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Cited by 22Steroid Chemistry and BiochemistryMuscle Physiology and DisordersHormonal and reproductive studies

Akt-dependent anabolic activity of natural and synthetic brassinosteroids in rat skeletal muscle cells

Open access
Esposito, Debora, Rathinasabapathy, Thirumurugan, Poulev, Alexander, Komarnytsky, Slavko, Raskin, Ilya·2011·Journal of Medicinal Chemistry
DOI 10.1021/jm200028hPMC PMC3128125
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Abstract

Brassinosteroids are plant-derived polyhydroxylated derivatives of 5α-cholestane, structurally similar to cholesterol-derived animal steroid hormones and insect ecdysteroids. In this study, we synthesized a set of brassinosteroid analogues of a natural brassinosteroid (22S,23S)-homobrassinolide (HB, 1), including (22S,23S)-homocastasterone (2), (22S,23S)-3α-fluoro-homobrasinolide (3), (22S,23S)-3α-fluoro-homocastasterone (4), (22S,23S)-7-aza-homobrassinolide (5), and (22S,23S)-6-aza-homobrassinolide (6) and studied their anabolic efficacy in the L6 rat skeletal muscle cells in comparison to other synthetic and naturally occurring brassinosteroids (22R,23R)-homobrassinolide (7), (22S,23S)-epibrassinolide (8), and (22R,23R)-epibrassinolide (9). Presence of the 6-keto group in the B ring and stereochemistry of 22α,23α-vicinal hydroxyl groups in the side chain were critical for the anabolic activity, possibly due to higher cytotoxicity of the 22β,23β-hydroxylated brassinosteroids. All anabolic brassinosteroids tested in this study selectively activated PI3K/Akt signaling pathway as evident by increased Akt phosphorylation in vitro. Plant brassinosteroids and their synthetic derivatives may offer a novel therapeutic strategy for promoting growth, repair, and maintenance of skeletal muscles.

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Written from the paper’s full text.

TL;DR

Natural and synthetic brassinosteroids were tested in L6 rat skeletal muscle cells, where the 6-keto group and 22α,23α-hydroxyl stereochemistry were critical for anabolic activity. Active compounds selectively increased Akt phosphorylation, and oral homobrassinolide altered muscle growth and metabolism genes in rats.

Key findings

  • In L6 rat skeletal muscle cells, (22S,23S)-homobrassinolide (HB) and (22S,23S)-homocastasterone increased protein synthesis by 37.2 ± 5.9% and 41.0 ± 2.7% (both p < 0.001), comparable to IGF-1 at 6.5 nM (42.5 ± 4.5%, p < 0.001). Removal of the 2α-hydroxyl group and C-3 fluorination (compounds 3–4) reduced activity by about 50% (24.6 ± 5.3% and 22.5 ± 2.7%, both p < 0.01), while 6-aza substitution (5) caused complete loss of protein synthesis activation and side-chain modifications (7–9) abolished activity.
  • HB, homocastasterone, and compounds 3–5 and 7 reduced protein degradation; 1–3 showed the strongest prevention, by more than 20%, compared with 10 nM insulin (20.2 ± 1.6%, p < 0.05). At 1 μM, HB decreased degradation by 8.2 ± 0.6% and compound 2 by 8.7 ± 1.7% above control (both p < 0.05).
  • Bioactive brassinosteroids stimulated Akt phosphorylation in rat skeletal muscle cell culture; HB and homocastasterone activated Akt after 1 h, slower than IGF-1 (within 10 min). Compound 5 was the only analogue inhibiting 3T3/NIH fibroblast proliferation dose-dependently, with IC50 12.5 μM; C-3 fluorination increased cytotoxicity versus 1–2.
  • In rats given 60 mg/kg HB daily for 24 d, lean body mass increased 14.2%; muscle gene expression showed Adra1d 6.5-fold, Igfbp1 2.5-fold, Srebf1 2.5-fold, Fbp2 4.5-fold, Igf2 1.4-fold, Myod1 2.1-fold, Myf5 1.3-fold, Myf6 1.3-fold, and Myog 1.7-fold changes.

Methods

  • The study synthesized HB analogues 2–6 and compared them with natural/synthetic brassinosteroids 7–9, using L6 rat skeletal muscle cells for protein synthesis ([3H]-phenylalanine incorporation), protein degradation (acid-soluble [3H]-phenylalanine release), MTT viability, and Western blot for phospho-Akt/total Akt.
  • Animal work used six-week-old male Wistar rats randomized into two groups (n=6), gavaged daily for 24 d with vehicle or 60 mg/kg HB; gastrocnemius muscle RNA was analyzed by rat insulin signaling PCR array and RT-PCR for selected genes.
  • Statistics used one-way ANOVA with Dunnett’s multiple comparison test, significance at p < 0.05, means ± SEM, and nonlinear regression for IC50.

Limitations

  • The paper does not state a limitations section. Evident from the text: the main anabolic assays are in L6 rat skeletal muscle cells in vitro, with no human data; the in vivo gene-expression analysis used pooled RNA for the PCR array and only n=6 rats per group; some cytotoxicity data in L6 myotubes are reported as “data not shown”; and the receptor mediating brassinosteroid effects in mammalian muscle is described as unknown.

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Integrity signals

Registry

Crossref · 10.1021/jm200028h

Cites

43 of 43 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 24 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Compounds 1-3 showed the strongest prevention of protein degradation, by more than 20%.

    “1–3 showed the strongest prevention of protein degradation, by more than 20%, that compared favorably with 10 nM insulin treatment that served as a positive control in this assay (20.2 ± 1.6%, p<0.05).”
    Quote in the paperSubstantial claim
  • Findingown data

    Replacement of the 6-carbonyl group with an amine in compound 5 resulted in a complete loss of activation of protein synthesis.

    “Replacement of 7-oxalactone group with amine in the B ring of 6 reduced biological activity by half, while a similar replacement of the 6-carbonyl group with amine in 5 resulted in a complete loss of activation of protein synthesis.”
    Quote in the paperNot a substantial claim
  • Findingown data

    All brassinosteroids and their analogues showed no toxicity in fully differentiated L6 rat skeletal myotubes up to 30 μM as established by the MTT assay and cytological observations.

    “All brassinosteroids and their analogues showed no toxicity in fully differentiated L6 rat skeletal myotubes up to 30 μM as established by the MTT assay and cytological observations (data not shown).”
    Quote in the paperSubstantial claim
  • Findingown data

    Bioactive brassinosteroids stimulated phosphorylation of Akt in rat skeletal muscle cell culture.

    “Consistent with the results obtained with the [3H]-phenylalanine incorporation assay, bioactive brassinosteroids stimulated phosphorylation of Akt in rat skeletal muscle cell culture (Figure 4).”
    • Also stated in: § Discussion · plain
      “Consistent with the results obtained with the [3H]-phenylalanine incorporation assays, bioactive brassinosteroids stimulated phosphorylation of Akt in rat skeletal muscle cell culture (Figure 4).”
    Quote in the paperSubstantial claim
  • Findingown data

    At 1 μM, compound 2 suppressed protein degradation by 8.7 ± 1.7% above control levels (p < 0.05).

    “At the lower concentration, 2 at 1 μM suppressed protein degradation by 8.7 ± 1.7% above control levels (p < 0.05).”
    Quote in the paperNot a substantial claim
  • May combine two claims. 25
  • Classified as probably not a substantial claim. 39
  • The quote only partly states this claim. 1

  • May combine two claims.

    Findingown data

    Compound 5 was the only brassinosteroid analogue that inhibited cell proliferation in a dose-dependent manner with IC50 of 12.5 μM in 3T3/NIH murine fibroblast cells.

    “5 was the only brassinosteroid analogue that inhibited cell proliferation in a dose-dependent manner with IC50 of 12.5 μM.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    HB at concentrations of 0.3-20 μM inhibited protein degradation dose-dependently and its activity reached plateau between 3 and 10 μM.

    “Among the most active compounds in this assay, HB at concentrations of 0.3–20 μM, inhibited protein degradation dose-dependently and its activity reached plateau between 3 and 10 μM (Figure 2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HB, homocastasterone (2), and brassinosteroid analogs 3-5 and 7 reduced protein degradation in L6 rat skeletal muscle cells in vitro, but the potency of their activities differed according to their structure.

    “HB, homocastasterone (2), as well as brassinosteroid analogs 3–5 and 7 reduced protein degradation in vitro, but the potency of their activities differed according to their structure.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Compound 2 at concentrations of 0.3-30 μM inhibited protein degradation dose-dependently and its activity reached plateau at 10 μM.

    “2 at concentrations of 0.3–30 μM inhibited protein degradation dose-dependently and its activity reached plateau at 10 μM.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both HB and (22S,23S)-homocastasterone responses on protein synthesis approached saturation between 10 and 20 μM, with maximum increases of 36.9 ± 2.9% and 40.7 ± 4.9% (p<0.01), respectively.

    “Both responses approached saturation between 10 and 20 μM, with the maximum increases of 36.9 ± 2.9% and 40.7 ± 4.9% (p<0.01), respectively (Figure 2A).”
    Quote in the paperNot a substantial claim

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

    Findingown data

    No changes in expression of Eif2b1 were noted.

    “No changes in expression of Eif2b1 were noted.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Both HB and (22S,23S)-homocastasterone treatments resulted in significant activation of Akt after 1 h, a much slower response than IGF-1, which phosphorylates Akt within 10 min.

    “Both HB and (22S,23S)-homocastasterone treatments resulted in significant activation of Akt after 1 h, a much slower response than that produced by IGF-1, which phosphorylates Akt within 10 min.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Of the 84 genes responsible for insulin signaling, PI3K and MAPK pathways, carbohydrate metabolism, and cell cycle regulation, two subsets of genes were higher expressed in HB-treated group than in the control group, but the magnitude of the difference varied.

    “Of the 84 genes responsible for insulin signaling, PI3K and MAPK pathways, carbohydrate metabolism, and cell cycle regulation, two subsets of genes were higher expressed in HB-treated group than in the Ctr group, but the magnitude of the difference varied (Figure 5A).”
    • Also stated in: § Discussion · hedged
      “The results indicated that HB potently stimulated two sets of genes involved in muscle cell growth and carbohydrate metabolism.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    The (22S,23S)-28-homobrassinolide (HB) is one of the most active brassinosteroids in inducing plant growth in various plant bioassay systems.

    “The (22S,23S)-28-homobrassinolide (HB, Figure 1) is one of the most active brassinosteroids in inducing plant growth in various plant bioassay systems 7.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] Takatsuto S., 1984, J. Chem. Soc., Perkin Trans. source

      Not checked in the free run.

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

    Premisecitation

    In the rice leaf lamina inclination bioassay, 5α-configuration is required for optimum brassinosteroid activity, but the B-ring tolerates considerable variation provided a polar functional group is maintained.

    “Recent structure-activity studies of brassinosteroids in the rice leaf lamina inclination bioassay have revealed that 5α-configuration is required for optimum activity, but the B-ring tolerates considerable variation, providing that the presence of a polar functional group is maintained 17.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] Andersen D. L., 2001, J. Org. Chem. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Orally applied HB produced significant anabolic effects and improved physical fitness in healthy animals with minimal androgenic effects.

    “In the previous study we found that orally applied HB produced significant anabolic effects and improved physical fitness in healthy animals with minimal androgenic effects 20.”
    • Also stated in: § Discussion · plain
      “Previously we reported that orally applied HB produced significant anabolic effects and improved physical fitness in healthy animals with minimal androgenic effects 20.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Esposito, D.; Rathinasabapathy, T.; Poulev, A.; Komarnytsky, S.; Raskin, I.Anabolic effect of plant brassinosteroid. Unpublished results.

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Akt is the key intermediate in the IGF-1 signaling pathway that modulates downstream targets known to regulate protein synthesis and degradation.

    “Akt is the key intermediate in the IGF-1 signaling pathway that modulates downstream targets known to regulate protein synthesis and degradation 21.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] Hajduch E., 1998, Diabetes source

      Not checked in the free run.

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

    Premisecitation

    (2α,3α)- and (22R,23R)-vicinal diol moieties are required for optimum bioactivity of brassinosteroids in plants.

    “Structure-activity studies have also demonstrated that the (2α, 3α)- and (22R, 23R)-vicinal diol moieties are required for optimum bioactivity in plants 18.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Baron D. L., 1998, Phytochemistry source

      Not checked in the free run.

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

    Premisecitation

    Igfbp1 has been proposed as an acute regulator of IGF-1 bioactivity.

    “Igfbp1 has been proposed as an acute regulator of IGF-1 bioactivity 35 and its elevation in association with HB treatment is unclear.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] Lee P. D., 1997, Proc. Soc. Exp. Biol. Med. source

      Not checked in the free run.

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

    Premisecitation

    IGF-2 expression during skeletal muscle differentiation is regulated at the transcriptional level, and signaling through the IGF-1 receptor by locally produced IGF-2 defines a pathway critical for normal muscle growth and regeneration.

    “IGF-2 expression during skeletal muscle differentiation is regulated at the transcriptional level 33, and signaling through the IGF-1 receptor by locally produced IGF-2 defines a pathway that is critical for normal muscle growth and regeneration 34.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [33] Kou K., 1993, Mol. Endocrinol.

      Not checked in the free run.

    • cites [34] Alzhanov D. T., 2010, J. Biol. Chem. source

      Not checked in the free run.

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

    Premisecitation

    Epibrassinolide is more active than homobrassinolide in plants.

    “Many papers have dealt with the relationship between the side chain structure and brassinolide activity in plants, suggesting that epibrassinolide is more active than homobrassinolide 22.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [22] Zullo M. A. T., 2002, Braz. J. Plant Physiol. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    HB treatment (60 mg/kg body weight daily to healthy rats fed normal diet for 24 d) was associated with a 14.2% increase in lean body mass and improved physical fitness of untrained rats.

    “HB treatment (60 mg/kg body weight daily to healthy rats fed normal diet for 24 d) was associated with a 14.2% increase in the lean body mass and the improved physical fitness of untrained rats (limb grip strength was measured using digital force gauge) 20.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Esposito, D.; Rathinasabapathy, T.; Poulev, A.; Komarnytsky, S.; Raskin, I.Anabolic effect of plant brassinosteroid. Unpublished results.

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Replacement of the 3-hydroxy function by a 3-fluoro group yielded compounds active at the rice lamina inclination assay, but not as active as their parent compounds.

    “Similarly, replacement of 3-hydroxy function by a 3-fluoro group yielded compounds active at the rice lamina inclination assay, but not as active as their parent compounds 27.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Galagovsky L. R., 2001, Phytochemistry source

      Not checked in the free run.

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

    Premisecitation

    The α1 adrenergic receptors mediate endogenous functions of catecholamines, which involve coupling to G proteins followed by activation of phospholipase Cβ and protein kinase C.

    “The α1 adrenergic receptors mediate endogenous functions of catecholamines, which involve coupling to G proteins followed by activation of phospholipase Cβ and protein kinase C 31.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [31] Strosberg A. D., 1995, Obes. Res. source

      Not checked in the free run.

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

    Premisecitation

    Plant brassinolide activity was significantly reduced in 7-aza-homobrassinolide, while 6-aza-7-oxo-homobrassinolide was inactive.

    “This is similar to plant brassinolide activity that was significantly reduced in 7-aza-homobrassinolide 25, while 6-aza-7-oxo-homobrassinolide was inactive 26.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [25] Takatsuto S., 1987, Chem. Pharm. Bull. source

      Not checked in the free run.

    • cites [26] Anastasia M., 1984, Gazz. Chim. Ital.

      Not checked in the free run.

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

    Premisecitation

    A similar delayed Akt response has been reported for ecdysteroids.

    “A similar delayed Akt response has been reported for ecdysteroids 30.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [30] Gorelick-Feldman J., 2008, J. Agric. Food Chem. source

      Not checked in the free run.

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

    Premisecitation

    Adra1d may potentially regulate muscle survival and differentiation.

    “Recently, it has also been suggested that Adra1d may potentially regulate muscle survival and differentiation 32.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Saini A., 2010, Cell. Physiol. Biochem. source

      Not checked in the free run.

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

    Premisecitation

    Activation of Akt in skeletal muscle leads to rapid muscle hypertrophy accompanied by improved metabolism.

    “Activation of Akt in skeletal muscle leads to rapid muscle hypertrophy 37 accompanied by improved metabolism 38.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [37] Lai K. M., 2004, Mol. Cell. Biol. source

      Not checked in the free run.

    • cites [38] Izumiya Y., 2008, Cell Metab. source

      Not checked in the free run.

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

    Study design and conductown data

    Rats were randomized into 2 groups (n=6).

    “six weeks old male Wistar rats (Charles River Laboratories, MA) fed normal diet (#5001 Rodent Chow diet, Purina, St. Louis, MO), were randomized into 2 groups (n=6) and gavaged daily for 24 d with either 1 ml of vehicle (5% DMSO in corn oil) or 60 mg/kg body weight of HB.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    MyoD induces cell differentiation by activating muscle specific genes and is important in the switch from cellular proliferation to differentiation.

    “This factor induces cell differentiation by activating muscle specific genes and is important in the switch from cellular proliferation to differentiation 40.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Solomon A. M., 2006, J. Endocrinol. source

      Not checked in the free run.

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

    Study design and conductown data

    The purity of compound 2b was confirmed by TLC and ESI-MS.

    “The purity of the compound was confirmed by TLC using Hexane: EtOAc (80:20) solvent (Rf = 0.7) and ESI-MS (m/z): 431.56 (M+1).”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The purity of compound 3 was confirmed by TLC and ESI-MS.

    “The purity of the compound was confirmed by TLC using Hexane: EtOAc (2:1) solvent (Rf = 0.26) and ESI-MS (m/z): 481.30 (M+1).”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The purity of compound 4 was confirmed by TLC, ESI-MS, and 1H NMR.

    “The purity of the compound was confirmed by TLC using Hexane: EtOAc (2:1) solvent (Rf = 0.58) and ESI-MS (m/z): 465.34 (M+1) and 1H NMR (500 MHz, CDCl3): δ = 0.71 (s, 3H), 0.74 (s, 3H), 0.88 (d, 3H, J = Hz), 0.96 (m, 6H), 1.04 (d, 3H, J = 6.9 Hz), 2.32 (dd, 1H, J = 13.1, 4.5 Hz), 2.63 (dd, 1H, J = 12.6, 3.0 Hz), 3.61 (d, 2H, J = 5.0 Hz), 4.91 (d, 1H, J = 48.3).”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    Protein degradation measurements were normalized to total cell protein.

    “Specific radioactivity of protein-free phenylalanine was quantified using liquid scintillation counter LS 6500 (Beckman Coulter, Fullerton, CA) and normalized to mg of total cell protein determined by BCA protein assay (Pierce Biotechnology, Rockford, IL).”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    Structures and purity (>95%) of all compounds were confirmed by mass spectrometry, 1H NMR, and 13C NMR.

    “Structures and purity (>95%) of all compounds were confirmed by Varian 1200 L (Varian, Palo Alto, CA) triple quadrupole mass detector with electrospray ionization (ESI) interface using a Dionex Acclaim RSLC 120 C18 reverse phase column (150 mm × 2.1 mm, 2.2 μm), 1H NMR, and 13C NMR spectra recorded on Varian 400, 500 MHz & Bruker Avance 950 MHz spectrophotometer.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Western blot analysis used equal amounts of protein (50 μg).

    “Equal amounts of protein (50 μg) from the supernatants were separated on 10% SDS polyacrylamide gels and blotted onto the nitrocellulose membrane.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    The purity of compound 5 was confirmed by TLC, ESI-MS, and 1H NMR.

    “The purity of the compound was confirmed by TLC using CHCl3: CH3OH (90:10) solvent (Rf = 0.2) and ESI-MS (m/z): 494.32 and 1H NMR (500MHz, CD3OD): δ = 0.76 (s, 3H), 0.86 (s, 3H), 0.96 (m, 6H), 1.03 (d, 3H, J = 6.9 Hz), 2.05 (m, 1H), 2.13 (dtd, 1H, J = 9.4, 6.9, 2.4 Hz), 2.21 (d, 1H, J = 13.3 Hz), 2.44 (m, 1H), 3.55 (dd, 2H, J = 8.5, 5.6 Hz), 3.60 (ddd, 1H, J = 7.0, 6.0, 3.3 Hz), 3.73 (dd, 1H, J =”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Inducible activation of Akt is sufficient to increase skeletal muscle mass and force without satellite cell activation.

    “Even though inducible activation of Akt is sufficient to increase skeletal muscle mass and force without satellite cell activation 39, we also evaluated effect of HB supplementation on expression of the genetically-determined transcriptional programs regulated by myogenic transcription factors Myod1, Myf5, Myf6, and Myog.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Blaauw B., 2009, FASEB J. source

      Not checked in the free run.

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

    Study design and conductnone given

    IC50 was calculated by nonlinear regression curve analysis.

    “The 50% inhibitory concentration (IC50) was calculated by a nonlinear regression curve analysis.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The purity of compound 3b was confirmed by TLC and ESI-MS.

    “The purity of the compound was confirmed by TLC using CHCl3: CH3OH (90:10) solvent (Rf = 0.57) and ESI-MS (m/z): 662.68 (M+1).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data

    Protein degradation experiments used insulin as positive control.

    “Cells were washed twice with PBS to remove the non-incorporated trace and treated for 4 h with vehicle (0.1% ethanol), increasing concentrations of brassinosteroids, or 10 nM of insulin in serum-free medium.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The purity of compound 3a was confirmed by TLC and ESI-MS.

    “The purity of the compound was confirmed by TLC using Hexane: EtOAc (2:1) solvent (Rf = 0.3) and ESI-MS (m/z): 647.58 (M+1).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Cell viability was measured by MTT assay in triplicate.

    “Cell viability was measured by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in triplicate essentially as described 42 and quantified spectrophotometrically at 550 nm using a microplate reader (Molecular Devices, Sunnyvale, CA).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [42] Mosmann T., 1983, J. Immunol. Methods source

      Not checked in the free run.

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

    Study design and conductown data

    Protein synthesis experiments were performed in triplicate with IGF-1 as positive control.

    “Fully differentiated myotubes were washed with serum-free DMEM and treated in triplicate with vehicle (0.1% ethanol), increasing concentrations of HB, or 6.5 nM of insulin-like growth factor-1 (IGF-1) as positive control.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Protein synthesis measurements were normalized to total protein.

    “Specific radioactivity of protein-bound phenylalanine was quantified using liquid scintillation counter LS 6500 (Beckman Coulter, Fullerton, CA) and normalized to mg of total protein determined by BCA protein assay (Pierce Biotechnology, Rockford, IL).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    After 7-9 days of culture, L6 myoblasts had fused into multinucleated myotubes.

    “After 7–9 days of culture the myoblasts had fused into multinucleated myotubes 41.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [41] Mandel J. L., 1974, Nature source

      Not checked in the free run.

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

    Study design and conductnone given

    RNA quantity and purity were determined using NanoDrop.

    “Total RNA was isolated using Trizol, its quantity and purity were determined using a NanoDrop (Nanodrop Technologies, Wilmington, DE).”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    Animal experiment was approved by Rutgers IACUC in an AAALAC accredited facility.

    “Animal experiment was performed according to procedures approved by the Rutgers Institutional Animal Care and Use Committee in the AAALAC accredited animal care facility as described previously 20.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Data were analyzed by one-way ANOVA with treatment as a factor, with Dunnett's post hoc test and significance at p < 0.05.

    “Unless otherwise noted, data were analyzed by one-way ANOVA with treatment as a factor. Post hoc analyses of differences between individual experimental groups were made using the Dunnett’s multiple comparison test. Significance was set at p < 0.05.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    The purity of compound 2a was confirmed by TLC and ESI-MS.

    “The purity of the compound was confirmed by TLC using Hexane: EtOAc (90:10) solvent (Rf = 0.28) and ESI-MS: (m/z) 429.32 (M+1).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Total Akt antibodies served as loading controls in Western blots.

    “After being stripped, the same blots were probed with total Akt antibodies to serve as loading controls.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    The purity of compound 6 was confirmed by TLC, ESI-MS, and 1H NMR.

    “The purity of the compound was confirmed by TLC using CHCl3: CH3OH (90:10) solvent (Rf = 0.22); ESI-MS (m/z): 494.30 (M+1) and 1H NMR (500MHz, CD3OD): δ = 0.76 (s, 3H), 0.86 (d, 3H, J = 6.9 Hz), 0.90 (s, 3H), 0.95 (dd, 3H, J = 14.8, 7.2 Hz), 1.03 (d, 3H, J = 6.9 Hz), 2.05 (m, 1H), 2.13 (dtd, 1H, J = 9.3, 6.8, 2.4 Hz), 3.01 (m, 1H), 3.08 (m, 2H), 3.56 (m, 1H), 3.92 (s, 1H).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Concentrations of test reagents that showed no changes in cell viability compared with vehicle (0.1% ethanol) were selected for further studies.

    “The concentrations of test reagents that showed no changes in cell viability compared with that of the vehicle (0.1% ethanol) were selected for further studies.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    The purity of compound 4c was confirmed by TLC and ESI-MS.

    “The purity of the compound was confirmed by TLC using CHCl3: EtOAc (2:1) solvent (Rf = 0.45) and ESI-MS (m/z): 662.68 (M+1).”
    Quote in the paperNot a substantial claim
49 of 49

  • The paper relies on this without support.

    The L6 rat skeletal muscle cell line is a valid model for studying anabolic effects in skeletal muscle in vivo.

    “The bioactivity of HB and its analogues was evaluated by measuring increase in protein synthesis in the L6 rat skeletal muscle cells in vitro.”

  • The paper relies on this without support.

    Age- or disease-related skeletal muscle atrophy is a condition that requires novel therapeutic agents.

    “Importance of identifying novel agents that influence muscle growth, development, or regeneration with possible therapeutic application for the age or disease-related skeletal muscle atrophy prompted us in this study to explore, for the first time, the structure-activity relationship between HB and its natural and synthetic analogues in their effects on protein synthesis and degradation in rat ske”

  • The paper relies on this without support.

    The effects of brassinosteroids on protein synthesis and degradation in L6 cells are predictive of their anabolic effects in skeletal muscle in vivo.

    “Plant brassinosteroids and their synthetic derivatives may offer a novel therapeutic strategy for promoting growth, repair, and maintenance of skeletal muscles.”
3 of 3

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?

  • Anabolic, hypoglycaemic and wound-healing effects are associated with brassinosteroids in rodents.

    “since anabolic, hypoglycaemic and wound-healing effects are also associated with brassinosteroids in rodents [213-215]”

    From Ecdysteroids (2021) · cites this paper as [213]

  • Beta-ecdysone increases muscle weight in rodents.

    “As it increases muscle weight in rodents [26-28], βEcd has been referred to as an “anabolic” naturally-derived supplement [29].”

    From Prevention of glucocorticoid induced bone changes with beta-ecdysone (2015) · cites this paper as [27]

  • Beta-ecdysone is reported to promote muscle cell growth via insulin and PI3K/Akt signaling.

    “Mechanistically, βEcd is reported to promote muscle cell growth via insulin and PI3K/Akt signaling and is reported to inhibit NF-kB activation in a cancer cell line [21, 25, 27, 61].”

    From Prevention of glucocorticoid induced bone changes with beta-ecdysone (2015) · cites this paper as [27]

  • Beta-ecdysone is reported to inhibit NF-kB activation in a cancer cell line.

    “Mechanistically, βEcd is reported to promote muscle cell growth via insulin and PI3K/Akt signaling and is reported to inhibit NF-kB activation in a cancer cell line [21, 25, 27, 61].”

    From Prevention of glucocorticoid induced bone changes with beta-ecdysone (2015) · cites this paper as [27]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • HB is assigned conflicting side-chain stereochemistry

    Minor

    The paper identifies HB (compound 1) as (22S,23S) throughout the study, but the chemicals section assigns the purchased HB compound (22R,23R) stereochemistry. The distinction is central to its comparison of stereoisomers.

  • The B-ring lactone is called both unnecessary and critical

    Minor

    The discussion says the lactone moiety is unnecessary because HB and homocastasterone have similar anabolic activity, but later calls the B-ring lactone critical for anabolic activity.

  • Compound 5 is described as both retaining and replacing its 6-carbonyl

    Minor

    The protein-synthesis results describe compound 5 as having its 6-carbonyl group replaced with an amine. The discussion instead identifies compound 5 as 6-oxo-7-aza, which retains an oxygen-containing group at position 6.

3 of 3

Cites

Flagged first, then by citations

Research works printed but not linked: 5

  1. Kulaeva O. N., 2009, Brassinosteroids, 141
  2. Massey A. P., 2003, Synthesis, 3, 426
  3. Kuzmitsky B. B., 1991, Tech. Rep.—Acad. Sci. Belarus, Minsk, 1
  4. Anastasia M., 1984, Gazz. Chim. Ital., 114, 159
  5. Kou K., 1993, Mol. Endocrinol., 7, 291

Cited by

Sorted by citations

Related

Authors
Esposito, D., Rathinasabapathy, T., Poulev, A., Komarnytsky, S., & Raskin, I.
Affiliations
  • Biotech Center, SEBS, Rutgers University, 59 Dudley Road, New Brunswick, New Jersey 08901, United States
  • Rutgers, The State University of New Jersey (US)
Corresponding author
Komarnytsky, Slavko
ORCID
Venue
Journal of Medicinal Chemistry
Publisher
American Chemical Society (ACS)
Published
2011-05-26
Volume
54
Issue
12
Pages
4057-4066
ISSN
  • 0022-2623 (ISSN-L)
  • 1520-4804
DOI
10.1021/jm200028h
PMC
PMC3128125
OpenAlex
W2064372365
Cited by
22
References
43
Citations by year
2026: 2 · 2025: 1 · 2024: 1 · 2022: 1 · 2021: 5 · 2020: 2 · 2016: 1 · 2015: 2 · 2014: 2 · 2013: 4 · 2012: 1
FWCI
0.91
Type
journal article
Language
en
Open access
Locations
  • Journal of Medicinal Chemistry (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
National Center for Complementary and Integrative Health (P50AT002776)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Steroid Chemistry and Biochemistry
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Hormonal and reproductive studies
Keywords
brassinosteroids, homobrassinolide, brassinosteroid analogues, skeletal muscle anabolism, L6 myoblasts, PI3K/Akt signaling, Akt phosphorylation, stereochemistry, epibrassinolide, muscle growth, muscle repair
MeSH terms
Animals, Cell Line, Cell Survival, Cholestanones, Phosphorylation, Stereoisomerism, Structure-Activity Relationship, Muscle, Skeletal, Anabolic Agents, Proto-Oncogene Proteins c-akt, Mice, Rats

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.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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