Phenethyl isothiocyanate suppresses protein degradation through the Akt pathway in the skeletal muscle of rats

Abstract

Phenethyl isothiocyanate (PEITC) is an aromatic compound found in cruciferous vegetables. We previously have shown that it induces glucose utilization through Akt activation in mouse skeletal muscle cells, C2C12 myotubes. The Akt pathway regulates protein metabolism. However, the effects of PEITC on muscle protein metabolism remain unclear. This study investigated whether PEITC regulates the degradation of muscle protein in isolated rat skeletal muscles. Treatment with PEITC induced the phosphorylation of Akt in the rat extensor digitorum longus (EDL) muscles. PEITC suppressed the release of 3-methylhistidine (MeHis), a marker of protein degradation in the EDL muscles. Moreover, it tended to suppress the expression of atrogin-1, a muscle specific ubiquitin ligase. Akt inhibition diminished the suppressive effect of PEITC on MeHis release and atrogin-1 expression. These findings suggest that PEITC regulates muscle protein metabolism through Akt. PEITC may therefore serve as a dietary component with useful effects on skeletal muscle metabolism.

Introduction

Skeletal muscles are essential for maintaining and changing body position, locomotion, and metabolic functions (Lee and Jun, 2019; Severinsen et al., 2020). Muscle atrophy occurs when the rate of protein degradation exceeds the rate of protein synthesis. Protein degradation in skeletal muscles is caused by various pathological and physiological conditions that have a significant impact on quality of life (Fanzani et al., 2012). Moreover, skeletal muscle contributes to approximately 40 % of the body mass and is a major site of glucose and energy metabolisms (Frontera et al., 2015). Therefore, the preservation of muscle mass and function plays a crucial role in maintaining optimal health.

The ubiquitin-proteasome system (UPS) is a major regulatory mechanism of protein degradation in skeletal muscles. Ubiquitin ligases play a major role in the regulation of the UPS (Rom and Reznick, 2016). Muscle-specific ubiquitin ligases, muscle atrophy F-box proteins (atrogin-1), and muscle RING finger-1 (MuRF-1) are expressed in the skeletal muscle during cancer cachexia, aging, diabetes, disuse, and nutritional conditions (Bodine et al., 2001; Fanzani et al., 2012; Rom and Reznick, 2016). Activation of Akt by anabolic physiological factors such as insulin-like growth factor-1 (IGF-1) and insulin regulates atrogin-1 expression; it suppresses atrogin-1 expression and myofibrillar protein degradation (Sacheck et al., 2004; Sandri et al., 2004). Therefore, the Akt pathway plays an important role in protein metabolism.

Dietary components also play a role in the regulation of protein metabolism, and Akt activation is involved in their mechanism of action. Dietary Lys decreased myofibril protein degradation associated with Akt activation in rats fed with a low protein diet and in senescence-accelerated mouse-prone 8 (SAMP8) mice (Sato et al., 2015; Sato et al., 2017). Similar results have been reported for plant-derived phytochemicals. Oligonol administration increased the phosphorylation of Akt and suppressed the expression of atrogin-1 and MuRF-1 in the skeletal muscles of SAMP8 mice (Chang et al., 2019). Otsuka et al. (2019) reported that quercetin increases Akt phosphorylation and reduces dexamethasone-induced atrogin-1 and MuRF-1 expression in C2C12 cells. The isothiocyanates present in cruciferous vegetables have potential health benefits and affect muscle protein metabolism. Sulforaphane, a bioactive compound found in broccoli, attenuates muscle atrophy via Akt activation in C2C12 myotubes (Son et al., 2017). We previously showed that phenethyl isothiocyanate (PEITC), a compound found in watercress, activates the Akt pathway to induce glucose uptake in C2C12 myotubes (Chiba et al., 2019). As Akt is also an important regulator of protein metabolism, PEITC may have a regulatory effect on protein degradation. However, its precise effect on protein metabolism remains unclear. Therefore, we investigated the suppressive effects of PEITC on muscle protein degradation in isolated rat skeletal muscles. Our results suggest that PEITC inhibits skeletal muscle degradation, potentially contributing to the improvement of systemic metabolic function.

Materials and Methods

Reagents  PEITC was purchased from LKT Laboratories (P2508, purity ≥ 99 %, St. Paul, MN, USA). Anti-Akt (#4691), anti-pAkt (Ser473) (#4060), anti-pAkt (Thr308) (#9275), anti-phospho-p44/42 Map kinase (Thr202/Tyr204) (#9101), anti-phospho-p38 MAPK (Thr180/Tyr182) (#4511), and anti-GAPDH (#5174) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-p38 MAPK (A-12) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-ERK antibody was obtained from BD Transduction Laboratories (Franklin Lakes, NJ, USA). Anti-atrogin-1 antibody (AP2041) was purchased from PhosphoSolutions (Aurora, CO, USA). All the other reagents were of analytical grade.

Animals  Four-week-old male Wistar rats were obtained from Japan SLC, Inc. (Shizuoka, Japan). The rats were individually housed in stainless steel wire cages and were maintained at 22 °C and 55 % relative humidity, with a 12 h light/dark cycle (6:00–18:00). They had free access to tap water and a 20 % casein diet based on AIN-93G. All animal protocols were approved by the Iwate University Animal Research Committee (approval number: A202104) and performed in accordance with the guidelines for animal experiments at Iwate University. After 3–4 days of acclimation, the rats were fasted overnight to induce proteolysis in the skeletal muscle and then euthanized. The extensor digitorum longus (EDL) and soleus muscles were removed from the hind legs and subjected to an ex vivo incubation as previously described (Sugawara et al., 2009) to measure the intracellular signaling responses and proteolysis rates.

Sample preparation for western blotting and proteolytic assessment in isolated skeletal muscles  Isolated EDL and soleus muscles were pre-incubated in a Krebs-Ringer bicarbonate (KRB) buffer supplemented with 10 mM glucose under 95 % O₂–5 % CO₂ at 37 °C for 30 min. The muscles were moved to a new KRB buffer supplemented with 10 mM glucose and various concentration of PEITC (0, 20, 30, and 40 µM) with Akt inhibitor (Akt 1/2 kinase inhibitor, 1 µM, Sigma-Aldrich, St. Louis, MO, USA) or without for 3 h. After incubation, the muscles were removed from the KRB buffer and homogenized in 20 volumes of buffer solution (50 mM HEPES-NaOH (pH 7.6), 10 mM Na₃VO₄, 10 mM sodium pyrophosphate, 100 mM NaF, 2mM EDTA, 2 % (w/v) Triton X-100, and 2 mM phenylmethylsulfonyl fluoride). The homogenates were centrifuged and the supernatants were subjected to western blot analysis. The KRB buffer, from which the muscles were removed, was used to measure the rate of MeHis release and aspartate aminotransferase (AST) levels.

Determination of aspartate aminotransferase (AST) levels  The KRB buffer incubated with skeletal muscles of rats was used to examine tissue damage levels. The AST activities in the KRB buffer were determined by the enzymatic method using the Transaminase CII Test Wako (FUJIFILM Wako, Osaka, Japan).

Western blotting  Equal amounts of protein were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (FUJIFILM Wako). The membrane was immersed in blocking buffer (1 % polyvinylpyrrolidone in 20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 1 mM EDTA, and 0.1 % Tween 20 [TBS-T]) or 1 % skim milk at room temperature for 1 h. Next, it was incubated with primary antibodies overnight at 4 °C. The membrane was then incubated with the appropriate secondary antibody, i.e., anti-rabbit or anti-mouse IgG, conjugated with horseradish peroxidase at room temperature for 1 h. Immunoreactive proteins were detected using a chemiluminescence system and a lumino image analyzer (ImageQuant LAS 4000, GE Healthcare) and subsequently analyzed using NIH Image.

Measurement of 3-methylhistidine (MeHis) release  The amount of MeHis in the KRB buffer incubated with skeletal muscles of rats was determined through high-performance liquid chromatography as previously described (Sato et al., 2013).

Statistical analysis  All data are expressed as the mean ± SE. One-way analysis of variance and Tukey’s post-hoc tests were performed using GraphPad InStat (ver. 3.0a, GraphPad Software, Inc., San Diego, CA, USA) to compare groups with comparable variances. p < 0.05 was considered statistically significant.

Results

Effects of PEITC on Akt activation in isolated EDL and soleus muscles of rats  We first investigated whether PEITC regulates Akt activation in skeletal muscles isolated from fasted rats. The ex vivo incubation method used to evaluate proteolytic activity in isolated skeletal muscles was applied to this analysis (Sugawara et al., 2009). Akt activation was observed when isolated EDL and soleus muscles were incubated with several concentrations of PEITC in a Krebs-Ringer bicarbonate buffer, with the highest response observed at 30 µM in EDL muscles. However, the response was less clear in isolated soleus muscle (Fig. 1a and 1b). As shown in Fig. 2, there were no difference in AST levels in the KRB incubation buffer. Therefore, the difference in Akt response in the EDL and soleus muscles is not likely to be related to the condition of isolated muscles.

Fig. 1

Comparison of Akt phosphorylation in response to PEITC stimulation in rat soleus and EDL muscles.

Isolated soleus or EDL muscles were incubated with PEITC (0, 20, 30, or 40 µM) for 3 h in a Krebs-Ringer bicarbonate buffer. Homogenates were prepared as described in the Materials and Methods. Equal amounts of protein were analyzed through western blotting using anti-phospho Akt (Ser473), anti-phospho Akt (Thr308), and anti-Akt antibodies. (a) The Akt phosphorylation levels in the soleus muscle. (b) The Akt phosphorylation levels in the EDL muscle. Data are expressed as the mean ± SE (n = 4). Significant differences are indicated by different letters (p < 0.05).

Fig. 2

Effects of PEITC on the release of tissue injury markers in the incubation medium from rat soleus and EDL muscles.

Isolated soleus or EDL muscles were incubated with PEITC (0, 20, 30, or 40 µM) for 3 h in a Krebs-Ringer bicarbonate buffer. The level of aspartate aminotransferase (AST) activity in the incubation medium from the soleus (a) or EDL muscles (b) was evaluated as a tissue injury marker, as described in the Materials and Methods. Data are expressed as the mean ± SE (n = 4).

The role of Akt activation on PEITC-induced proteolysis suppression in isolated EDL muscles  As Akt activation was clearly observed in EDL muscles stimulated with PEITC, we evaluated the effects of Akt inhibition on the suppressive activity of PEITC on protein degradation in isolated EDL muscles. The phosphorylation of Akt stimulated with PEITC was suppressed when EDL muscles were treated with the Akt1/2 kinase inhibitor (Akti, 1 µM) (Fig. 3a). We further measured effects of PEITC on the rate of MeHis release from isolated skeletal muscles. MeHis is a post-translationally modified, non-recyclable amino acid that is mainly located in actin and myosin in myofibrils. Therefore, the rate of MeHis release can be used as a marker of protein degradation. The rate of MeHis release was suppressed in isolated EDL muscles stimulated with PEITC. The suppressive activity of PEITC on MeHis release from isolated EDL muscles was reduced by the inhibition of Akt activation (Fig. 3b). As shown in Fig. 3c, atrogin-1 expression tended to decrease with PEITC treatment and was slightly increased by inhibition of Akt compared to PEITC treatment alone. However, these changes in atrogin-1 expression were not statistically significant. These results suggest that PEITC suppresses protein degradation via Akt activation in isolated EDL muscles of rats.

Fig. 3

The role of Akt activation on PEITC-mediated suppression of protein degradation in rat EDL muscles.

Isolated EDL muscles were incubated with 30 µM PEITC in the presence or absence of an Akt inhibitor (1 µM, Akti) for 3 h in a Krebs-Ringer bicarbonate buffer. Homogenates were prepared as described in the Materials and Methods. Equal amounts of protein were analyzed through western blotting using anti-phospho Akt (Ser473), anti-phospho Akt (Thr308), and anti-Akt (a), and anti-atrogin-1 and anti-GAPDH (c) antibodies. The rate of protein degradation in the EDL muscles was evaluated based on the concentration of released MeHis in the medium, as described in the Materials and Methods (b). Data are expressed as the mean ± SE (n = 6 for western blotting, n = 6–10 for MeHis release). Significant differences are indicated by different letters (p < 0.05).

Effects of PEITC on ERK and p38 MAPK pathways in isolated rat EDL muscles  We previously reported that PEITC induces ERK activation in addition to Akt activity in C2C12 myotubes (Chiba et al., 2019). Here we investigated whether PEITC also activates other signaling pathways in isolated EDL muscles. ERK phosphorylation was observed upon PEITC stimulation (Fig. 4b). Furthermore, PEITC significantly induced p38 MAPK activation (Fig. 4c). These results suggest that PEITC induces several intracellular signal responses in the ex vivo analysis of isolated EDL muscles.

Fig. 4

The effect of PEITC on ERK and p38 MAPK activities in rat EDL muscles.

Isolated EDL was incubated with PEITC (0, 20, 30, or 40 µM) for 3 h in a Krebs-Ringer bicarbonate buffer. Homogenates were prepared as described in the Materials and Methods. Equal amounts of protein were analyzed using western blotting. (a) Representative blots. (b) ERK phosphorylation levels in the EDL muscles. (c) Phosphorylation levels of p38 MAPK in the EDL muscles. Data are expressed as the mean ± SE (n = 4). Significant differences are indicated by different letters (p < 0.05).

Discussion

We have previously shown that PEITC induces glucose uptake through Akt activation in C2C12 myotubes (Chiba et al., 2019). In addition, Akt regulates protein and glucose metabolism. However, the involvement of PEITC-induced Akt activation in muscle protein metabolism remains unclear. The present study demonstrates that PEITC suppresses protein degradation through the Akt pathway in rat skeletal muscles (Fig. 5).

Fig. 5

Summary of present study.

PEITC, a dietary isothiocyanate found in cruciferous vegetables, suppresses protein breakdown in rat skeletal muscle via Akt activation.

Several studies have shown that isothiocyanates regulate skeletal muscle mass maintenance. The physiological effects of sulforaphane, an isothiocyanate found in cruciferous vegetables, have been well investigated in skeletal muscles. Dietary sulforaphane restores age-associated decline in muscle strength and satellite cell activity in the skeletal muscles of aged mice (Bose et al., 2020). Muscular dystrophy in Duchenne muscular dystrophy mice was suppressed by the administration of sulforaphane, and the muscle mass of the EDL and soleus muscles increased compared with that in control mice (Sun et al., 2015). In addition, a decrease in the cell diameter of serum-starved C2C12 myotube cells was mitigated by treatment with sulforaphane (Moon et al., 2020). These results were attributed to increased antioxidative activity through sulforaphane-induced activation of the Nrf2 pathway and reduction of oxidative stress associated with age, disease, and starvation. Furthermore, sulforaphane and PEITC can activate Akt. Son et al. (2017) reported that Akt activation by sulforaphane suppressed dexamethasone-induced atrogin-1 mRNA expression in C2C12 myotube cells. These results are consistent with those of the present study. However, they did not provide sufficient evidence to establish that Akt activation by sulforaphane was necessary for its inhibitory effect on muscle atrophy (Son et al., 2017). To our knowledge, our study is the first to demonstrate that PEITC suppresses muscle protein degradation via the Akt pathway.

In the present study, in addition to Akt activation, we observed the activation of other signaling pathways, such as p38 MAPK and ERK, in the EDL muscle stimulated with PEITC (Fig. 4). These signaling pathways may also regulate the suppressive effects of PEITC on protein degradation. For instance, p38 MAPK is involved in muscle atrophy under various catabolic states. Hindlimb unloading and cast immobilization of the hindlimb induce muscle atrophy associated with the expression of atrogin-1 and MuRF-1 and the activation of p38 MAPK (Derbre et al., 2012; Kim et al., 2009). The TNF-α-stimulated expression of atrogin-1 is increased in C2C12 myotubes. This expression is suppressed by inhibition of p38 MAPK (Li et al., 2005). ERK activation is also involved in muscle protein atrophy. The simulation of microgravity through 3D-clinorotation induces the expression of atrogin-1 and MuRF-1, along with the phosphorylation of ERK, in C2C12 myotubes. Catechins and quercetin treatments reduce both atrogin-1 and MuRF-1 expression and ERK activation induced by 3D-clinorotation (Hemdan et al., 2009). Penna et al. (2010) reported an increase in ERK phosphorylation and atrogin-1 expression in the gastrocnemius muscle of mice bearing the C26 carcinoma, revealing that the inhibition of ERK activation prevents muscle wasting in tumor-bearing mice. These findings imply that the activation of p38 MAPK and ERK may negatively affect the suppressive effect of PEITC on protein degradation. However, the effects of p38 MAPK and ERK on muscle protein metabolism are controversial, and the activation of these pathways may be associated with muscle growth and maintenance of muscle mass (Perdiguero et al., 2007; Shi et al., 2009). Therefore, further studies are required to elucidate the roles of these kinases in PEITC-regulated muscle protein metabolism.

We have evaluated the effects on regulation of protein metabolism in skeletal muscles of various animal models by measuring the release of MeHis from isolated skeletal muscles in KRB buffer (Sato et al., 2015; Sato et al., 2017; Sugawara et al., 2009). In the present study, we applied the incubation method to analyze the effect of PEITC on protein degradation in rat skeletal muscles incubated in PEITC-supplemented KRB buffer. As shown in results, isolated EDL muscles exhibited a significant response to PEITC treatment, i.e., PEITC induced several signaling molecules such as Akt and MAPKs, and suppressed the release of MeHis from isolated EDL muscles of rats through Akt activation. However, comparable reactions were not observed in isolated rat soleus muscles. Differences in the responses of the EDL and soleus muscles have also been observed in previous ex vivo studies. Soleus muscles exhibited a greater response to IL-6 release by a cytokine stimulant (a mixture of phorbol 12-myristate 13-acetate and ionomycin). In addition, they exhibited a higher glucose uptake by osteocalcin than EDL muscles (Liang et al., 2018; Lin et al., 2018). Contraction-stimulated glucose uptake and AMPK phosphorylation are higher in the EDL muscle than the soleus muscle (Jensen et al., 2009). Similarly, changes in atrogin-1 mRNA expression during the transition between fed and fasted states were more prominent in the EDL muscle than in the soleus muscle (Holeček et al., 2017). These differences in response to various stimuli may be due to variations in the metabolic characteristics of oxidative (soleus) or glycolytic (EDL) muscles. Thus, the observed results may also be attributed to the differences in muscle types. However, Li et al. (2018) showed that both the EDL and soleus muscles respond to insulin stimulation, and that the Akt activation and glucose uptake are induced at the similar levels. Therefore, further investigation is needed to clarify the different responses to PEITC in both muscles because phosphorylation of Akt and MAPKs upon PEITC stimulation was observed in some soleus muscles.

In conclusion, PEITC suppressed MeHis release and tended to decrease atrogin-1 expression in isolated EDL muscle of rats. Moreover, the effects of PEITC on proteolysis in skeletal muscles are dependent on Akt activation. Therefore, PEITC may serve as a dietary component with useful effects on the regulation of protein metabolism in skeletal muscle.

Author contributions  Y.I. designed the study; S.I., M.S., and A.E. performed the experiments; S.I., M.S., A.E., Y.I., and T.N. analyzed the data and discussed the results; and S.I., M.S., A.E., and Y.I. wrote the manuscript. All the authors have read and approved the final manuscript.

Acknowledgements  This work was supported in part by the Grant-in-Aid for Scientific Research (C) (JSPS KAKENHI Grant numbers JP21K05419) to Y.I.

Conflict of interest  There are no conflicts of interest to declare.

References

•  Bodine,  S.C.,  Latres,  E.,  Baumhueter,  S.,  Lai,  V.K.,  Nunez,  L.,  Clarke,  B.A.,  Poueymirou,  W.T.,  Panaro,  F.J.,  Na,  E.,  Dharmarajan,  K.,  Pan,  D.M.,  DeChiara,  T.M.,  Stitt,  T.N.,  Yancopoulos,  G.D., and  Glass,  D.J. (2001). Identification of ubiquitin ligases required for skeletal muscle atrophy. Science, 294, 1704–1708. doi: 10.1126/science.1065874
•  Bose,  C.,  Alves,  I.,  Singh,  P.,  Palade,  P.T.,  Carvalho,  E.,  Børsheim,  E.,  Jun,  S.R.,  Cheema,  A.,  Boerma,  M.,  Awasthi,  S., and  Singh,  S.P. (2020). Sulforaphane prevents age-associated cardiac and muscular dysfunction through Nrf2 signaling. Aging Cell, 19, e13261. doi: 10.1111/acel.13261
•  Chang,  Y.C.,  Chen,  Y.T.,  Liu,  H.W.,  Chan,  Y.C.,  Liu,  M.Y.,  Hu,  S.H.,  Tseng,  W.T.,  Wu,  H.L.,  Wang,  M.F., and  Chang,  S.J. (2019). Oligonol alleviates sarcopenia by regulation of signaling pathways involved in protein turnover and mitochondrial quality. Mol. Nutr. Food Res., 63, e1801102. doi: 10.1002/mnfr.201801102
•  Chiba,  M.,  Ito,  Y., and  Nagasawa,  T. (2019). Phenethyl isothiocyanate stimulates glucose uptake through the Akt pathway in C2C12 myotubes. Biosci. Biotechnol. Biochem., 83, 1319–1328. doi: 10.1080/09168451.2019.1594675
•  Derbre,  F.,  Ferrando,  B.,  Gomez-Cabrera,  M.C.,  Sanchis-Gomar,  F.,  Martinez-Bello,  V.E.,  Olaso-Gonzalez,  G.,  Diaz,  A.,  Gratas-Delamarche,  A.,  Cerda,  M., and  Viña,  J. (2012). Inhibition of xanthine oxidase by allopurinol prevents skeletal muscle atrophy: role of p38 MAPKinase and E3 ubiquitin ligases. PLoS One, 7, e46668. doi: 10.1371/journal.pone.0046668
•  Fanzani,  A.,  Conraads,  V.M., and  Penna,  F.M. (2012). Molecular and cellular mechanisms of skeletal muscle atrophy: an update. J. Cachexia Sarcopenia Muscle, 3, 163–179. doi: 10.1007/s13539-012-0074-6
•  Frontera,  W.R. and  Ochala,  J. (2015). Skeletal muscle: a brief review of structure and function. Calcif. Tissue Int., 96, 183–195. doi: 10.1007/s00223-014-9915-y
•  Hemdan,  D.I.,  Hirasaka,  K.,  Nakao,  R.,  Kohno,  S.,  Kagawa,  S.,  Abe,  T.,  Harada-Sukeno,  A.,  Okumura,  Y.,  Nakaya,  Y.,  Terao,  J., and  Nikawa,  T. (2009). Polyphenols prevent clinorotation-induced expression of atrogenes in mouse C2C12 skeletal myotubes. J. Med. Invest., 56, 26–32. doi: 10.2152/jmi.56.26
•  Holeček,  M. and  Mičuda,  S. (2017). Amino acid concentrations and protein metabolism of two types of rat skeletal muscle in postprandial state and after brief starvation. Physiol. Res., 66, 959–967. doi: 10.33549/physiolres.933638
•  Jensen,  T.E.,  Maarbjerg,  S.J.,  Rose,  A.J.,  Leitges,  M., and  Richter,  E.A. (2009). Knockout of the predominant conventional PKC isoform, PKCalpha, in mouse skeletal muscle does not affect contraction-stimulated glucose uptake. Am. J. Physiol. Endocrinol. Metab., 297, E340–348. doi: 10.1152/ajpendo.90610.2008
•  Kim,  J.,  Won,  K.J.,  Lee,  H.M.,  Hwang,  B.Y.,  Bae,  Y.M.,  Choi,  W.S.,  Song,  H.,  Lim,  K.W.,  Lee,  C.K., and  Kim,  B. (2009). p38 MAPK participates in muscle-specific RING finger 1-mediated atrophy in cast-immobilized rat gastrocnemius muscle. Korean J. Physiol. Pharmacol., 13, 491–496. doi: 10.4196/kjpp.2009.13.6.491
•  Lee,  J.H. and  Jun,  H.S. (2019). Role of myokines in regulating skeletal muscle mass and function. Front. Physiol., 10, 42. doi: 10.3389/fphys.2019.00042
•  Li,  Y.P.,  Chen,  Y.,  John,  J.,  Moylan,  J.,  Jin,  B.,  Mann,  D.L., and  Reid,  M.B. (2005). TNF-alpha acts via p38 MAPK to stimulate expression of the ubiquitin ligase atrogin1/MAFbx in skeletal muscle. FASEB J., 19, 362–370. doi: 10.1096/fj.04-2364com
•  Li,  Z.,  Rasmussen,  M.L.,  Li,  J.,  Olguin,  C.H.,  Knudsen,  J.R.,  Søgaard,  O.,  Madsen,  A.B., and  Jensen,  T.E. (2018). Low- and high-protein diets do not alter ex vivo insulin action in skeletal muscle. Physiol. Rep., 6, e13798. doi: 10.14814/phy2.13798
•  Liang,  A.P.,  Drazick,  A.T.,  Gao,  H., and  Li,  Y. (2018). Skeletal muscle secretion of IL-6 is muscle type specific: Ex vivo evidence. Biochem. Biophys. Res. Commun., 505, 146–150. doi: 10.1016/j.bbrc.2018.09.042
•  Lin,  X.,  Parker,  L.,  Mclennan,  E.,  Zhang,  X.,  Hayes,  A.,  McConell,  G.,  Brennan-Speranza,  T.C., and  Levinger,  I. (2018). Uncarboxylated osteocalcin enhances glucose uptake ex vivo in insulin-stimulated mouse oxidative but not glycolytic muscle. Calcif. Tissue Int., 103, 198–205. doi: 10.1007/s00223-018-0400-x
•  Moon,  J.Y.,  Kim,  D.J., and  Kim,  H.S. (2020). Sulforaphane ameliorates serum starvation-induced muscle atrophy via activation of the Nrf2 pathway in cultured C2C12 cells. Cell Biol. Int., 44, 1831–1839. doi: 10.1002/cbin.11377
•  Otsuka,  Y.,  Egawa,  K.,  Kanzaki,  N.,  Izumo,  T.,  Roji,  T., and  Shibata,  H. (2019). Quercetin glycosides prevent dexamethasone-induced muscle atrophy in mice. Biochem. Biophys. Rep., 18, 100618. doi: 10.1016/j.bbrep.2019.100618
•  Penna,  F.,  Costamagna,  D.,  Fanzani,  A.,  Bonelli,  G.,  Baccino,  F.M., and  Costelli,  P. (2010). Muscle wasting and impaired myogenesis in tumor bearing mice are prevented by ERK inhibition. PLoS One, 5, e13604. doi: 10.1371/journal.pone.0013604
•  Perdiguero,  E.,  Ruiz-Bonilla,  V.,  Serrano,  A.L., and  Muñoz-Cánoves,  P. (2007). Genetic deficiency of p38alpha reveals its critical role in myoblast cell cycle exit: the p38alpha-JNK connection. Cell Cycle, 6, 1298–1303. doi: 10.4161/cc.6.11.4315
•  Rom,  O. and  Reznick,  A.Z. (2016). The role of E3 ubiquitinligases MuRF-1 and MAFbx in loss of skeletal muscle mass. Free Radic. Biol. Med., 98, 218–230. doi: 10.1016/j.freeradbiomed.2015.12.031
•  Sacheck,  J.M.,  Ohtsuka,  A.,  McLary,  S.C., and  Goldberg,  A.L. (2004). IGF-I stimulates muscle growth by suppressing protein breakdown and expression of atrophy-related ubiquitin ligases, atrogin-1 and MuRF1. Am. J. Physiol. Endocrinol. Metab., 287, E591–601. doi: 10.1152/ajpendo.00073.2004
•  Sandri,  M.,  Sandri,  C.,  Gilbert,  A.,  Skurk,  C.,  Calabria,  E.,  Picard,  A.,  Walsh,  K.,  Schiaffino,  S.,  Lecker,  S.H., and  Goldberg,  A.L. (2004). Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell, 117, 399–412. doi: 10.1016/s0092-8674(04)00400-3
•  Sato,  T.,  Ito,  Y., and  Nagasawa,  T. (2013). Regulation of skeletal muscle protein degradation and synthesis by oral administration of lysine in rats. J. Nutr. Sci. Vitaminol., 59, 412–419. doi: 10.3177/jnsv.59.412
•  Sato,  T.,  Ito,  Y., and  Nagasawa,  T. (2015). Dietary L-lysine suppresses autophagic proteolysis and stimulates Akt/mTOR signaling in the skeletal muscle of rats fed a low-protein diet. J. Agric. Food Chem., 63, 8192–8198. doi: 10.1021/acs.jafc.5b03811
•  Sato,  T.,  Ito,  Y., and  Nagasawa,  T. (2017). L-Lysine suppresses myofibrillar protein degradation and autophagy in skeletal muscles of senescence-accelerated mouse prone 8. Biogerontology, 18, 85–95. doi: 10.1007/s10522-016-9663-7
•  Severinsen,  M.C.K. and  Pedersen,  B.K. (2020). Muscle-organ crosstalk: The emerging roles of myokines. Endocr. Rev., 41, 594–609. doi: 10.1210/endrev/bnaa016
•  Shi,  H.,  Scheffler,  J.M.,  Zeng,  C.,  Pleitner,  J.M.,  Hannon,  K.M.,  Grant,  A.L., and  Gerrard,  D.E. (2009). Mitogen-activated protein kinase signaling is necessary for the maintenance of skeletal muscle mass. Am. J. Physiol. Cell Physiol., 296, C1040–1048. doi: 10.1152/ajpcell.00475.2008
•  Son,  Y.H.,  Jang,  E.J.,  Kim,  Y.W., and  Lee,  J.H. (2017). Sulforaphane prevents dexamethasone-induced muscle atrophy via regulation of the Akt/Foxo1 axis in C2C12 myotubes. Biomed. Pharmacother., 95, 1486–1492. doi: 10.1016/j.biopha.2017.09.002
•  Sugawara,  T.,  Ito,  Y.,  Nishizawa,  N., and  Nagasawa,  T. (2009). Regulation of muscle protein degradation, not synthesis, by dietary leucine in rats fed a protein-deficient diet. Amino Acids, 37, 609–616. doi: 10.1007/s00726-008-0180-0
•  Sun,  C.,  Yang,  C.,  Xue,  R.,  Li,  S.,  Zhang,  T.,  Pan,  L.,  Ma,  X.,  Wang,  L., and  Li,  D. (2015). Sulforaphane alleviates muscular dystrophy in mdx mice by activation of Nrf2. J. Appl. Physiol., 118, 224–237. doi: 10.1152/japplphysiol.00744.2014