Dietary Genistein Prevents Denervation-Induced Muscle Atrophy in Male Rodents via Effects on Estrogen Receptor-a 1 – 3

Background: Genistein has high estrogenic activity. Previous studies have shown beneficial effects of estrogen or hormone replacement therapy on muscle mass and muscle atrophy. Objective: We investigated the preventive effects and underlying mechanisms of genistein on muscle atrophy. Methods: In Expt. 1, maleWistar rats were fed a diet containing no genistein [control (CON)] or 0.05% genistein (GEN; wt: wt diet) for 24 d. On day 14, the sciatic nerve in the left hind leg was severed, and the right hind leg was sham-treated. In Expt. 2, male C57BL6J mice were subcutaneously administered a vehicle (Veh group) or the estrogen receptor (ER) antagonist ICI 182,780 (ICI group) via an osmotic pump for 27 d, and each group was subsequently fed CON or GEN diets from day 3 to day 27. Muscle atrophy was induced on day 17 as in Expt. 1. In Expt. 3, male C57BL6J mice were subcutaneously administered vehicle or a selective ER agonist—ER-a [4,4#,4#-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT)] or ER-b [2,3-bis(4-hydroxyphenyl)-propionitrile (DPN)]—or genistein (GEN-sc-i) via an osmotic pump for 13 d, and muscle atrophywas induced on day 3 as in Expt. 1. The ratio of denervated soleusmuscleweight to sham-operated soleus muscle weight (d/s ratio) was used as the index of muscle atrophy. Results: Expt. 1: The d/s ratio in the GEN groupwas 20%higher than that in the CON group (P< 0.05). Expt. 2: The d/s ratio in the Veh-GEN group was 14% higher than that in the Veh-CON group (P < 0.05), although there was no significant difference between ICI-CON and ICI-GEN groups (P = 0.69). Expt. 3: The d/s ratio in the PPT-treated groupwas 20% greater than that in the Veh group (P < 0.05), but DPN and GEN-sc-i had no effect on the d/s ratio (P $ 0.05 compared with vehicle). Conclusion: Genistein intake mitigated denervation-induced soleus muscle atrophy. ER-a was related to the preventive effect of genistein on muscle atrophy. J Nutr 2016;146:1147–54.


Introduction
The maintenance of skeletal muscle mass is regulated by the balance between muscle protein synthesis and degradation.The synthesis of muscle protein is controlled by several hormones (including growth hormones and insulin) as well as proteins and amino acids in the diet (1).Skeletal muscle atrophy occurs as a result of enhanced protein degradation, which can be induced by several factors, including low mechanical stress, fasting, steroid treatment, cachexia, and excess oxidative stress.
ligases.Their respective genes, Atrogin1 and Murf1, are upregulated in atrophied muscle and are responsible for the acceleration of the ubiquitin-proteasome pathway in disused atrophied muscle.Atrogin1 and Murf1 knockout mice showed resistance to denervation-induced muscle atrophy (3).The expressions of these genes are regulated by several pathways including the insulin and insulin-like growth factor I (IGF-I) pathways ( 4) and the NF-kB cells pathway (5).
Genistein, a flavonoid that is present at high concentrations in soybeans, has several physiologic actions including estrogenlike and antioxidative activities (6,7).A large part of genisteinÕs physiologic functions has been attributed to its estrogenic activity, because genistein can bind to estrogen receptors (ERs) (6,8).Several effects of estrogen (17-estradiol) have been identified in skeletal muscle (9).Beneficial effects of estrogen or hormone replacement therapy on muscle mass or muscle atrophy have been shown in rodents and in postmenopausal women (10,11).ERs are present in skeletal muscle, and the ER subtypes ER-a and ER-b (12) play notable roles in the differentiation of myoblasts and inflammatory status (13,14).These findings suggest that the estrogenic activity of genistein has a beneficial effect on muscle atrophy.The molecular effects of genistein on muscle loss are currently unclear, however.
Here we investigated the molecular effects of genistein dietary intervention on muscle atrophy in rodent models of denervation-induced atrophy.We report that the dietary intake of genistein suppressed muscle atrophy via the ER-a-mediated modulation of Atrogin1 and Murf1.

Methods
Animal experiments.Rats and mice (as described below) were obtained from Charles River Japan and housed individually in cages in a room maintained at 23 6 2°C and 50% 6 10% humidity on a 12-h light (0800-2000)-dark cycle.They were allowed free access to their diet and water.Each rodentÕs food intake and body weight were monitored throughout the experiment.All surgeries and dissections were performed with the rodents under anesthesia by using isoflurane (Merck Animal Health) or Somnopentyl (Kyoritsu Seiyaku).This study was approved by the Animal Care and Use Committee of the University of Tokyo (approval P12-673), and the rodents were treated in accordance with the committeeÕs guidelines.
Expt. 1. Ten-week-old male Wistar rats were divided into 2 groups (n = 8/group) with equal mean body weights.The control (CON) group was fed a modified AIN-93G diet in which the soybean oil was replaced with corn oil.The genistein (GEN) group was fed an AIN-93G diet containing 0.5 g genistein/kg diet (LC Laboratories) (Supplemental Table 1) for 24 d.On day 14, muscle atrophy was induced by excising the sciatic nerve in the left leg of the rat, and sham surgery was performed for the right leg of each rat during the same operation.Ten days after the operation, the rats were killed by the administration of pentobarbital sodium.The soleus muscle, gastrocnemius muscle, and tibialis anterior muscle from both the right and left legs were collected, weighed, snap-frozen in liquid nitrogen, and stored at 280°C until further analysis.The ratio of denervated soleus muscle weight to sham-operated soleus muscle weight (d/s ratio) was used as the index of muscle atrophy in all experiments.
Expt. 2. Ten-week-old male C57BL6J mice were divided into 4 groups (n = 12/group) with equal mean body weights.Mice were fed the 0% (CON) or 0.05% GEN diet described above and administered vehicle (Veh; 50% DMSO/polyethylene glycol 600; Veh-CON and Veh-GEN, respectively) or the pure ER antagonist ICI 182,780 (ICI; Sigma-Aldrich; ICI-CON or ICI-GEN, respectively).Each solution was administered subcutaneously by using an osmotic pump (Alzet; 1.04 mg/h) throughout the experimental periods.The dose of ICI was equivalent to 1.00 mg/(kg body weight Á d), which has been reported to almost completely repress the estrogen-induced increase of uterine weight in ovariectomized rats (15).Each diet was initiated 3 d after the osmotic pump was implanted.Muscle denervation and tissue collection were performed in the same manner as in Expt. 1 at days 17 and 27, respectively.Tocris Bioscience], and the GEN group received solvent (50% DMSO/ saline) in a subcutaneous injection (sc-i).Genistein (GEN-sc-i) binds both ERs, but it binds more ly to ER-b than to ER-a (16).Each solution described below was administered as described in Expt. 2 for 13 d, at a rate of 1.04 mg/h.The dosages of PPT, DPN, and GEN-sc-i in Expt. 3 were equivalent to 1.00 mg/(kg body weight Á d), which has been reported to increase uterine weight in ovariectomized rats and to possess cardiac muscular protective properties (17,18).The PPT mice consumed the AIN-93G diet ad libitum.The other 3 groups were  Total RNA extraction and real-time RT-PCR.Total RNA from the frozen soleus muscle was isolated by using TRIzol (Invitrogen), as previously described (19).The integrity of the RNA was verified by using the ribosomal RNA 28S to 18S ratio (>2) obtained from agarose-gel electrophoresis.One microgram of total RNA was reverse-transcribed at 37°C by using the PrimeScript TR Enzyme (Takara).A real-time RT-PCR was performed by using a real-time PCR detection system (Takara Bio).The primer sequences are given in Supplemental Table 2.
DNA microarray analysis and data analysis.We conducted a cDNA microarray analysis as previously described (19).We used the GeneChip Rat Genome 230 2.0 Array (Affymetrix) to analyze the gene expression profiles of the RNA samples.
Array images were analyzed with the GeneChip Operating Software (GCOS version 1.1; Affymetrix) to obtain the gene expression ratios between the denervated control and the sham control, as well as those for the denervated CON and the denervated GEN groups.The probe sets that expressed an increase or decrease and those in which at least a higher signal value was observed to be present were used for the subsequent analyses.We analyzed the selected probe sets by performing an Ingenuity Pathway Analysis to map the molecular interaction of the genes (20).The data discussed in this study have been deposited in the National Center for Biotechnology InformationÕs Gene Expression Omnibus (GEO) (21) and are accessible at the GEO Series (accession GSE77121) (22).
Statistical analysis.Results are expressed as means 6 SEMs.We determined whether the data showed equal or biased variation by performing LeveneÕs test or an F test.If there was a significant difference (P < 0.05), data were logarithmically transformed.When the data showed equal variation, they were analyzed by 2-factor ANOVA (Expt.1: denervation 3 genistein intake; Expt.2: genistein intake 3 ICI treatment; Expt.3: denervation 3 ER agonist treatment) or by 3-factor ANOVA (Expt.2: denervation 3 genistein intake 3 ICI treatment).
When an interaction was significant, TukeyÕs test was used as a post hoc analysis.The gene expression data on Atrogin1 and Murf1 in Expt.1 and Foxo1 in Expt. 3 were analyzed by the Kruskal-Wallis test with the Steel-Dwass test for a post hoc analysis, because the log-transformed data had biased variation.The data on the d/s ratio in Expt. 1 were analyzed by unpaired StudentÕs t test for a comparison between 2 groups (CON and GEN groups).In Expt.3, the data on the d/s ratio were analyzed by DunnettÕs test for a comparison between the Veh group and each selective ER agonist group (PPT, DPN, or GEN-sc-i).Differences were considered significant at P < 0.05.All statistical analyses were performed with the use of Ekuseru-Toukei, a statistical analysis software (Social Survey Research Information Co., Ltd.).

Results
Genistein prevented the denervation-induced soleus muscle loss (Expt.1).Rat body weights (CON: 434 6 19 g; GEN: 439 6 14 g) and food intakes (CON: 425 6 21 g; GEN: 433 6 16 g) were not changed by genistein intake.The soleus muscle mass in each group was decreased by denervation (Figure 1A).The d/s ratio in the GEN group was significantly higher than that in the CON group (P < 0.05) (Figure 1B), showing that genistein suppressed denervation-induced soleus muscle atrophy.The d/s ratios of the gastrocnemius muscle (CON: 58.1 6 1.9; GEN: 58.6 6 2.1) and the tibialis anterior muscle (CON: 59.6 6 4.2; GEN: 57.6 6 2.0) were not affected by genistein intake.The expressions of Atrogin1 and Murf1 in the soleus muscle in the CON group were significantly increased by denervation (P = 0.014 and 0.021, respectively), whereas those in the GEN group were not significantly increased by denervation (P = 0.48 and 0.21, respectively) (Figure 1C, D).
Among these genes, the expression levels (data expressed as log 2 ratios, denervated GEN compared with denervated CON) of Foxo1 (20.6), Skp1 (20.4), and Ube2j1 (20.3) were lower than the respective values in the denervated CON soleus muscle.The amount of FOXO1 protein was significantly increased by denervation, and the elevation was significantly suppressed by genistein intake (denervated-CON compared with denervated-GEN; P < 0.05) (Figure 2B).
Genistein intake regulated the expression of ER-targeted genes and ER and ER protein (Expt.1).According to the results of a transcription factor analysis in the Ingenuity Pathway Analysis, the expression of many ER-targeted genes in denervated GEN soleus muscle was changed compared with that of denervated CON soleus muscle (Figure 3).The amounts of ER-a and ER-b proteins were significantly increased and decreased by denervation, respectively (P < 1.00 3 10 211 and P < 0.01, respectively) (Figure 2C, D).These alterations were not affected by genistein intake (P = 0.29 and 0.58, respectively).
Genistein intake prevented muscle atrophy through the modulation of ER-a (Expts. 2 and 3).The d/s ratios in the Veh-GEN group were higher than those in the Veh-CON group (P = 0.0018) (Figure 4A).On the other hand, there was no significant difference between the d/s ratios in the ICItreated groups (ICI-CON compared with ICI-GEN; P = 0.69).The effect of genistein on Foxo1 expression in soleus muscle was attenuated by the ICI treatment (P-interaction < 0.05) (Figure 4D).
We next examined the effects of agonists of ER-a and ER-b to address which of the ER subtypes is involved in the denervationinduced muscle atrophy and the regulation of muscle atrophyrelated genes (Figure 5).The d/s ratios of the soleus muscle in the PPT group but not the DPN and GEN-sc-i groups were significantly higher than those in the Veh group (P = 0.02) (Figure 5A).Denervation-induced upregulations of Atrogin1 expression in the soleus muscles of the Veh, DPN, and GEN-sc-i groups were not observed in the PPT-treated mice (P-interaction < 0.05, denervation compared with sham; Veh: P < 0.0001; PPT: P = 0.165; DPN: P < 0.0001) (Figure 5B).

Discussion
The maintenance of muscle mass in the elderly is important to prevent falls and to avoid the deterioration of quality of life.Some food factors such as quercetin and b-carotene have been reported to prevent muscle loss, an effect that has been attributed to their antioxidative activities (23,24).Hormone replacement therapy has also been shown to have positive effects on muscle mass retention in aging women (10).These reports indicated that some flavonoids, such as isoflavones, with estrogenic activity may have a preventive effect on muscle atrophy.The present study is the first, to our knowledge, to show in animal-based experiments that dietary genistein intake can prevent a denervation-induced loss of soleus muscle through the suppression of FOXO1, which is the transcriptional factor of Atrogin1 and Murf1, and that ER-a is involved in the preventive effects of genistein intake (Figure 6).
However, we did not observe this preventive effect of genistein in the gastrocnemius muscle or the tibialis anterior muscle.Gustafsson et al. (25) reported that ER concentrations in the rabbit soleus muscle were higher than those in gastrocnemius and plantaris muscles.This suggests that a difference in the expression of ER between the muscles is one of the reasons for the greater effects of genistein in soleus muscle than in gastrocnemius and tibialis anterior muscles.
It is generally accepted that muscle fibers can be classified into 2 main types: slow-twitch (type I) muscle fibers and fast-twitch (type II) muscle fibers.Fast-twitch fibers are further categorized into type IIa and type IIb fibers.Soleus muscle fibers are mainly composed of slow-twitch muscle fibers (26), whereas more than half of the gastrocnemius muscle fibers and the tibialis anterior muscle fibers are fast-twitch muscle fibers.Dehority et al. (27) reported that denervation-and tail suspension-induced muscle loss showed differential responses in a muscle fiber typedependent manner.In addition, Ogawa et al. (24) reported that b-carotene suppressed soleus muscle atrophy but not gastrocnemius muscle atrophy.
These reports indicate that the induction of muscle atrophy and its prevention depend on the muscle fiber types.The soleus muscle has a higher capillary density than the gastrocnemius muscle (28), which may lead to a higher bioavailability of active food components in the soleus muscle than in the gastrocnemius muscle.In the present study, we determined the expressions of myosin heavy-chain isoforms.They were not changed by genistein intake, although denervation decreased these expressions (data not shown).We therefore speculate that genistein intake did not affect the population of fiber types in atrophied soleus muscles in our rodent models.
We also observed that among the upstream genes of Atrogin1 and Murf1 that were regulated by denervation, the expression levels of Foxo1, Skp1, and Ube2j1 were downregulated by genistein intake.Although SKP1 and UBE2J1 have been reported to bind to ATROGIN1 and MURF1 (29)(30)(31), their effects on muscle atrophy are unclear.FOXO1 is known as a transcriptional factor regulating Atrogin1 and Murf1 genes (32).Kamei et al. (33) reported that musclespecific Foxo1 overexpression in mice was linked to muscle atrophy.Thus, that the denervation-induced upregulations of Atrogin1 and Murf1 were not observed in the GEN group is likely attributable to the lower Foxo1 expression.
It is well known that genistein binds to ERs and then regulates their transcriptional activity in several cell types (6,8).The results of the present study revealed that genistein intake altered many ER-target genes in denervated soleus muscle (Figure 3).This alteration was not observed in the sham-treated muscles.In addition, denervation increased the concentration of ER-a and decreased that of ER-b.These results suggest that an increase in ER-a protein concentration by denervation may accelerate the binding of genistein to the ER-a protein.
Our findings further show that treatment with an ER antagonist canceled the preventive effects of genistein on muscle atrophy (Figure 4) and that treatment with an ER-a agonist suppressed the loss of soleus muscle (Figure 5).The results of our study support the findings of a study by Brown et al. (34), in which female ERa knockout mice had decreased soleus muscle weight in relation to body weight.However, another report indicated that the activation of ER-a inhibited muscle differentiation and induced muscle loss in C2C12 myoblasts and ovariectomized female mice (13).The difference between our results and theirs may due to the use of the different ligands (genistein and b-estradiol) as well as different experimental models (35).
In our study, genistein intake inhibited a denervation-induced elevation in FOXO1.Moreover, the induction of Foxo1 expression by denervation was not observed when an ER-a agonist was administered, although the difference was not significant (Figure 5D).These results suggest that the genistein intake downregulated the Foxo1 expression via the modulation of ER-a.Schuur et al. (36) observed that ER-a bound to FOXO1, and Foulds et al. (37) reported that the binding of ERa to FOXO1 is dependent on ER ligands and that ER-a-FOXO1 complex binds to estrogen-responsive elements.The expression of Foxo1 is autoregulated through the binding of FOXO1 to its promoter at the FOXO DNA binding site (38).Taken together, these findings indicate that the modulation of ER-a might suppress the transcriptional activity of FOXO1 via recruitment of FOXO1 to ER-a.
In conclusion, our present findings showed, for the first time to our knowledge, that genistein intake has protective activities against denervation-induced muscle loss.Because the activation of ER-a prevented the denervation-induced soleus muscle loss, ER-a is a probable target of genistein in exerting protective action against muscle atrophy.

FIGURE 1
FIGURE 1 Relative soleus muscle mass (A), d/s ratio (B), and mRNA levels of Atrogin1 (C) and Murf1 (D) in sham-operated or denervated soleus muscles of rats fed CON or GEN diets for 24 d (Expt.1).Values are means 6 SEMs, n = 8.Soleus muscle weight data in panel A were tested by 2-factor ANOVA.Labels in panels C and D sharing a common letter indicate a significant difference, P , 0.05 (Steel-Dwass test).*Different from CON, P , 0.05 (unpaired StudentÕs t test).NS, P $ 0.05.Atrogin1, F-box protein 32; BW, body weight; CON, control; Den, denervation; d/s ratio, ratio of weights of the denervated soleus muscle relative to those of the sham-operated soleus muscle; GEN, genistein; Murf1, muscle RING finger 1; 18s rrna, 18s ribosomal RNA.

FIGURE 6
FIGURE 6 Schematic diagram of the effects of Den and GEN intake on muscle atrophy.Den caused muscle loss via the induction of Atrogin1 and Murf1, to which an increase in Foxo1 expression is attributed.The amounts of ER-a and ER-b increased and decreased, respectively, by Den (upper panel).GEN prevented muscle atrophy via ER-a (lower panel).Atrogin1, F-box protein 32; Den, denervation; ER, estrogen receptor; Foxo1, forkhead box O1; GEN, genistein; Murf1, muscle RING finger 1.