U.S. patent application number 16/922432 was filed with the patent office on 2020-11-05 for muscular atrophy-inducing agent using hypometabolism-inducing substance t1am, and use thereof in treating muscular hypertrophy.
The applicant listed for this patent is UNIVERSITY INDUSTRY FOUNDATION, YONSEI UNIVERSITY WONJU CAMPUS. Invention is credited to Inho CHOI, Hyun Woo JU, Kyoungsook PARK.
Application Number | 20200345662 16/922432 |
Document ID | / |
Family ID | 1000004957499 |
Filed Date | 2020-11-05 |
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United States Patent
Application |
20200345662 |
Kind Code |
A1 |
CHOI; Inho ; et al. |
November 5, 2020 |
MUSCULAR ATROPHY-INDUCING AGENT USING HYPOMETABOLISM-INDUCING
SUBSTANCE T1AM, AND USE THEREOF IN TREATING MUSCULAR
HYPERTROPHY
Abstract
A screening method for a drug for treating muscular atrophy is
proposed. The method may include the steps of: (a) inducing
muscular atrophy by administering to a normal cell or a normal
animal a hypometabolism-inducing substance selected from the group
consisting of 3-iodothyronamine (T1AM), [D-Ala2, D-Leu5] enkephalin
(DADLE), 5'-adenosine monophosphate (5'-AMP), and hydrogen sulfide
(H2S); (b) treating a candidate substance in the cell or animal
treated with the hypometabolism-inducing substance; (c) evaluating
the degree of improvement or treatment of muscular atrophy in the
cell or animal treated with the candidate substance; and (d)
determining the candidate substance as a drug for treating muscular
atrophy.
Inventors: |
CHOI; Inho; (Wonju-si,
KR) ; PARK; Kyoungsook; (Seoul, KR) ; JU; Hyun
Woo; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY INDUSTRY FOUNDATION, YONSEI UNIVERSITY WONJU
CAMPUS |
Wonju-si |
|
KR |
|
|
Family ID: |
1000004957499 |
Appl. No.: |
16/922432 |
Filed: |
July 7, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15576231 |
Nov 21, 2017 |
|
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PCT/KR2016/006955 |
Jun 29, 2016 |
|
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16922432 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 33/04 20130101;
A61K 38/08 20130101; G01N 33/5061 20130101; A61K 31/7076 20130101;
A61P 21/00 20180101; C12N 15/01 20130101; A61K 31/135 20130101 |
International
Class: |
A61K 31/135 20060101
A61K031/135; A61K 31/7076 20060101 A61K031/7076; A61K 33/04
20060101 A61K033/04; A61P 21/00 20060101 A61P021/00; C12N 15/01
20060101 C12N015/01; G01N 33/50 20060101 G01N033/50; A61K 38/08
20060101 A61K038/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 29, 2015 |
KR |
10-2015-0091807 |
Jun 2, 2016 |
KR |
10-2016-0068882 |
Claims
1. A screening method for a drug for treating muscular atrophy,
comprising: (a) inducing muscular atrophy by administering to a
normal cell or a normal animal a hypometabolism-inducing substance
selected from the group consisting of 3-iodothyronamine (T1AM),
[D-Ala2, D-Leu5] enkephalin (DADLE), 5'-adenosine monophosphate
(5'-AMP), and hydrogen sulfide (H.sub.2S); (b) treating a candidate
substance in the cell or animal treated with the
hypometabolism-inducing substance; (c) evaluating the degree of
improvement or treatment of muscular atrophy in the cell or animal
treated with the candidate substance; and (d) determining the
candidate substance as a drug for treating muscular atrophy.
2. The screening method of claim 1, wherein in step (d), as
compared to a control treated with DMSO, physiological saline,
sterilized distilled water, carboxymethyl cellulose or phosphate
buffered saline (PBS), when the group treated with the candidate
substance has at least one result selected from the group
consisting of: an increase in size of myotubes or size of muscle; a
decrease in expression ratio of p-AMPK/AMPK; an increase in
expression ratio of p-Akt1/Akt1; an increase in expression ratio of
p-S6K/S6K; an increase in expression ratio of p-FoxO1/FoxO1; an
increase in expression ratio of p-FoxO3/FoxO3; a decrease in
expression level of MuRF1; a decrease in activity of proteasome; an
increase in expression level of heat shock protein 72 (HSP72); and
an increase in expression level of .alpha.B-crystallin, the
candidate substance is determined as the drug for treating muscular
atrophy.
3. The screening method of claim 1, wherein the
hypometabolism-inducing substance in step(a) is 3-iodothyronamine
(T1AM).
4. The screening method of claim 3, wherein a dose of the
3-iodothyronamine (T1AM) is 0.1 .mu.M to 1000 .mu.M in the case of
the cell, and 10 to 500 mg/kg per unit weigh (kg) of the animal in
the case of the animal.
5. The screening method of claim 1, wherein the administering of
the hypometabolism-inducing substance is performed by a method
selected from the group consisting of oral administration,
intraperitoneal administration, intravenous administration,
intramuscular administration, subcutaneous administration, and
intradermal administration.
6. The screening method of claim 1, wherein the cell is a muscle
cell and, the animal is a vertebrate.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 15/576,231, filed Nov. 21, 2017, which is a national stage
application of PCT/KR2016/006955, filed Jun. 29, 2016, which claims
priority to KR 10-2015-0091807, filed Jun. 29, 2015 and KR
10-2016-0068882, filed Jun. 2, 2016, the entire disclosures of
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Technical Field
[0002] The present invention relates to a muscular atrophy-inducing
agent based on hypometabolism efficacy of 3-iodothyronamine (T1AM)
and a composition for preventing or treating muscular hypertrophy
including myotonia congenital, calf hypertrophy, myhre syndrome,
and myostatin-related muscular hypertrophy, or for facial muscle
shrinkage using the hypometabolism-inducing substance.
Description of Related Art
[0003] The muscular atrophy occurs in various pathological and
physiological conditions such as bodily injury, cancer cachexia in
cancer patients, muscle aging, long-term bed life, or space flight
as well as genetic disorders (e.g., Duchenne muscle dystrophy).
Amounts of muscle proteins such as actin and myosin are decreased,
and muscle mass and muscle strength are significantly decreased.
Accordingly, since the muscular atrophy has an effect on most of
activities from simple behaviors to routine tasks, exercises, and
even astronaut missions, a pharmacological rehabilitative medical
research to treat the muscular atrophy is important.
[0004] A first step for treating of the muscular atrophy is to
develop an appropriate model to induce the muscular atrophy. As an
animal model (in vivo), denervation and hindlimb suspension methods
have been mainly used. Treatment methods of dexamethasone which is
synthetic glucocorticoid, oxidizing substances (for example, active
oxygen such as H.sub.2O.sub.2), or the like have been used as
drugs. It has been known that the animal model and the drugs
activate signaling pathways which are associated with muscle
protein catabolism such as activation of forkhead box O (FoxO),
increased expression of ubiquitin E3 ligase and proteasome without
exception and simultaneously, inhibit signaling pathways (Akt1-S6K)
which are associated with muscle protein anabolism (Shimizu et al.,
2011). Further, it has been known that the expression of chaperone
proteins (e.g., heat shock proteins) that help protein biogenesis,
repair of damage, and the like during the muscular atrophy is also
decreased (Gwag et al. 2009).
[0005] When describing the mechanism of the muscular
atrophy-inducing agents, according to recent reports, it is known
that the dexamethasone as a steroid hormone-based substance having
an anti-inflammatory effect binds to a glucocorticoid receptor (GR)
and activates a proteolytic signaling pathway of FoxO-proteasome to
induce the muscular atrophy. It is reported that oxidative
substances such as hydrogen peroxide damage the sarcoplasmic
reticulum membrane and the mitochondrial membrane, and the released
Ca.sup.2+ and cytochrome C accelerate the activation of calpain
proteases to induce the muscular atrophy (McClung et al. 2009).
[0006] In addition, the muscular hypertrophy is a disease caused
when the balance of muscle protein synthesis and degradation breaks
down, and as typical examples, there are myotonia congenita, calf
hypertrophy, myhre syndrome, myostatin-related muscular
hypertrophy, and the like. Among these diseases, particularly, the
myostatin-related muscular hypertrophy is a symptom caused by
breakdown of a myostatin gene associated with the muscle protein
degradation. Myostatin serves to inhibit a muscle protein synthesis
pathway (e.g., Akt1-mTOR) and increases the activity of a muscle
protein degradation pathway (e.g., SMAD-proteasome), but when this
gene is broken, the balance of muscle mass retention is broken and
thus the muscular hypertrophy occurs.
[0007] Meanwhile, 3-iodothyronamine (T1AM) is a derivative of
thyroid hormones T3 and T4 and a hypometabolism-inducing substance
that may be generated in the body. It has been found that a pico
mole of 3-iodothyronamine is present in most of the rodent tissue
samples (brain, liver, heart, kidney, muscle, etc.) and the human
blood (Zucchi R et al., 2006). In addition, 3-iodotronamine is a
synthesizable substance and a preparing method thereof is disclosed
in U.S. Pat. Nos. 6,979,750 and 7,321,065 and Korean Patent
Registration No. 1,112,731, which is a prior patent of the inventor
of the present application, and the 3-iodotronamine can be
mass-produced to be easily used for industrial use.
[0008] The present inventors found that muscular atrophy may be
induced by treating a hypometabolism-inducing substance according
to protein expression levels associated with generation and
inhibition of muscle proteins and a change in size of myotube cells
and intend to provide a new concept of muscular atrophy study model
which is different from existing methods using the
hypometabolism-inducing substance, and a muscle hyperthrophy
treating agent through a muscular atrophy inhibition effect or a
composition for facial muscle shrinkage usable for Botox.
BRIEF SUMMARY OF THE INVENTION
[0009] An object of the present invention is to provide a muscular
atrophy study model as a novel muscular atrophy inducing method
which is different from a denervation method, a hindlimb suspension
method, and a method for treating dexamethasone as synthetic
glucorticoid, an oxidizing agent (e.g., active oxygen such as
H.sub.2O.sub.2), or the like in the related art. Herein, the study
model may include cell, tissue, and animal models.
[0010] Another object of the present invention is to provide a
pharmaceutical composition or a health food for preventing or
treating muscular hypertrophy using a hypometabolism-inducing
substance of the present invention.
[0011] Yet another object of the present invention is to provide a
composition for facial muscle shrinkage usable for Botox using a
hypometabolism-inducing substance of the present invention.
[0012] In order to achieve the objects, an exemplary embodiment of
the present invention provides a muscular atrophy-inducing agent
containing as an active ingredient a hypometabolism-inducing
substance selected from the group consisting of 3-iodothyronamine
(T1AM), [D-Ala2,D-Leu5] enkephalin (DADLE), 5'-adenosine
monophosphate (5'-AMP), and hydrogen sulfide (H.sub.2S), by
targeting an animal model, a muscular atrophy study model including
inducing muscular atrophy by administering the muscular
atrophy-inducing agent to cells and animals, and a drug screening
method for preventing or treating muscular atrophy based on the
muscular atrophy study model.
[0013] Another exemplary embodiment of the present invention
provides a pharmaceutical composition for preventing or treating
muscular hypertrophy using the hypometabolism-inducing
substance.
[0014] Yet another exemplary embodiment of the present invention
provides a health food for preventing or treating muscular
hypertrophy using the hypometabolism-inducing substance.
[0015] Still another exemplary embodiment of the present invention
provides a composition for facial muscle shrinkage using the
hypometabolism-inducing substance.
[0016] According to the present invention, the muscular atrophy
inducing model may provide an economic muscular atrophy study model
by using a hypometabolic compound enabling mass production and may
be usefully used by verification for screening a drug for
preventing or treating muscular atrophy. Further, since the
hypometabolic compound significantly activates muscle protein
degradation, the hypometabolic compound may be usefully used as a
drug for treating muscular hypertrophy or a composition for facial
muscle shrinkage.
BRIEF DESCRIPTION OF THE DRAWING
[0017] FIGS. 1A-1B illustrate comparison of diameters of C2C12
myotubes between a T1AM treated group and a control: FIG. 1A
illustrates a representative photograph of myotubes taken with an
Axiovert 200 optical microscope (magnification: 200.times.0, in
which a pair of arrows indicates locations where diameters of the
myotubes are measured (black bar=25 .mu.m). FIG. 1B is a graph
illustrated by measuring the diameters of myotubes between two
groups. Data: Mean.+-.SEM (n=3; 96 cells/group), *: It means that
there is a significant difference between the two groups
[independent samples t-test, P<0.05)].
[0018] FIGS. 2A-2C illustrate comparison of AMPK activities of
C2C12 myotubes between a T1AM treated group and a control: FIG. 2A
illustrates a result of Immunoblotting analysis for expression of
p-AMPK and AMPK. FIG. 2B is a graph illustrating expression levels
of p-AMPK and AMPK as densitometric quantitation, and FIG. 2C is a
graph illustrating an expression ratio of p-AMPK/AMPK as
densitometric quantitation [mean.+-.SEM (n=6), *, P<0.05].
[0019] FIGS. 3A-3E illustrates comparison of expression of Akt1 and
S6K of C2C12 myotubes between a T1AM treated group and a control:
FIG. 3A illustrates a result of Immunoblotting analysis for
expression of Akt1 and S6K. FIGS. 3B and 3C are graphs illustrating
expression levels of p-Akt1 and Akt1 and an expression ratio of
p-Akt1/Akt1, respectively. FIGS. 3D and 3E are graphs illustrating
expression levels of p-S6K and S6K and an expression ratio of
p-S6K/S6K, respectively [mean.+-.SEM (n =6), *, P<0.05].
[0020] FIGS. 4A-4F illustrate comparison of expression of FoxO1 and
FoxO3 of C2C12 myotubes between a T1AM treated group and a control:
FIG. 4A illustrates a result of immunoblotting analysis for
expression of FoxO1 and FoxO3. FIG. 4B is a photograph taken as an
analysis result of immunofluorescence staining for FoxO1 and FoxO3
with a confocal microscope. FIGS. 4C and FIG. 4D are graphs
illustrating expression levels of p-FoxO1 and FoxO1 and an
expression ratio of p-FoxO1/FoxO1, respectively. FIG. 4E and FIG.
4F are graphs illustrating expression levels of p-FoxO1 and FoxO1
and an expression ratio of p-FoxO3/FoxO3, respectively [mean.+-.SEM
(n=6), *, P<0.05].
[0021] FIGS. 5A-5D illustrates comparison of expression of MuRF1
and MAFbx of C2C12 myotubes between a T1AM treated group and a
control: A illustrates a result of immunoblotting analysis for
expression of FoxO1 and FoxO3. FIG. 5B and FIG. 5C are graphs
illustrating densitometric quantitation for expression levels of
MuRF1 and MAFbx. FIG. 5D illustrates chymotrypsin-like activity of
26S, which is determined through cell-based luminescence analysis
and expressed as a relative light unit (RLU). Actual
chymotrypsin-like activity was determined from <total
RLUs--background RLUs>in each analysis [mean.+-.SEM (n=6), *,
P<0.05].
[0022] FIGS. 6A-6D illustrates comparison of expression of
chaperone of C2C12 myotubes between a T1AM treated group and a
control: A illustrates a result of immunoblotting analysis of
expression of heat shock protein 72 (HSP72), HSP60 and
.alpha.B-crystallin. FIG. 6B and FIG. 6D are graphs illustrating
densitometric quantitation for an expression level of each
chaperone protein [mean.+-.SEM (n=6), *, P<0.05].
[0023] FIG. 7 is a schematic diagram of signaling pathways
associated with synthesis and degradation of muscle proteins
according to T lAM treatment.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention relates to a muscular atrophy-inducing
agent containing as an active ingredient a hypometabolism-inducing
substance selected from the group consisting of 3-iodothyronamine
(T1AM), [D-Ala2,D-Leu5] enkephalin (DADLE), 5'-adenosine
monophosphate (5'-AMP), and hydrogen sulfide (H.sub.2S), by
targeting an animal model, a method for preparing a muscular
atrophy study model including inducing muscular atrophy by treating
or administering the muscular atrophy-inducing agent, a sturdy
model prepared according to the method, a method for using drug
screening for preventing or treating muscular atrophy using the
muscular atrophy study model, and a for preventing or treating
muscular hypertrophy using the hypometabolic substance as an active
ingredient or a composition for facial muscle shrinkage using the
hypometabolic substance as an active ingredient. The present
inventors found the fact that the hypometabolism-inducing substance
inhibited a muscle protein synthesis mechanism and activated a
degradation mechanism through related protein expression and a
change in size of myotubes and completed the present invention.
[0025] Hereinafter, the present invention will be described in more
detail.
[0026] The present invention provides a muscular atrophy-inducing
agent containing as an active ingredient a hypometabolism-inducing
substance selected from the group consisting of 3-iodothyronamine
(T1AM), [D-Ala2,D-Leu5] enkephalin (DADLE), 5'-adenosine
monophosphate (5'-AMP), and hydrogen sulfide (H.sub.2S).
[0027] In one embodiment of the present invention, the
hypometabolism-inducing substance may be more particularly
3-iodothyronamine (T1AM).
[0028] In one embodiment of the present invention, in the study
model, as a cell line, a muscle cell line may be used, general
muscle cell lines or muscle fibers used in the art may be used, and
for example, C2C12 muscle cells and the like may be used.
[0029] In one embodiment of the present invention, in the study
model, the animal may be vertebrate animals, more specifically
vertebrate animals except for humans, and for example, may include
rodents including mice, rats, and hamsters, rabbits, horses, cows,
dogs, cats, monkeys, guinea pigs, and the like.
[0030] Further, the present invention provides a method for
preparing a muscular atrophy study model including inducing
muscular atrophy by administering to a normal animal a
hypometabolism-inducing substance selected from the group
consisting of 3-iodothyronamine (T1AM), [D-Ala2,D-Leu5] enkephalin
(DADLE), 5'-adenosine monophosphate (5'-AMP), and hydrogen sulfide
(H.sub.2S).
[0031] In one embodiment of the present invention, the
hypometabolism-inducing substance may be more particularly
3-iodothyronamine (T1AM), and a dose of the T1AM may be
appropriately adjusted. For example, when T1AM is administered
intraperitoneally, if the dose of T1AM is less than 10 mg/kg per
unit weight (kg) of an administered animal, it is difficult to
cause muscular atrophy, and if the dose exceeds 500 mg/kg, the
animal dies. As a result, the close of T1AM may be 10 to 500 mg/kg,
more specifically 20 to 250 mg/kg, and more specifically 25 to 100
mg/kg, per unit body weight (kg) of animal, but may be
appropriately adjusted according to the condition of the animal and
experimental conditions.
[0032] In one embodiment of the present invention, the treatment
concentration in the cells may be 0.1 .mu.M to 1000 .mu.M, but may
be appropriately adjusted according to the amount of cells,
conditions of the cells, and experimental conditions.
[0033] In one embodiment of the present invention, the
administration of the hypometabolism-inducing substance may be
performed by a general administration method, such as oral
administration, intraperitoneal administration, intravenous
administration, intramuscular administration, subcutaneous
administration, or intradermal administration, but the
administration method is not limited thereto. Further, the
hypometabolism-inducing substance may be administered by any device
which is movable to a target cell as an active substance.
[0034] In one embodiment of the present invention, the number of
dose times of the hypometabolism-inducing substance may be one to
two or more times per day, but the number of dose times may be
controlled according to the dose of the hypometabolism-inducing
substance.
[0035] In one embodiment of the present invention, the degree of
muscular atrophy in the muscular atrophy animal model may be
adjusted by controlling a dose of the hypometabolism-inducing
substance or an exposure time in vivo of the
hypometabolism-inducing substance, and this may be performed based
on the degree of muscular atrophy increasing in proportion to the
dose or the exposure time in vivo.
[0036] Further, the present invention provides a muscular atrophy
cell model or animal model prepared by the method.
[0037] In one embodiment of the present invention, the muscular
atrophy study model may show the following results from (a) to (j)
as compared to a normal by the administration of
hypometabolism-inducing substance, which was confirmed by a muscle
cell experiment:
[0038] (a) Decrease in size of myotubes or size of muscle;
[0039] (b) Increase in expression ratio of p-AMPK/AMPK;
[0040] (c) Decrease in expression ratio of p-Akt1/Akt1;
[0041] (d) Decrease in expression ratio of p-S6K/S6K;
[0042] (e) Decrease in expression ratio of p-FoxO1/FoxO1;
[0043] (f) Decrease in expression ratio of p-FoxO3/FoxO3;
[0044] (g) Increase in expression level of MuRF1;
[0045] (h) Increase in activity of proteasome;
[0046] (i) Decrease in expression level of heat shock protein 72
(HSP72); and
[0047] (j) Decrease in expression level of .alpha.B-crystallin
[0048] Accordingly, the muscular atrophy study model may have at
least one characteristic selected from the group consisting of (a)
a decrease in size of myotubes or size of muscle; (b) an increase
in expression ratio of p-AMPK/AMPK; (c) a decrease in expression
ratio of p-Akt1/Akt1; (d) a decrease in expression ratio of
p-S6K/S6K; (e) a decrease in expression ratio of p-FoxO1/FoxO1; (f)
a decrease in expression ratio of p-FoxO3/FoxO3; (g) an increase in
expression level of MuRF1; (h) an increase in activity of
proteasome; (i) a decrease in expression level of heat shock
protein 72 (HSP72); and (j) a decrease in expression level of
.alpha.B-crystallin, as compared to a normal.
[0049] In one embodiment of the present invention, (a) to (j) may
be characteristics compared to the normal after 5 to 10 days of
administration of the hypometabolism-inducing substance.
[0050] In one embodiment of the present invention, when 0.1 .mu.M
to 1000 .mu.M of T1MA as the hypometabolism-inducing substance is
treated to the myotubes, with respect to (a) above, the size of the
myotubes may be decreased by 0.01 to 0.20 times as compared to the
size of the normal myotubes. With respect to (b) above, the
expression ratio of p-AMPK/AMPK in the muscular atrophy study model
may be increased by 0.01 to 2.5 times as compared to the expression
ratio of p-AMPK/AMPK in the normal model. With respect to (c)
above, the expression ratio of p-Akt1/Akt1 in the muscular atrophy
study model may be decreased by 0.01 to 1.0 times as compared to
the expression ratio of p-Akt1/Akt1 in the normal model. With
respect to (d) above, the expression ratio of p-S6K/S6K in the
muscular atrophy study model may be decreased by 0.01 to 1.0 times
as compared to the expression ratio of p-S6K/S6K in the normal
model. With respect to (e) above, the expression ratio of
p-FoxO1/FoxO1 in the muscular atrophy study model may be decreased
by 0.01 to 1.0 times as compared to the expression ratio of
p-FoxO1/FoxO1 in the normal model. With respect to (f) above, the
expression ratio of p-FoxO3/FoxO3 in the muscular atrophy study
model may be decreased by 0.01 to 0.8 times as compared to the
expression ratio of p-FoxO3/FoxO3 in the normal model. With respect
to (g) above, the expression level of MuRF1 in the muscular atrophy
study model may be increased by 0.01 to 2.5 times as compared to
the expression level of MuRF1 in the normal model. With respect to
(h) above, the activity of proteasome in the muscular atrophy study
model may be increased by 0.01 to 2.0 times as compared to the
activity of proteasome in the normal model. The proteasome may be
26S proteasome, and the activity thereof can be measured using a
method known as a method for measuring the activity of proteasome.
For example, the activity of proteasome may be analyzed through
chemotrypsin-like activity. With respect to (i) above, the
expression level of heat shock protein 72 (HSP72) in the muscular
atrophy study model may be increased by 0.01 to 0.15 times as
compared to the expression level of heat shock protein 72 (HSP72)
in the normal model. With respect to (j) above, the expression
level of .alpha.B-crystallin in the muscular atrophy study model
may be decreased by 0.01 to 1.0 times as compared to the expression
level of .alpha.B-crystallin in the normal model. The expression
levels of AMPK, phospho-AMPK (p-AMPK), FoxO1, p-FoxO1, FoxO3,
p-FoxO3, Akt1, p-Akt1, S6K, p-S6K, MuRF1, HSP72, and
.alpha.B-crystallin in (b) to (g), (i), and (j) may be measured by
using methods known as a protein analysis method. For example, the
expression levels may be measured by an immunoblotting method.
[0051] The muscular atrophy study model may be used as a study
model for accurate muscular atrophy studies, but may also be useful
as verification for screening of a drug of preventing or treating
muscular atrophy.
[0052] Accordingly, the present invention provides a method for
screening a drug for treating muscular atrophy including: treating
a candidate substance in the muscular atrophy study model;
[0053] and determining the candidate substance as the drug for
treating muscular atrophy by evaluating the improved or treated
degree of the muscular atrophy in the sturdy model treated with the
candidate substance.
[0054] In one embodiment of the present invention, the candidate
substance is a substance capable of treating muscular atrophy and
includes chemicals, oligonucleotides, peptides, genes, proteins,
and the like, without limitation.
[0055] In one embodiment of the present invention, the degree of
improvement or treatment of muscular atrophy may be evaluated by
comparing at least one of the following indicators (1) to (10) with
that of the control:
[0056] (1) Size of myotubes or size of muscle;
[0057] (2) Expression ratio of p-AMPK/AMPK;
[0058] (3) Expression ratio of p-Akt1/Akt1;
[0059] (4) Expression ratio of p-S6K/S6K;
[0060] (5) Expression ratio of p-FoxO1/FoxO1;
[0061] (6) Expression ratio of p-FoxO3/FoxO3;
[0062] (7) Expression level of MuRF1;
[0063] (8) Activity of proteasome;
[0064] (9) Expression level of heat shock protein 72 (HSP72);
and
[0065] (10) Expression level of .alpha.B-crystallin
[0066] With respect to indicator (1) above, the degree of
improvement or treatment of muscular atrophy may be evaluated by
comparing changes in the size of the myotubes or the size of the
muscle in the group treated with the candidate substance and the
control. When the size of the myotubes or the muscle in the group
treated with the candidate substance is increased compared to that
of the control, the candidate substance may be determined as a drug
for treating muscular atrophy.
[0067] With respect to indicator (2) above, the degree of
improvement or treatment of muscular atrophy may be evaluated by
comparing expression ratios of p-AMPK/AMPK in the group treated
with the candidate substance and the control. When the expression
ratio of p-AMPK/AMPK in the group treated with the candidate
substance is decreased compared to that of the control, the
candidate substance may be determined as a drug for treating
muscular atrophy.
[0068] With respect to indicator (3) above, the degree of
improvement or treatment of muscular atrophy may be evaluated by
comparing expression ratios of p-Akt1/Akt1 in the group treated
with the candidate substance and the control. When the expression
ratio of p-Akt1/Akt1 in the group treated with the candidate
substance is increased compared to that of the control, the
candidate substance may be determined as a drug for treating
muscular atrophy.
[0069] With respect to indicator (4) above, the degree of
improvement or treatment of muscular atrophy may be evaluated by
comparing expression ratios of p-S6K/S6K in the group treated with
the candidate substance and the control. When the expression ratio
of p-S6K/S6K in the group treated with the candidate substance is
increased compared to that of the control, the candidate substance
may be determined as a drug for treating muscular atrophy.
[0070] With respect to indicator (5) above, the degree of
improvement or treatment of muscular atrophy may be evaluated by
comparing expression ratios of p-FoxO1/FoxO1 in the group treated
with the candidate substance and the control. When the expression
ratio of p-FoxO1/FoxO1 in the group treated with the candidate
substance is increased compared to that of the control, the
candidate substance may be determined as a drug for treating
muscular atrophy.
[0071] With respect to indicator (6) above, the degree of
improvement or treatment of muscular atrophy may be evaluated by
comparing expression ratios of p-FoxO3/FoxO3 in the group treated
with the candidate substance and the control. When the expression
ratio of p-FoxO3/FoxO3 in the group treated with the candidate
substance is increased compared to that of the control, the
candidate substance may be determined as a drug for treating
muscular atrophy.
[0072] With respect to indicator (7) above, the degree of
improvement or treatment of muscular atrophy may be evaluated by
comparing expression levels of MuRF1 in the group treated with the
candidate substance and the control. When the expression level of
MuRF1 in the group treated with the candidate substance is
decreased compared to that of the control, the candidate substance
may be determined as a drug for treating muscular atrophy.
[0073] With respect to indicator (8) above, the degree of
improvement or treatment of muscular atrophy may be evaluated by
comparing proteasome activities in the group treated with the
candidate substance and the control. When the activity of
proteasome in the group treated with the candidate substance is
decreased compared to that of the control, the candidate substance
may be determined as a drug for treating muscular atrophy.
[0074] With respect to indicator (9) above, the degree of
improvement or treatment of muscular atrophy may be evaluated by
comparing expression levels of hot shock protein 72 (HSP72) in the
group treated with the candidate substance and the control. When
the expression level of hot shock protein 72 (HSP72) in the group
treated with the candidate substance is increased compared to that
of the control, the candidate substance may be determined as a drug
for treating muscular atrophy.
[0075] With respect to indicator (10) above, the degree of
improvement or treatment of muscular atrophy may be evaluated by
comparing expression levels of .alpha.B-crystallin in the group
treated with the candidate substance and the control. When the
expression level of .alpha.B-crystallin in the group treated with
the candidate substance is increased compared to that of the
control, the candidate substance may be determined as a drug for
treating muscular atrophy.
[0076] Accordingly, in the determining, when the group treated with
the candidate substance is compared to the control, in the case of
at least one result selected from the group consisting of an
increase in size of myotubes or size of muscle; a decrease in
expression ratio of p-AMPK/AMPK; an increase in expression ratio of
p-Akt1/Akt1; an increase in expression ratio of p-S6K/S6K; an
increase in expression ratio of p-FoxO1/FoxO1; an increase in
expression ratio of p-FoxO3/FoxO3; a decrease in expression level
of MuRF1; a decrease in activity of proteasome; an increase in
expression level of heat shock protein 72 (HSP72); and an increase
in expression level of .alpha.B-crystallin, the candidate substance
may be determined as a drug for treating muscular atrophy.
[0077] The control refers to a group treated with an excipient of
the drug for treating muscular atrophy instead of the candidate
substance, and for example, the control may be a group consisting
of dimethyl sulfoxide (DMSO), physiological saline, sterilized
distilled water, carboxymethyl cellulose or phosphate buffered
saline (PBS).
[0078] The present invention provides a screening method for a drug
for preventing muscular atrophy including: treating or
administering a candidate substance to normal cells or a normal
animal; treating or administering a hypometabolism-inducing
substance to the cells or the animal;
[0079] and determining the candidate substance as the drug for
preventing muscular atrophy by evaluating the degree of muscular
atrophy in the cells or the animal treated with the
hypometabolism-inducing substance.
[0080] In one embodiment of the present invention, the candidate
substance is the same as described above.
[0081] In one embodiment of the present invention, the degree of
muscular atrophy may be evaluated by comparing at least one of the
following indicators (1) to (10) with that of the control:
[0082] (1) Size of myotubes or size of muscle;
[0083] (2) Expression ratio of p-AMPK/AMPK;
[0084] (3) Expression ratio of p-Akt1/Akt1;
[0085] (4) Expression ratio of p-S6K/S6K;
[0086] (5) Expression ratio of p-FoxO1/FoxO1;
[0087] (6) Expression ratio of p-FoxO3/FoxO3;
[0088] (7) Expression level of MuRF1;
[0089] (8) Activity of proteasome;
[0090] (9) Expression level of heat shock protein 72 (HSP72);
and
[0091] (10) Expression level of .alpha.B-crystallin
[0092] With respect to indicator (1) above, the degree of muscular
atrophy may be evaluated by comparing changes in the size of the
myotubes or the size of the muscle in the group treated with the
candidate substance and the control. When the size of the myotube
or the muscle in the group treated with the candidate substance is
increased compared to that of the control, the candidate substance
may be determined as a drug for preventing muscular atrophy.
[0093] With respect to indicator (2) above, the degree of muscular
atrophy may be evaluated by comparing expression ratios of
p-AMPK/AMPK in the group treated with the candidate substance and
the control. When the expression ratio of p-AMPK/AMPK in the group
treated with the candidate substance is decreased compared to that
of the control, the candidate substance may be determined as a drug
for preventing muscular atrophy.
[0094] With respect to indicator (3) above, the degree of muscular
atrophy may be evaluated by comparing expression ratios of
p-Akt1/Akt1 in the group treated with the candidate substance and
the control. When the expression ratio of p-Akt1/Akt1 in the group
treated with the candidate substance is increased compared to that
of the control, the candidate substance may be determined as a drug
for preventing muscular atrophy.
[0095] With respect to indicator (4) above, the degree of muscular
atrophy may be evaluated by comparing expression ratios of
p-S6K/S6K in the group treated with the candidate substance and the
control. When the expression ratio of p-S6K/S6K in the group
treated with the candidate substance is increased compared to that
of the control, the candidate substance may be determined as a drug
for preventing muscular atrophy.
[0096] With respect to indicator (5) above, the degree of muscular
atrophy may be evaluated by comparing expression ratios of
p-FoxO1/FoxO1 in the group treated with the candidate substance and
the control. When the expression ratio of p-FoxO1/FoxO1 in the
group treated with the candidate substance is increased compared to
that of the control, the candidate substance may be determined as a
drug for preventing muscular atrophy.
[0097] With respect to indicator (6) above, the degree of muscular
atrophy may be evaluated by comparing expression ratios of
p-FoxO3/FoxO3 in the group treated with the candidate substance and
the control. When the expression ratio of p-FoxO3/FoxO3 in the
group treated with the candidate substance is increased compared to
that of the control, the candidate substance may be determined as a
drug for preventing muscular atrophy.
[0098] With respect to indicator (7) above, the degree of muscular
atrophy may be evaluated by comparing expression levels of MuRF1 in
the group treated with the candidate substance and the control.
When the expression level of MuRF1 in the group treated with the
candidate substance is decreased compared to that of the control,
the candidate substance may be determined as a drug for preventing
muscular atrophy.
[0099] With respect to indicator (8) above, the measurement of the
activity of proteasome is the same as that of (h) described above,
and the degree of muscular atrophy may be evaluated by comparing
activities of proteasome in the group treated with the candidate
substance and the control. When the activity of proteasome in the
group treated with the candidate substance is decreased compared to
that of the control, the candidate substance may be determined as a
drug for preventing muscular atrophy.
[0100] With respect to indicator (9) above, the degree muscular
atrophy may be evaluated by comparing expression levels of hot
shock protein 72 (HSP72) in the group treated with the candidate
substance and the control. When the expression level of hot shock
protein 72 (HSP72) in the group treated with the candidate
substance is increased compared to that of the control, the
candidate substance may be determined as a drug for preventing
muscular atrophy.
[0101] With respect to indicator (10) above, the degree of muscular
atrophy may be evaluated by comparing expression levels of
.alpha.B-crystallin in the group treated with the candidate
substance and the control. When the expression level of
.alpha.B-crystallin in the group treated with the candidate
substance is increased compared to that of the control, the
candidate substance may be determined as a drug for preventing
muscular atrophy.
[0102] The expression levels of AMPK, phospho-AMPK (p-AMPK), FoxO1,
p-FoxO1, FoxO3, p-FoxO3, Akt1, p-Akt1, S6K, p-S6K, MuRF1, HSP72,
and .alpha.B-crystallin proteins The expression levels in (2) to
(7), (9), and (10) may be measured using methods known as a protein
analysis method. For example, the expression levels may be measured
by immunoblotting.
[0103] Therefore, in the determining, when the group treated with
the candidate substance is compared to the control, in the case of
at least one result selected from the group consisting of an
increase in size of myotubes or size of muscle; a decrease in
expression ratio of p-AMPK/AMPK; an increase in expression ratio of
p-Akt1/Akt1; an increase in expression ratio of p-S6K/S6K; an
increase in expression ratio of p-FoxO1/FoxO1; an increase in
expression ratio of p-FoxO3/FoxO3; a decrease in expression level
of MuRF1; a decrease in activity of proteasome; an increase in
expression level of heat shock protein 72 (HSP72); and an increase
in expression level of .alpha.B-crystallin, the candidate substance
may be determined as a drug for preventing muscular atrophy.
[0104] The control refers to a group treated with an excipient of
the drug for preventing muscular atrophy instead of the candidate
substance, and for example, the control may be a group consisting
of dimethyl sulfoxide (DMSO), physiological saline, sterilized
distilled water, carboxymethyl cellulose or phosphate buffered
saline (PBS).
[0105] In the present specification, the normal cells or the normal
animal refer to cells or animals without muscular atrophy. For
example, the animal may be an animal that is the same species as
the muscular atrophy model and does not have muscular atrophy
raised in the same or similar environment.
[0106] The present invention provides a pharmaceutical composition
for preventing or treating muscular hypertrophy, containing as an
active ingredient a hypometabolism-inducing substance selected from
the group consisting of 3-iodothyronamine (T1AM), [D-Ala2,D-Leu5]
enkephalin (DADLE), 5'-adenosine monophosphate (5'-AMP), and
hydrogen sulfide (H.sub.2S).
[0107] In one embodiment of the present invention, the
hypometabolism-inducing substance may be more particularly
3-iodothyronamine (T1AM).
[0108] In one embodiment of the present invention, the muscular
hypertrophy includes myotonia congenital, calf hypertrophy, myhre
syndrome, and myostatin-related muscular hypertrophy.
[0109] In one embodiment of the present invention, since the
hypometabolism-inducing substance of the present invention induces
muscular atrophy by inhibiting activity of Akt1-S6K involved in
muscle protein synthesis and activating FoxO-proteasome involved in
muscle protein degradation, the hypometabolism-inducing substance
can be used as a drug which may replace the role of myostatin and
may be used for treatment of various muscle hypertrophies including
myostatin-related muscular hypertrophy caused by binding of
myostatin.
[0110] The present invention includes all of its pharmaceutically
acceptable salt and solvates, hydrates, racemates, or stereoisomers
capable of being prepared therefrom as well as the
hypometabolism-inducing substance of the present invention.
[0111] The hypometabolism-inducing substance of the present
invention may be used in a form of its pharmaceutically acceptable
salt and as the salt, acid additional salts formed by free
pharmaceutically acceptable acid are useful. The acid additional
salts are obtained from inorganic acids such as hydrochloric acid,
nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid,
hydriodic acid, nitrous acid or phosphorous acid and non-toxic
organic acids such as aliphatic mono and dicarboxylate,
phenyl-substituted alkanoate, hydroxy alkanoate and alkandioate,
aromatic acids, aliphatic and aromatic sulfonic acids. The
pharmaceutically non-toxic salt includes sulfate, fatigue sulfate,
bisulfate, sulfite, bisulfite, nitrate, phosphate, mono-hydrogen
phosphate, dihydrogen phosphate, meta-phosphate, pyrophosphate
chloride, bromide, iodide, fluoride, acetate, propionate succinate,
decanoate, caprylate, acrylate, formate, isobutyrate, caprate,
heptanoate, propionic oleate, oxalate, malonate, succinate,
suberate, sebacate, fumarate, maleate , butyne-1,4-dioate,
hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,
dinitro benzoate, hydroxybenzoate, methoxybenzoate, phthalate,
terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene
sulfonate, xylene sulfonate, phenylacetate, phenyl propionate,
phenyl butyrate, citrate, lactate, hydroxybutyrate, glycollate,
maleate, tartrate, methanesulfonate, propanesulfonate,
naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.
[0112] The acid additional salt according to the present invention
may be prepared by a general method, for example, dissolving the
hypometabolism-inducing substance of the present invention in a
large amount of acid aqueous solution and precipitating the salt by
using a water-miscible organic solvent, for example, methanol,
ethanol, acetone, or acetonitrile. Further, the salt which is dried
or precipitated by evaporating the solvent or a large amount of
acid from the mixture may also be prepared through
suction-filtering.
[0113] Further, a pharmaceutically acceptable metal salt may be
prepared by using base. An alkali metal or alkaline earth metal
salt is obtained by, for example, dissolving the compound in a
large amount of alkali metal hydroxide or alkaline earth metal
hydroxide solution and filtering an insoluble compound salt and
then evaporating and drying a filtrate. In this case, the metal
salt is pharmaceutically suitable to prepare sodium, potassium or
calcium salts. Further, the silver salt corresponding thereto is
obtained by reacting alkali metal or alkaline earth metal salts
with an appropriate silver salt (for example, silver nitrate).
[0114] When the composition is formulated, the formulation is
prepared by using diluents or excipients, such as a filler, an
extender, a binding agent, a wetting agent, a disintegrating agent,
and a surfactant, which are generally used.
[0115] A solid formulation for oral administration includes a
tablet, a pill, a powder, a granule, a capsule, a troche agent, or
the like, and the solid formulation may be prepared by mixing at
least one excipient, for example, starch, calcium carbonate,
sucrose or lactose, gelatin, or the like with at least one
hypometabolism-inducing substance of the present invention.
Further, lubricants such as magnesium stearate talc may be used in
addition to simple excipients. A liquid formulation for oral
administration may use a suspension, a solution, an emulsion, a
syrup, and the like, and may include various excipients, for
example, a wetting agent, a sweetener, an aromatic agent, a
preserving agent, and the like in addition to water and liquid
paraffin, as simple diluents which are commonly used.
[0116] A formulation for parenteral administration includes a
sterile aqueous solution, a non-aqueous solution, a suspension, an
emulsion, a lyophilizing agent, a suppository, and the like.
[0117] As the non-aqueous solution and the suspension, propylene
glycol, polyethylene glycol, vegetable oils such as olive oil,
injectable ester such as ethyl oleate, and the like may be used. As
a matter of the suppository, witepsol, macrogol, tween 61, cacao
butter, laurin, glycerol, gelatin, and the like may be used.
[0118] The composition according to the present invention is
administered with a pharmaceutically effective dose. In the present
invention, the "pharmaceutically effective dose" refers to a amount
which is sufficient to treat the diseases at a reasonable
benefit/risk ratio applicable to medical treatment, and an
effective dose level may be determined according to elements
including a kind of disease of the patient, the severity, activity
of a drug, sensitivity to a drug, a time of administration, a route
of administration, and an emission rate, duration of treatment, and
simultaneously used drugs and other elements well-known in the
medical field. The composition of the present invention may be
administered as an individual therapeutic agent or administered in
combination with other therapeutic agents, sequentially or
simultaneously administered with existing therapeutic agents, and
administered singly or multiply. It is important to administer an
amount capable of obtaining a maximum effect with a minimal amount
without side effects by considering the above elements and the
amount may be easily determined by those skilled in the art.
[0119] Particularly, the effective dose of the composition
according to the present invention may vary according to age,
gender, and weight of the patient, and generally administered by
0.1 mg to 100 mg per weight 1 kg, preferably administered by 0.5 mg
to 10 mg daily or every other day, or administered one to three
times per day. However, since the effective dose may be decreased
or increased depending on the route of administration, the severity
of obesity, gender, weight, age, and the like, the dose is not
limited to the scope of the present invention in any way.
[0120] The present invention provides a health food for preventing
or treating muscular hypertrophy, containing as an active
ingredient a hypometabolism-inducing substance selected from the
group consisting of 3-iodothyronamine (T1AM), [D-Ala2,D-Leu5]
enkephalin (DADLE), 5'-adenosine monophosphate (5'-AMP), and
hydrogen sulfide (H.sub.2S).
[0121] In one embodiment of the present invention, since the
hypometabolism-inducing substance of the present invention induces
muscular atrophy by inhibiting activity of Akt1-S6K involved in
muscle protein synthesis and activating FoxO-proteasome involved in
muscle protein degradation, the hypometabolism-inducing substance
can be used as a drug which may replace the role of myostatin and
may be used for health foods for preventing or improving various
muscle hypertrophies including myostatin-related muscular
hypertrophy caused by binding of myostatin.
[0122] Kinds of foods which are added with the
hypometabolism-inducing substance of the present invention are not
particularly limited. Examples of the foods which may be added with
the materials include drinks, meat, sausages, bread, biscuits, rice
cakes, chocolate, candies, snacks, cookies, pizza, ramen noodles,
other noodles, gums, dairy products including ice cream, various
soups, beverages, alcohol drinks, vitamin complex, milk products,
milk dairy products, and the like, and include all health
functional foods in the accepted meaning.
[0123] The hypometabolism-inducing substance of the present
invention may be added to the food as it is or may be used together
with other food or food ingredients, and may be appropriately used
according to general methods. A mixing amount of active ingredients
may be appropriately determined according to a purpose of use (for
prevention or improvement) thereof. Generally, the amount of
compound in the health functional food may be added with 0.1 to 90
parts by weight with respect to the entire food weight. However, in
the case of long-term administration for health and hygiene or
health control, the amount may be the range or less. Since there is
no problem in terms of safety, the active ingredients may be used
with the amount in the range or more.
[0124] In the health food composition according to the present
invention, other ingredients are not particularly limited except
for containing the compound as the required ingredient at the
indicated ratio, and like a general beverage, various flavoring
agents, natural starches, or the like may be contained as an
additional ingredient. Examples of the aforementioned natural
carbohydrates include general sugars, such as monosaccharides, for
example, glucose, fructose, and the like; disaccharides, for
example, maltose, sucrose, and the like; and polysaccharides, for
example, dextrin, cyclodextrin, and the like, and sugar alcohols,
such as xylitol, sorbitol, and erythritol. As the flavoring agent
other than the above examples, natural flavoring agents (thaumatin,
stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and
synthetic flavoring agents (saccharin, aspartame, etc.) may be
advantageously used. A ratio of the natural carbohydrate may be
generally about 1 to 20 g and preferably about 5 to 10 g per 100 g
of the composition of the present invention.
[0125] Further, the health food composition according to the
present invention may contain various nutrients, vitamins, minerals
(electrolytes), flavoring agents such as synthetic flavoring agents
and natural flavoring agents, coloring agents and thickening agents
(cheese, chocolate, etc.), pectic acid and salts thereof, alginic
acid and salts thereof, organic acid, a protective colloidal
thickener, a pH adjusting agent, a stabilizer, a preservative,
glycerin, alcohol, a carbonic acid agent used in a carbonated
drink, or the like. Besides, the health food composition of the
present invention may include pulps for preparing natural fruit
juice and fruit juice drinks, and vegetable drinks.
[0126] These ingredients may be used independently or in
combination. The ratio of such additives is not limited, but is
generally selected in the range of 0.1 to about 20 parts by weight
per 100 parts by weight of the hypometabolism-inducing substance of
the present invention.
[0127] Further, the present invention provides a pharmaceutical
composition for facial muscle shrinkage containing a
hypometabolism-inducing substance of the present invention as an
active ingredient.
[0128] In one embodiment of the present invention, since the
hypometabolism-inducing substance of the present invention induces
muscular atrophy by inhibiting activity of Akt1-S6K involved in
muscle protein synthesis and activating FoxO-proteasome involved in
muscle protein degradation, the hypometabolism-inducing substance
may be usefully used as the composition for facial muscle shrinkage
which may be used for Botox.
[0129] Hereinafter, the present invention will be described in more
detail through Experimental Examples according to the present
invention, but the scope of the present invention is not limited to
Experimental Examples to be described below and the like.
Experimental Examples
1. Experimental Substances and Method
1) Chemicals and Storage Solutions
[0130] T1AM was chemically synthesized (Korean Patent Registration
No. 1,112,731) and dissolved in dimethyl sulfoxide (DMSO; SIGMA,
Missouri, US) at a storage concentration of 0.75 and 1 M. A DMEM
(Welgene, Dalseogu, Daegu, Korea) medium was used, and nonidet
P-40, a complete mini protease inhibitor, and a phosphatase
inhibitor cocktail were purchased from Roche. A RIPA buffer
solution (11% Nonidet P-40, 1% sodium deoxycholate, 150 mM NaCl, 10
mM sodium phosphate [pH 7.4], 2 mM EDTA, 50 mM NaF, 0.2 mM
Na.sub.3VO.sub.4, 40 mM HEPES [pH 7.4], 0.7% CHAPS, 1% SDS, and
protease inhibitor cocktail) was used for protein extraction. An
ECL system purchased from GE Healthcare (Fairfield, Conn., USA) and
stored at 4 and a restore western blot stripping buffer purchased
from Thermo Scientific (Rockford, Ill., USA) were used for
immunoblot analysis. Rabbit anti-phospho-AMPK (at Thr172)), AMPK,
phospho-FoxO1 (Ser256), FoxO1, phospho-FoxO3 (Ser253), FoxO3,
HSP27, .alpha.B-crystallin, phosphor-S6K (Thr389), S6K,
phospho-Akt1 (Ser473), and Akt1 polyclonal antibodies were
purchased from Cell Signaling Technology (Beverly, Calif., USA) and
used. Rabbit anti-muscle RING-finger protein-1 (MuRF1) and F-Box
Only Protein 32 (MAFbx/atrogen) polyclonal antibody were purchased
from Santa Cruz Biotechnology (Santa Cruz, Calif., USA) and used,
and mouse anti-heat shock proteins (HSP) 90, 72, and 60 were
purchased from Stressgen (Victoria, BC, Canada) and used. Mouse
anti-glyceraldehydes-3-phosphate dehydrogenase (GAPDH) antibodies
were purchased from Abcam (Cambridge, UK) and HRP-conjugated
anti-mouse IgG and anti-rabbit IgG were purchased from Cell
Signaling Technology and used.
2) Cell Culture
[0131] C2C12 myoblasts were purchased from American Type Culture
Collection (Rockville, Md., USA) and cultured in a DMEM medium
containing 4,500 mg/L glucose supplemented with 10% fetal bovine
serum (Hyclone, Logan, Utah, USA) and 1% antibiotics/antimycotics
Gibco, Burlington, Ontario, Canada). The myoblasts were stored
under conditions of 37.degree. C. and 5% CO.sub.2. The myoblasts
were grown on a 6-well culture plate for immunoblot analysis and
measurement of diameters of myotubes. The myoblasts were maintained
in each well for 5 days by replacing the medium with a
differentiation medium (DMEM containing 2% horse serum and 1%
antibiotics/antimycotics) at about 80% confluent state and induced
to be differentiated into myotubes. The medium was replaced with a
new medium every two days.
3) Measurement of Cell Size
[0132] To verify the effect of T1AM on the size of C2C12 myotubes,
the cells were fixed with 4% paraformaldehyde and photographed at
200.times. magnification on an Axiovert 200 optical microscope. For
analysis, the cells were divided into 9 fractions in order to
randomly select the cells. The diameter of each myotube was
measured using Image J software (NIH, Frederick, Md., USA).
4) Immunoblot Analysis
[0133] The cells were obtained with a RIPA buffer, degraded by
repeated suction through a 21 gauge needle, and the transferred to
a 1.5 mL microtube. A sample was cultured on ice for 5 minutes and
centrifuged at 13,000 rpm at 4.degree. C. for 10 minutes. A
supernatant was obtained with whole-cell soluble lysates and the
protein concentration was determined through Bradford assay. To
detect AMPK, phospho-AMPK (p-AMPK), FoxO1, p-FoxO1, FoxO3, p-FoxO3,
Akt1, p-Akt1, S6K, p-S6K, MuRF1, MAFbx, HSP90, HSP72, HSP60, HSP27,
.alpha.B-crystallin, and GAPDH, a total of 30 .mu.g of proteins was
electrophoresed on 8 to 10% SDS-PAGE.
[0134] The proteins were electrophoretically transferred from the
gel to a nitrocellulose membrane. The membrane reacted with a
blocking buffer (1X TBS, 0.5% Tween-20 with 5% w/v nonfat dry milk)
for 1 hour at room temperature and then washed with 10 mL TBST
three times every 10 minutes. Thereafter, the membranes reacted
with a primary antibody diluted appropriately with 10 mL TBST
(1:500 to 1:10,000) overnight at 4.degree. C. The membrane reacted
with a HRP-conjugated secondary antibody for detection of bound
proteins in 10 mL TBST at room temperature for 1 hour by stirring
and then washed with 10 mL TBST three times every 10 minutes. An
immunocomplex was detected by the ECL system (GE Healthcare,
Fairfield, Conn., USA) and the obtained bands were quantified by
ImageJ 1.47t software (NIH, MD, USA). The protein density was
normalized by the density of GAPDH. To detect the GAPDH, the
membrane was washed with TBST three times for every 10 minutes and
then cultured in a restore buffer for 30 minutes at room
temperature to be stripped.
5) Immunofluorescence and Confocal Microscope
[0135] The cells on each 6-well plate were washed three times with
1.times. PBS and fixed with 4% paraformaldehyde for 30 minutes at
room temperature. Thereafter, the cells were then treated with 0.2%
Tritin X-100 for 10 minutes on ice to ensure permeability and
blocked from the 1.times. PBS with 3% BSA. The cells were stained
with primary antibodies against FoxO1 and FoxO3 diluted at 1:100 in
1.times. PBS, respectively, and reacted with Alexa 488-conjugated
secondary antibody diluted at 1:1,000. Finally, the cells were
washed three times with 1.times. PBS and then a mounting medium
containing DAPI (Vector Laboratories, Burlingame, Calif., USA) was
dropped on the cells. Fluorescent-labeled cells were detected with
a Carl Zeiss LSM750 confocal microscope (Jena, Germany).
6) Analysis of Activity of 26S Proteasome
[0136] Two groups of myotubes were trypsinized and then washed with
a fresh differentiation medium. Among three determinants of
proteasome activities (trypsin-, chemotrypsin- and caspase-like
activities), the chemotrypsin-like activity is regarded as
representative of the protease capacity of the proteasome. The
chemotrypsin-like activity was determined according to the
manufacturer's protocol using a Promega Proteasome-Glo cell-based
luminescence assay kit (Promega, Madison, Wis., USA) by
approximately 7,500 cells measured by a cell counter (Biorad,
Hercules, Calif., USA) in 50 .mu.l of the differentiation medium.
To confirm the specificity of the analysis, a partial sample
containing the same number of cells was pretreated with a
proteasome inhibitor, epoxomicin, at a concentration of 10 .mu.M
for 30 minutes. The chemotrypsin-like activity was measured by the
same process and the result was used as a background signal for
analysis. The luminescence was measured with a GloMax 20/20
Luminometer (Promega).
7) Statistical Analysis
[0137] All values corresponding to the measurement result were
represented by mean.+-.SEM. A difference between the groups in
means for biochemical measurement (e.g., AMPK, Akt1, etc.) was
verified by an independent sample t-test. Statistical analysis was
performed using SPSS/PC+, and significance was determined at
P=0.05.
2. Experimental Result
1) Muscular Atrophy Effect of T1AM in Muscle Cells
[0138] To determine whether T1AM induced muscular atrophy in C2C12
myotubes, cells were photographed under a phase contrast microscope
(FIG. 1A) and the diameter was measured at 200.times. magnification
(FIG. 1B).
[0139] As a result, as shown in FIGS. 1A-1B, it was shown that when
75 .mu.M of T1AM was treated for 6 hours, the size of myotube was
decreased by 0.13 times as compared to a vehicle control
(16.97.+-.0.32 m).
2) Increase in AMPK Phosphorylation in T1AM-Treated Cells
[0140] As shown in FIGS. 2A-2C, it was shown that the AMPK
phosphorylation was remarkably increased (2.7 times) in the
T1AM-treated group compared to the control in the immunoblotting
analysis, whereas the total expression levels of AMPK were similar
between the two groups. As a result, the expression ratio of
p-AMPK/AMPK in the T1AM-treated group was 2.0 times higher than
that of the control (FIG. 2C).
3) Down-Regulation Of Anabolic Signaling Activity in T1AM-Treated
Cells
[0141] As shown in FIGS. 3A-3E, it was shown that a phosphorylation
level of Akt1 was significantly down-regulated in the T1AM-treated
group compared to the control, but the non-phosphorylation level
between the two groups was similar to each other. Accordingly, the
expression ratio of p-Akt1/Akt1 in the T1AM-treated group was 0.45
times lower than that of the control (FIG. 3C). Further, the p-S6K
level was lowered by T1AM treatment and as a result, the expression
ratio of p-S6K/S6K in the T1AM-treated group was 0.53 times lower
than that of the control (FIG. 3E).
4) Down-Regulation of p-FoxO1 and p-FoxO3 in T1AM Treated Cells
[0142] As shown in FIGS. 4A-4F, it was shown that the total
expression of FoxO1 in the T1AM-treated group was 2.5 times higher
than that of the control (in Ser256), whereas the phosphorylation
level between the two groups was similar. It was shown that the
expression ratio of p-FoxO1/FoxO1 was 0.66 times lowered in the
T1AM-treated group (FIG. 4D). On the other hand, it was shown that
the total expression of FoxO3 was not different between the
T1AM-treated group and the control, but the p-FoxO3 level was 0.58
times lowered in the T1AM-treated group. Thus, the expression ratio
of p-FoxO3/FoxO3 in the T1AM-treated group was 0.39 times lower
than of the control (FIG. 4F).
5) Up-Regulation of MuRF1 Expression and Proteasome Activity
[0143] As shown in FIGS. 5A-5D, among experimented catabolic
signaling markers, the expression of MuRF1 in the T1AM-treated
group was 1.8 times higher than that in the control (FIGS. 5A and
5B), whereas the expression of MAFbx was not affected by the T1AM
treatment (FIGS. 5A and 5C). The chymotrypsin-like activity, one of
the major catabolic properties of proteasome, in the T1AM-treated
group was 1.5 times higher than that of the control (FIG. 5D).
6) Decrease in Expression of HSP72 and .alpha.B-crystallin in T1AM
Treated Cells
[0144] As shown in FIGS. 6A-6D, it was shown that the expression
levels of HSP72 and .alpha.B-crystallin in the T1AM-treated group
were 0.89 times and 0.63 times down-regulated compared to the
control, whereas a difference in HSP60 expression between the two
groups was not statistically significant.
3. Conclusion
[0145] The activity of FoxOs is known to be regulated by an
antagonistic effect of AMPK and Akt1. That is, a decrease in
expression ratio of p-FoxO/FoxO corresponds to up-regulated p-AMPK
and corresponds to down-regulated p-Akt1. This induces protein
degradation as one of the catabolism. As seen from the above
experimental results, AMPK, FoxO1, FoxO3, MuRF1 and proteasome
involved in the muscle protein degradation mechanism are activated
by T1AM mediated hypometabolism, whereas AKT1, S6K, heat shock
protein 72 (HSP72), and .alpha.B-crystallin involved in the muscle
protein synthesis mechanism are inactivated. Therefore, the
hypometabolism-inducing substance according to the present
invention, particularly T1AM, induces hypometabolism to inhibit
energy metabolism and activating the catabolism, thereby activating
the protein associated with the muscle protein degradation
mechanism and inhibiting the proteins associated with the muscle
protein synthesis mechanism, and as a result, the sizes of the
myoblasts are decreased.
* * * * *