PreprintCited by 11Renin-Angiotensin System StudiesHormonal and reproductive studiesEstrogen and related hormone effects

20-Hydroxyecdysone activates the protective arm of the renin angiotensin system via Mas receptor

Open access
Lafont, René, Raynal, Sophie, Serova, Maria, Didry-Barca, Blaise, Guibout, Louis, Latil, Mathilde, Dilda, Pierre J., Dioh, Waly +1 more·2020·bioRxiv (Cold Spring Harbor Laboratory)

Abstract

20-Hydroxyecdysone (20E) is a steroid hormone that plays a key role in insect development through nuclear ecdysone receptors (EcRs) and at least one membrane GPCR receptor (DopEcR) and displays numerous pharmacological effects in mammals. However, its mechanism of action is still debated, involving either an unidentified GPCR or the estrogen ERβ receptor. The goal of our study was to better understand 20E mechanism of action. A mouse myoblast cell line (C2C12) and the gene expression of myostatin (a negative regulator of muscle growth) was used as a reporter system of anabolic activity. Experiments using protein-bound 20E established the involvement of a membrane receptor. 20E-like effects were also observed with Angiotensin-(1-7), the endogenous ligand of Mas. Additionally, the effect on myostatin gene expression was abolished by Mas receptor knock-down using small interfering RNA (siRNA) or pharmacological inhibitors. 17β-Estradiol (E2) also inhibited myostatin gene expression, but protein-bound E2 was inactive, and E2 activity was not abolished by angiotensin-(1-7) antagonists. A mechanism involving cooperation between Mas receptor and a membrane-bound palmitoylated estrogen receptor is proposed. The possibility to activate the Mas receptor with a safe steroid molecule is consistent with the pleiotropic pharmacological effects of ecdysteroids in mammals and indeed this mechanism may explain the close similarity between angiotensin-(1-7) and 20E effects. Our findings open a lot of possible therapeutic developments by stimulating the protective arm of the renin-angiotensin-aldosterone system (RAAS) with 20E.

AI snapshot

Written from the paper’s full text.

TL;DR

20-Hydroxyecdysone (20E) inhibits myostatin gene expression and stimulates protein synthesis in C2C12 myotubes through a membrane-initiated mechanism involving the Mas receptor, the angiotensin-(1-7) receptor. The authors propose that 20E activates the protective arm of the renin-angiotensin system via Mas, with cooperation from a non-nuclear membrane estrogen receptor.

Key findings

  • 20E increased de novo protein synthesis in differentiated C2C12 myotubes, with a maximal effect of +27% at 5 µM (p < 0.001); IGF-1 (100 ng/mL) increased incorporation by +20% (p < 0.001).
  • 20E dose-dependently inhibited myostatin gene expression, reaching significance from 0.5 µM; the membrane-impermeable 20E-HSA conjugate retained activity similar to free 20E at 10 µM 20E-equivalents.
  • Mas antagonists A1 and A779, and MAS siRNA knockdown, abolished the inhibitory effects of both 20E and angiotensin-(1-7) on myostatin expression, while IGF-1 effects were unchanged.
  • 20E did not bind AR, ERα, or ERβ up to 100 µM, and a screen of 45 GPCRs and 5 nuclear receptors at 10 µM 20E was negative; E2 inhibited myostatin from 0.1 to 1 µM, but E2-CMO-BSA was inactive (−8%, ns) and E2 activity was not blocked by A779.

Methods

  • C2C12 mouse myoblast cells were differentiated into myotubes and used for protein synthesis assays ([3H]-leucine incorporation) and qRT-PCR of myostatin and MAS.
  • Treatments included 20E, IGF-1, angiotensin-(1-7), Mas antagonists A1/A779, MAS siRNA, 17β-estradiol (E2), E2-CMO, E2-CMO-BSA, and 17α-estradiol.
  • Binding studies used radioligand competition for AR, ERα, ERβ, and GR up to 100 µM 20E, plus a fixed-concentration screen of 45 GPCRs and 5 nuclear receptors; statistics used ANOVA/Dunnett, Kruskal-Wallis/Dunn, t-test, or Mann-Whitney as appropriate.

Limitations

  • The paper states that the results do not unambiguously identify the primary target and that the specific membrane estrogen receptor form involved remains open.
  • The paper states that demonstrating the proposed functional Mas–estrogen receptor association will require membrane fractionation combined with immunoprecipitation techniques.
  • The paper notes that clinical evidence for 20E effectiveness in humans remains limited; evident from the text, the experimental work is in vitro using the C2C12 cell line, with no in vivo or human data reported here.

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

Integrity signals

Registry

Crossref · 10.1101/2020.04.08.032607

Cites

164 of 164 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 15 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    The maximal 20E effect on protein synthesis is +27% (p<0.001) at 5 μM.

    “The maximal effect (+ 27 %; p < 0.001) was measured with 5 μM of 20E”
    Quote in the paperNot a substantial claim
  • Findingown data

    The inhibition of myostatin gene expression by 20E reaches significance from 0.5 μM.

    “This inhibition reached significance from 0.5 µM 20E (Fig. 1B).”
    Quote in the paperNot a substantial claim
  • Findingown data

    20E treatment of C2C12 myotubes causes a dose-dependent increase in de novo protein synthesis versus untreated conditions.

    “A dose-dependent increase in protein synthesis was observed in response to 20E treatment of C2C12 myotubes versus untreated conditions (Fig. 1A).”
    Quote in the paperNot a substantial claim
  • Findingown data

    10 μM 20E is notably less efficient (+11%, ns) than 5 μM for protein synthesis.

    “while a treatment with a higher concentration of 20E (10 μM) appeared to be notably less efficient (+11%, ns) than the previous concentration tested.”
    Quote in the paperNot a substantial claim
  • Findingown data

    The 20E effect on protein synthesis is significant from 0.5 μM to 5 μM.

    “20E effect was significant from 0.5 μM to 5 μM.”
    Quote in the paperNot a substantial claim
  • Classified as probably not a substantial claim. 8
  • May combine two claims. 9
  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    IGF-1 (100 ng/mL) improves [3H]-Leu incorporation by +20% (p<0.001) in C2C12 myotubes.

    “IGF-1 (100 ng/mL), employed as positive control (30) displayed, as expected, an improvement in [ 3 H]-Leu incorporation (+20%, p < 0.001).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [30] source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    E2-CMO-BSA conjugate is inactive and does not significantly decrease myostatin expression (-8%, ns).

    “By contrast, E2-CMO-BSA-conjugate was inactive (Fig. 4B) and did not significantly decrease myostatin expression (-8 %, ns).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The E2-CMO-BSA conjugate is membrane-impermeable.
  • May combine two claims.

    Findingown data

    E2-CMO significantly inhibits myostatin gene expression (-39%, p<0.05) with the same trend as IGF-1 positive control (-47%, p<0.01).

    “E2-CMO significantly inhibited myostatin gene expression (-39%, p < 0.05) with the same trend as IGF-1 positive control (-47%, p < 0.01).”
    Quote in the paperSubstantial claim
  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    17α-estradiol inhibits myostatin gene expression in differentiated C2C12 cells.

    “Inhibitory activity of 17α-estradiol (αE2) on myostatin gene expression.”
    Quote in the paperNot a substantial claim
  • May combine two claims.

    Interpretationreasoning

    The effects of 20E on C2C12 cells involve both Mas receptor and a non-nuclear estradiol receptor.

    “Our data combined with those previously available from the literature allow us to conclude that the effects of 20E on C2C12 cells involve both Mas receptor and a nonnuclear estradiol receptor.”
    Quote in the paperSubstantial claim
  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    C2C12 cells were maintained at low passage (3-20 passages) for all experiments to maintain the differentiation potential of the cultures.

    “C2C12 cells were maintained at low passage (3-20 passages) for all experiments to maintain the differentiation potential of the cultures.”
    Quote in the paperNot a substantial claim
  • May combine two claims.

    Interpretationown data

    20E does not bind the concerned ER receptor, and activation of this estrogen receptor by 20E is secondary to Mas activation.

    “This allows us to conclude that 20E does not bind the concerned ER receptor, and that the activation of this estrogen receptor by 20E is secondary to Mas activation.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The 20E-HSA conjugate is membrane-impermeable.
  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The clinical evidence for 20E effectiveness in humans remains limited at the moment.

    “While many effects have been described on animals (10,12,13), the clinical evidence for 20E effectiveness in humans remains limited at the moment (14)(15)(16).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [14] 2019, Ecdysteroids as non-conventional anabolic agent: performance enhancement by ecdysterone supplementation in humans, Arch Toxicol source

      Not checked in the free run.

    • cites [15] 1988, Combined application of preparation Ecdysten and product Bodrost during training in cyclic sports, Scientific Sports Bulletin

      Not checked in the free run.

    • cites [16] 2015, Eine neue Alternative für die Prävention und Therapie postmenopausaler Erkrankungen, insbesondere des metabolischen Syndroms, J. Gynäkol. Endokrinol

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The presence of a bulky protein does not prevent 20E activity, which is an additional argument for interaction of 20E with a membrane receptor.

    “This results demonstrates that the presence of a bulky protein does not prevent 20E activity and is an additional argument for the interaction of 20E with a membrane receptor, as proposed by Gorelick-Feldman et al. (2010).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Gorelick-Feldman et al. (2010)

      Not checked in the free run.

    • Rests on an assumption: The 20E-HSA conjugate is membrane-impermeable.
  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationcitation

    20E is devoid of any feminizing activity and is probably inactive on estrogen-dependent cancer cells.

    “Thus 20E is devoid of any feminizing activity (13,56) and is probably inactive on estrogen-dependent cancer cells.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [13] 2010, β-Ecdysone has bone protective but no estrogenic effects in ovariectomized rats, Phytomedicine source

      Not checked in the free run.

    • cites [56] 1974, Crustecdysone is without estrogenic or antiestrogenic activity in the rat, Experientia source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    The myostatin transcript bioassay was developed and standardized in order to assess ecdysteroid activity.

    “The myostatin transcript bioassay was developed and standardized in order to assess ecdysteroid activity (31).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [31] 2012, <i>In Vitro</i> Characterization of the Efficacy and Safety Profile of a Proprietary Ajuga Turkestanica Extract, Chinese Medicine source

      Not checked in the free run.

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

    Premisecitation

    Several data favour an action on membranes through a GPCR receptor, whereas other ones suggest the involvement of a nuclear receptor, the estrogen receptor ERβ.

    “Several data favour an action on membranes through a GPCR receptor (17), whereas other ones suggest the involvement of a nuclear receptor, the estrogen receptor ERβ (18,19).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [17] source

      Not checked in the free run.

    • cites [18] 2018, Pharmacology of doping agents – mechanisms promoting muscle hypertrophy, AIMS Molecular Science

      Not checked in the free run.

    • cites [19] 2014, Estrogen receptor beta is involved in skeletal muscle hypertrophy induced by the phytoecdysteroid ecdysterone, Mol Nutr Food Res source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    17β-Estradiol-mediated myostatin inhibition is not linked with a transmembrane receptor.

    “17β-Estradiol-mediated myostatin inhibition is not linked with a transmembrane receptor and is not blunted by a Mas antagonist.”
    Quote in the paperSubstantial claim
  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Gorelick-Feldman et al. concluded that the membrane 20E receptor belongs to the GPCR family and proposed a mechanism of transduction involving an unidentified GPCR and a membrane calcium channel.

    “They concluded that the membrane 20E receptor belongs to the GPCR family and proposed a mechanism of transduction involving an unidentified GPCR and a membrane calcium channel (Supporting Fig. S1).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [17] source

      Not checked in the free run.

14 of 14
  • The paper relies on this without support.

    Myostatin is a major autocrine regulator that inhibits muscle growth in mammals.

    “Myostatin is a major autocrine regulator that inhibits muscle growth in mammals.”
  • The paper relies on this without support.

    The C2C12 mouse myoblast cell line is a valid model for studying muscle anabolism and 20E effects on muscle.

    “The C2C12 mouse myoblast cell line (29) was purchased from ATCC (CRL-1772).”
  • The paper relies on this without support.

    The Ang 1-7 antagonists A1 and A779 are specific for the Mas receptor.

    “As expected, this inhibition by Ang 1-7 (10 µM) was totally abolished by specific Ang 1-7 antagonists (A1 or A779, 10 µM) (Fig. 3A).”
  • The paper relies on this without support.

    The E2-CMO-BSA conjugate is membrane-impermeable.

    “We used a membrane-impermeable conjugate of E2 made of bovine serum albumin (BSA) and a carboxymethyloxime (CMO) linker.”
  • The paper relies on this without support.

    The 20E-HSA conjugate is membrane-impermeable.

    “a membraneimpermeable derivative of 20E was produced (Supporting Fig. S2).”
  • The paper relies on this without support.

    The mechanism of action of ecdysteroids on mammals/humans has not been elucidated.

    “In spite of more than 40 years of research, the mechanism of action of these molecules on mammals/humans has not been elucidated, as only diverging reports are available for the moment.”
  • The paper relies on this without support.

    20E is safe for therapeutic use.

    “The possibility to activate the Mas receptor with a safe steroid molecule is consistent with the pleiotropic pharmacological effects of ecdysteroids in mammals”
7 of 7

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Cites

Flagged first, then by citations

2 of the 166 references couldn’t be matched to a known paper.

Research works printed but not linked: 25

  1. 1988, Combined application of preparation Ecdysten and product Bodrost during training in cyclic sports, Scientific Sports Bulletin, 2, 29
  2. 1995, Mechanisms of the early effect of biologically active oxysterones calcitriol and ecdysterone, modulation of intracellular pools of arachidonic acid and products of its oxidative metabolism, Ukrainian Biokhim Zhurnal, 67, 45
  3. 1999, The C(27)-steroid hormones ecdysterone and calcitriol activate the phosphoinositide messenger cascade in its membrane phase of action, Ukrainian Biokhim Zhurnal, 71, 27
  4. 1988, Effects of methandrostenolone and ecdysterone on physical endurance of animals and protein metabolism in the skeletal muscles, Farmakol Toksikol, 6, 57
  5. 2012, Quinoa extract enriched in 20-hydroxyecdysone protects mice from diet-induced obesity and modulates adipokines expression, Obesity, 20, 270, 10.1038/oby.2011.257 · DOI · Paper page
  6. 2008, β-Ecdysterone induces osteogenic differentiation in mouse mesenchymal stem cells and relieves osteoporosis, Biol Pharm Bull, 31, 2245, 10.1248/bpb.31.2245 · DOI · Paper page
  7. 2010, Ecdysteroids elicit a rapid Ca2+ flux leading to Akt activation and increased protein synthesis in skeletal muscle cells, Steroids, 70, 632
  8. 10.1152/ajpendo.90772.2008 · DOI · Paper page
  9. 10.1161/circresaha.113.301077 · DOI
  10. 2013, Effect of ecdysterone on the expression of toll-like receptor 4 and surfactant protein A in lung tissue of rats with acute lung injury, Infect Inframm Rep, 14, 22
  11. 2009, Effect of ecdysterone on injury of lipid peroxidation following focal cerebral ischemia in rats, Zhongguo Yaoye, 18, 12
  12. 10.1002/mnfr.201300806 · DOI · Paper page
  13. 10.5604/20831862.1144420 · DOI · Paper page
  14. 2009, Cardiac anti-fibrotic effects of angiotensin (1-7) effects of angiotensin (1-7) mimetic in aged mice, Hypertension, 53, 1116
  15. 2010, β-Ecdysone has bone protective but no estrogenic effects in ovariectomized rats, Phytomedicine, 17, 884, 10.1016/j.phymed.2010.03.021 · DOI · Paper page
  16. 2010, The administration of the agonist of angiotensin-(1-7), AVE0991, improved inflammation and proteinuria in experimental nephrotic syndrome, Pediatr Nephrol, 25, 1795
  17. 2012, Possible mechanism of the cardio-renal effects of AVE-0991, a nonpeptide Mas-receptor agonist, in diabetic rats, JRAAS, 13, 334
  18. 2017, Mas receptor is involved in the estrogen-receptor induced nitric oxide-dependent vasorelaxation, Biochemical Pharmacology, 129, 67, 10.1016/j.bcp.2017.01.012 · DOI
  19. 2019, Protective effect of 20-hydroxyecdysone against lipopolysaccharides-induced acute lung injury in mice, Journal of Pharmaceutics and Drug Research, 2, 109
  20. 1992, An experimental study of the hepatoprotective properties of phytoecdysteroids and nerobol in carbon tetrachloride - induced liver injury, Eksp Klin Farmakologiya, 55, 61
  21. Tchoukouegno Ngueu S. 2013. Bioactivity of plant secondary metabolites. Estrogenic, cytotoxic and anabolic effects on estrogen target organs of an extract of Erythrinaexcelsa and ecdysterone. PhD Thesis, Univ Cologne.
  22. 10.1096/fj.11-194779 · DOI · Paper page
  23. 1998, Effects of ecdysterone on rat lung reperfusion injury, Zhongguo Yaolixue Tongbao (Chinese Pharmaceutical Bulletin), 14, 256
  24. 2013, Protective effect of 20-hydroxyecdysone on diabetic hepathopathy of rats, Xiandai Yufang Yixue, 40, 4031
  25. 2010, Effects of ecdysterone on early stage diabetic nephropathy in streptozotocin-induced diabetic rats, Zhongguo Xinyao Yu Lingchuan Zazhi, 29, 842

Cited by

Sorted by citations

Authors
Lafont, R., Raynal, S., Serova, M., Didry-Barca, B., Guibout, L., Latil, M., Dilda, P. J., Dioh, W., & Veillet, S.
Affiliations
  • Centre National de la Recherche Scientifique (FR)
  • Sorbonne Université (FR)
Corresponding author
Dilda, Pierre J.
Venue
bioRxiv (Cold Spring Harbor Laboratory)
Publisher
openRxiv
Published
2020-04-10
OpenAlex
W3015408723
Cited by
11
References
166
Citations by year
2024: 1 · 2023: 1 · 2022: 2 · 2021: 7
Type
posted content
Language
en
Open access
Locations
bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
Funders
Université de Bordeaux
Topics
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Renin-Angiotensin System Studies
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Hormonal and reproductive studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Estrogen and related hormone effects
Keywords
20-hydroxyecdysone, ecdysteroids, Mas receptor, angiotensin-(1-7), renin-angiotensin system, myostatin, estrogen receptor beta, muscle growth, C2C12 myoblasts

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