acid in the blood, which may, in turn, lead to physical fatigue.¹⁾

We have previously studied fatigue in mice subjected to forced walking as a model of physical fatigue and found that the mice model exhibited a marked decrease in spontaneous locomotor activity after forced walking; however, the administration of a tonic or liver hydrolysate (LH) ameliorated this decrease,^2,3) suggesting the model to be useful for evaluating anti-fatigue effects. We have also demonstrated that LH exerts anti-fatigue effects by decreasing lactate levels via AMP-activated protein kinase (AMPK) activation in the soleus muscle of mice.²⁾

This study focused on globin digest (GD), a substance rich in amino acids and peptides; GD is a peptide mixture with a molecular weight of 100–1500 and protein and free amino acid contents of 91 and 8%, respectively, and is obtained by the enzymatic degradation of porcine globin protein. Furthermore, GD inhibits elevated triglyceride levels.⁴⁾ The possibility that valine (Val)-Val-tyrosine (Tyr)-proline (Pro) (VVYP), a component of GD, may play a role in reducing its triglycerides has also been explored.⁴⁾ However, the effects of GD on physical fatigue remain unclear.

Therefore, this study investigated the effects of GD on physical fatigue and its mechanisms using behavioral, pharmacological, and biochemical analyses.

MATERIALS AND METHODS

All experiments were approved by the Ethics Committee of Animal Experiments at Tohoku Medical and Pharmaceutical University (Approval Number: 21035-cn) and conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Efforts were made to minimize animal suffering and reduce the number of animals used.

Animals

Male ddY mice (age, 6–7 weeks; weight, 26–28 g; Japan SLC, Shizuoka, Japan) were used for all experiments (total, n = 301; behavioral test, n = 250; measurement of blood glucose and lactate, n = 29; Western blotting analysis, n = 22). Mice were housed in cages (5–6 mice/cage) under steady conditions (23 ± 1 °C, 55 ± 5%, 12/12 h light/dark cycle with lights on at 7:00 a.m.) with free access to food or water. Behavioral test was performed between 9:00 and 17:00 h. All mice were numbered using earmarks and randomly divided into groups using a table of random numbers.

Compounds

GD (MG Pharma Inc., Osaka, Japan) was dissolved in drinking water and administered orally (per os (p.o.)) at a volume of 0.1 mL/10 g mouse body weight using a 1 mL syringe with an oral probe. VVYP (MG Pharma Inc.) was dissolved in saline and injected intraperitoneally (i.p.) at a volume of 0.1 mL/10 g body weight.

Forced Walking Test

Forced walking was conducted, as previously described.^2,5) Briefly, 10 mice were subjected to forced walking at room temperature (23 ± 1 °C) in a cylindrical cage (37 cm diameter), which was rotated on a horizontal axis at 2.0 rpm using an electric motor, providing a walking speed of 2.3 m/min for 3 h. Control mice were placed in a cylindrical cage without rotation. Following forced walking for 3 h, mice were individually placed in a multichannel Supermex activity box (Muromachi Kikai, Co., Ltd., Tokyo, Japan) and allowed to adapt for 15 min before GD or VVYP administration. Locomotor activity was measured every 15 min for 90 min. The Supermex instrument can monitor minute movements in all three planes of motion (sagittal, coronal, and horizontal) as one movement owing to its IR sensor with multiple Fresnel lenses, which can be moved close enough to the cage to capture multidirectional locomotor alterations in a single mouse. The Supermex instrument was connected to a behavioral analysis system (CompACT AMS, Muromachi Kikai) that can interpret each movement as one count. Therefore, vertical movements, such as jumping, and horizontal movements, such as walking and running, could be counted. Locomotor activity was measured between 12:00 and 16:00 h during the light phase.

Blood Glucose and Lactate Assays

After 3 h of forced walking, the mice were allowed to adapt to the environment for 15 min. Next, water or GD was administered po, and after 15 min, blood was obtained from the tail vein of the mice to measure blood glucose and lactate levels. The Freestyle Precision Neo (Abbott Japan LLC, Tokyo, Japan) was used to measure blood glucose levels, and Lactate Pro 2 (Arkray Inc., Kyoto, Japan) was used to measure blood lactate levels.

Western Blotting

Mice were divided into four groups (forced walking 3 h [−]/water, forced walking 3 h [−]/GD [0.1 g/kg], forced walking 3 h [+]/water, and forced walking 3 h [+]/GD [0.1 g/kg]) and sacrificed by decapitation 15 min after the last water or GD administration. Protein extraction from the soleus muscle and Western blotting were performed as described previously.²⁾ After electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes and incubated with a blocking solution (10 mM Tris–HCl [pH 7.4], 100 mM NaCl, 0.01% Tween 20, and 5% skim milk). The membranes were probed with antibodies against phospho- (p-) AMPK (1 : 1000; Cell Signaling Technology, Danvers, MA, U.S.A.) and total- (t-) AMPK (1 : 1000; Cell Signaling Technology) overnight at 4 °C. Next, the membranes were washed with the blocking solution without milk, incubated with horseradish peroxidase-conjugated secondary antibody (1 : 5000; Cell Signaling Technology) for 2 h, and visualized using an enhanced chemiluminescence Western blotting detection reagent (Amersham Life Science). Band density was measured using densitometry (Image J 1.43 µ, National Institute of Health).

Statistical Analysis

The results of the experiments are expressed as mean ± standard error of the mean (S.E.M.). The significance of differences was determined using one- or two-way ANOVA, followed by the Tukey–Kramer test for multiple group comparisons. The criterion for significance was set at p-values <0.05. In some cases, where the main effect was significant without an interaction effect, an exploratory and limited pairwise post-hoc comparison consistent with our a priori hypothesis was performed. All statistical analyses were performed by investigators who were not the experimenters to avoid bias and ensure blinding.

RESULTS

GD Showed an Anti-fatigue Effect in the Forced Walking Test

Mice showed significantly reduced locomotor activity after the forced walking test, while repeated administration of GD (0.1 g/kg) for 5 d (p = 0.0290, Fig. 1F) improved this change. Moreover, this effect of GD was observed 15 min after the last administration (Fig. 1G). However, this effect was not observed during twice-daily or 3-d administration of GD (Figs. 1B, D).

Fig. 1. Effect of Globin Digest (GD) on Locomotor Activity after Forced Walking for 3 h

(A, C, E) Time course of each experimental protocol. (B, D, F) Total locomotor activity. (G) Time-dependent change in locomotor activity. Bars represent means ± standard error of the mean (S.E.M.). One-way ANOVA: F (4, 55) = 4.27, p = 0.0044, (B); F (4, 59) = 9.118, p < 0.0001, (D); F (5, 78) = 9.67, p < 0.0001, (F). Two-way ANOVA: time: F (6, 280) = 24.46, p < 0.0001; treatment: F (2, 280) = 6.893, p = 0.0012; interaction: F (12, 280) = 2.574, p = 0.0030, (G). **: p < 0.01 vs. control group without forced walking. #: p < 0.05, ##: p < 0.01 vs. control group with forced walking (n = 9–16 per group).

Treatment with GD Prevented the Forced Walking-Induced Increasing Blood Lactate and Enhanced AMPK Activity in the Soleus Muscle

After the forced walking test, the mice exhibited decreased blood glucose (p = 0.0141, Fig. 2B) and increased blood lactate (p = 0.0002, Fig. 2C) levels when compared to the control group (without forced walking); however, the increased blood lactate level reduced by repeated GD administration (0.1 g/kg; p = 0.0007, Fig. 2C). In addition, repeated GD administration significantly increased p-AMPK levels in the soleus muscle after forced walking when compared with the control group (p = 0.0002, Fig. 2D).

Fig. 2. Effect of GD on the Changes in Blood Glucose (B) and Lactate (C) Concentration and Phosphorylated (p-) AMPK Levels (D) in the Soleus Muscle after Forced Walking

(A) Time course of each experimental protocol. Quantification of values for p-AMPK normalized with total (t-) AMPK levels in the soleus muscle. Bars represent means ± S.E.M. Two-way ANOVA: group: F (1, 26) = 15.7, p = 0.0005; treatment: F (1, 26) = 0.3818, p = 0.5420; interaction: F (1, 26) = 0.1944, p = 0.6630, (B). Group: F (1, 25) = 12.73, p = 0.0015; treatment: F (1, 25) = 6.395, p = 0.0181; interaction: F (1, 25) = 10.55, p = 0.0033, (C). Group: F (1, 18) = 24.08, p = 0.0001; treatment: F (1, 18) = 38.92, p < 0.0001; interaction: F (1, 18) = 2.1, p = 0.1645, (D). *: p < 0.05, **: p < 0.01 vs. control group without forced walking. ##: p < 0.01 vs. control group with forced walking (n = 5–9 per group).

VVYP Showed an Anti-fatigue Effect in the Forced Walking Test

Repeated administration of VVYP (1.0 mg/kg) for five days prevented the forced walking-induced fatigue-like behavior (p = 0.0363, Fig. 3B).

Fig. 3. Effect of Val-Val-Tyr-Pro on Locomotor Activity after Forced Walking for 3 h

Bars represent means ± S.E.M. (A) Time course of each experimental protocol. (B) Total locomotor activity. One-way ANOVA: F (3, 37) = 6.041, p = 0.0019, (B). **: p < 0.01 vs. control group without forced walking. #: p < 0.05 vs. control group with forced walking (n = 9–13 per group).

DISCUSSION

This study showed that GD exhibited an anti-fatigue effect in mice by AMPK activation in the soleus muscle.

We have previously demonstrated that the administration of anti-fatigue compounds high in peptides and amino acids, such as LH and peptides, obtained from enzymatic degradation of mackerel before and after the forced walking tests, induces fatigue reduction in mice^2,5,6); however, sole administration of either before or after the forced walking test was ineffective. Therefore, in this study, water or GD was administered orally before and after the forced walking test. Locomotor activity markedly decreased after 3 h of forced walking; however, the fatigue-like behavior was attenuated by repeated administration of GD (0.1 g/kg) for 5 d (Fig. 1F). No change in locomotor activity was observed during twice-daily or 3-d administration of GD (Figs. 1B, D), suggesting that repeated administration is necessary for the anti-fatigue effects of GD to be exerted.

Blood glucose uptake by the muscle suppresses fatigue, and lactate production by the muscle via anaerobic glycolysis is one of the key indicators of physical fatigue during intense exercise.^7–9) AMPK activation decreases the lactate levels by suppressing lactate production-related protein expression, including lactate dehydrogenase and monocarboxylate transporter 4.¹⁰⁾ Several studies have demonstrated that AMPK phosphorylation in the soleus muscle is associated with anti-fatigue effects in mice.^2,5) Therefore, this study investigated the association between the anti-fatigue effect of GD and blood glucose and lactate levels and AMPK activity in the soleus muscle. Blood and muscle samples were obtained 15 min after water and GD oral administration following forced walking, as the anti-fatigue effect of GD was observed 15 min after the second GD treatment (Fig. 1G). After forced walking, the mice exhibited decreased blood glucose (Fig. 2B) and increased blood lactate levels (Fig. 2C), and the increased blood lactate level was prevented by repeated GD administration (Fig. 2C). Moreover, repeated GD administration significantly increased p-AMPK levels in the soleus muscle after forced walking (Fig. 2D). Given that VVYP in GD exerts inhibitory effects on triglycerides,⁴⁾ its possible association with anti-fatigue effect was investigated. Repeated VVYP treatment (1.0 mg/kg) exerted an anti-fatigue effect in mice (Fig. 3B). These results suggest that the anti-fatigue effect of GD is associated with AMPK activation through the inhibition of blood lactate accumulation and that VVYP, a component of GD, is important for the anti-fatigue effect in mice. However, the anti-fatigue effect of VVYP requires further investigation. Moreover, there is a large discrepancy between the effective dose of GD (0.1 g/kg) and that of VVYP (1.0 mg/kg), one of the components of GD, in this study. Previous studies using reversed-phase ODS LC had confirmed the presence of VVYP (3.7 mg /g) in GD.⁴⁾ Based on the results of that study, we determined the dosage of VVYP and conducted experiments. We believe that the results of this study reflect VVYP in the GD, although the dosage of VVYP (1 mg/kg) is slightly higher.

In conclusion, this study demonstrated that GD attenuated physical fatigue and concomitantly activated AMPK activity by reducing blood lactate accumulation. Thus, GD exhibited potential as a supplement with anti-fatigue effects.

Acknowledgments

The authors thank Dr. Yuka Sasakawa and Mr. Yasuo Sumida of MG Pharma Inc. for their assistance. This work was funded by JSPS KAKENHI (Grant Numbers: 21K15351 and 22K06866).

Conflict of Interest

Kotaro Yamada is an employee of ROHTO Pharmaceutical Co., Ltd. (Osaka, Japan).

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