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Cited by 31Muscle Physiology and DisordersPhytochemical Studies and BioactivitiesHormonal and reproductive studies

Continuous infusion of 20-hydroxyecdysone increased mass of triceps brachii in C57BL/6 mice

Open access
Cheng, Diana M., Kutzler, Louis W., Boler, Dustin D., Drnevich, Jenny, Killefer, John, Lila, Mary Ann·2013·Phytotherapy Research
DOI 10.1002/ptr.4679PMC PMC3410052
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Abstract

Phytoecdysteroids have been attributed with numerous pharmacological properties in animals, including increasing muscle mass, and 20‐hydroxyecdysone (20E) is one of the most abundant phytoecdysteroids produced by plants. In this study, the physiological and gene expression effects of 20E were analyzed in C57BL/6 mice given a continuous infusion of saline or 20E (5 mg/kg/day) for 5 or 15 days using subcutaneously implanted Alzet® osmotic pumps. The masses of the total body, muscle groups and organs were determined. There was a significant increase ( p = 0.01) in the mass of triceps brachii in mice treated with 20E for 5 days (115 ± 8 mg) compared with mice treated with saline for 5 days (88 ± 3 mg), however, there were no differences in the other measured parameters. To determine potential mechanisms of 20E in skeletal muscle, Illumina's Mouse Whole Genome‐6 v2.0 Expression BeadChips were used to evaluate changes in gene expression of the triceps brachii after 20E infusion. Ingenuity Pathways Analysis was used to identify genes with the most evidence for differential expression, of which, 16 genes involved in the skeletal and muscular system were identified. Overall, the data suggest that 20E does not have potent anabolic properties, however, a muscle‐specific increase was observed and genes were identified to provide an explanation for the muscle accretion. Copyright © 2012 John Wiley & Sons, Ltd.

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Written from the paper’s full text.

TL;DR

Continuous infusion of 20-hydroxyecdysone (20E; 5 mg/kg/day) via subcutaneous osmotic pumps for 5 or 15 days in C57BL/6J mice significantly increased triceps brachii mass at 5 days but did not alter other measured muscle, organ, or body masses. Microarray analysis found no genes significant after FDR correction, and the authors conclude that 20E does not have potent anabolic properties, though a muscle-specific effect was observed.

Key findings

  • At 5 days, 20E-treated mice had greater triceps brachii mass (115 +/− 8 mg) than saline-treated mice (88 +/− 3 mg; p = 0.01). There were no differences in other muscle groups (tibialis anterior, biceps femoris, gastrocnemius), body mass, organ mass, or average daily feed intake, and no differences at 15 days.
  • Microarray analysis of triceps brachii found no beadtypes significantly different after False Discovery Rate correction for 20E vs. saline at either 5 or 15 days. Using a raw p-value cutoff of 0.01, 159 beadtypes were selected at 5 days and 224 at 15 days; IPA mapped these to 104 and 147 network-eligible genes, respectively, and identified 16 genes in skeletal and muscular system categories.
  • 20E was not detected in hydrolyzed plasma samples, possibly due to low plasma sample volume (< 155 μL) and concentration below the limit of detection. Pump reservoir volumes remaining were approximately 160 and 90 μL for the 5- and 15-day treatments, respectively.
  • Overall, the data suggest that 20E does not have potent anabolic properties, but a muscle-specific increase in triceps brachii mass was observed.

Methods

  • Adult male C57BL/6J mice (12 weeks old) were weight-balanced into treatment groups (n = 9–10). Alzet osmotic pumps were subcutaneously implanted to continuously infuse saline or 20E at 5 mg/kg/day for 5 or 15 days. Total body, selected muscle groups, and organs were extracted and weighed; statistical significance was assessed by ANOVA comparing 20E vs. saline at each time point.
  • RNA was isolated from triceps brachii, pooled into groups of three biological replicates (except 15-day saline, which had two), with three pools analyzed per treatment using Illumina MouseWG-6 v2.0 Expression BeadChips. Differential expression was assessed with limma, FDR correction was applied, and genes with raw p < 0.01 were analyzed with Ingenuity Pathways Analysis.

Limitations

  • The paper states that 20E was not detected in plasma, possibly because of low plasma sample volume (< 155 μL) and concentrations below the limit of detection.
  • The paper states that no genes were differentially expressed after FDR correction and that this may be due to the complexity of muscle tissue, individual variation, timing, and hormonal interferences that may confound the mild influence of 20E on skeletal muscle in vivo.
  • The paper notes that studies of 20E growth-promoting effects in mammals are inconsistent, possibly due to varying routes of administration, doses, and rodent strains, and that further research optimizing dose, duration, and route is needed. Evident from the text: the 15-day saline microarray pool had only two biological replicates, fewer than the other pools.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1002/ptr.4679

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 10 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    IPA identified sixteen genes in the categories of skeletal and muscular system development and function and skeletal and muscular disorders.

    “In the categories of skeletal and muscular system development and function and skeletal and muscular disorders, IPA identified sixteen genes presented in Table 2.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The genes selected at raw p < 0.01 (159 at 5 days, 224 at 15 days) include true differentially expressed genes related to 20E treatment, despite no genes passing FDR correction.
  • Findingown data

    Continuous infusion of 20-hydroxyecdysone (20E) at 5 mg/kg/day for 5 days significantly increased triceps brachii mass in C57BL/6 mice compared with saline (115 ± 8 mg vs 88 ± 3 mg, p = 0.01).

    “There was a significant increase (p = 0.01) in the mass of triceps brachii in mice treated with 20E for 5 d (115 +/− 8 mg) compared to mice treated with saline for 5 d (88 +/− 3 mg), however, there were no differences in the other measured parameters.”
    • Also stated in: § Body Tissue Analysis · plain
      “There was a significant increase in the mass of the triceps brachii in the mice treated with 20E for five days (115 +/− 8 mg) compared to mice treated with saline for five days (88 +/− 3 mg) (p = 0.01).”
    • Also stated in: § Discussion · plain
      “Results showed a significant increase in the mass of the triceps brachii in mice treated with 20E for five days compared to mice treated with saline for five days and no differences in masses between treatment groups in the other parameters (Table 1).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The 20E infused subcutaneously at 5 mg/kg/day was bioavailable and reached skeletal muscle in mice despite not being detected in plasma.
    • Rests on an assumption: The statistically significant increase in triceps brachii mass at 5 days is not a false positive arising from multiple comparisons among measured tissues.
  • Interpretationown data

    Overall, the data suggest that 20E does not have potent anabolic properties in C57BL/6 mice under continuous infusion at 5 mg/kg/day for 5 or 15 days.

    “Overall, the data suggests that 20E does not have potent anabolic properties, however, a muscle-specific increase was observed and genes were identified to provide an explanation for the muscle accretion.”
    • Also stated in: § Discussion · boosted
      “Administration of 20E by continuous infusion did not demonstrate potent anabolic properties, however, there was a statistically significant increase in the mass of the triceps brachii.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The 20E infused subcutaneously at 5 mg/kg/day was bioavailable and reached skeletal muscle in mice despite not being detected in plasma.
    • Rests on an assumption: The statistically significant increase in triceps brachii mass at 5 days is not a false positive arising from multiple comparisons among measured tissues.
    • Rests on an assumption: The increase in triceps brachii mass reflects an anabolic effect on skeletal muscle rather than fluid accumulation or other non-muscle changes.
  • Study design and conductown data

    Administration of 20E by infusion was confirmed by measuring the volume of solution remaining in the osmotic pump reservoirs (approximately 160 μL for 5-day and 90 μL for 15-day treatments).

    “Administration of 20E by infusion was confirmed by measuring the volume of 20E solution or saline remaining in the osmotic pump reservoirs; approximately 160 and 90 μL for the five and fifteen day treatments, respectively.”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 9
  • May combine two claims. 7

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingreasoning

    20E was not detected in hydrolyzed plasma samples from mice infused with 20E.

    “However, 20E was not detected in the hydrolyzed plasma samples possibly due to the low plasma sample volume (< 155 μL) and concentration of 20E that was below the limit of detection.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    20E infusion at 5 mg/kg/day for 5 or 15 days did not change total body mass, organ mass, or average daily feed intake compared with saline in C57BL/6 mice.

    “Additionally, results showed no significant differences in body mass, organ mass or average daily feed intake between the treatment groups.”
    • Also stated in: § Discussion · plain
      “In this study, 20E infusion (5 mg/kg/day) for five or fifteen days did not result in an increase in total body mass.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The 20E infused subcutaneously at 5 mg/kg/day was bioavailable and reached skeletal muscle in mice despite not being detected in plasma.

  • May combine two claims.

    Findingown data

    20E infusion at 5 mg/kg/day for 5 or 15 days did not change the masses of tibialis anterior, biceps femoris, or gastrocnemius compared with saline in C57BL/6 mice.

    “However, there were no differences in masses of the other muscle groups (tibialis anterior, biceps femoris and gastrocnemius) and also no differences in mice treated with 20E compared to saline for the longer duration of fifteen days.”
    • Also stated in: § Discussion · plain
      “no differences in masses between treatment groups in the other parameters (Table 1).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The 20E infused subcutaneously at 5 mg/kg/day was bioavailable and reached skeletal muscle in mice despite not being detected in plasma.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Using a raw p-value cutoff of 0.01, 159 beadtypes were selected for the response to 20E at 5 days and 224 beadtypes for the response at 15 days.

    “A raw p-value cutoff of 0.01 was used, which resulted in 159 beadtypes for the response to 20E at five days and 224 beadtypes for the response to 20E at fifteen days.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    IPA mapped the selected beadtypes to 104 network eligible genes for the 5-day response and 147 network eligible genes for the 15-day response.

    “Beadtypes meeting the raw p-value cutoff of 0.01 were input into IPA, which mapped them to 104 network eligible genes for the response to 20E at five days and 147 network eligible genes for the response to 20E at fifteen days.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The genes selected at raw p < 0.01 (159 at 5 days, 224 at 15 days) include true differentially expressed genes related to 20E treatment, despite no genes passing FDR correction.

  • May combine two claims.

    Findingown data

    Neither the response to 20E at 5 days nor at 15 days had any beadtypes significantly different after False Discovery Rate correction in the triceps brachii microarray analysis.

    “Neither the response to 20E (20E vs. saline) at five days nor the response to 20E at fifteen days had any beadtypes on the array that were significantly different after False Discovery Rate (FDR) correction (Benjamini & Hochberg, 1995).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Benjamini & Hochberg, 1995

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    20E stimulated protein synthesis in vitro in C2C12 murine myotubes and in vivo in rats given a single daily dose of 5 mg/kg for 10 days.

    “20E stimulated protein synthesis in vitro in C2C12 murine myotubes as well as in vivo in rats given a single daily dose of 5 mg/kg for 10 days (Gorelick-Feldman et al., 2008; Syrov, 2000).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Gorelick-Feldman et al., 2008

      Not checked in the free run.

    • cites Syrov, 2000

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Mice were randomly assigned to weight-balanced treatment groups (n = 9-10).

    “Following a one week acclimation period, mice were weighed and randomly assigned to weight balanced treatment groups (n = 9–10).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    20E increased the mass of tibialis anterior and the size of soleus and extensor digitorum longus, and stimulated muscle fiber growth in regenerating soleus muscles.

    “Additionally, 20E increased the mass (tibialis anterior) and size (soleus and extensor digitorum longus) of skeletal muscle and stimulated muscle fiber growth in regenerating soleus muscles (Syrov, 2000; Tóth et al., 2008)”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Syrov, 2000

      Not checked in the free run.

    • cites Tóth et al., 2008

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationown data

    The IPA-identified genes involved in skeletal muscle development and function are candidate leads for further investigation of the mechanism of 20E in skeletal muscle.

    “genes were identified to provide an explanation for the muscle accretion.”
    • Also stated in: § Discussion · hedged
      “IPA identified genes involved in skeletal muscle development and function that may be lead candidates for further investigation.”
    • Also stated in: § Discussion · hedged
      “Genes and biological pathways identified by IPA provide potential leads on the mechanism of 20E influence.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The genes selected at raw p < 0.01 (159 at 5 days, 224 at 15 days) include true differentially expressed genes related to 20E treatment, despite no genes passing FDR correction.
    • Rests on an assumption: The candidate genes identified in triceps after 20E infusion are related to the observed muscle mass increase.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Others have indicated no difference in body mass with 20E treatment with daily venous injections of 20E (1 mg/kg) for 3 weeks in mice.

    “Conversely, others have indicated no difference in body mass with 20E treatment with daily veinous injections of 20E (1 mg/kg) for 3 weeks in mice (Gao et al., 2008).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Gao et al., 2008

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Growth-promoting effects of phytoecdysteroids have been reported for mice, rats, sheep, pigs, and Japanese quail after ingestion of phytoecdysteroids.

    “Growth-promoting effects have been reported for mice, rats, sheep, pigs, and Japanese quail after ingestion of PE, implying a potentially valuable application in improving livestock production (Lafont and Dinan, 2003).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lafont and Dinan, 2003

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In vitro studies using C2C12 murine myotubes suggested that 20E may induce an intracellular calcium flux to activate Akt and increase protein synthesis through a GPCR-PLC-PI3K pathway.

    “In vitro studies using C2C12 murine myotubes suggested that 20E may induce an intracellular calcium flux to activate Akt and increase protein synthesis through a G-protein coupled receptor-phospholipase c-phosphoinositide-3-kinase (GPCR-PLC-PI3K) pathway (Gorelick-Feldman et al., 2010).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Gorelick-Feldman et al., 2010

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Toth et al. (2008) reported an increase in body mass with daily subcutaneous injections of 20E (5 mg/kg) for 8 days in rats.

    “Toth et al. (2008) reported an increase in body mass with daily subcutaneous injections of 20E (5 mg/kg) for 8 days in rats.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Toth et al. (2008)

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    After caudal vein injection of 20E at 50 mg/kg in mice, approximately 90% of 20E was eliminated in 30 min and the effective half-time was 8.15 min.

    “After caudal vein injection of 20E at 50 mg/kg, approximately 90% of 20E was eliminated in 30 min and the effective half-time was 8.15 min (Dzhukharova et al., 1987).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Dzhukharova et al., 1987

      Not checked in the free run.

15 of 15

  • The paper relies on this without support.

    The 20E infused subcutaneously at 5 mg/kg/day was bioavailable and reached skeletal muscle in mice despite not being detected in plasma.

    “However, 20E was not detected in the hydrolyzed plasma samples possibly due to the low plasma sample volume (< 155 μL) and concentration of 20E that was below the limit of detection.”

  • The paper relies on this without support.

    The genes selected at raw p < 0.01 (159 at 5 days, 224 at 15 days) include true differentially expressed genes related to 20E treatment, despite no genes passing FDR correction.

    “This did not mean there were no genes with differential expression due to 20E treatment, just that the model was not able to distinguish them from the expected false positives of testing 23,709 beadtypes.”
    “A raw p-value cutoff of 0.01 was used, which resulted in 159 beadtypes for the response to 20E at five days and 224 beadtypes for the response to 20E at fifteen days.”

  • The paper relies on this without support.

    The statistically significant increase in triceps brachii mass at 5 days is not a false positive arising from multiple comparisons among measured tissues.

    “Statistical significance was determined by comparing the 20E treatment group with saline control at each time point by analysis of variance (ANOVA) using SAS version 9.2 for Windows (SAS Institute Inc., Cary, NC, USA). A probability of p < 0.05 was considered statistically significant.”
    “There was a significant increase in the mass of the triceps brachii in the mice treated with 20E for five days (115 +/− 8 mg) compared to mice treated with saline for five days (88 +/− 3 mg) (p = 0.01).”

  • The paper relies on this without support.

    The 20E solution remained stable and active in the osmotic pumps over the 5- and 15-day infusion periods.

    “Pumps supplied a dose of 5 mg/kg/day of 20E dissolved in saline (0.9% NaCl) or saline alone (control), with a flow rate of 0.5 μl/hr.”
    “Administration of 20E by infusion was confirmed by measuring the volume of 20E solution or saline remaining in the osmotic pump reservoirs; approximately 160 and 90 μL for the five and fifteen day treatments, respectively.”

  • The paper relies on this without support.

    The increase in triceps brachii mass reflects an anabolic effect on skeletal muscle rather than fluid accumulation or other non-muscle changes.

    “To determine whether 20E conferred an anabolic effect on skeletal muscle, muscle groups were weighed in mice infused with 20E or saline (Table 1).”
    “Overall, the data suggests that 20E does not have potent anabolic properties, however, a muscle-specific increase was observed and genes were identified to provide an explanation for the muscle accretion.”

  • The paper relies on this without support.

    The candidate genes identified in triceps after 20E infusion are related to the observed muscle mass increase.

    “To determine potential mechanisms of 20E in skeletal muscle, Illumina’s Mouse Whole Genome-6 v2.0 Expression BeadChips were used to evaluate changes in gene expression of the triceps brachii after 20E infusion.”
    “genes were identified to provide an explanation for the muscle accretion.”
6 of 6

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References 30 · Cited by 31

Research works printed but not linked: 2

  1. Báthori M, 2001, Separation methods for phytoecdysteroids, LC‐GC Europe, 14, 626
  2. Stopka P, 1999, Effect of insect hormone, 20‐hydroxyecdysone on growth and reproduction in mice, Acta Soc Zool Bohemicae, 63, 367

Authors
Cheng, D. M., Kutzler, L. W., Boler, D. D., Drnevich, J., Killefer, J., & Lila, M. A.
Affiliations
University of Illinois Urbana-Champaign (US)
Corresponding author
Lila, Mary Ann
ORCID
Venue
Phytotherapy Research
Publisher
Wiley
Published
2012-04-12
Volume
27
Issue
1
Pages
107-111
ISSN
  • 0951-418X (ISSN-L)
  • 1099-1573
DOI
10.1002/ptr.4679
PMC
PMC3410052
OpenAlex
W1928230478
Semantic Scholar
f773edc534e0765d909db3042400fe006e2d3a5d
Cited by
31
References
30
Citations by year
2026: 2 · 2025: 5 · 2024: 1 · 2023: 2 · 2022: 2 · 2021: 6 · 2020: 1 · 2019: 1 · 2018: 2 · 2017: 2 · 2016: 1 · 2015: 3 · 2014: 1 · 2013: 2
FWCI
0.77
Type
journal article
Language
en
Open access
Licence
onlinelibrary.wiley.com/termsAndConditions#vor (vor)
Locations
  • Phytotherapy Research (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Institutes of Health
  • Fogarty International Center (U01TW006674)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Phytochemical Studies and Bioactivities
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Hormonal and reproductive studies
Keywords
20-hydroxyecdysone, phytoecdysteroids, triceps brachii, muscle mass, skeletal muscle hypertrophy, gene expression, anabolic effects, C57BL/6 mice, continuous infusion
MeSH terms
Animals, Ecdysterone, Male, Mice, Inbred C57BL, Organ Size, Gene Expression, Muscle, Skeletal, Oligonucleotide Array Sequence Analysis, Anabolic Agents, Mice, Transcriptome

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