Evaluating the effect of 20-hydroxyecdysone (20HE) on mechanistic target of rapamycin complex 1 (mTORC1) signaling in the skeletal muscle and liver of rats
Open accessAbstract
Phytoecdysteroids such as 20-hydroxyecdysone (20HE) are nutritional supplements marketed as enhancers of lean body mass. In this study the impact of 20HE ingestion on protein kinase B/Akt-mechanistic target of rapamycin complex 1 signaling in the skeletal muscle and liver of male rats was found to be limited. Bioavailability of 20HE, whether consumed alone or with leucine, also remained low at all doses ingested. Additional work is necessary to clarify 20HE mechanism of action in vivo.
AI snapshot
Written from the paper’s full text.
TL;DR
Acute oral 20-hydroxyecdysone (20HE) did not activate Akt–mTORC1 signaling in skeletal muscle or liver of male rats across dose-response and time-course studies. Bioavailability of 20HE remained low whether given alone or with leucine, and 20HE slightly dampened leucine-stimulated mTORC1 signaling in skeletal muscle.
Key findings
- In Study 1, 20HE doses of 0, 10, 50, and 200 mg/kg did not significantly alter phosphorylation of Akt, mTOR, or 4E-BP1 at 30 min in skeletal muscle or liver.
- In Study 2, 200 mg/kg 20HE over 30, 60, 120, and 240 min produced no changes in Akt, mTOR, or 4E-BP1 phosphorylation in muscle or liver, except 4E-BP1 phosphorylation was reduced at 240 min in skeletal muscle only.
- Serum 20HE was low and similar across doses in 3% DMSO (~0.45 ng/µl) and over the time course (0.3–0.5 ng/µl). With Labrasol, serum 20HE was 0.74 ± 0.076 ng/µl with 20HE + leucine versus 1.52 ± 1.23 ng/µl with 20HE alone.
- Leucine alone significantly increased mTOR and 4E-BP1 phosphorylation in skeletal muscle and liver; 20HE alone did not alter mTORC1 signaling and slightly dampened leucine-stimulated mTORC1 in skeletal muscle. Neither leucine nor 20HE altered Akt phosphorylation at 30 min alone or in combination.
Methods
- Overnight-fasted 4–8 week old male Sprague-Dawley rats (n=5–6 per group) were randomized to three designs: a 20HE dose response (0, 10, 50, 200 mg/kg, euthanized 30 min post-gavage), a 200 mg/kg 20HE time course (pre-gavage, 30, 60, 120, 240 min), and acute 20HE (200 mg/kg) versus L-leucine (1.35 g/kg) alone or combined, euthanized 30 min post-gavage.
- Serum leucine was analyzed by HPLC with OPA/FMOC derivatization; 20HE bioavailability was quantified by LC-MS after butanol extraction.
- Immunoblot analysis measured phosphorylated Akt (Thr308), mTOR (Ser2448), and 4E-BP1 in skeletal muscle and liver; data were analyzed by ANOVA with Tukey’s post hoc test, significance at P ≤ 0.05.
Limitations
- The paper states that 20HE bioavailability was low and appeared insensitive to increasing doses, that the mechanism of 20HE action remains an open question, and that follow-up time courses with calories and/or carbohydrates are warranted.
- The paper notes that the early time points were chosen based on prior work, and that alternate mechanisms independent of mTOR require future investigation.
- Evident from the text: the study is acute and uses only male rats, with small group sizes (n=5–6); Study 3 inconsistently refers to “mice” in the results despite the methods describing rats, and no direct measures of muscle growth, lean mass, or protein synthesis are reported.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
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Claims the paper makes
0 of 14 cited sources checked · 0 supported · 0 flagged
- Findingown data
Bioavailability of 20HE remained low at all doses ingested, whether consumed alone or with leucine.
“Bioavailability of 20HE, whether consumed alone or with leucine, also remained low at all doses ingested.”
- Also stated in: § Study 1: 20HE Dose Response · plain
“Serum 20HE concentrations were detectible only in rats gavaged with 20HE but remained similarly low across all doses (~0.45 ng/µl).”
- Also stated in: § DISCUSSION · hedged
“The bioavailability of 20HE was quite low in these studies and appeared insensitive to increasing doses.”
Quote in the paperSubstantial claim - Also stated in: § Study 1: 20HE Dose Response · plain
- Findingown data
In the current study, provision of leucine in combination with 20HE results in a dampening of mTORC1 signaling despite elevated circulating leucine.
“Interestingly, in the current study provision of leucine in combination with 20HE results in a dampening of mTORC1 signaling despite elevated circulating leucine.”
Quote in the paperSubstantial claim - Findingown data
In Study 3, leucine alone significantly increased phosphorylation of mTOR and 4E-BP1 in both skeletal muscle and liver.
“In agreement with previous reports, leucine alone significantly increased phosphorylation of mTOR and 4E-BP1 in both skeletal muscle (Figure 1, H-I) and liver (Figure 2, H-I).”
Quote in the paperSubstantial claim - Findingown data
In Study 1, none of the doses of 20HE significantly altered phosphorylation states of Akt, mTOR or 4E-BP1 at 30 minutes after ingestion in either skeletal muscle or liver.
“None of the doses administered significantly altered the phosphorylation states of Akt, mTOR or 4E-BP1 at 30 minutes after ingestion in either the skeletal muscle (Figure 1, A-C) or liver (Figure 2, A-C).”
Quote in the paperSubstantial claim - Findingown data
In Study 2, phosphorylation of 4E-BP1 was reduced at 240 min in skeletal muscle only.
“No changes in the phosphorylation status of Akt, mTOR or 4E-BP1 as compared to untreated controls were noted in either skeletal muscle (Figure 1, D-F) or liver (Figure 2, D-F) over the 4 h time course except phosphorylation of 4E-BP1 was reduced at 240 min in skeletal muscle only (Figure 1F).”
Quote in the paperNot a substantial claim
- Findingown data
In Study 3, neither leucine nor 20HE altered phosphorylation of Akt in liver or muscle at 30 min when gavaged alone or in combination.
“Neither leucine nor 20HE altered phosphorylation of Akt in liver or muscle at 30 min when gavaged alone or in combination (Figure 1G, 2G).”
Quote in the paperSubstantial claim - Findingown data
Quantification of 20HE from plasma samples showed minimal differences in bioavailability among the doses at 30 minutes when delivered in 3% DMSO in saline.
“Quantification of 20HE from plasma samples showed there were minimal differences in bioavailability among the doses at 30 minutes when delivered in 3% DMSO in saline.”
Quote in the paperNot a substantial claim - Findingown data
After gavage in 3% DMSO in saline, 20HE levels in blood ranged between 0.3-0.5 ng/μl.
“Quantification of 20HE from plasma showed 20HE levels in blood ranged between 0.3-0.5 ng/µl after gavage when delivered in 3% DMSO in saline.”
Quote in the paperNot a substantial claim - Premisecitation
20HE is not structurally close enough to androgens to bind to intracellular steroid receptors in humans.
“Despite being classified as a steroid, 20HE is not structurally close enough to androgens to be able to bind to intracellular steroid receptors in humans (Bathori et al. 2008).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Bathori et al. 2008
Not checked in the free run.
- Premisecitation
In that in vitro study, the effects of 20HE were suppressed by inhibitors of GPCR, phospholipase C (PLC) and phosphoinositide kinase-3 (PI3K).
“In this study, the effects of 20HE were suppressed by inhibitors of GPCR, phospholipase C (PLC) and phosphoinositide kinase-3 (PI3K).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Gorelick-Feldman et al. 2010
Not checked in the free run.
- Premisecitation
One human trial was unable to substantiate the anabolic potential of 20HE.
“but one human trial was unable to substantiate the anabolic potential of 20HE (Wilborn et al. 2006).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Wilborn et al. 2006
Not checked in the free run.
- Interpretationown data
If 20HE is to augment the action of another nutrient, it is not likely to be an amino acid-driven pathway.
“Thus, if 20HE is to augment the action of another nutrient, it is not likely to be an amino acid-driven pathway.”
Quote in the paperSubstantial claim - Premisecitation
An in vitro study showed that ecdysteroids increase protein synthesis in muscle cells through an influx of Ca2+ and subsequent activation of protein kinase B/Akt.
“An in vitro study showed that ecdysteroids increase protein synthesis in muscle cells through an influx of Ca 2+ and subsequent activation of protein kinase B/Akt (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.
- May combine two claims. 11
- Classified as probably not a substantial claim. 10
May combine two claims.
Findingown dataIn Study 2, no changes in phosphorylation status of Akt, mTOR or 4E-BP1 compared to untreated controls were noted in either skeletal muscle or liver over the 4 h time course, except 4E-BP1 phosphorylation was reduced at 240 min in skeletal muscle only.
“No changes in the phosphorylation status of Akt, mTOR or 4E-BP1 as compared to untreated controls were noted in either skeletal muscle (Figure 1, D-F) or liver (Figure 2, D-F) over the 4 h time course except phosphorylation of 4E-BP1 was reduced at 240 min in skeletal muscle only (Figure 1F).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIn Study 3, 20HE bioavailability appeared to be improved by Labrasol, though with great variation.
“While leucine concentrations rose significantly regardless of 20HE consumption, 20HE bioavailability appeared to be improved by Labrasol, though with great variation, for serum levels of 20HE measured 0.74 ± 0.076 ng/µl in rats gavaged with 20HE + leucine compared with 1.52 ± 1.23 ng/µl in rats gavaged with 20HE alone.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataIn Study 3, serum leucine concentrations rose significantly in rats gavaged with leucine alone and in mice gavaged with leucine plus 20HE compared to control.
“Serum leucine concentrations rose significantly in rats gavaged with leucine alone and in mice gavaged with leucine plus 20HE (both P<0.05 as compared to control).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataIn male rats, acute ingestion of 20HE does not activate Akt phosphorylation or mTORC1 signaling in skeletal muscle or liver.
“In this study the impact of 20HE ingestion on protein kinase B/Akt-mTORC1 signaling in the skeletal muscle and liver of male rats was found to be limited.”
- Also stated in: § Study 1: 20HE Dose Response · plain
“None of the doses administered significantly altered the phosphorylation states of Akt, mTOR or 4E-BP1 at 30 minutes after ingestion in either the skeletal muscle (Figure 1, A-C) or liver (Figure 2, A-C).”
- Also stated in: § DISCUSSION · plain
“study results did not support this idea, showing that 20HE does not acutely activate Akt phosphorylation or mTORC1 signaling in hind limb muscle or liver.”
Quote in the paperSubstantial claim- Also stated in: § Study 1: 20HE Dose Response · plain
May combine two claims.
Findingown dataUsing Labrasol improved 20HE bioavailability but did not result in improved activation of the Akt-mTORC1 pathway at the time points examined.
“While using an excipient such as Labrasol improved bioavailability, this did not result in improved activation of the Akt-mTORC1 pathway at the time points examined.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataSerum 20HE concentrations were detectible only in rats gavaged with 20HE but remained similarly low across all doses (~0.45 ng/μl).
“Serum 20HE concentrations were detectible only in rats gavaged with 20HE but remained similarly low across all doses (~0.45 ng/µl).”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
PremisecitationIt is suggested that 20HE binds to G protein coupled receptors (GPCR) on the plasma membrane.
“Instead, it is suggested that 20HE binds to G protein coupled receptors (GPCR) on the plasma membrane (Bathori et al. 2008).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Bathori et al. 2008
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Premisenone givenOral ingestion of amino acids, particularly leucine, stimulates mTORC1 assembly and signaling via a PI3K-Akt-independent mechanism.
“Oral ingestion of amino acids, and in particular leucine, also stimulates mTORC1 assembly and signaling but in a PI3K-Akt-independent mechanism, allowing for a convergence of amino acid and insulin/IGF-I signaling on mTORC1 upon meal feeding”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim. May combine two claims.
PremisecitationIngestion of 20HE reportedly increases the size of muscle fibers in rats and increased lean body mass in sheep, pigs and quail.
“Ingestion of 20HE reportedly increases the size of muscle fibers in rats (Toth et al. 2008) as well as increased lean body mass in sheep, pigs and quail (Chermnykh et al. 1988;Lafont and Dinan 2003) and is associated with antidiabetic effects in obese mice (Kizelsztein et al. 2009).”
Quote in the paperSubstantial claim0 of 3 sources checked- cites Toth et al. 2008
Not checked in the free run.
- cites Chermnykh et al. 1988
Not checked in the free run.
- cites Lafont and Dinan 2003 source
Not checked in the free run.
Classified as probably not a substantial claim. May combine two claims.
PremisecitationOral administration of 1.35 g/kg body weight L-leucine activates mTORC1 signaling in skeletal muscle between 15-60 minutes following ingestion without altering Akt phosphorylation.
“Previous work by this lab and others show that oral administration of 1.35 g/kg body weight L-leucine activates mTORC1 signaling in skeletal muscle between 15-60 minutes following ingestion without altering Akt phosphorylation (Anthony et al. 2001a).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Anthony et al. 2001a
Not checked in the free run.
Classified as probably not a substantial claim.
PremisecitationOther work has described anti-obesity and anti-diabetic effects of 20HE in mice via reduced hepatic glucose production in association with increased Akt phosphorylation.
“Separate from its effects in muscle, other work has described anti-obesity and anti-diabetic effects of 20HE in mice via reduced hepatic glucose production in association with increased Akt phosphorylation (Kizelsztein et al. 2009).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Kizelsztein et al. 2009
Not checked in the free run.
Classified as probably not a substantial claim.
PremisecitationStimulation of muscle protein synthesis by insulin and/or IGF-I is mediated via PI3K-Akt activation of mTORC1 assembly and signal transduction.
“Stimulation of muscle protein synthesis by insulin and/or insulin-like growth factor I (IGF-I) is mediated via PI3K-Akt activation of mammalian target of rapamycin complex 1 (mTORC1) assembly and signal transduction (Laplante and Sabatini 2012).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Laplante and Sabatini 2012
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenImmunoblots included a tissue sample from rats injected with IGF-I as a positive control for Akt phosphorylation.
“Immunoblots included a tissue sample from rats injected with IGF-I as a positive control for Akt phosphorylation.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationmTORC1 is one of the most important cellular signaling hubs in the body, regulating cell growth, autophagy, and proliferation.
“Certainly, among the pathways affected by nutritional status, mTORC1 is one of the most important cellular signaling hubs in the body, regulating cell growth, autophagy, and proliferation (Laplante and Sabatini 2012).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Laplante and Sabatini 2012
Not checked in the free run.
Classified as probably not a substantial claim. May combine two claims.
Study design and conductnone givenOvernight fasted 4-8 week old male Sprague-Dawley rats (n=5-6 per group) were randomized to one of three study designs.
“Overnight fasted 4-8 week old male Sprague-Dawley rats (n=5-6 per group) were randomized to one of three study designs: 1) gavaged with 0, 10, 50, 200 mg/kg 20HE and euthanized 30 min post-gavage; 2) gavaged with 200 mg/kg 20HE or excipient and euthanized at pre-gavage or 30 min, 60 min, 120 min, 240 min post-gavage, and 3) administered excipient or 200 mg/kg 20HE alone or in combination with 1.35”
Quote in the paperSubstantial claimMay combine two claims.
PremisecitationThe choice to focus on early time points was based on previous work showing activation of Akt by 20HE within 4 h and activation of mTORC1 by leucine within 1 h.
“The choice to focus on early time points was based on previous work showing activation of Akt by 20HE within 4 h and activation of mTORC1 by leucine within 1 h (Anthony et al. 2001a;Gorelick-Feldman et al. 2010).”
Quote in the paperSubstantial claim0 of 2 sources checked- cites Anthony et al. 2001a
Not checked in the free run.
- cites Gorelick-Feldman et al. 2010
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAll experiments were reviewed and approved by the Institutional Animal Care and Use Committee at Rutgers University.
“All rats were cared for in accordance with the Guide to the Care and Use of Laboratory Animals and all experiments were reviewed and approved by the Institutional Animal Care and Use Committee at Rutgers University.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
There are no studies assessing phosphorylation of Akt by 20HE in vivo.
“There are no studies assessing phosphorylation of Akt by 20HE in vivo.”
The paper relies on this without support.
The molecular pathway mediating 20HE's anabolic potential remains unknown.
“The molecular pathway mediating 20HE's anabolic potential remains unknown.”
The paper relies on this without support.
It is unknown if reported changes in hepatic glucose production by 20HE are linked in any way to acute activation of Akt.
“It is unknown if reported changes in hepatic glucose D r a f t 4 production by 20HE are linked in any way to acute activation of Akt.”
The paper relies on this without support.
It is unknown if 20HE activates downstream anabolic signaling separate from metabolic signaling in liver.
“Furthermore, it is unknown if 20HE activates downstream anabolic signaling separate from metabolic signaling in liver.”
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?
A limited, if any, effect was found when 20E was fed to rats and a pool of skeletal muscles was analysed for protein kinase B/Akt level as a target of rapamycin.
“In contrast, a limited, if any, effect was found when 20E was fed to rats, and a pool of skeletal muscles had been analysed for protein kinase B/Akt level as a target of rapamycin.”
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 [16]
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.
Study 3 animals are called mice and rats
MinorThe Study 3 results say mice received the treatments, but the study design identifies the animals as male Sprague-Dawley rats.
“To compare the impact of 20HE ingestion to that of leucine, mice were orally administered either 20HE (200 mg/kg), leucine (1.35 g/kg), the combination together, or excipient alone.”
“Overnight fasted 4-8 week old male Sprague-Dawley rats (n=5-6 per group) were randomized to one of three study designs: 1) gavaged with 0, 10, 50, 200 mg/kg 20HE and euthanized 30 min post-gavage; 2) gavaged with 200 mg/kg 20HE or excipient and euthanized at pre-gavage or 30 min, 60 min, 120 min, 240 min post-gavage, and 3) administered excipient or 200 mg/kg 20HE alone or in combination with 1.35 g/kg L-leucine and euthanized 30 min post-gavage.”
The citation lists haven’t been fetched yet
References 19 · Cited by 25
Research works printed but not linked: 1
- Chermnykh N.S., 1988, Farmakol. Toksikol., 51, 57
- Authors
- Anthony, T. G., Mirek, E. T., Bargoud, A. R., Phillipson-Weiner, L., DeOliveira, C. M., Wetstein, B., Graf, B. L., Kuhn, P. E., & Raskin, I.
- Affiliations
- Rutgers, The State University of New Jersey (US)
- Department of Nutritional Sciences, Rutgers University, New Brunswick, NJ 08901, USA.
- Venue
- Applied Physiology, Nutrition, and Metabolism
- Publisher
- Canadian Science Publishing
- Published
- 2015-12
- Volume
- 40
- Issue
- 12
- Pages
- 1324-1328
- ISSN
- 1715-5312 (ISSN-L)
- 1715-5320
- OpenAlex
- W1888193095
- Cited by
- 25
- References
- 19
- Citations by year
- 2026: 2 · 2025: 1 · 2024: 3 · 2023: 1 · 2021: 8 · 2020: 2 · 2019: 3 · 2018: 3 · 2017: 1 · 2016: 1
- FWCI
- 0.89
- Type
- journal article
- Language
- en
- Open access
- green
- hdl.handle.net
- Locations
- Applied Physiology, Nutrition, and Metabolism (publishedVersion)
- PubMed (publishedVersion)
- TSpace (University of Toronto) (acceptedVersion)
- Rutgers University Community Repository (Rutgers University) (submittedVersion)
- Funders
- U.S. Department of Agriculture (NC-1184)
- New Jersey Agricultural Experiment Station
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Muscle metabolism and nutrition
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › PI3K/AKT/mTOR signaling in cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
- Keywords
- 20-hydroxyecdysone, phytoecdysteroids, mTORC1 signaling, skeletal muscle, liver, bioavailability, leucine, lean body mass, dietary supplements
- MeSH terms
- Mechanistic Target of Rapamycin Complex 1, Animals, Biological Availability, Dose-Response Relationship, Drug, Ecdysterone, Leucine, Liver, Male, Phosphorylation, Signal Transduction, Rats, Sprague-Dawley, Muscle, Skeletal, Dietary Supplements, Multiprotein Complexes, Proto-Oncogene Proteins c-akt, TOR Serine-Threonine Kinases
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