Combined intake of astaxanthin, β-carotene, and resveratrol elevates protein synthesis during muscle hypertrophy in mice

Accepted Manuscript

Combined intake of astaxanthin, β -carotene, and resveratrol elevates protein synthesis during muscle hypertrophy in mice Aki Kawamura M.Sc. , Wataru Aoi Ph.D. , Ryo Abe M.Sc. , Yukiko Kobayashi Ph.D. , Sayori Wada M.D., Ph.D. , Masashi Kuwahata Ph.D. , Akane Higashi M.D., Ph.D. PII: DOI: Article Number: Reference:

S0899-9007(19)30120-0 https://doi.org/10.1016/j.nut.2019.110561 110561 NUT 110561

To appear in:

Nutrition

Received date: Revised date: Accepted date:

15 February 2019 23 April 2019 12 May 2019

Please cite this article as: Aki Kawamura M.Sc. , Wataru Aoi Ph.D. , Ryo Abe M.Sc. , Yukiko Kobayashi Ph.D. , Sayori Wada M.D., Ph.D. , Masashi Kuwahata Ph.D. , Akane Higashi M.D., Ph.D. , Combined intake of astaxanthin, β -carotene, and resveratrol elevates protein synthesis during muscle hypertrophy in mice, Nutrition (2019), doi: https://doi.org/10.1016/j.nut.2019.110561

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Highlights ・Combined intake of astaxanthin, β-carotene, and resveratrol accelerated muscle hypertrophy. ・Along with the muscle hypertrophy, protein synthesis signaling was activated.

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・These effects might be mediated by oxidative stress reduction.

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Combined intake of astaxanthin, β-carotene, and resveratrol elevates protein synthesis during muscle hypertrophy in mice Aki Kawamura M.Sc.a,b, Wataru Aoi Ph.D.a,*, Ryo Abe M.Sc.a,c, Yukiko Kobayashi Ph.D.a,

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Sayori Wada M.D., Ph.D.a, Masashi Kuwahata Ph.D.a , Akane Higashi M.D., Ph.D.a

Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences,

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Kyoto Prefectural University, Kyoto, Japan

Sports Science Research Promotion Center, Nippon Sport Science University, Tokyo, Japan

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Wakayama Medical University Hospital, Wakayama, Japan

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[email protected]

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*Corresponding author: Wataru Aoi, Tel.: +81757035417, Fax: +81757035417, E-mail address:

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Running head: Combined antioxidants accelerate muscle hypertrophy

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Word count: 4985

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Number of figures and tables: 5 figures Authors contribution: A.K. and W.A. designed and coordinated the study. A.K., W.A., and R.A. contributed to designing and performed experiments. A.K., W.A., Y.K., S.W., M.K., and A.H. analyzed and evaluated data. A.K. and W.A. wrote the manuscript with input from other authors. All authors critically reviewed and approved the final version of manuscript.

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Abstract Objective: The antioxidant factors, astaxanthin, β-carotene, and resveratrol, have a potential effect on protein synthesis in skeletal muscle and a combined intake may have a greater

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cumulative effect than individual intake. We investigated the combined effects on skeletal muscle mass and protein metabolic signaling during hypertrophic process from atrophy in mice.

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Research Methods and Procedures: Male ICR mice were divided into 5 diet groups consisting of 7 animals: normal, astaxanthin, β-carotene, resveratrol, and all 3 antioxidants. Equal concentrations (0.06% (w/w)) of the respective antioxidants were included in the diet of each

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group. In the mixed group, 3 antioxidants were added in equal proportion. One leg in all mice

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was casted for 3 weeks to induce muscle atrophy. After removal of the cast, the mice were fed each diet for 2 weeks. The muscle tissues were collected, weighed, and examined for protein

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metabolism signaling and oxidative damage.

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Results: The weight of soleus muscle was increased in the astaxanthin, β-carotene, and

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resveratrol groups to a greater extent in comparison to in the normal group; this was accelerated by intake of the mix antioxidants (p=0.007). Phosphorylation levels of mTOR and p70S6K in the muscle were higher in the mixed antioxidant group than in the normal group (p=0.025， p=0.020). The carbonylated protein concentration was lower in the mixed antioxidant group than in the normal group (p=0.021).

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Conclusions: These results suggest that a combination of astaxanthin, β-carotene, and resveratrol, even in small amounts, promoted protein synthesis during the muscle hypertrophic process following atrophy.

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Keywords: antioxidant, muscle hypertrophy, protein synthesis, oxidative stress, combined

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intake

Introduction

To increase muscle mass, it is important to increase protein content by modulating protein

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metabolism. Namely, accelerating protein synthesis and preventing protein catabolism promote

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muscle hypertrophy. Resistance training is the most established way to enhance protein synthesis. It is well known that appropriate nutrition accelerates the improvement of muscle

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strength and mass by resistance training. Specifically, sufficient intake of dietary protein

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improves exercise-induced protein synthesis more than poor intake below the recommended

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dietary intake [1]. In addition to protein, several amino acids and peptides can also stimulate muscle protein synthesis [2-5]. A combined intake of carbohydrate and protein is more effective in increasing muscle protein by stimulating insulin secretion [6]. However, it is unclear whether micronutrients with antioxidant capacity can stimulate muscle hypertrophy during resistance training.

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The effect of oxidative stress on protein metabolism in skeletal muscle has been investigated. Chronic oxidative stress, which is associated with disuse atrophy, induces proteolysis of the muscle [7]. Dietary antioxidant intervention has been shown to prevent

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protein catabolism during atrophy [8-10]. In contrast, the evidence that oxidative stress is involved in the process of protein synthesis has been limited. However, some antioxidants can

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stimulate protein synthesis signaling. Oral administration of β-carotene, a carotenoid, increased the phosphorylation level of p70S6 kinase (p70S6K) and promoted protein synthesis [11]. Resveratrol, a polyphenol, has been shown to stimulate insulin growth factor-1-mediated

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signaling, in particular Akt and ERK 1/2 protein, and promote hypertrophy in cultured muscle

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cells [12]. Another study showed that resveratrol activated phosphorylation level of Akt, mammalian target of rapamycin (mTOR), p70S6K, 4E-BP1, and increased muscle mass [13].

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Astaxanthin, a xanthophyll carotenoid, also modulated muscle protein metabolism and

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accelerated strength in animals and humans [14-16]. Thus, we hypothesized that these 3

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antioxidants can promote muscle mass by stimulating protein anabolism during hypertrophy. Various antioxidants are localized at different cellular positions in the living body. Their

antioxidant functions are balanced/maintained by electron transfer. In many cases, the antioxidant capacity is higher with the combined intake of several antioxidants compared to that with individual intake [17, 18]. Thus, the combined intake of several antioxidants, even in

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small amounts, may exert more antioxidant functions as reduced oxidative maker, i.e. carbonylated proteins than individual intake in greater amounts. Therefore, we hypothesized that combined intake of astaxanthin, β-carotene, and resveratrol may efficiently enhance

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muscle protein synthesis. Here, we investigated the effect of combined antioxidant intake on protein anabolic signals using a mouse model that show muscle hypertrophy after

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immobilization-induced atrophy.

Material and Methods

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Animals and experimental design

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Care of all animals in the study complied with the guidelines of the Japanese Council on Animal Care and was approved by the Committee for Animal Research of the Kyoto Prefectural

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University (KPU028526). The experimental design is shown in Fig. 1. A total of 44 ICR male

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mice (7 weeks old; Shimizu Laboratory Supplies Co., Ltd., Kyoto, Japan) were acclimatized for

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1 week in an air-conditioned (22 ± 2°C) room with a 12:12-h light-dark cycle (lights on from 0800 to 2000). Then, characteristic of 9 mice were measured immediately after casting, and the other mice were divided into 5 groups with 7 mice each: normal, astaxanthin, β-carotene, resveratrol, and mix antioxidant (mixture of three antioxidants) diet groups. According to the previous study [19], the knee and ankle joints of one hindlimb were fixed with cast, and muscle

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atrophy was induced. After 3 weeks, the cast was removed, and soleus and gastrocnemius muscles from both legs of the 9 mice were weighed. The remaining 5 groups of mice maintained their diets for 2 weeks after cast removal. Each antioxidant (astaxanthin, Fuji

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Chemical Industries Co., Ltd., Toyama, Japan; β-carotene, Sigma-Aldrich, St. Louis, MO, USA; resveratrol, BGG Japan Co. Ltd., Tokyo, Japan) was added to the baseline diet (MF, Shimizu

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Laboratory Supplies Co., Ltd., Kyoto, Japan) containing protein, 23.1 g%; fat, 5.1 g%; carbohydrate, 55.3 g%; retinol, 1283 IU%; α-tocopherol, 9.1 mg%; and ascorbic acid, 4 mg%. Referring to previous studies that showed improvement of skeletal muscle functions in rodents

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[11, 20, 21], the effective concentration of 3 antioxidants was estimated to be 0.02% and the

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mix antioxidant diet was prepared with equal proportion of 0.02% each (total concentration 0.06%). Then, equal concentrations (0.06%) of the respective antioxidants were included in the

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diet of each group. Plasma concentration of each antioxidant is assumed to be elevated after

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supplementation, from information of previous studies [22-24]. There was no difference in food

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intake between groups. After 2 weeks, the body weight (B.W.) of all mice was measured, euthanized under anesthesia, and then blood and soleus and gastrocnemius muscles from both legs were collected. After measuring the weight of the muscle tissues, the tissues were stored at -80 °C until biochemical experiments.

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Western blotting The soleus muscles were homogenized using stainless beads and a crushing machine (Shakeman 3, BMS, Tokyo, Japan) in lysis buffer (CellLytic MT Cell Lysis Reagent;

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Sigma-Aldrich) added protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Protein was extracted from the muscle tissue by

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centrifugation at 16,000 rpm for 15 min at 4°C. Protein concentration of the supernatant was measured using the BCA method (BCA Protein Assay Kit; Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were separated by SDS-polyacrylamide gel

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electrophoresis and then transferred into nitrocellulose membranes (iBlot® Gel Transfer Stacks

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Nitrocellulose; Thermo Fisher Scientific). Subsequently, the blots were incubated overnight at 4°C with primary antibodies against phospho-mTOR (Ser2481), total mTOR, phospho-p70S6K

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(Thr389), total p70S6K, phospho-Akt (Ser473), total Akt, phospho-FoxO1 (Ser256), total

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FoxO1 (Cell Signaling Technology, Beverly, MA, USA), and glyceraldehyde-3-phosphate

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dehydrogenase (GAPDH) (Abcam Co., Ltd., Cambridge, MA, USA). Then, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence substrate (Chemi-Lumi One Super; Nacalai Tesque). The detected bands were visualized by image analysis (LuminoGraphⅢ, ATTO Corporation, Tokyo, Japan) and signal intensities were quantified using an image analyzer (Image Saver 6, ATTO Corporation).

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Carbonylated protein Carbonylated protein concentration in protein lysate obtained from soleus muscle tissues

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was measured using the enzyme-linked immunosorbent assay kit (BioCell Corporation Ltd.,

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Auckland, New Zealand), according to the manufacturer’s instruction.

Statistical analysis

All data are expressed as mean ± standard errors. IBM SPSS Statistics for Windows

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version 18 (IBM Japan Inc., Tokyo, Japan) was used for the statistical analysis. Differences

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between multiple groups were evaluated using a 1-way or 2-way analysis of variance. When significant interactions were detected, multiple comparison tests were conducted using the

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Dunnett’s or the Tukey tests. Differences within a group were evaluated using paired t-tests. A

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p-value of < 0.05 was considered to be statistically significant.

Results

1. Skeletal muscle mass Immediately after cast removal, weights of both the gastrocnemius and soleus muscles were markedly lower in the casted leg than in the control leg (p < 0.001) (Fig. 2A, B). Two

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weeks after the removal, the soleus weight was significantly lower in the casted leg (0.20 ± 0.01 mg/g B.W.) than in the control leg (0.27 ± 0.01 mg/g B.W.) in the normal diet group (p = 0.010) (Fig. 3A). In contrast, it was significantly higher in the casted leg (0.24 ± 0.01 mg/g B.W.) than

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in the control leg (0.20 ± 0.01 mg/g B.W.) in the mix antioxidant diet group (p = 0.027) and not significantly different between both legs in other antioxidant groups. In addition, the ratio of

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relative weight of the casted leg to the control leg was significantly higher in the mix antioxidant diet group (17.1 ± 5.7%, p = 0.007) and astaxanthin diet group (9.8 ± 10.0%, p = 0.026) than in the normal diet group (-25.6 ± 6.6%) (Fig. 3B). In contrast to no significant

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difference in the weight of gastrocnemius muscle between groups, gastrocnemius muscle weight

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from the casted leg was significantly smaller than that from the control leg in all groups (Fig.

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3C, D). Thus, further biochemical analyses were performed for the soleus muscle.

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2. Protein metabolic signaling in the skeletal muscle

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Total protein concentration (μg/mg wet weight) in the casted soleus muscle was not significantly different between groups (normal, 60.5 ± 2.4; astaxanthin, 55.3 ± 2.1; β-carotene, 59.5 ± 3.3; resveratrol, 63.0 ± 3.3; mix antioxidant, 62.0 ± 3.0). In addition, there was no significant difference when it was compared with the protein concentration from the control muscle (normal, 54.9 ± 2.4; astaxanthin, 62.2 ± 3.1; β-carotene, 66.3 ± 4.1; resveratrol, 61.7 ±

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5.4; mix antioxidant, 66.9 ± 3.8). Thus, these results suggest that muscle hypertrophy was caused by the increase in protein contents, but not by changes in other components, such as water. We then measured protein metabolism-related signaling factors in the soleus muscle from

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the casted leg.

Phosphorylation of mTOR, a key anabolic signal factor, was significantly higher in the mix

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antioxidant diet group (244 ± 54%) than in the normal diet group (100 ± 23%) (p = 0.025) (Fig. 4A). The down-stream factor of mTOR, phospho-p70S6K was also significantly higher in the mix antioxidant diet group (149 ± 15%) than in the normal group (100 ± 11%) (p = 0.020) (Fig.

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4B). No significant difference in phospho-Akt level were found between groups (Fig. 4C). In

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contrast, phosphorylation of FoxO1, a key catabolic signal factor, was not significantly different

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in antioxidant treatment groups in comparison to the normal group (Fig. 4D).

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3. Carbonylated protein in the skeletal muscle

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Carbonylated protein, an oxidative damage marker, was measured in the soleus muscle from the casted leg. The concentration of carbonylated protein was significantly lower in the mix antioxidant group (3.59 ± 0.09 nmol/mg protein) but not in other antioxidant groups compared with that in the normal group (3.94 ± 0.05 nmol/mg protein, p = 0.021) (Fig. 5).

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Discussion Many previous studies have examined the effect of dietary antioxidants intervention on protein catabolism and muscle atrophy [8-10]. In contrast, their effects on protein synthesis and

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hypertrophy have not yet been clarified. We examined whether specific antioxidants can have protein anabolic effect during the recovery from atrophy. Although the weight of the soleus

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muscle of the casted leg recovered for over 2 weeks of hypertrophic period after cast removal, it was still lower than that of the control leg under the baseline diet condition. However, no differences between control and casted legs were observed in the astaxanthin, β-carotene, and

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resveratrol diet groups. Furthermore, in the group that received three antioxidants, the weight of

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muscle of the casted leg was rather higher than that of the control leg. Repetitive mechanical overload to skeletal muscle induces hypertrophy. As an adaptation to resistance exercise training,

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the common mechanism is associated with muscle hypertrophy during recovery after

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immobilization. Thus, the muscle weight can surpass that of the normal group, along with the

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timing of recovery and hypertrophic activity. The results suggest that the supplementation of mixed antioxidants stimulates protein synthesis, resulting in muscle hypertrophy beyond the basal level. These results firstly indicated that astaxanthin, β-carotene, and resveratrol promoted muscle hypertrophy during recovery after atrophy, and this effect was accelerated by combined intake of these antioxidants.

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Several signal transduction pathways are involved in protein synthesis and degradation. Phosphorylation levels of Akt, mTOR, and p70S6K, key molecules in the protein synthesis pathway, are increased during the muscle hypertrophic period in resistance training [25]. Muscle

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contraction accompanying exercise promotes growth factors and then activates Akt, resulting in activation of the downstream mTOR/p70S6K signal [2, 26-28]. In contrast, FoxO1, the key

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molecule in proteolysis is activated in bedridden, inactive, and space environment [29]. It activates the downstream MuRF-1, Atrogin-1, and ubiquitin-proteasome system, promotes protein degradation, and induces atrophy [30, 31]. We showed that combined intake of

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astaxanthin, β-carotene, and resveratrol increases the phosphorylation level of mTOR and

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p70S6K but not FoxO1, suggesting that these compounds promoted protein synthesis. Because the phosphorylation level of Akt was not changed by antioxidant diet, other upstream pathways

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can be involved in the activation of mTOR/p70S6K. Phosphorylation of mTOR and p70S6K is

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not always mediated by the activation of Akt. Hara et al. [2] revealed that amino acids

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phosphorylate S6K and 4EBP1 without activating the insulin signaling pathway in cultured cells, which are mediated by Rag, Vps34, and MAP4K3, putative intracellular amino acid sensors. Along with the results in muscle weight and anabolic signaling, carbonylated protein, a

typical oxidative marker, was lower in the mix antioxidant diet group than the normal diet group. This marker has been used in many studies to examine oxidative stress level in skeletal muscle

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in various experimental conditions [32-34]. Furthermore, protein carbonylation is an irreversible posttranslational modification of proteins and the modified protein can be inactivated or catabolized. Thus, elevation of carbonylated protein shows protein dysfunction as well as higher

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oxidative stress. It is well known that chronic oxidative stress stimulates proteolysis and induces muscle atrophy. In addition, several studies suggest that protein synthesis signal can be

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negatively regulated by excessive oxidative stress [35-37]. A high level of oxidative stress inhibits anabolic signaling by reducing phosphorylation of mTOR and 4EBP1 in cultured muscle cells [38]. In this case, antioxidants might have a positive effect that activates the signal

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transduction by scavenging excess ROS. Here, the mix antioxidant diet had the largest effect on

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muscle hypertrophy even with an equal amount of total antioxidant in the diet, which supports the concept that it activates protein synthesis signaling via increased antioxidant capacity.

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Antioxidants have different characteristics in the living body. They accumulate in various

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cellular components and scavenge radicals with the individual characteristics. Additionally,

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some antioxidants could be changed to prooxidants in particular conditions such as high-dose intake of a single compound. Thus, radicals are efficiently eliminated by contracted redox chain reaction when several compounds are ingested in combination. Indeed, previous studies have shown, in various experimental models, that oxidative stress in humans and mice is lower with combined intake of antioxidants than with individual intake [39, 40]. In the muscle tissues after

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exercise, combined intake of antioxidants also reduced oxidative stress compared to individual intake [41]. Lipid-soluble astaxanthin and β-carotene are easily accumulated in plasma and mitochondrial membranes. Astaxanthin scavenges radicals both inside and outside the

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membrane sites since it penetrates across the membrane [42]. β-carotene rather works in the membrane since it exists in a bundled form in the membrane [42]. In contrast, resveratrol works

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at the cytosol and intracellular components [43]. These individual characteristics might lead to higher antioxidant effect even with intake of a small dose.

The production of ROS is larger in slow-twitch fiber, which is enriched in the soleus

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muscle, than in fast-twitch fiber. It contains high density of mitochondria, a major source of

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ROS with energy production [44]. Several studies reported that dietary antioxidant was effective in reduction of ROS and ameliorated atrophy in the soleus muscle more than the gastrocnemius

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muscle, which contains more fast-twitch fibers [7-11]. In this study, the hypertrophic effect of

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antioxidant intake was observed in the soleus muscle, which might easily generate the

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antioxidant effects.

Phytochemical compounds that have antioxidant capacity, have individual effects apart

from antioxidant properties. Astaxanthin, β-carotene, and resveratrol which have positive effects on protein synthesis. In contrast, some reports have shown that several antioxidants including vitamin C and E did not change [45, 46] or rather negatively [47-50] regulate the metabolism in

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exercise conditions. Although the reason for the difference in compounds is unclear, we cannot deny that their protein synthesis effects were not caused by antioxidant effects. Namely, both hypertrophic and antioxidant effects might independently occur in parallel. Further studies

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should be required to clarify whether the protein synthesis enhanced by the 3 compounds is mediated by reduction of oxidative stress. In addition, this study was performed in a limited

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condition, e.g., dose and type of compounds. An investigation on the effects of other antioxidants and their combination is also needed. Considerably, our findings support that combined intake of antioxidants is beneficial for muscle metabolism in the hypertrophic period.

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Conclusion

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This study examined the effect of antioxidants intake on protein synthesis and muscle mass during the hypertrophic period after immobilization-induced atrophy. Astaxanthin, β-carotene,

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and resveratrol increased protein synthesis signaling and muscle mass, and the combined intake

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of antioxidants showed the greatest effect. These effects might be mediated by oxidative stress

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reduction.

Acknowledgements This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (No.17H02176).

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Conflict of Interest The authors declare that there is no conflict of interest associated with this manuscript.

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Figure legends Fig. 1. Experimental design Fig. 2. Muscle weight after cast removal

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Soleus (A) and gastrocnemius (B) muscle weight per body weight after cast removal. Data are presented as mean ± standard errors, n = 9. Within-group comparison was conducted using

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paired t-test. ** p < 0.01. Fig. 3. Comparison of muscle weight in hypertrophic period

Soleus (A) and gastrocnemius (C) muscle weight per body weight in control and casted legs.

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White bar shows the soleus or gastrocnemius muscle weight per body weight in the control leg

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and black bar shows the that of the casted leg. Comparisons within-group and between-group were conducted using two-way analysis of variance and Tukey test. Muscle mass was corrected

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by body weight. Relative weight of the casted leg compared to control leg in the soleus (B) and

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gastrocnemius (D) muscles. Between-group comparison was conducted using one-way analysis

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of variance and Dunnett’s test. Data are presented as mean ± standard errors, n = 7. * p < 0.05, ** p < 0.01.

Fig. 4. Comparison of protein metabolic signaling of the casted leg in hypertrophic period Phospho-mTOR (A), phospho-p70S6K (B), phospho-Akt (C), and phospho-FoxO1 (D) levels

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are expressed as relative value to the normal. Data are presented as mean ± standard errors, n = 6-7. Between-group comparison was conducted using one-way analysis of variance and

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Dunnett’s test. * p < 0.05.

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Fig. 5. Comparison of carbonylated protein concentrations in the soleus muscle of the casted leg in hypertrophic period

Data are presented as mean ± standard errors, n = 6-7. Between-group comparison was

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conducted using one-way analysis of variance and Dunnett’s test. * p < 0.05.

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Atrophy

Hypertrophy

Leg fixation with a cast for 3 weeks

Control or antioxidant diet for 2 weeks

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Acclimatization for 1 week

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Removal of the cast Measurement 1

Measurement 2

Control group

Astaxanthin group

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β-carotene group

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Figure 1

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Resveratrol group Mix group

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B Gastrocnemius weight (mg/g B.W.)

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Figure 2

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Soleus weight (mg/g B.W.)

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Figure 3

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Cont r ol

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Phospho-FoxO1 Total FoxO1 GAPDH

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Total mTOR

Figure 4

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* 5.0

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Carbonylated protein (nmol/mg)

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Figure 5

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