Effect of 20-hydroxyecdysone on proteolytic regulation in skeletal muscle atrophy
Open accessAbstract
BACKGROUND/AIM: 20-Hydroxyecdystone (20E) is an ecdysteroid hormone which controls molting and reproduction in arthropods. 20E also produces a variety of effects in vertebrates, including enhancing protein synthesis and skeletal muscle regeneration. The effect of 20E on disuse muscle atrophy has not been reported to date. This study examined the proteolytic regulation of 20E in tenotomized rat slow soleus and fast plantaris muscles. MATERIALS AND METHODS: Male Wistar rats were randomly divided into three groups: sedentary control (CON), tenotomy without 20E treatment (TEN), and tenotomy with treatment of 5 mg/kg BW of 20E (TEN+20E). The TEN+20E group was administered 20E via subcutaneous injection to the right thigh for 7 days after tenotomy. RESULTS: 20E treatment tended to attenuate disuse muscle atrophy and reduced ubiquitination only in soleus muscle. CONCLUSION: 20E treatment alleviates skeletal muscle atrophy partially mediated by ubiquitinate pathway, dependent on the muscle phenotype.
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Written from the paper’s full text.
TL;DR
In tenotomized rats, 20E treatment tended to attenuate soleus muscle atrophy and reduced high-molecular-weight ubiquitin conjugates only in soleus, not plantaris. It did not affect body weight, myofibrillar protein content, fiber cross-sectional area, or MURF1 protein expression.
Key findings
- Tenotomy decreased soleus absolute and relative wet weight by 58.97% and 54.4%, respectively, and plantaris absolute and relative wet weight by 18.85% and 11.25%, respectively, versus control.
- 20E treatment lessened tenotomy-induced soleus wet-weight loss (49.13% vs. 58.97% reduction in TEN+20E vs. TEN), but had no additional effect on myofibrillar protein content, fiber CSA, or body weight in either muscle.
- Tenotomy increased high-molecular-weight ubiquitin conjugates in soleus and plantaris (151.1% and 128.3%, respectively, vs. control); 20E attenuated this by 63.1% in soleus only, with no effect in plantaris.
- MURF1 protein expression was not significantly affected by tenotomy or 20E in either soleus or plantaris.
Methods
- Adult male Wistar rats (10 weeks old, approximately 290–310 g) were randomly assigned to control (CON), tenotomy without 20E (TEN), or tenotomy with 20E (TEN+20E), with n=8 per group; 20E was given at 5 mg/kg BW subcutaneously into the right thigh for 7 days after Achilles tendon tenotomy, and rats were sacrificed 24 h after the last treatment.
- Soleus and plantaris muscles were collected for wet weight, myofibrillar protein content, Western blot analysis of ubiquitin conjugates and MURF1, and H&E histology for fiber cross-sectional area.
- Statistical analysis used one-way ANOVA with Student-Newman-Keuls post hoc test, or Kruskal-Wallis with Dunn’s test when variance homogeneity was not assumed, with significance at p<0.05.
Limitations
- The paper states that further study is required to determine the optimal dose and duration of 20E administration and to measure plasma 20E concentration throughout the experiment.
- The paper notes that MURF1 protein may have already returned to baseline by 8 days after tenotomy, and that other proteolytic mechanisms and time courses need further investigation.
- Evident from the text: some outcomes used smaller samples (fiber CSA n=3/group, MURF1 n=5/group, ubiquitin n=6–7/group), only male Wistar rats and a single 20E dose/time point were studied, and protein synthesis/degradation rates were not directly measured.
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Claims the paper makes
0 of 22 cited sources checked · 0 supported · 0 flagged
- Findingown data
Tenotomy significantly reduced mean fiber cross-sectional area (CSA) in soleus and plantaris muscles compared with CON.
“In contrast, the muscle fibers revealed smaller in size associated with the significant (p<0.01)reduction of mean fiber CSA in soleus and plantaris muscles of tenotomized groups when compared with the CON group.”
Quote in the paperSubstantial claim - Findingown data
Tenotomy significantly reduced myofibrillar protein content in both soleus and plantaris muscles compared with CON.
“Corresponding with the muscle wet weight, the myofibrillar protein content was significantly reduced in both soleus and plantaris muscles (p<0.01 and p<0.05, respectively) in TEN compared with CON groups.”
Quote in the paperSubstantial claim - Findingown data
Tenotomy significantly increased high molecular weight (HMW) ubiquitin conjugates in both soleus and plantaris muscles compared with CON (151.1% and 128.3%, respectively).
“As expected, tenotomy significant (p<0.01)increased high molecular weight (HMW.)ubiquitin conjugates in both soleus and plantaris muscles (151.1% and 128.3%, respectively) compared to the CON group.”
Quote in the paperSubstantial claim - Findingown data
20E treatment did not alleviate tenotomy-induced reduction of fiber CSA in soleus or plantaris muscles.
“However, 20E treatment did not alleviate the tenotomyinduced reduction of fiber CSA (Figure 2).”
- Also stated in: § Discussion · plain
“In this study, 20E treatment had no effect on fiber CSA in tenotomy-induced muscle atrophy.”
Quote in the paperSubstantial claim - Also stated in: § Discussion · plain
- Findingown data
20E treatment had no additional effect on myofibrillar protein content in tenotomized soleus or plantaris muscles.
“However, 20E treatment had no additional effect on tenotomized muscle.”
Quote in the paperSubstantial claim
- Premisecitation
20E stimulates dose-dependent [3H] leucine incorporation into muscle proteins of C2C12 and isolated human skeletal cells mediating protein kinase B (AKT) activation.
“20E stimulates in dosedependent manner [ 3 H] leucine incorporation into muscle proteins of C2C12 and isolated human skeletal cells mediating protein kinase B (AKT) activation (17).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [17]
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- Premisecitation
20E enhances muscle hypertrophy of different fiber types in a muscle-specific fashion.
“and 20E enhances muscle hypertrophy of different fiber types in a muscle-specific fashion (15).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [15]
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- Premisecitation
Disuse muscle atrophy is primarily caused by an increase in the rate of protein degradation rather than a decrease in the rate of protein synthesis.
“However, previous compelling evidence has indicated that disuse muscle atrophy is primarily caused by the increase in the rate of protein degradation rather than a decrease in the rate of protein synthesis (4).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [4]
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- Premisecitation
Microarray analysis of tenotomized muscle in mice demonstrated increased expression of genes involved in lysosomal protein degradation (e.g. cathepsin) and calcium-activated protease.
“Microarray analysis of tenotomized muscle in mice demonstrated increased expression of genes involved in lysosomal protein degradation (e.g.cathepsin) (1) and calcium-activated protease (34) in mouse tenotomized muscle.”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [1]
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- cites [34]
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- Premisecitation
Slow-twitch fibers are more sensitive to inactivity or microgravity than fast-twitch fibers.
“Additionally, there is accumulating evidence that slow-twitch fibers are more sensitive to inactivity or microgravity than fast-twitch fibers (19,20)”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [19]
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- cites [20]
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- Premisecitation
The dose of 5 mg/kg BW of 20E is an effective dose to promote regeneration in rat skeletal muscle.
“In this study, the dose of 5 mg/kg BW of 20E was applied to the rat, since the dose-dependent effect of this compound on the muscle fiber growth has been shown and this was an effective dose to promote regeneration in rat skeletal muscle (15).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [15]
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- Premisecitation
The half-life of 20E in rodents is short, e.g., 8 min 15 sec in mice.
“Thus, the half-life of this compound in rodents is short e.g. 8 min 15 sec in mice (11).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [11]
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- May combine two claims. 12
- Classified as probably not a substantial claim. 8
May combine two claims.
Findingown dataTenotomy for 8 days induced muscle atrophy in both soleus and plantaris muscles, with greater atrophy in the predominantly slow soleus than in the predominantly fast plantaris.
“There were significant (p<0.01)decreases in the absolute and relative wet weight of soleus muscles (by 58.97% and 54.4%, respectively) 8 days after Achilles tendon transection compared to the CON group.Additionally in the tenotomized groups, the absolute and relative plantaris wet weights decreased by 18.85% and 11.25%, respectively, as compared with the CON group.”
- Also stated in: § Discussion · plain
“Our findings that transection of the Achilles tendon for 8 days induced muscle atrophy in both soleus and plantaris muscles and this atrophic effect was greater in the predominantly slow soleus muscle than the predominantly fast plantaris muscle were in accordance with other literature (20).”
Quote in the paperSubstantial claim- Also stated in: § Discussion · plain
May combine two claims.
Findingown data20E treatment attenuated tenotomy-induced loss of soleus muscle wet weight; the reduction was less in the TEN+20E group (49.13%) than in the TEN group (58.97%).
“Interestingly, 20E treatment attenuated tenotomy-induced muscle loss in soleus muscle, the reduction of muscle wet weight was less in the TEN+20E group when compared with the TEN group (49.13% vs. 58.97%)(Figure 1C and D).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataMURF1 protein expression in soleus and plantaris muscles was not significantly affected by tenotomy or by 20E.
“Surprisingly, MURF1 protein expression in soleus and plantaris muscles was not significantly affected by tenotomy nor by 20E (Figure 4A and B).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown data20E treatment attenuated HMW-ubiquitin conjugates by 63.1% in soleus of tenotomized rats, but not in plantaris muscles.
“Interestingly, the level of HMW-ubiquitin conjugates was attenuated by 63.1% in soleus of tenotomized rat treated with 20E, but this effect was not observed in the plantaris muscles (Figure 3A and B).”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
PremisecitationMURF1 protein is mostly up-regulated in various models of muscle atrophy including immobilization, denervation, and hindlimb suspension.
“Nevertheless, the finding that tenotomy did not alter protein expression of MURF1, muscle-specific E3 ligase, in both muscles examined was unexpected, given that this protein is mostly up-regulated in various models of muscle atrophy including immobilization, denervation, and hindlimb suspension (33).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [33]
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PremisecitationEcdysteroids, insect hormones, have an anabolic effect on skeletal muscle with no side-effects in mammals, including humans.
“There is evidence that ecdysteroids, insect hormones, have an anabolic effect on skeletal muscle (6) with no side-effects in mammals, including humans (7).”
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Interpretationreasoning5 mg/kg BW of 20E treatment may be an effective dose in regenerating soleus fiber CSA but is not sufficient for increasing CSA in atrophied muscle.
“Thus, 5 mg/kg BW of 20E treatment may be an effective dose in regenerating soleus fiber CSA but is not sufficient for increasing the CSA in atrophied muscle.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [15]
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PremisecitationThe ubiquitin-proteasome system (UPS) represents the major intracellular proteolysis machinery responsible for degradation of major contractile proteins and contributes significantly to muscle atrophy in animal and human models.
“Although several proteolytic systems are involved in protein turnover in skeletal muscle, recent studies suggested that the ubiquitin-proteasome system (UPS) represents the major intracellular proteolysis machinery responsible for the degradation of major contractile proteins, and contributes significantly to muscle atrophy in both animal and human models (1,5).”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [1]
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- cites [5]
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PremisecitationAnabolic action of 20E includes increased fiber size in a muscle-specific manner, as well as increasing the myonuclear number in both normal and regenerating muscles in rat.
“Anabolic action of 20E includes increased fiber size in a muscle-specific manner, as well as increasing the myonuclear number in both of normal and regenerating muscles in rat (15).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [15]
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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Premisecitation20E affects certain major metabolic pathways such as protein synthesis, and lipid and carbohydrate metabolism.
“Notably, 20E affects certain major metabolic pathways such as protein synthesis, and lipid and carbohydrate metabolism (13).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [13]
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Interpretationown data20E treatment alleviates tenotomy-induced skeletal muscle atrophy in predominantly slow-twitch muscle, partially mediated by the ubiquitin-proteasome pathway, in rat skeletal muscle.
“In conclusion, we demonstrated that 20E attenuated tenotomy-induced muscle atrophy in predominantly slowtwitch muscle, partially mediated by ubiquitin proteasome pathway in rat skeletal muscle.”
- Also stated in: § Abstract · plain
“20E treatment alleviates skeletal muscle atrophy partially mediated by ubiquitinate pathway, dependent on the muscle phenotype.”
Quote in the paperSubstantial claim- Also stated in: § Abstract · plain
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Premisecitation20E has low toxicity in mammals: the LD50 is 6.4 g/kg in mice for intraperitoneal injection and more than 9 g/kg when given orally.
“20E has low toxicity in mammals: the lethal dose 50 (LD 50 ) is 6.4 g/kg in mice for intraperitoneal injection and more than 9 g/kg when given orally (9).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [9]
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Study design and conductreasoningAnimals were pair-fed to eliminate confounding factors of food intake between groups.
“In order to eliminate confounding factors of food intake between groups, animals were pair-fed with standard food and water.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataThe physical and spectroscopic data of the isolated 20E were in accordance with literature values.
“The physical and spectroscopic (nuclear magnetic resonance and mass spectra) data of the isolated compound were found to be in accordance with the literature values (22,23).”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [22]
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PremisecitationMURF1 mRNA tended to increase to the highest level after 3 days and then declined at 7 days in ambulatory muscles following immobilization.
“Furthermore, a recent study demonstrated that the MURF1 mRNA tended to increase to the highest level at after 3 days and then declined at 7 days in the ambulatory muscles following immobilization (35).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [35]
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Study design and conductown dataRats were randomly assigned to three groups (CON, TEN, TEN+20E).
“Rats were randomly assigned to three groups (n=8 in each group): controls (CON) received vehicle [olive oil plus dimethyl sulfoxide (DMSO; not exceeding 10% of volume injected)]; tenotomy without 20E treatment (TEN), which received vehicle; and tenotomy with 20E treatment (TEN+20E) received 20E at a dose of 5 mg/kg BW diluted in vehicle.”
Quote in the paperSubstantial claimMay combine two claims.
Premisecitation20E is rapidly eliminated by various routes (bile, urinary, feces) with low toxicity in mammals.
“20E was found to be rapidly eliminated by various routes e.g.bile, urinary and feces with low toxicity in mammals (12).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [12]
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The paper relies on this without support.
The effect of 20E on disuse muscle atrophy had not been reported before this study.
“The effect of 20E on disuse muscle atrophy has not been reported to date.”
The paper relies on this without support.
The response of the muscle proteolytic pathway to 20E is unknown.
“Although 20E has anabolic action, the response of the muscle proteolytic pathway to this compound is still unknown.”
The paper relies on this without support.
There is no single effective method to completely cure muscle atrophy.
“To date, in spite of many attempts, there is no single effective method to completely cure muscle atrophy.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
After cutting the Achilles tendon, high-molecular-weight ubiquitin conjugates decreased in the soleus but not plantaris, suggesting attenuation of proteolysis depending on muscle type.
“After cutting the Achilles tendon, the level of high-molecular-weight ubiquitin conjugates was decreased in the soleus but not in the plantaris muscle, suggesting attenuation of proteolysis depending on muscle type [15].”
From Molecular Targets of 20-Hydroxyecdysone in Mammals, Mechanism of Action: Is It a Calorie Restriction Mimetic and Anti-Aging Compound? (2025) · cites this paper as [15]
The results section (‘Effect of 20E on components of the UPS after tenotomy’) says tenotomy increased high-molecular-weight ubiquitin conjugates in both soleus and plantaris muscles, contrary to a decrease after tendon cutting. The soleus-only decrease occurred in tenotomized rats treated with 20-hydroxyecdysone, compared with tenotomy alone; the Discussion (p. 874) likewise attributes the muscle-specific effect to treatment. (LLM, conf=0.90)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measured20E appears to decrease protein breakdown and increase protein synthesis.
“Although the mechanism of this anabolic effect after detraining is not fully understood, 20E appears to decrease protein breakdown [18] and increase protein synthesis [14].”
Muscle atrophy was alleviated by 20E in the soleus in the tenotomized leg of adult male rats.
“Muscle atrophy was alleviated by 20E in the soleus in the tenotomized leg of adult male rats.”
From Molecular Targets of 20-Hydroxyecdysone in Mammals, Mechanism of Action: Is It a Calorie Restriction Mimetic and Anti-Aging Compound? (2025) · cites this paper as [15]
The signaling pathway of the ubiquitin-proteasome was suggested to be involved in the ecdysteroids-induced increase in protein synthesis, together with a downstream activation of the PI3K/Akt pathway.
“the signaling pathway of the ubiquitin-proteasome was suggested to be involved in the ecdysteroids-induced increase in protein synthesis [28,29,30], together with a downstream activation of the PI3K/Akt pathway [31].”
Ecdysteroids have shown anabolic effects in vertebrates and humans.
“Interestingly, ecdysteroids, a class of hormones found in arthropods responsible for molting and reproduction, have shown anabolic effects in vertebrates and humans [5,6,7].”
From Phytoecdysteroids and Anabolic Effect of Atriplex dimorphostegia: UPLC-PDA-MS/MS Profiling, In Silico and In Vivo Models (2023) · cites this paper as [5]
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Self-citationDenben, B., Sripinyowanich, S., Ruangthai, R., & Phoemsapthawee, J. (2023) · Sports · doi:10.3390/sports11090175 · Cited by 6 · see citing works
“In addition, a previous study demonstrated that 20E attenuated tenotomy-induced muscle atrophy in predominantly slow-twitch muscle in rat skeletal muscle [18].”
BackgroundKnížková, K., Cihlářová, P., & Kuchař, M. (2025) · Food Analytical Methods · doi:10.1007/s12161-025-02805-y · Cited by 5 · see citing works
Dini, I. (2020) · Nuts and Seeds in Health and Disease Prevention · doi:10.1016/b978-0-12-818553-7.00016-4 · Cited by 2 · see citing works
Semenova, A. A., Igoshkina, A. D., Cherepanova, A. A., Mikina, N. V., Stepanova, A. E., Krasnoshchekova, O. E., Sharapov, V. A., Савченко, Р. Г., Parfenova, L. V., & Dubinin, M. V. (2025) · Biocell · doi:10.32604/biocell.2025.061798 · Cited by 1 · see citing works
Suhatcho, K., Yingyongnarongkul, B., Kumpun, S., & Srikuea, R. (2023) · Tropical Journal of Pharmaceutical Research · doi:10.4314/tjpr.v22i10.11
Metabolic and Hormonal Modulation by Phytoecdysteroids in Mammals
Lafont, R. (2026) · Springer eBooks · doi:10.1007/978-981-95-8423-9_3
- Authors
- HIRUNSAI, M., YIMLAMAI, T., & SUKSAMRARN, A.
- Affiliations
- Mahidol University (TH)
- Chulalongkorn University (TH)
- Ramkhamhaeng University (TH)
- Venue
- In Vivo
- Published
- 2016-11-04
- Volume
- 30
- Issue
- 6
- Pages
- 869-878
- ISSN
- 0258-851X (ISSN-L)
- 1791-7549
- OpenAlex
- W2547866881
- Semantic Scholar
- ab9f59a2e395d282dcb495e7db26548687c1193c
- Cited by
- 13
- References
- 32
- Citations by year
- 2026: 1 · 2025: 3 · 2023: 4 · 2021: 2 · 2020: 1 · 2017: 2
- FWCI
- 0.35
- Type
- journal article
- Language
- en
- Open access
- diamond
- doi.org
- Locations
- In Vivo (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Mahidol University
- Thailand Research Fund (DBG 5980003)
- Faculty of Science, Mahidol University
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Muscle metabolism and nutrition
- Health Sciences › Medicine › Physiology › Nutrition and Health in Aging
- Keywords
- 20-hydroxyecdysone, ecdysteroids, skeletal muscle atrophy, disuse atrophy, tenotomy, ubiquitination, proteolytic regulation, soleus muscle, plantaris muscle, muscle fiber type, protein synthesis
- MeSH terms
- Animals, Ecdysterone, Injections, Subcutaneous, Male, Muscular Atrophy, Random Allocation, Blotting, Western, Signal Transduction, Rats, Wistar, Muscle, Skeletal, Ubiquitination, Ubiquitinated Proteins, Tenotomy, Proteolysis
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
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