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

10 Apr 2020-bioRxiv (Cold Spring Harbor Laboratory)-

TL;DR: 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.

Abstract20-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 ER 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. 17-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.

Topics: Estrogen receptor (57%), Receptor (57%), Mechanism of action (56%), Myostatin (55%), 20-Hydroxyecdysone (55%)

Summary (3 min read)

INTRODUCTION

  • Steroids in animal and plant kingdoms Steroid hormones are sterol derivatives widespread in animals and plants, where they are involved in the control of plenty of physiological processes.
  • They include for example vertebrate sex hormones (progestagens, estrogens, androgens), insect moulting hormones , as well as plant growth hormones .
  • This means that the rigid carbon skeleton of sterols is particularly suitable to generate a very large number of derivatives, which differ by the carbon number and/or the position of various substituents (mainly hydroxyl or keto groups) 2 (1).
  • All these receptors belong to the family of GPCR/7TD receptors.
  • Moreover, intact or truncated forms or the steroid nuclear receptors are bound to the plasma membrane and do not act there as transcription factors(7,8).

Ecdysteroid effects on vertebrates

  • Ecdysteroids are a large family of steroids initially discovered in arthropods and later in plants (9).
  • They are present in many plant species where they can reach concentrations of up to 2-3 % of the plant dry weight, and they are expected to protect plants against phytophagous insects.
  • With the aim to use these molecules for crop protection, toxicological studies were performed on mammals, which unexpectedly concluded to both their lack of toxicity (oral LD50 > 9 g/kg) and their “beneficial” effects, e.g. anti-diabetic and anabolic properties (10).
  • Such effects have to be linked with the presence of large amounts of phytoecdysteroids in several plants used worldwide by traditional medicine.
  • At the moment, numerous effects have been reported, allowing to consider ecdysteroids as some kind of “universal remedy”(11).

How do ecdysteroids work?

  • 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.
  • Some evidence for a direct binding was provided by in silico modelling (22), but this certainely does not represent a definite proof, as the result may strongly depend on the model used, and an opposite conclusion was drawn by Lapenna et al. (23).
  • More recently, Gorelick-Feldman et al. (17) used a pharmacological approach with various inhibitors (e.g. pertussis toxin).
  • The above pharmacological arguments appear strong enough to consider that the cell membrane is (maybe not exclusively) a site of action of 20E.
  • The present experiments have been undertaken in an attempt to identify the/one GPCR involved in 20E effects, and to understand its estrogen-like effects using gene silencing or different pharmacological approaches.

Chemicals

  • Except otherwise mentioned, all the reagents and chemicals were from Sigma (Saint-Quentin Fallavier, France).
  • Peptides such as angiotensin-(1-7), A779 (Asp-Arg-Val-Tyr-IleHis-D-Ala) and A1 (Asp-Arg-Val-Tyr-Ile-HisD-Pro) were custom-prepared by the IBPS peptide synthesis platform (Sorbonne University, Paris, France).
  • 20- Hydroxyecdysone (20E) was obtained from Chemieliva Pharmaceutical (Chongqing, China) or from Patheon (Regensburg, Germany) and had a purity of 96.5-97%.

Preparation of HSA-conjugated 20- hydroxyecdysone

  • Analyses were performed in a 4700 MALDI TOF/TOF proteomics analyzer (Applied Biosystems).
  • Laser acceleration is set at 20kV and the default laser fluency was set at 2,000 and modified according to the signal-to-noise quality.
  • The matrix used was α-cyano-4-hydrocinnamic Acid (HCCA).
  • The mass shift of HSA around 5 kDa after coupling indicates that 9 molecules of 20E derivatives are coupled to each albumin molecule.

Cell culture

  • Except otherwise mentioned, culture media, serum, antibiotics and supplements were from Life technologies (Villebon-sur-Yvette, France).
  • Cell confluency was always kept below or equal to ~80%.
  • For all experiments, cells were first seeded at 30,000 cells per well in 24-well plates.
  • To induce differentiation, C2C12 at ~80% confluency in proliferation medium were shifted to DMEM medium supplemented with either 2% FBS or 2% horse serum.

Protein synthesis (3H-leucine incorporation)

  • The cell soluble fraction-associated radioactivity was then counted using Wallac Microbeta 1450-021 TriLux Luminometer Liquid Scintillation Counter (Wallac EG&G, Gaithersburg, MD, USA) and protein quantification was performed using the colorimetric Lowry method.
  • 4 Myostatin and MAS gene expression assays Cells were plated at a density of 30,000 cells per well in 24-well plates and were grown overnight in 5 % CO2 at 37°C.
  • RNAs were converted into cDNAs with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, ThermoFisher, Villebon-surYvette, France) before performing a quantitative PCR using iTaq SybrGreen (Biorad, Marnes-la Coquette, France).
  • Primer sequences used are described in Table 1.

SiRNA MAS assay

  • After 3 days of differentiation, cells were transfected either with scramble SiRNA (10 nM) or MAS-1 SiRNA (10 nM) according to manufacturer’s instructions (Origene Technologies, Rockville, MD, USA).
  • Two days after transfection, myotubes were treated with either DMSO or IGF-1 or 20E or angiotensin-(1-7) for 6 h.
  • At the end of incubation, RNA was extracted and analyzed by QRT-PCR as described above.

Binding studies

  • The affinity of 20E for human nuclear steroid receptors such as androgen receptor (AR), estrogen receptors alpha and beta (ERα, ERβ) and glucocorticoid receptor (GR) was determined by radioligand binding assays (CEREP/Eurofins).
  • The selective ligands [3H]methyltrienolone, [3H]-estradiol, [3H]dexamethasone were employed on cells expressing either human endogenous or recombinant AR, ERα or β, or GR, respectively.
  • Inhibition of control specific binding was determined, and IC50 and Ki were calculated when possible.
  • Radioligand binding assays were performed according to manufacturer instructions employing 3H- or 125I-labelled specific ligands of each receptor (SafetyScreen87 Panel, Panlabs, Taipei, Taiwan).

Statistical analyses

  • Statistical analysis was performed using Graph Pad Prism® Software.
  • Anova followed by a Dunnett t-test or a Kruskal Wallis followed by a Dunn’s test when the variances significantly differed have been performed.
  • To evaluate the significance of differences between two groups, the choice of parametric Student ttest or non-parametric Mann-Whitney test was based on the normality or non-normality of data distribution, respectively (D’Agostino & Pearson test).

RESULTS

  • 20-Hydroxyecdysone stimulates muscle anabolism 20-Hydroxyecdysone (20E) effects were investigated on pre-established murine myotubes (following 6 days of differentiation).
  • The inhibition of myostatin gene expression was then employed as a readout for 20E activity.
  • In a similar way to what was observed with antagonists (Fig. 3A), down regulation of Mas receptor reversed 20E or Ang1-7 effects on myostatin gene expression (Fig. 3B).
  • Thus, while the above binding studies seem to exclude canonical nuclear forms of ERs, the question remains open for the membrane ones.

DISCUSSION

  • The authors 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.
  • 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.
  • Experiments on C2C12 cells treated with diarylheptanoid compounds (HPPH) provide very important informations(50,51).
  • Interestingly, the authors would thus be in the presence of both a complex between aldosterone and angiotensin II AT1 receptors and, symmetrically, of a complex between estradiol and angiotensin-(1-7).
  • Mas receptors and displaying opposite physiological effects.

CONCLUSION:

  • Based on these above findings, a pharmaceutical grade preparation of 20E (BIO101) has been developed and is presently being assayed in a phase 2 clinical trial for treating sarcopenia, (a double-blind, placebo controlled, randomized interventional clinical trial (SARA-INT), ClinicalTrials #NCT03452488).
  • The authors are confident that the beneficial effects of 20E/BIO101 will also be further established on e.g. lungs, kidneys and cardiovascular pathologies and could offer new therapeutic strategies.

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1
20-Hydroxyecdysone activates the protective arm of the renin angiotensin
system via Mas receptor
René Lafont
1,2
, Sophie Raynal
1
, Maria Serova
1
, Blaise Didry-Barca
1
, Louis Guibout
1
,
Mathilde Latil
1
, Pierre J. Dilda
1*
, Waly Dioh
1
, Stanislas Veillet
1
1
Biophytis, Sorbonne Université BC9, 4 place Jussieu, 75005 Paris, France.
2
Sorbonne Université, CNRS - Institut de Biologie Paris Seine (BIOSIPE), 75005 Paris,
France
*Corresponding author: Pierre J. Dilda
Email: pierre.dilda@biophytis.com
Running title: 20E, a new Mas receptor activator
Keywords: 20-Hydroxyecdysone (20E), Mas receptor, renin-angiotensin-aldosterone system
(RAAS), ecdysteroid, G protein-coupled receptor (GPCR), estrogen, muscle, myoblast
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.
17-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.
INTRODUCTION
Steroids in animal and plant kingdoms
Steroid hormones are (chole)sterol
derivatives widespread in animals and plants,
where they are involved in the control of plenty
of physiological processes. They include for
example vertebrate sex hormones
(progestagens, estrogens, androgens), insect
moulting hormones (ecdysteroids), as well as
plant growth hormones (brassinosteroids). This
means that the rigid carbon skeleton of sterols
is particularly suitable to generate a very large
number of derivatives, which differ by the
carbon number and/or the position of various
substituents (mainly hydroxyl or keto groups)
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted April 10, 2020. ; https://doi.org/10.1101/2020.04.08.032607doi: bioRxiv preprint

2
(1). In addition to hormones, sterols give rise to
bile acids/alcohols, initially considered as
emulsifiers facilitating lipid digestion, but
nowadays also known as important signalling
molecules acting on specific receptors (2).
Diversity of steroid mechanisms of action
Our concepts on (steroid) hormone
mechanism of action has evolved. In the
classical scheme, steroid hormones interact
with nuclear receptors and the complex formed
regulates the transcriptional activity of target
genes, which promoters contain specific
sequences (hormone-responsive elements). But
steroids also act at cell membrane level where
they elicit rapid non-transcriptional effects.
Among the identified steroid membrane
receptors, we may mention vertebrate
GPER1/GPR30 (a membrane estrogen receptor
(3), TGR5 (a bile acid membrane receptor
(4), MARRS (a calcitriol receptor (5) or
drosophila DopEcR (a dopamine and ecdysone
membrane receptor (6). All these receptors
belong to the family of GPCR/7TD receptors.
Moreover, intact or truncated forms or the
steroid nuclear receptors are bound to the
plasma membrane and do not act there as
transcription factors(7,8).
Ecdysteroid effects on vertebrates
We are especially interested by the
pharmacological effects of ecdysteroids on
mammals. Ecdysteroids are a large family of
steroids initially discovered in arthropods
(zooecdysteroids) and later in plants
(phytoecdysteroids) (9). They are present in
many plant species where they can reach
concentrations of up to 2-3 % of the plant dry
weight, and they are expected to protect plants
against phytophagous insects.
With the aim to use these molecules for
crop protection, toxicological studies were
performed on mammals, which unexpectedly
concluded to both their lack of toxicity (oral
LD
50
> 9 g/kg) and their “beneficial” effects,
e.g. anti-diabetic and anabolic properties (10).
Such effects have to be linked with the presence
of large amounts of phytoecdysteroids in
several plants used worldwide by traditional
medicine. At the moment, numerous effects
have been reported, allowing to consider
ecdysteroids as some kind of “universal
remedy”(11). While many effects have been
described on animals (10,12,13), the clinical
evidence for 20E effectiveness in
humans remains limited at the moment
(14-16).
How do ecdysteroids work?
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. 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).
There is in fact no direct evidence for
the binding of 20E to nuclear estrogen (or
androgen) receptors (12,13). The evidence of
ERβ involvement in 20E effect is based on the
use of specific pharmacological activators or
inhibitors of ERs, the former being able to
mimic and the latter to inhibit the effects of
20E on target cells such as osteoblasts (20) or
myoblasts (19). These studies however do not
bring proofs for a direct 20E binding, as ER
receptor could be activated indirectly, and
even in the absence of ligand, e.g. by
phosphorylation (21). Some evidence for a
direct binding was provided by in silico
modelling (22), but this certainely does not
represent a definite proof, as the result may
strongly depend on the model used, and an
opposite conclusion was drawn by Lapenna et
al. (23).
Evidence for membrane effects of 20E
is based on early studies showing the
rapid modulation of several second
messengers (cAMP, cGMP, IP3, DAG, Ca
2+
)
in target cells (24-26) and on the fact that
20E bound to metallic nanoparticles,
preventing its entrance in target cells, is still
active (27). More recently, Gorelick-Feldman
et al. (17) used a pharmacological
approach with various inhibitors (e.g.
pertussis toxin). 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).
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted April 10, 2020. ; https://doi.org/10.1101/2020.04.08.032607doi: bioRxiv preprint

3
The above pharmacological arguments
appear strong enough to consider that the cell
membrane is (maybe not exclusively) a site of
action of 20E. The present experiments have
been undertaken in an attempt to identify
the/one GPCR involved in 20E effects, and to
understand its estrogen-like effects using gene
silencing or different pharmacological
approaches.
MATERIALS AND METHODS
Chemicals
Except otherwise mentioned, all the reagents
and chemicals were from Sigma (Saint-Quentin
Fallavier, France). Peptides such as
angiotensin-(1-7), A779 (Asp-Arg-Val-Tyr-Ile-
His-D-Ala) and A1 (Asp-Arg-Val-Tyr-Ile-His-
D-Pro) were custom-prepared by the IBPS
peptide synthesis platform (Sorbonne
University, Paris, France). 20-
Hydroxyecdysone (20E) was obtained from
Chemieliva Pharmaceutical (Chongqing,
China) or from Patheon (Regensburg,
Germany) and had a purity of 96.5-97%.
Preparation of HSA-conjugated 20-
hydroxyecdysone
22-succinyl-20E was prepared according to
Dinan et al.(28). Coupling the 20E derivative to
human serum albumin (HSA) was performed
according to a method provided by Dr J-P
Delbecque (JP Delbecque and M. de Reggi,
personal communication). The 20E-HSA
conjugate (Supporting Fig. S2) was analyzed
by mass spectrometry to determine the number
of 20E molecules coupled to each HSA
molecule. Analyses were performed in a 4700
MALDI TOF/TOF proteomics analyzer
(Applied Biosystems). The 20E conjugate was
studied in linear mode, positive ion mode. Laser
acceleration is set at 20kV and the default laser
fluency was set at 2,000 and modified according
to the signal-to-noise quality. The matrix used
was α-cyano-4-hydrocinnamic Acid (HCCA).
Samples were prepared following the dried-
droplet method, 1 µL of a mixture of 1 µL of
matrix (10 mg/mL) and 1 µL of sample was
spotted and dried with gaseous nitrogen. The
mass shift of HSA around 5 kDa after coupling
indicates that 9 molecules of 20E derivatives are
coupled to each albumin molecule.
Cell culture
The C2C12 mouse myoblast cell line (29) was
purchased from ATCC (CRL-1772). Except
otherwise mentioned, culture media, serum,
antibiotics and supplements were from Life
technologies (Villebon-sur-Yvette, France). All
cultures contained 100 U/mL of penicillin, and
100 μg/mL of streptomycin and are maintained
in a 5% CO
2
, 95% air humidified atmosphere at
37°C. For C2C12 proliferation, cells were
maintained in DMEM medium containing
4.5 g/L glucose supplemented with 10% FBS.
C2C12 cells were maintained at low passage (3-
20 passages) for all experiments to maintain the
differentiation potential of the cultures. Cell
confluency was always kept below or equal to
~80%. For all experiments, cells were first
seeded at 30,000 cells per well in 24-well plates.
To induce differentiation, C2C12 at ~80%
confluency in proliferation medium were
shifted to DMEM medium supplemented with
either 2% FBS or 2% horse serum.
Protein synthesis (
3
H-leucine incorporation)
C2C12 cells were grown on 24-well
plates at a density of 30,000 cells/well in 0.5 mL
of growth medium (DMEM 4.5 g/L glucose
supplemented with 10% fetal bovine serum).
Twenty-four hours after plating, the
differentiation induction into multinucleated
myotubes was carried out in DMEM 4.5 g/L
glucose containing 2% fetal bovine serum.
After 5 days, cells were pre-incubated in Krebs
medium 1 h at 37°C before being incubated in
DMEM media without serum for 2.5 h in the
presence of radiolabeled leucine (5 µCi/mL)
and DMSO (control condition) or Insulin
Growth Factor (IGF-1, 100 ng/mL) or 20E (0.1
0.5 1 5 -10 µM). At the end of incubation,
supernatants were discarded and cells were
lysed in 0.1 N NaOH for 30 min. The cell
soluble fraction-associated radioactivity was
then counted using Wallac Microbeta 1450-021
TriLux Luminometer Liquid Scintillation
Counter (Wallac EG&G, Gaithersburg, MD,
USA) and protein quantification was performed
using the colorimetric Lowry method.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted April 10, 2020. ; https://doi.org/10.1101/2020.04.08.032607doi: bioRxiv preprint

4
Myostatin and MAS gene expression assays
Cells were plated at a density of 30,000
cells per well in 24-well plates and were grown
overnight in 5 % CO
2
at 37°C. On day 5 of
differentiation, treatments were carried out for
6 h. At the end of incubation, RNAs were
extracted and purified using the RNAzol
(Eurobio, Les Ulis, France). RNAs were
converted into cDNAs with High-Capacity
cDNA Reverse Transcription Kit (Applied
Biosystems, ThermoFisher, Villebon-sur-
Yvette, France) before performing a
quantitative PCR using iTaq SybrGreen
(Biorad, Marnes-la Coquette, France).
Q-RT-PCRs were then performed using a
7900HT Fast Real-Time PCR detection system
(Applied Biosystems) and standard qPCR
program (1 cycle 95°C 15 min followed by 40
cycles 95°C 15s and 60°C 1 min). QRT-PCR
master mix contained the 100 ng cDNA
samples and a set of primers at final
concentration of 200 nM designed into two
different exons and described below. The
quality of RNA was checked using the
nanodrop™ technology (ThermoFisher) when
necessary.
The relative differences in gene expression
levels between treatments were expressed as
increases or decreases in cycle time [Ct]
numbers compared to the control group where
the [Ct] value of each gene was normalized to
the beta actin gene or hypoxanthine guanine
phosphoribosyl transferase (HPRT) gene.
Results of gene expression were expressed in 2
-
∆∆CT
after normalization with house-keeping
genes. Primer sequences used are described in
Table 1.
SiRNA MAS assay
Cells were plated at a density of 10,000
cells per well in 24-well plates. After 3 days of
differentiation, cells were transfected either
with scramble SiRNA (10 nM) or MAS-1
SiRNA (10 nM) according to manufacturer’s
instructions (Origene Technologies, Rockville,
MD, USA). Two days after transfection,
myotubes were treated with either DMSO or
IGF-1 or 20E or angiotensin-(1-7) for 6 h. At
the end of incubation, RNA was extracted and
analyzed by QRT-PCR as described above.
Binding studies
The affinity of 20E for human nuclear
steroid receptors such as androgen receptor
(AR), estrogen receptors alpha and beta (ERα,
ERβ) and glucocorticoid receptor (GR) was
determined by radioligand binding assays
(CEREP/Eurofins). The selective ligands [
3
H]-
methyltrienolone, [
3
H]-estradiol, [
3
H]-
dexamethasone were employed on cells
expressing either human endogenous or
recombinant AR, ERα or β, or GR, respectively.
20E was used at concentrations up to 100 μM as
a potential competitor. Inhibition of control
specific binding was determined, and IC
50
and
K
i
were calculated when possible. Additionally,
a receptor screen was carried out on 45 GPCR
and 5 nuclear receptors at a fixed concentration
of 20E (10 μM). Radioligand binding assays
were performed according to manufacturer
instructions employing
3
H- or
125
I-labelled
specific ligands of each receptor
(SafetyScreen87 Panel, Panlabs, Taipei,
Taiwan).
Statistical analyses
Statistical analysis was performed
using Graph Pad Prism® Software. Anova
followed by a Dunnett t-test or a Kruskal Wallis
followed by a Dunn’s test when the variances
significantly differed have been performed. To
evaluate the significance of differences between
two groups, the choice of parametric Student t-
test or non-parametric Mann-Whitney test was
based on the normality or non-normality of data
distribution, respectively (D’Agostino &
Pearson test). The results are considered
significant at p-value <0.05 (*), <0.01(**),
<0.001 (***).
RESULTS
20-Hydroxyecdysone stimulates muscle
anabolism
20-Hydroxyecdysone (20E) effects were
investigated on pre-established murine
myotubes (following 6 days of differentiation).
An anabolic effect was investigated through de
novo protein synthesis assay. A dose-dependent
increase in protein synthesis was observed in
response to 20E treatment of C2C12 myotubes
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted April 10, 2020. ; https://doi.org/10.1101/2020.04.08.032607doi: bioRxiv preprint

5
versus untreated conditions (Fig. 1A). IGF-1
(100 ng/mL), employed as positive control (30)
displayed, as expected, an improvement in [
3
H]-
Leu incorporation (+20%, p < 0.001). 20E
effect was significant from 0.5 μM to 5 μM. The
maximal effect (+ 27 %; p < 0.001) was
measured with 5 μM of 20E, while a treatment
with a higher concentration of 20E (10 μM)
appeared to be notably less efficient (+11%, ns)
than the previous concentration tested.
Myostatin is a major autocrine regulator that
inhibits muscle growth in mammals. The
myostatin transcript bioassay was developed
and standardized in order to assess ecdysteroid
activity (31). IGF-1 (100 ng/mL) used as a
positive control demonstrated a significant
inhibition of myostatin gene expression (57% of
untreated control cells, p<0.001, Fig. 1B). A
dose-dependent and partial inhibition of
myostatin gene expression was observed in
response to 20E treatment at concentrations
comprised between 0.001 and 10 µM. This
inhibition reached significance from 0.5 µM
20E (Fig. 1B). The inhibition of myostatin gene
expression was then employed as a readout for
20E activity.
20-Hydroxyecdysone acts on cell membranes
In order to confirm that 20E acts primarily on
cell membrane, or if it needs to penetrate into
the cell to exert its effects, a membrane-
impermeable derivative of 20E was produced
(Supporting Fig. S2). We compared the effects
of free 20E and its 22S-HSA conjugate on
myostatin gene expression at the concentration
of 10 µM 20E-equivalents (Fig. 2). We
observed that the membrane-impermeable 20E-
derivative retained an activity similar to that of
free 20E. 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).
20-Hydroxyecdysone acts via a GPCR-type
receptor
The GPCR hypothesis is based on the inhibition
of 20E effects by pertussis toxin, but there are
still numerous possible GPCR candidates. We
selected a set of GPCR receptors based on
available literature and using different criteria
corresponding to well-established effects of
20E: (i) involvement in the control of muscle
cells activity and glycaemia/insulin sensitivity,
and (ii) ability to reduce fat mass gain in high-
fat fed animals (32,33). A set of GPCRs was thus
selected including TGR5 (bile acids receptor -
(4), GPER/GPR30 (estradiol, aldosterone
receptors -(3,6), LPA1 (lysophosphatidic acids
receptor -(34), APJ (apelin receptor -(35),
OXTR (oxytocin receptor -(36), AVPR1
(vasopressin receptor -(37), MrgD (alamandine
receptor - (38), MARRS (vitamin D3 receptor -
(5) and Mas (angiotensin-(1-7) receptor -(39).
The possible interaction of 20E with those
receptors was assayed using different
approaches according to available
methodologies: (1) in silico binding when 3D
structures were available, (2) in vitro direct
binding studies by competition with a
radioactive natural ligand, (3) comparison of the
effects of agonists with those of 20E on C2C12
cells or (4) effect of known antagonists on the
response of C2C12 cells to 20E. Using this
approach, the only receptor which gave positive
data was Mas, the receptor of angiotensin-(1-7).
20-Hydroxyecdysone and Angiotensin-(1-7)
act via Mas receptor activation
Using the myostatin gene expression assay, a
pharmacological approach was emloyed to
compare the effects of the endogenous Mas
receptor agonist (angiotensin-(1-7)) with those
of 20E on C2C12 cells in the presence and
absence of known antagonists.
Angiotensin-(1-7) (Ang 1-7, Supporting Fig.
S3) as well as 20E (Fig. 1B) partially inhibits
myostatin gene expression in a dose-dependent
manner. 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).
Interestingly, 20E inhibitory effects on
myostatin gene expression was also fully
reverted by the same antagonists (Fig. 3A)
suggesting that inhibition of myostatin gene
expression by 20E (or Ang 1-7) is mediated by
the receptor of Ang 1-7. In contrast, the effect
of IGF-1, which acts through its own receptor
(insulin-like growth factor 1 receptor; IGF1R)
remains unchanged in the presence or in the
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted April 10, 2020. ; https://doi.org/10.1101/2020.04.08.032607doi: bioRxiv preprint

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Journal ArticleDOI
Abstract: COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has reached pandemic proportions with negative impacts on global health, the world economy and human society. The clinical picture of COVID-19, and the fact that Angiotensin converting enzyme 2 (ACE2) is a receptor of SARS-CoV-2, suggests that SARS-CoV-2 infection induces an imbalance in the renin-angiotensin system (RAS). We review clinical strategies that are attempting to rebalance the RAS in COVID-19 patients by using ACE inhibitors, angiotensin receptor blockers, or agonists of angiotensin-II receptor type 2 or Mas receptor (MasR). We also propose that the new MasR activator BIO101, a pharmaceutical grade formulation of 20-hydroxyecdysone that has anti-inflammatory, anti-fibrotic and cardioprotective properties, could restore RAS balance and improve the health of COVID-19 patients who have severe pneumonia.

6 citations


Journal ArticleDOI
Abstract: The current Severe Acute Respiratory Syndrome disease caused by Coronavirus-2 (SARS-CoV-2) has been a serious strain on the healthcare infrastructure mainly due to the lack of a reliable treatment option. Alternate therapies aimed at symptomatic relief are currently prescribed along with artificial ventilation to relieve distress. Traditional medicine in the form of Ayurveda has been used since ancient times as a holistic treatment option rather than targeted therapy. The practice of Ayurveda has several potent herbal alternatives for chronic cough, inflammation, and respiratory distress which are often seen in the SARS-CoV-2 infection. In this study we have used the aqueous extracts of Tinospora cordifolia (willd.) Hook. f. and Thomson in the form of Giloy Ghanvati, as a means of treatment to the SARS-CoV-2 spike-protein induced disease phenotype in a humanized zebrafish model. The introduction of spike-protein in the swim bladder transplanted with human lung epithelial cells (A549), caused an infiltration of pro-inflammatory immune cells such as granulocytes and macrophages into the swim bladder. There was also an increased systemic damage as exemplified by renal tissue damage and increased behavioral fever in the disease induction group. These features were reversed in the treatment group, fed with three different dosages of Giloy Ghanvati. The resultant changes in the disease phenotype were comparable to the group that were given the reference compound, Dexamethasone. These findings correlated well with various phyto-compounds detected in the Giloy Ghanvati and their reported roles in the viral disease phenotype amelioration.

5 citations


Journal Article
Abstract: It is established that ecdysterone (20-hydroxyecdysone) in concentration of 10(-8) M and calcitriol (1 alpha, 25-dihydroxyvitamin D3) in concentration 10(-12) M evoke in the studied tissues-targets of rats (brain, heart, liver, intestine mucosa) rapid (0.5-2 min) transit (2.5-4 times) increase of the intracellular pools of arachidonic acid, eicosanoid--total peptidoleukotrienes C4/D4/E4 (2.5-6 times), and, especially prostaglandins--stable metabolite prostacyclin 6-ketoprostaglandin F1 alpha (10-400 times). Ecdysterone in concentration 10(-11)-10(-9) inhibits arachidonic cascade in the brain and heart. Activation of arachidonic cascade and further increase of biosynthesis of cGMP are considered as a single negative link of autoregulation of activity of phospholipid-dependent signal system of cells and ecdysterone as a possible efficient modulator intracellular pools of eicosanoid in the tissues of warm-blooded animals.

4 citations


Journal ArticleDOI
Abstract: There is growing interest in the pharmaceutical and medical applications of 20-hydroxyecdysone (20E), a polyhydroxylated steroid which naturally occurs in low but very significant amounts in invertebrates, where it has hormonal roles, and in certain plant species, where it is believed to contribute to the deterrence of invertebrate predators. Studies in vivo and in vitro have revealed beneficial effects in mammals: anabolic, hypolipidemic, anti-diabetic, anti-inflammatory, hepatoprotective, etc. The possible mode of action in mammals has been determined recently, with the main mechanism involving the activation of the Mas1 receptor, a key component of the renin-angiotensin system, which would explain many of the pleiotropic effects observed in the different animal models. Processes have been developed to produce large amounts of pharmaceutical grade 20E, and regulatory preclinical studies have assessed its lack of toxicity. The effects of 20E have been evaluated in early stage clinical trials in healthy volunteers and in patients for the treatment of neuromuscular, cardio-metabolic or respiratory diseases. The prospects and limitations of developing 20E as a drug are discussed, including the requirement for a better evaluation of its safety and pharmacological profile and for developing a production process compliant with pharmaceutical standards.

2 citations


Journal ArticleDOI
Abstract: Since December 2019, coronavirus disease 2019 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has changed our lives. Elderly, and those with comorbidities represent the vast majority of patients hospitalized with severe COVID-19 symptoms, including acute respiratory disease syndrome, and cardiac dysfunction. Despite a huge effort of the scientific community, improved treatment modalities limiting the severity and mortality of hospitalized COVID-19 patients, are still required. Here, we compare the effectiveness of virus- and patients-centred strategies to reduce COVID-19 mortality. We also discuss the therapeutic options that might further reduce death rates associated with the disease in the future. Unexpectedly, extensive review of the literature suggests that SARS-CoV-2 viral load seems to be associated neither with the severity of symptoms nor with mortality of hospitalized patients with COVID-19. This may explain why, so far, virus-centred strategies using antivirals aiming to inhibit the viral replicative machinery, have failed to reduce COVID-19 mortality in patients with respiratory failure. By contrast, anti-inflammatory treatments without antiviral capacities but centred on patients, such as dexamethasone or Tocilizumab®, reduce COVID-19 mortality. Finally, since the spike protein of SARS-CoV-2 binds to Angiotensin Converting Enzyme 2 (ACE2) and inhibits its function, we explore the different treatment options focussing on rebalancing the Renin-Angiotensin System (RAS). This new therapeutic strategy could hopefully further reduce the severity of respiratory failure and limit COVID-19 mortality in elderly patients.

1 citations


References
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Journal ArticleDOI
01 Dec 1977-Nature
TL;DR: The present report describes the isolation of a cloned population of myogenic cells, derived from adult dystrophic mouse muscle, that can proliferate and differentiate in cell culture.
Abstract: THE muscular dystrophies are a group of hereditary disorders manifested by a progressive wasting of the skeletal muscles. In spite of extensive studies, the nature of the primary lesion is unknown (for review see ref. 1). Because of the complex interaction between tissues, it is difficult to study this question in vivo. Therefore attempts have been made to investigate this question in cultures of dystrophic muscles of human or animal origin. Tissue explants as well as monolayer primary cell cultures contain, in addition to the myogenic cells, a heterogeneous cell population, the composition of which might differ in normal and dystrophic muscle cultures. It is difficult in such experiments to distinguish between properties intrinsic to the myogenic cells and effects exerted by other cell types. Indeed, previous experiments have yielded conflicting conclusions2–6. We therefore tested the possibility of obtaining cell cultures consisting of pure populations of myogenic cells obtained from dystrophic muscles. The present report describes the isolation of a cloned population of such cells, derived from adult dystrophic mouse muscle, that can proliferate and differentiate in cell culture.

1,948 citations


"20-Hydroxyecdysone activates the pr..." refers methods in this paper

  • ...Cell culture The C2C12 mouse myoblast cell line (29) was purchased from ATCC (CRL-1772)....

    [...]


Journal ArticleDOI
TL;DR: It is shown here that TGR5 signaling induces intestinal glucagon-like peptide-1 (GLP-1) release, leading to improved liver and pancreatic function and enhanced glucose tolerance in obese mice, and suggested that pharmacological targeting of T GR5 may constitute a promising incretin-based strategy for the treatment of diabesity and associated metabolic disorders.
Abstract: TGR5 is a G protein-coupled receptor expressed in brown adipose tissue and muscle, where its activation by bile acids triggers an increase in energy expenditure and attenuates diet-induced obesity. Using a combination of pharmacological and genetic gain- and loss-of-function studies in vivo, we show here that TGR5 signaling induces intestinal glucagon-like peptide-1 (GLP-1) release, leading to improved liver and pancreatic function and enhanced glucose tolerance in obese mice. In addition, we show that the induction of GLP-1 release in enteroendocrine cells by 6alpha-ethyl-23(S)-methyl-cholic acid (EMCA, INT-777), a specific TGR5 agonist, is linked to an increase of the intracellular ATP/ADP ratio and a subsequent rise in intracellular calcium mobilization. Altogether, these data show that the TGR5 signaling pathway is critical in regulating intestinal GLP-1 secretion in vivo, and suggest that pharmacological targeting of TGR5 may constitute a promising incretin-based strategy for the treatment of diabesity and associated metabolic disorders.

1,223 citations


"20-Hydroxyecdysone activates the pr..." refers background or methods in this paper

  • ...20E-like effects were also observed with Angiotensin-(1-7), the endogenous ligand of Mas....

    [...]

  • ...myostatin gene expression, but proteinbound E2 was inactive, and E2 activity was not abolished by angiotensin-(1-7)...

    [...]

  • ...Angiotensin-(1-7) (Ang 1-7, Supporting Fig....

    [...]

  • ...A set of GPCRs was thus selected including TGR5 (bile acids receptor (4), GPER/GPR30 (estradiol, aldosterone receptors -(3,6), LPA1 (lysophosphatidic acids receptor -(34), APJ (apelin receptor -(35), OXTR (oxytocin receptor -(36), AVPR1 (vasopressin receptor -(37), MrgD (alamandine receptor - (38), MARRS (vitamin D3 receptor (5) and Mas (angiotensin-(1-7) receptor -(39)....

    [...]

  • ...Two days after transfection, myotubes were treated with either DMSO or IGF-1 or 20E or angiotensin-(1-7) for 6 h....

    [...]


Journal ArticleDOI
TL;DR: Evidence is provided that estrogen-induced Erk-1/-2 activation occurs independently of known estrogen receptors, but requires the expression of the G protein-coupled receptor homolog, GPR30.
Abstract: Estrogen rapidly activates the mitogen-activated protein kinases, Erk-1 and Erk-2, via an as yet unknown mechanism. Here, evidence is provided that estrogen-induced Erk-1/-2 activation occurs independently of known estrogen receptors, but requires the expression of the G protein-coupled receptor homolog, GPR30. We show that 17beta-estradiol activates Erk-1/-2 not only in MCF-7 cells, which express both estrogen receptor alpha (ER alpha) and ER beta, but also in SKBR3 breast cancer cells, which fail to express either receptor. Immunoblot analysis using GPR30 peptide antibodies showed that this estrogen response was associated with the presence of GPR30 protein in these cells. MDA-MB-231 breast cancer cells (ER alpha-, ER beta+) are GPR30 deficient and insensitive to Erk-1/-2 activation by 17beta-estradiol. Transfection of MDA-MB-231 cells with a GPR30 complementary DNA resulted in overexpression of GPR30 protein and conversion to an estrogen-responsive phenotype. In addition, GPR30-dependent Erk-1/-2 activation was triggered by ER antagonists, including ICI 182,780, yet not by 17alpha-estradiol or progesterone. Consistent with acting through a G protein-coupled receptor, estradiol signaling to Erk-1/-2 occurred via a Gbetagamma-dependent, pertussis toxin-sensitive pathway that required Src-related tyrosine kinase activity and tyrosine phosphorylation of tyrosine 317 of the Shc adapter protein. Reinforcing this idea, estradiol signaling to Erk-1/-2 was dependent upon trans-activation of the epidermal growth factor (EGF) receptor via release of heparan-bound EGF (HB-EGF). Estradiol signaling to Erk-1/-2 could be blocked by: 1) inhibiting EGF-receptor tyrosine kinase activity, 2) neutralizing HB-EGF with antibodies, or 3) down-modulating HB-EGF from the cell surface with the diphtheria toxin mutant, CRM-197. Our data imply that ER-negative breast tumors that continue to express GPR30 may use estrogen to drive growth factor-dependent cellular responses.

1,210 citations


"20-Hydroxyecdysone activates the pr..." refers background in this paper

  • ...Five different E2 receptors have been described (not including splice variants) including 2 GPCRs(44): GPR30(45), and Gq-mER (46) – plus possibly another plasma-membrane associated receptor, ER-X(47)....

    [...]

  • ...Five different E2 receptors have been described (not including splice variants) including 2 GPCRs(44): GPR30(45), and GqmER (46) – plus possibly another plasmamembrane associated receptor, ER-X(47)....

    [...]

  • ...A set of GPCRs was thus selected including TGR5 (bile acids receptor - (4), GPER/GPR30 (estradiol, aldosterone receptors -(3,6), LPA1 (lysophosphatidic acids receptor -(34), APJ (apelin receptor -(35), OXTR (oxytocin receptor -(36), AVPR1 (vasopressin receptor -(37), MrgD (alamandine receptor - (38), MARRS (vitamin D3 receptor - (5) and Mas (angiotensin-(1-7) receptor -(39)....

    [...]

  • ...Our experiments with GPR30 agonists and antagonists exclude GPR30 from the candidates....

    [...]

  • ...Ruhs et al., (54) described an association between aldosterone receptor (MR) and GPER/GPR30: accordingly, some effects of aldosterone are inhibited by G15, a GPER inhibitor (55) and, most interestingly, they also showed an association between MR and the angiotensin II receptor AT1....

    [...]


OtherDOI
TL;DR: Enterohepatic circulation of bile acids from the liver to intestine and back to the liver plays a central role in nutrient absorption and distribution, and metabolic regulation and homeostasis.
Abstract: Bile acids are important physiological agents for intestinal nutrient absorption and biliary secretion of lipids, toxic metabolites, and xenobiotics. Bile acids also are signaling molecules and metabolic regulators that activate nuclear receptors and G protein-coupled receptor (GPCR) signaling to regulate hepatic lipid, glucose, and energy homeostasis and maintain metabolic homeostasis. Conversion of cholesterol to bile acids is critical for maintaining cholesterol homeostasis and preventing accumulation of cholesterol, triglycerides, and toxic metabolites, and injury in the liver and other organs. Enterohepatic circulation of bile acids from the liver to intestine and back to the liver plays a central role in nutrient absorption and distribution, and metabolic regulation and homeostasis. This physiological process is regulated by a complex membrane transport system in the liver and intestine regulated by nuclear receptors. Toxic bile acids may cause inflammation, apoptosis, and cell death. On the other hand, bile acid-activated nuclear and GPCR signaling protects against inflammation in liver, intestine, and macrophages. Disorders in bile acid metabolism cause cholestatic liver diseases, dyslipidemia, fatty liver diseases, cardiovascular diseases, and diabetes. Bile acids, bile acid derivatives, and bile acid sequestrants are therapeutic agents for treating chronic liver diseases, obesity, and diabetes in humans.

609 citations


"20-Hydroxyecdysone activates the pr..." refers background or methods in this paper

  • ...20E-like effects were also observed with Angiotensin-(1-7), the endogenous ligand of Mas....

    [...]

  • ...myostatin gene expression, but proteinbound E2 was inactive, and E2 activity was not abolished by angiotensin-(1-7)...

    [...]

  • ...Angiotensin-(1-7) (Ang 1-7, Supporting Fig....

    [...]

  • ...In addition to hormones, sterols give rise to bile acids/alcohols, initially considered as emulsifiers facilitating lipid digestion, but nowadays also known as important signalling molecules acting on specific receptors (2)....

    [...]

  • ...A set of GPCRs was thus selected including TGR5 (bile acids receptor (4), GPER/GPR30 (estradiol, aldosterone receptors -(3,6), LPA1 (lysophosphatidic acids receptor -(34), APJ (apelin receptor -(35), OXTR (oxytocin receptor -(36), AVPR1 (vasopressin receptor -(37), MrgD (alamandine receptor - (38), MARRS (vitamin D3 receptor (5) and Mas (angiotensin-(1-7) receptor -(39)....

    [...]


Journal ArticleDOI
TL;DR: An overview of the evidence for the cellular and physiological actions of GPR30 in estrogen-dependent processes and the relationship of G PR30 with classical estrogen receptors is provided.
Abstract: Steroids play an important role in the regulation of normal physiology and the treatment of disease. Steroid receptors have classically been described as ligand-activated transcription factors mediating long-term genomic effects in hormonally regulated tissues. It is now clear that steroids also mediate rapid signaling events traditionally associated with growth factor receptors and G protein–coupled receptors. Although evidence suggests that the classical steroid receptors are capable of mediating many of these events, more recent discoveries reveal the existence of transmembrane receptors capable of responding to steroids with cellular activation. One such receptor, GPR30, is a member of the G protein–coupled receptor superfamily and mediates estrogen-dependent kinase activation as well as transcriptional responses. In this review, we provide an overview of the evidence for the cellular and physiological actions of GPR30 in estrogen-dependent processes and discuss the relationship of GPR30 with classica...

544 citations


"20-Hydroxyecdysone activates the pr..." refers background or methods in this paper

  • ...20E-like effects were also observed with Angiotensin-(1-7), the endogenous ligand of Mas....

    [...]

  • ...myostatin gene expression, but proteinbound E2 was inactive, and E2 activity was not abolished by angiotensin-(1-7)...

    [...]

  • ...Angiotensin-(1-7) (Ang 1-7, Supporting Fig....

    [...]

  • ...A set of GPCRs was thus selected including TGR5 (bile acids receptor (4), GPER/GPR30 (estradiol, aldosterone receptors -(3,6), LPA1 (lysophosphatidic acids receptor -(34), APJ (apelin receptor -(35), OXTR (oxytocin receptor -(36), AVPR1 (vasopressin receptor -(37), MrgD (alamandine receptor - (38), MARRS (vitamin D3 receptor (5) and Mas (angiotensin-(1-7) receptor -(39)....

    [...]

  • ...Two days after transfection, myotubes were treated with either DMSO or IGF-1 or 20E or angiotensin-(1-7) for 6 h....

    [...]


Frequently Asked Questions (1)
Q1. What have the authors contributed in "20-hydroxyecdysone activates the protective arm of the renin angiotensin system via mas receptor" ?

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. This means that the rigid carbon skeleton of sterols is particularly suitable to generate a very large number of derivatives, which differ by the carbon number and/or the position of various substituents ( mainly hydroxyl or keto groups ) ( which was not certified by peer review ) is the author/funder.