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Cited by 9Muscle Physiology and DisordersHormonal and reproductive studiesMuscle metabolism and nutrition

20-Hydroxyecdysone Boosts Energy Production and Biosynthetic Processes in Non-Transformed Mouse Cells

Open access
Shuvalov, Oleg, Kirdeeva, Yulia, Fefilova, Elizaveta, Daks, Alexandra, Fedorova, Olga, Parfenyev, Sergey, Nazarov, Alexander, Vlasova, Yulia +2 more·2024·Antioxidants
DOI 10.3390/antiox13111349PMC PMC11591052
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Abstract

20-Hydroxyecdysone (20E) is an arthropod steroid hormone that possesses a number of beneficial pharmacological activities in humans, including anabolic, antioxidant, hypoglycemic, cardioprotective, hepatoprotective, neuroprotective, and antineoplastic properties, etc. While several studies have explored the anabolic activity of 20E in muscle cells, they have concentrated on its effects on myofibril size, protein biosynthesis intensity, and myostatin expression, without assessing energy metabolism. In this research, we have demonstrated that 20E boosts both catabolism and anabolism, coupling energy-producing and biosynthetic metabolic processes in mouse myoblasts and fibroblasts in the same way. Using a transcriptomic approach, we identified the 20E-mediated up-regulation of genes involved in different metabolic processes. Further experiments revealed that 20E increased the levels of enzymes involved in glycolysis and one-carbon metabolism. It also increased the uptake of glucose, glycolysis, respiration, the production of ATP, and global protein biosynthesis in mouse myoblasts and fibroblasts. This phenomenon involves the PI3K/AKT/mTOR signaling pathway. Taken together, the observed 20E-dependent upregulation of energy metabolism may be the main reason for 20E’s well-known anabolic activity.

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Written from the paper’s full text.

TL;DR

20-Hydroxyecdysone (20E) treatment of non-transformed mouse C2C12 myoblasts and NIH/3T3 fibroblasts coordinately up-regulates energy-producing and biosynthetic metabolism, including glycolysis, respiration, ATP production, glucose uptake, and global protein synthesis. The effects are associated with PI3K/AKT/mTOR activation and may explain 20E’s anabolic activity.

Key findings

  • RNA-seq in C2C12 myoblasts treated with 1 μM 20E for 24 h identified 768 differentially expressed genes: 424 up-regulated and 344 down-regulated; top up-regulated genes included c-Myc, AP-1 components (Jun, Fos, Atf3), Egr1/2/3, and mitochondrial respiratory chain genes.
  • 20E increased glucose uptake up to 1.5 times and ATP production 1.2–1.5 times in both cell lines; Seahorse profiling showed increased glycolysis and especially respiration, with respiration up to 1.5 times in C2C12 and 2 times in NIH/3T3 at 1 μM, and glycolysis up-regulated by 1.6 times.
  • 20E increased global protein biosynthesis by 15–32% depending on concentration and cell line, measured by SUnSET.
  • 20E induced phospho-AKT (S473) and phospho-mTOR (S2448); the PI3K inhibitor LY294002 prevented the 20E-mediated up-regulation of glycolysis and respiration.

Methods

  • In vitro study using mouse C2C12 myoblasts and NIH/3T3 embryonic fibroblasts; 20E was dissolved in DMSO and compared with DMSO controls.
  • RNA-seq was performed on C2C12 cells treated with 1 μM 20E for 24 h with six biological replicates; differential expression used FDR ≤ 0.05 and LogFC ≥ 0.1. Additional assays included real-time PCR, Western blot, glucose uptake, ATP production, Seahorse XF Real-Time ATP Rate Assay, and SUnSET.
  • Statistical analysis used Student’s t-test for N = 3 experiments and Mann–Whitney U-test for Seahorse experiments with N = 5; significance was p < 0.05.

Limitations

  • The paper states that the dependence between log2 expression fold change and log2 transcript length remained unexplained despite checks for RNA degradation and bias corrections; the authors corrected for it cautiously by subtracting the linear regression and taking minimum absolute values.
  • The paper notes that the molecular mechanisms of 20E in mammals remain enigmatic, that mammals lack the ecdysone receptor, and that proposed targets such as MAS1 and estrogen receptor cannot alone explain activity in ER-negative cells.
  • Evident from the text: the study is limited to two non-transformed mouse cell lines in vitro, with no in vivo validation; the authors also state that other biosynthetic pathways, such as the HMP shunt/PPP, remain to be studied.

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

Integrity signals

Registry

Crossref · 10.3390/antiox13111349

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 55 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    20E preferentially increased rather than repressed gene expression in C2C12 myoblasts.

    “Interestingly, based on the LogFC values (Table S2), it seems that 20E preferentially increased rather than repressed the expression of genes.”
    Quote in the paperNot a substantial claim
  • Findingown data

    20E increased glucose uptake by up to 1.5 times in C2C12 myoblasts and NIH/3T3 fibroblasts.

    “The results in Figure 5A,B show that 20E increased glucose uptake by up to 1.5 times.”
    Quote in the paperSubstantial claim
  • Findingown data

    The greatest increase in respiration (up to 1.5 times for C2C12 and 2 times for NIH/3T3) was observed upon treatment with 1 μM of 20E, followed by 10 μM and 100 μM.

    “The greatest increase in respiration (up to 1.5 and 2 times for C2C12 and NIH/3T3, respectively) was observed upon treatment with 1 μM of 20E, followed by 10 μM and 100 μM.”
    Quote in the paperSubstantial claim
  • Findingown data

    Among the top genes up-regulated by 20E in C2C12 myoblasts were the transcription factors c-Myc and ATF3.

    “Moreover, two transcriptional factors that are critical for global metabolic regulation—c-Myc and ATF3—were identified among the top genes up-regulated by 20E (Figure 1).”
    Quote in the paperNot a substantial claim
  • Findingown data

    20E increased ATP production 1.2-1.5 times in both C2C12 and NIH/3T3 cell lines.

    “In addition, 20E increased ATP production 1.2–1.5 times in both cell lines studied (Figure 5C,D).”
    Quote in the paperSubstantial claim
  • May combine two claims. 13
  • Classified as probably not a substantial claim. 17

  • May combine two claims.

    Findingown data

    20E activated mTOR, as detected by its active form phospho-mTOR (S2448), which results from phosphorylation by AKT.

    “Importantly, the master regulator of anabolic processes, mTOR, was also activated, because we detected its active form, phospho-mTOR (S2448), which results from phosphorylation by AKT.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    20E treatment strongly induced up-regulation of both glycolysis and respiration, but it was not able to overcome the LY294002-mediated inhibitory effect on both metabolic processes in mouse myoblasts and fibroblasts.

    “Figure 10 and Figure 11 demonstrate that, although 20E treatment strongly induced the up-regulation of both glycolysis and respiration, it was not able to overcome the LY294002- mediated inhibitory effect on both metabolic processes in mouse myoblasts and fibroblasts.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    20E significantly up-regulated the expression of all studied genes (glut1, hk2, ldha, psat1, phgdh, psph, shmt2, mthfd2, c-Myc, ATF3) in both C2C12 and NIH/3T3 cells.

    “The relative real-time PCR data showed that 20E significantly up-regulated the expression of all the genes studied in both cell lines (Figure 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    20E increased ATP production in both glycolysis and oxidative phosphorylation (glycoATP and mitoATP), with a greater increase in glycoATP in C2C12 myoblasts and a greater increase in mitoATP in fibroblasts.

    “Using SeaHorse to look at ATP production revealed that, even though 20E increased ATP production in both glycolysis and oxidative phosphorylation (glycoATP and mitoATP), it increased glycoATP production more in C2C12 myoblasts and increased mitoATP production more in fibroblasts (Figure 6D and Figure 7D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The 20E-induced expression of metabolic genes was not concentration-dependent and was observed across the whole range of concentrations (0.1-100 μM) tested.

    “Importantly, 20E-induced expression of metabolic genes was not concentration-dependent, and the effect was observed in the whole range of concentrations (0.1–100 µM) tested.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    20E causes a 20E-mediated increase in both glycolysis and respiration.

    “In line with the molecular data obtained, we have demonstrated for the first time a 20E-mediated increase in both glycolysis and respiration.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The present study demonstrates that 20E induces the activation of both AKT (S473) and mTOR (S2448).

    “In accordance with this finding, the present study demonstrates that 20E induces the activation of both AKT (S473) and mTOR (S2448).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    20E improves lipid and glucose metabolism in the liver and heart.

    “A number of in vitro and in vivo studies have demonstrated the 20E-mediated improvement of lipid and glucose metabolism in the liver and the heart [17,18,19,20,21].”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [17] Kizelsztein, 2009, 20-Hydroxyecdysone decreases weight and hyperglycemia in a diet-induced obesity mice model, Am. J. Physiol.-Endocrinol. Metab. source

      Not checked in the free run.

    • cites [18] Baev, A.Y., Charishnikova, O.S., Khasanov, F.A., Nebesnaya, K.S., Makhmudov, A.R., Rakhmedova, M.T., Khushbaktova, Z.A., Syrov, V.N., and Levitskaya, Y.V. (2022). Ecdysterone prevents negative effect of acute immobilization stress on energy metabolism of rat liver mitochondria. J. Steroid Biochem. Mol. Biol., 219. source

      Not checked in the free run.

    • cites [19] Marschall, M.J., Ringseis, R., Gessner, D.K., Grundmann, S.M., Most, E., Wen, G., Maheshwari, G., Zorn, H., and Eder, K. (2021). Effect of ecdysterone on the hepatic transcriptome and lipid metabolism in lean and obese Zucker rats. Int. J. Mol. Sci., 22. source

      Not checked in the free run.

    • cites [20] Buniam, J., Chukijrungroat, N., Rattanavichit, Y., Surapongchai, J., Weerachayaphorn, J., Bupha-Intr, T., and Saengsirisuwan, V. (2020). 20-Hydroxyecdysone ameliorates metabolic and cardiovascular dysfunction in high-fat-high-fructose-fed ovariectomized rats. BMC Complement. Med. Ther., 20. source

      Not checked in the free run.

    • cites [21] Chen, 2006, Effect of ecdysterone on glucose metabolism in vitro, Life Sci. source

      Not checked in the free run.

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

    Premisecitation

    20E has mild, natural anabolic activity in humans/mammals that does not affect androgen receptors.

    “20E is best known for its mild, natural anabolic activity that does not affect androgen receptors [3,4].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [3] Dinan, L., Dioh, W., Veillet, S., and Lafont, R. (2021). 20-Hydroxyecdysone, from plant extracts to clinical use: Therapeutic potential for the treatment of neuromuscular, cardio-metabolic and respiratory diseases. Biomedicines, 9. source

      Not checked in the free run.

    • cites [4] Isenmann, 2019, Ecdysteroids as non-conventional anabolic agent: Performance enhancement by ecdysterone supplementation in humans, Arch. Toxicol. source

      Not checked in the free run.

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

    Premisecitation

    ATF3 coordinates serine and nucleotide metabolism.

    “and also coordinates serine and nucleotide metabolism [40]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Martinez, 2021, ATF3 coordinates serine and nucleotide metabolism to drive cell cycle progression in acute myeloid leukemia, Mol. Cell source

      Not checked in the free run.

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

    Premisecitation

    ATF3 is an important regulator of glucose and lipid metabolism in various tissues.

    “The literature data suggest that ATF3 also is an important regulator of glucose and lipid metabolism in various tissues [43].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] Hu, S., Zhao, X., Li, R., Hu, C., Wu, H., Li, J., Zhang, Y., and Xu, Y. (2022). Activating transcription factor 3, glucolipid metabolism, and metabolic diseases. J. Mol. Cell Biol., 14. source

      Not checked in the free run.

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

    Premisecitation

    Glycolysis supplies one-carbon metabolism through serine biosynthesis from 3-phosphoglycerate.

    “Glycolysis supplies one-carbon metabolism through serine biosynthesis from 3-phosphoglycerate [37,49].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [37] Petrova, B., Maynard, A.G., Wang, P., and Kanarek, N. (2023). Regulatory mechanisms of one-carbon metabolism enzymes. J. Biol. Chem., 299. source

      Not checked in the free run.

    • cites [49] Pan, 2020, Serine, glycine and one-carbon metabolism in cancer, Int. J. Oncol. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    20E activity observed in TNBC cell lines that lack ER argues that the wide range of pharmacological activities of 20E in tissues of different origins cannot be determined by only MAS1 and ER targets.

    “It should be noted, however, that we observed 20E activity also in triple-negative breast cancer (TNBC) cell lines that lack ER [41], arguing that the wide range of pharmacological activities of 20E in tissues of different origins cannot be determined by only these targets.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [41] Shuvalov, O., Fedorova, O., Tananykina, E., and Barlev, N. (2020). An arthropod hormone, ecdysterone, inhibits the growth of breast cancer cells via different mechanisms. Front. Pharmacol., 11. source

      Not checked in the free run.

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

    Premisecitation

    One-carbon metabolism links glycolysis with biosynthetic processes that result in DNA and RNA biosynthesis, protein biosynthesis, and maintenance of the redox and methylation states of cells.

    “As serine is derived from glycolysis intermediate 3-phosphoglycerate, one-carbon metabolism links glycolysis with different biosynthetic processes that result in DNA and RNA biosynthesis, protein biosynthesis, and the maintenance of the redox and methylation states of cells [51,52].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [51] Lionaki, E., Ploumi, C., and Tavernarakis, N. (2022). One-carbon metabolism: Pulling the strings behind aging and neurodegeneration. Cells, 11. source

      Not checked in the free run.

    • cites [52] Li, 2024, Amino acid metabolism, redox balance and epigenetic regulation in cancer, FEBS J. source

      Not checked in the free run.

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

    Premisecitation

    c-Myc is the master regulator of the metabolic network, affecting different metabolic processes and transactivating almost all genes that code for enzymes of glycolysis and one-carbon metabolism.

    “c-Myc is known as the master regulator of the metabolic network, affecting different metabolic processes and transactivating almost all genes that code for enzymes of glycolysis and one-carbon metabolism [35,36,37].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [35] Dong, 2020, Regulation of cancer cell metabolism: Oncogenic MYC in the driver’s seat, Signal Transduct. Target. Ther. source

      Not checked in the free run.

    • cites [36] Goetzman, E.S., and Prochownik, E.V. (2018). The role for Myc in coordinating glycolysis, oxidative phosphorylation, glutaminolysis, and fatty acid metabolism in normal and neoplastic tissues. Front. Endocrinol., 9. source

      Not checked in the free run.

    • cites [37] Petrova, B., Maynard, A.G., Wang, P., and Kanarek, N. (2023). Regulatory mechanisms of one-carbon metabolism enzymes. J. Biol. Chem., 299. source

      Not checked in the free run.

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

    Premisecitation

    Pyruvate entering the citric cycle paves the way for glutamate, glutamine, asparagine, aspartate, and proline biosynthesis.

    “Furthermore, being introduced into the citric cycle, pyruvate paves the way for glutamate, glutamine, asparagine, aspartate, and proline biosynthesis [48].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [48] Fry, B., and Carter, J.F. (2019). Stable carbon isotope diagnostics of mammalian metabolism, a high-resolution isotomics approach using amino acid carboxyl groups. PLoS ONE, 14. source

      Not checked in the free run.

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

    Premisecitation

    ATF3 is considered a hub of the cellular adaptive-response network.

    “ATF3 is considered a ‘’hub of the cellular adaptive-response network’’ [39]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Ku, H.-C., and Cheng, C.-F. (2020). Master regulator activating transcription factor 3 (ATF3) in metabolic homeostasis and cancer. Front. Endocrinol., 11. source

      Not checked in the free run.

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

    Premisecitation

    Glycolysis provides all non-essential and conditionally essential amino acids.

    “Glycolysis provides all non-essential and conditionally essential amino acids [47].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Chandel, N.S. (2021). Amino acid metabolism. Cold Spring Harb. Perspect. Biol., 13. source

      Not checked in the free run.

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

    Premisecitation

    ATF3 regulates serine biosynthesis by increasing PHGDH, PSAT1, and PSPH.

    “regulates serine biosynthesis by increasing PHGDH, PSAT1, and PSPH [38]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] Li, 2021, ATF3 promotes the serine synthesis pathway and tumor growth under dietary serine restriction, Cell Rep. source

      Not checked in the free run.

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

    Premisecitation

    AP-1 acts together with the Estrogen Related Receptor to activate transcription of PGC-1α.

    “it was reported to act together with the Estrogen Related Receptor to activate transcription of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) [44].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44] Baresic, 2014, Transcriptional network analysis in muscle reveals AP-1 as a partner of PGC-1α in the regulation of the hypoxic gene program, Mol. Cell. Biol. source

      Not checked in the free run.

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

    Interpretationcitation

    These results are consistent with the literature data on the adaptogenic and anabolic activities of 20E.

    “These results are consistent with the literature data on the adaptogenic and anabolic activities of 20E [3].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] Dinan, L., Dioh, W., Veillet, S., and Lafont, R. (2021). 20-Hydroxyecdysone, from plant extracts to clinical use: Therapeutic potential for the treatment of neuromuscular, cardio-metabolic and respiratory diseases. Biomedicines, 9. source

      Not checked in the free run.

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

    Premisecitation

    Increasing the intensity of anabolism requires the coordinated up-regulation of a number of biosynthetic processes, for which glycolysis and one-carbon metabolism are crucial sources of structural units.

    “Increasing the intensity of anabolism requires the coordinated up-regulation of a number of biosynthetic processes, for which glycolysis and one-carbon metabolism are crucial sources of structural units [37,45,46].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [37] Petrova, B., Maynard, A.G., Wang, P., and Kanarek, N. (2023). Regulatory mechanisms of one-carbon metabolism enzymes. J. Biol. Chem., 299. source

      Not checked in the free run.

    • cites [45] Mato, J., Martínez-Chantar, M., Noureddin, M., and Lu, S. (2017). One-carbon metabolism in liver health and disease. Liver Pathophysiology, Elsevier. source

      Not checked in the free run.

    • cites [46] Ducker, 2017, One-carbon metabolism in health and disease, Cell Metab. source

      Not checked in the free run.

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

    Interpretationcitation

    This is consistent with previously obtained data on the 20E-mediated increase in protein synthesis in differentiated myotubes.

    “This is consistent with the previously obtained data on the 20E-mediated increase in protein synthesis in differentiated myotubes [22,24].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [22] Cohick, 2010, Ecdysteroids elicit a rapid Ca2+ flux leading to Akt activation and increased protein synthesis in skeletal muscle cells, Steroids source

      Not checked in the free run.

    • cites [24] Lafont, 2022, 20-Hydroxyecdysone activates the protective arm of the RAAS via the MAS receptor, J. Mol. Endocrinol. source

      Not checked in the free run.

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

    Premisecitation

    Chen and co-authors reported a 20E-dependent increase in glucose uptake in Hep2G hepatoma cell lines.

    “which was also reported by Chen and co-authors in Hep2G hepatoma cell lines [21].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [21] Chen, 2006, Effect of ecdysterone on glucose metabolism in vitro, Life Sci. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    20E boosts both catabolism and anabolism, coupling energy-producing and biosynthetic metabolic processes.

    “In fact, we have demonstrated that 20E boosts both catabolism and anabolism, coupling the energy-producing and biosynthetic metabolic processes.”
    • Also stated in: § 5. Conclusions · plain
      “In the present study, we observed the 20E-dependent up-regulation of both catabolism and anabolism, coupling energy-producing and biosynthetic metabolic processes in mouse myoblasts and fibroblasts.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    A number of studies have demonstrated important roles of both AKT and mTOR in up-regulating glycolysis and respiration in various tissues.

    “In turn, a number of studies have demonstrated the important roles of both AKT and mTOR in up-regulating glycolysis and respiration in various tissues [54,55,56,57,58].”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [54] Hung, 2017, Akt regulation of glycolysis mediates bioenergetic stability in epithelial cells, Elife source

      Not checked in the free run.

    • cites [55] Li, 2013, Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) signaling regulates mitochondrial biogenesis and respiration via estrogen-related receptor α (ERRα), J. Biol. Chem. source

      Not checked in the free run.

    • cites [56] Rosario, F.J., Gupta, M.B., Myatt, L., Powell, T.L., Glenn, J.P., Cox, L., and Jansson, T. (2019). Mechanistic target of rapamycin complex 1 promotes the expression of genes encoding electron transport chain proteins and stimulates oxidative phosphorylation in primary human trophoblast cells by regulating mitochondrial biogenesis. Sci. Rep., 9. source

      Not checked in the free run.

    • cites [57] Goo, C.K., Lim, H.Y., Ho, Q.S., Too, H.-P., Clement, M.-V., and Wong, K.P. (2012). PTEN/Akt signaling controls mitochondrial respiratory capacity through 4E-BP1. PLoS ONE, 7. source

      Not checked in the free run.

    • cites [58] Morita, 2015, mTOR coordinates protein synthesis, mitochondrial activity and proliferation, Cell Cycle source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    20E is low-toxic with an LD50 > 9 g/kg in mice.

    “as it is a low-toxic compound with an LD50 > 9 g/kg in mice [3].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [3] Dinan, L., Dioh, W., Veillet, S., and Lafont, R. (2021). 20-Hydroxyecdysone, from plant extracts to clinical use: Therapeutic potential for the treatment of neuromuscular, cardio-metabolic and respiratory diseases. Biomedicines, 9. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    20E has adaptogenic, anabolic, antioxidant, hepato-, cardio-, and neuroprotective activities in healthy non-cancer tissues.

    “because of its adaptogenic, anabolic, antioxidant, hepato-, cardio-, and neuroprotective activities in healthy non-cancer tissues [3,5,7,9]”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [3] Dinan, L., Dioh, W., Veillet, S., and Lafont, R. (2021). 20-Hydroxyecdysone, from plant extracts to clinical use: Therapeutic potential for the treatment of neuromuscular, cardio-metabolic and respiratory diseases. Biomedicines, 9. source

      Not checked in the free run.

    • cites [5] Lim, 2020, The insect molting hormone 20-hydroxyecdysone protects dopaminergic neurons against MPTP-induced neurotoxicity in a mouse model of Parkinson’s disease, Free. Radic. Biol. Med. source

      Not checked in the free run.

    • cites [7] Todorova, V., Ivanov, K., Delattre, C., Nalbantova, V., Karcheva-Bahchevanska, D., and Ivanova, S. (2021). Plant adaptogens—History and future perspectives. Nutrients, 13. source

      Not checked in the free run.

    • cites [9] Volodin, 2013, 20-Hydroxyecdysone--plant adaptogen: An anabolic effect, possible use in sports nutrition, Vopr. Pitan.

      Not checked in the free run.

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  • The paper relies on this without support.

    Mouse C2C12 myoblasts and NIH/3T3 fibroblasts are suitable models for studying 20E's effects on human/mammalian energy metabolism and anabolic activity.

    “In this research, we have demonstrated that 20E boosts both catabolism and anabolism, coupling energy-producing and biosynthetic metabolic processes in mouse myoblasts and fibroblasts in the same way.”

  • The paper relies on this without support.

    LY294002 specifically inhibits PI3K/AKT without relevant off-target effects on glycolysis or respiration.

    “we pre-treated C2C12 and NIH/3T3 cells with the well-known PI3K inhibitor LY294002 (20 µM), which prevents AKT activation”

  • The paper relies on this without support.

    Mammals lack the G-protein-coupled ecdysone receptor (EcR) responsible for 20E-mediated effects.

    “Notably, mammals, unlike insects, lack the G-protein-coupled ecdysone receptor (EcR) which is responsible for 20E-mediated effects.”

  • The paper relies on this without support.

    C2C12 cells are a well-characterized model of immortalized mouse myoblasts that help interpretation of molecular results in the context of muscle function and repair.

    “The latter are a well-characterized model of immortalized mouse myoblasts that help interpretation of the results obtained at the molecular level in the context of muscle function and repair.”

  • The paper relies on this without support.

    The metabolic functions of ATF3 in muscle tissues remain largely unknown.

    “The metabolic functions of ATF3 in muscle tissues remain largely unknown and need to be explored in further studies.”

  • The paper relies on this without support.

    The role of AP-1 in skeletal muscle metabolism is still enigmatic.

    “Likewise, the role of AP-1 in skeletal muscle metabolism is still enigmatic”

  • The paper relies on this without support.

    Anabolic activity necessitates a coordinated increase in energy production with improved biosynthetic processes.

    “Anabolic activity, of course, necessitates a coordinated increase in energy production with improved biosynthetic processes.”
7 of 7

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?

  • The PI3K/Akt/mTor is the major signalling pathway involved in increased protein synthesis and anti-oxidant effect, accompanied by increased C1 metabolism.

    “The authors, just like other researchers, found that the PI3K/Akt/mTor is the major signalling pathway involved in increased protein synthesis and anti-oxidant effect, accompanied by increased C1 metabolism as newly reported data.”

    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 [96]

    Results §§3.2, 3.4, and 3.5 report increased expression of one-carbon-metabolism enzymes, increased protein biosynthesis, and activation of PI3K/AKT/mTOR after 20E treatment in mouse cells. But the pathway experiments establish its importance for glycolysis and respiration, not that it is the major pathway responsible for the protein-synthesis increase or an antioxidant effect; the paper does not demonstrate that linked mechanism. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • In liver hepatocytes, 20E inhibits FAS in vivo; meanwhile, it promotes fatty acid degradation, showing the opposite response to that of myoblasts and fibroblasts in vitro.

    “In liver hepatocytes, 20E inhibits FAS in vivo; meanwhile, it promotes fatty acid degradation [90], showing the opposite response to that of myoblasts and fibroblasts in vitro [96].”

    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 [96]

    Results §3.3 reports that 20E increased glycolysis, respiration, and ATP production in cultured C2C12 myoblasts and NIH/3T3 fibroblasts. The paper does not report FAS activity or fatty acid degradation in those cells, so it does not establish that their fatty-acid response was opposite to the in-vivo hepatocyte response cited separately as [90]. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • Shuvalov et al. reported that 20E elevated LDH expression together with increased terminal oxidation.

    “Shuvalov et al. [96] reported that 20E elevated LDH expression together with increased terminal oxidation.”

    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 [96]

  • The scenario with the central role of ACL may imply that 20E acts similarly to calorie restriction.

    “The above scenario with the central role of ACL does not refute Shuvalov et al.’s suggestion [96], but it may imply that 20E acts similarly to calorie restriction.”

    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 [96]

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.

  • Puromycin incubation time differs between methods and results

    Major

    The SUnSET methods specify a 15-minute puromycin treatment, whereas the results describe a 30-minute incubation for the assay.

  • RNA-seq treatment dose is given in incompatible units

    Minor

    The methods give the RNA-seq 20E dose as 1 μM, a concentration, but the results give it as 1 μm, a unit of length.

2 of 2

The citation lists haven’t been fetched yet

References 63 · Cited by 9

Authors
Shuvalov, O., Kirdeeva, Y., Fefilova, E., Daks, A., Fedorova, O., Parfenyev, S., Nazarov, A., Vlasova, Y., Krasnov, G. S., & Barlev, N. A.
Affiliations
  • Russian Academy of Sciences (RU)
  • Federal Almazov North-West Medical Research Centre (RU)
  • Engelhardt Institute of Molecular Biology (RU)
  • Institute of Cytology of the Russian Academy of Sciences, St. Petersburg 194064, Russia
Corresponding author
Shuvalov, Oleg
ORCID
Venue
Antioxidants
Publisher
MDPI AG
Published
2024-11-02
Volume
13
Issue
11
Pages
1349
ISSN
2076-3921 (ISSN-L)
DOI
10.3390/antiox13111349
PMC
PMC11591052
OpenAlex
W4404047591
Cited by
9
References
63
Citations by year
2026: 4 · 2025: 4 · 2024: 1
FWCI
1.22
Type
journal article
Language
en
Open access
Licence
Locations
  • Antioxidants (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Nazarbayev University Repository (Nazarbayev University) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • RSF (#21-75-10138, # 211123CRP1601)
  • CRP (#21-75-10138, # 211123CRP1601)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Hormonal and reproductive studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Muscle metabolism and nutrition
Keywords
20-hydroxyecdysone, energy metabolism, glycolysis, one-carbon metabolism, protein synthesis, myoblasts, fibroblasts, PI3K/AKT/mTOR pathway, glucose uptake, ATP production, anabolic activity, transcriptomics

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