Composition for promoting muscle regeneration and muscle growth

Abstract

The present invention relates to use of a growth factor, augmenter of liver regeneration (GFER) protein or GFER protein derivative for enhancing muscle function and regulating metabolism for the prevention, amelioration or treatment of muscle loss and muscle-related diseases caused by aging.

Description

Composition for promoting muscle regeneration and muscle growth

[0001] The present invention relates to a composition for promoting muscle regeneration and muscle growth, comprising GFER (Growth Factor, Augmenter of Liver Regeneration) or a GFER protein derivative as an active ingredient, and its use. By enhancing muscle function and regulating metabolism, the composition can prevent, improve, or treat muscle loss due to aging and muscle-related diseases.

[0002] Sarcopenia, also known as muscle atrophy, is a progressive loss of muscle mass and strength, leading to decreased physical performance and increased frailty, a condition that significantly impacts older adults. Key mechanisms of muscle aging include decreased muscle production, impaired protein synthesis, and mitochondrial dysfunction. However, current treatments for sarcopenia primarily involve exercise and nutritional modifications. While oral probiotics that simultaneously inhibit activin II and myostatin exist, effective pharmacological treatments remain scarce.

[0003] The GFER (Growth Factor, Augmenter of Liver Regeneration) gene, also known as augmenter of liver regeneration (ALR), is essential for mitochondrial function and liver regeneration. Located on chromosome 16p13.3, GFER is a protein that plays a key role in oxidative phosphorylation and protects cells from apoptosis. The protein exists in various isoforms, with the short form involved in mitochondrial electron transport and redox reactions, and the long form involved in cytoplasmic functions. GFER promotes electron transport in the mitochondrial electron transport chain, which is crucial for ATP production, maintains mitochondrial membrane potential, thereby reducing oxidative stress. It is also known to promote liver regeneration by enhancing hepatocyte proliferation after liver damage.

[0004] The present inventors have made extensive efforts to discover a substance that can alleviate age-related muscle loss and metabolic dysregulation, and as a result, have discovered that administering GFER and GFER protein derivatives to muscles can increase the expression of muscle growth biomarkers, improve muscle morphological differentiation, increase muscle fusion index, and increase the level of muscle growth-related mRNA expression, thereby treating age-related muscle loss, muscle damage, muscle dysfunction, and metabolic diseases associated with sarcopenia, thereby completing the present invention.

[0005] One object of the present invention is to provide a composition for promoting muscle regeneration or muscle growth.

[0006] Another object of the present invention is to provide a composition for preventing, improving or treating muscle-related diseases.

[0007] Another object of the present invention is to provide a method for promoting muscle regeneration or muscle growth.

[0008] Another object of the present invention is to provide a method for preventing, improving or treating muscle-related diseases.

[0009] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0010] Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques have not been described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0011] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0012]

[0013] According to one embodiment of the present invention, the present invention relates to a composition for promoting muscle regeneration or muscle growth.

[0014] In the present invention, the composition may include, as an active ingredient, at least one selected from the group consisting of GFER (Growth Factor, Augmenter of Liver Regeneration) protein; and a gene encoding GFER protein.

[0015] In the present invention, the GFER protein may be represented by sequence number 1, and includes a protein having functionally equivalent properties to the protein.

[0016] In the composition of the present invention, a protein having functionally equivalent properties refers to a protein having at least 70%, preferably 80%, and more preferably 90% sequence homology with the amino acid sequence of the GFER protein as a result of addition, substitution, or deletion of amino acids, and having substantially equivalent physiological activity to the protein. As a non-limiting example, it may have a homology of 99% to less than 100%, 95% to less than 99%, 90% to less than 95%, 85% to less than 90%, or 80% to less than 85% with the amino acid sequence of the GFER protein, and all of these may be included without limitation within a range that is obvious to a person skilled in the art to exhibit the desired effect of the present invention.

[0017] In the present invention, the GFER protein is characterized in that it is a wild-type GFER protein or a GFER protein derivative having substantially equivalent physiological activity as a protein having functionally equivalent characteristics.

[0018] In the present invention, the term "GFER protein derivative" may refer to a GFER protein comprising an additional domain capable of enhancing the stability or half-life of the GFER protein in the body. For example, it may be a protein having an Fc domain or PEG conjugated to the N-terminus or C-terminus of the GFER protein. However, the term "GFER protein derivative" is not limited thereto and may include any domain known to improve the stability or half-life of the target protein.

[0019] In the present invention, the composition may be included as a gene encoding the GFER protein or an expression vector encoding the GFER protein, and may be specifically included in a form included in a recombinant expression vector, but is not limited thereto.

[0020] In the present invention, the gene encoding the GFER protein may be represented by SEQ ID NO: 2, and may be provided in the form of a vector that expresses the gene within a cell for use in gene therapy, etc. By injecting an expression vector containing the gene encoding the GFER protein, the expression level of the gene can be increased.

[0021] The recombinant expression vector of the present invention is a recombinant expression vector capable of expressing a desired protein or peptide in a desired host cell, and refers to a genetic construct containing essential regulatory elements operatively linked to express a gene insert. The expression vector contains expression regulatory elements such as an initiation codon, a stop codon, a promoter, and an operator. The initiation codon and the stop codon are generally considered to be part of a nucleotide sequence encoding a polypeptide, and must exhibit function in a subject when the genetic construct is administered, and must be in frame with the coding sequence. The promoter of the vector may be a constitutive or inducible promoter.

[0022] The recombinant expression vector of the present invention may be one in which a promoter and a base sequence constituting a gene encoding the GFER protein of the present invention, i.e., a polynucleotide, are operably linked. The term "operably linked" in the present invention refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA are functionally linked to perform a general function. For example, a promoter and a polynucleotide encoding a protein or RNA may be operably linked to affect the expression of the coding sequence. An operably linked vector with a recombinant expression vector can be produced using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art.

[0023] The vector that can be used as the backbone of the recombinant expression vector of the present invention is not particularly limited as long as it can produce the protein of the present invention, and includes, for example, plasmid DNA, phage DNA, commercially developed plasmids (pGEM® T vector, pET22b, pUC18, pBAD, pIDTSAMRT-AMP, etc.), Escherichia coli-derived plasmids (pYG601BR322, pGEX-4T-1, pET, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, AAAAAλgt11, λZAP, etc.), animal virus vectors (Retrovirus, Adenovirus, Vaccinia virus, etc.), insect virus vectors (Baculovirus, etc.), but are not limited thereto.

[0024] The host cell of the present invention includes an individual cell or cell culture that may be or has been the recipient of the vector(s) for incorporation of the polypeptide insert. The host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (morphologically or in genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutation. The host cell includes a cell that has been transfected in vivo with the polypeptide(s) of the present invention.

[0025] In the present invention, the host cell may include a mammalian, plant, insect, fungal or cellular cell, and for example, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella Typhimurium; fungal cells such as yeast cells and Pichia pastoris; insect cells such as Drozophylla and Spodoptera Sf9 cells; animal cells such as CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bow melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells), or PERC.6 (human retinal cells); Alternatively, the host cell may be a plant cell, preferably a human cultured cell line, such as a HEK cell line, but is not limited thereto, and any cell known to those skilled in the art that can be used as a host cell may be used.

[0026] In the present invention, the composition can treat or prevent muscle damage by regenerating or growing muscles by proliferating and activating the GFER protein, the gene encoding the protein itself, an expression vector containing the gene, or a host cell containing the expression vector, and administering the composition to a patient.

[0027] In the present invention, the composition acts on the process of differentiating myoblasts into myotubes to improve the speed of muscle synthesis and increase the expression of muscle formation genes, thereby ultimately improving muscle function through promotion of muscle regeneration and muscle growth.

[0028] In the present invention, when the composition is applied to sarcopenia, which is a condition in which muscle strength declines due to a decrease in skeletal muscle mass during aging, it can significantly alleviate age-related muscle decline and metabolic dysregulation, and improve not only muscle function but also body composition.

[0029] In the present invention, the composition can be used in the form of a pharmaceutical composition or a food composition.

[0030] The pharmaceutical composition of the present invention may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder or beverage, and the pharmaceutical composition may be characterized as being intended for humans.

[0031] The pharmaceutical composition of the present invention is not limited thereto, but may be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, as well as external preparations, suppositories, and sterile injection solutions, each according to a conventional method. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, fragrances, etc. for oral administration, and buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used for injections, and bases, excipients, lubricants, preservatives, etc. may be used for topical administration. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing it with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dose ampoules or multiple doses. In addition, it can be formulated in the form of solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0032] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0033] Routes of administration of the pharmaceutical composition of the present invention include, but are not limited to, intramuscular, oral, intravenous, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is particularly preferred.

[0034] The "parenteral" of the present invention includes intramuscular, subcutaneous, intradermal, intravenous, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Preferably, the pharmaceutical composition of the present invention may be administered in a form suitable for intramuscular administration, but is not limited thereto.

[0035] The pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, dosage form, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, a dragee, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0036] The food composition of the present invention can be manufactured into various food products, such as beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, etc. Since the food composition of the present invention is composed of plant extracts with almost no toxicity or side effects, it can be safely used even for long-term use for preventive purposes.

[0037] When the composition of the present invention is included in a food composition, the amount may be added in a ratio of 0.1 to 50% of the total weight.

[0038] Here, when the food composition is manufactured in the form of a beverage, there are no special restrictions other than containing the food composition in the indicated ratio, and various flavoring agents or natural carbohydrates, etc. may be contained as additional ingredients like in a typical beverage. That is, as a natural carbohydrate, it may include a monosaccharide such as glucose, a disaccharide such as fructose, a polysaccharide such as sucrose, a dextrin, a cyclodextrin, a typical sugar such as dextrin, a cyclodextrin, a sugar alcohol such as xylitol, sorbitol, and erythritol. As the flavoring agent, there may be mentioned a natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and a synthetic flavoring agent (saccharin, aspartame, etc.).

[0039] In addition, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0040] These components can be used independently or in combination. The proportions of these additives are not particularly critical, but are typically selected within the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

[0041]

[0042] According to another embodiment of the present invention, the present invention relates to a composition for preventing, improving, or treating muscle diseases.

[0043] In the present invention, the composition may include, as an active ingredient, at least one selected from the group consisting of GFER (Growth Factor, Augmenter of Liver Regeneration) protein; and a gene encoding GFER protein.

[0044] In the present invention, the GFER protein is characterized in that it is a GFER protein or a GFER protein derivative.

[0045] In the present invention, the composition can treat or prevent muscle-related diseases by regenerating or growing muscles by proliferating and activating the GFER protein, the gene encoding the protein itself, an expression vector containing the gene, or a host cell containing the expression vector, and administering the composition to a patient.

[0046] In the present invention, the muscle-related disease may be one selected from the group consisting of sarcopenia, muscle damage, muscle atrophy, muscle rigidity, hypotonia, myotonic dystrophy, muscular atrophy, myasthenia gravis, muscular degeneration, muscular dystrophy, and myositis, and specifically, may be one selected from the group consisting of sarcopenia, muscle damage, muscle atrophy, muscular dystrophy, muscular atrophy, muscular dystrophy, and muscular dystrophy, but is not limited thereto.

[0047] In the present invention, the composition can be used in the form of a pharmaceutical composition or a food composition.

[0048] In the present invention, the term "prevention" may include, without limitation, any act that can block symptoms caused by a muscle-related disease or inhibit or delay the progression of a muscle-related disease by using the composition of the present invention.

[0049] In the present invention, the term "improvement" may include, without limitation, any act in which symptoms caused by muscle-related diseases due to aging or muscle damage are improved or beneficially changed by using the composition of the present invention.

[0050] As used herein, "treatment" refers to a series of activities performed to alleviate or improve a desired disease. For the purposes of the present invention, treatment encompasses activities that suppress or delay muscle-related diseases, and may include, without limitation, any action that alleviates or benefits symptoms caused by muscle-related diseases.

[0051] In the composition for preventing, improving or treating the muscle-related disease of the present invention, the description of GFER, GFER protein, GFER protein derivative, pharmaceutical composition, food composition, etc. overlaps with what has been described above, and thus, in order to avoid excessive complexity of the specification, a detailed description thereof is omitted below.

[0052]

[0053] According to another embodiment of the present invention, the present invention relates to a method for promoting muscle regeneration or muscle growth.

[0054] The method of the present invention may include a step of administering to a subject in need of administration an effective amount of at least one selected from the group consisting of GFER (Growth Factor, Augmenter of Liver Regeneration) protein; and a gene encoding the GFER protein.

[0055] An example of the present invention may include, but is not limited to, a step of proliferating and activating the GFER protein, the gene encoding the protein itself, an expression vector including the gene, or a host cell including the expression vector, and administering the same in an effective amount.

[0056]

[0057] According to another embodiment of the present invention, the present invention relates to a method for preventing or treating muscle-related diseases.

[0058] The method of the present invention may include a step of administering to a subject in need of administration an effective amount of at least one selected from the group consisting of GFER (Growth Factor, Augmenter of Liver Regeneration) protein; and a gene encoding the GFER protein.

[0059] An example of the present invention may include, but is not limited to, a step of proliferating and activating the GFER protein, the gene encoding the protein itself, an expression vector containing the gene, or a host cell containing the expression vector, and administering the same in an effective amount.

[0060] In the method for promoting muscle regeneration or muscle growth of the present invention; and the method for preventing or treating muscle-related diseases, the description of the GFER protein, the gene encoding the GFER protein, the expression vector containing the gene, or the host cell containing the expression vector, etc., is the same as described above, and thus is omitted to avoid excessive complexity of the present specification.

[0061] In the present invention, the term “administration” means providing a predetermined composition of the present invention to a subject by any appropriate method.

[0062] In the present invention, the "subject" requiring the administration may include both mammals and non-mammals. Here, examples of mammals include, but are not limited to, humans, non-human primates such as chimpanzees, other apes, or monkey species; livestock animals such as cows, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, or cats; and laboratory animals such as rodents such as rats, mice, or guinea pigs. In addition, examples of non-mammals in the present invention may include, but are not limited to, birds or fish.

[0063] In the present invention, the formulation of the composition administered as described above is not particularly limited, and may be administered as a solid formulation, a liquid formulation, or an aerosol formulation for inhalation, and may be administered as a solid formulation intended to be converted into a liquid formulation for oral or parenteral administration immediately before use, and may be administered by being formulated in the form of, for example, oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, but is not limited thereto.

[0064] In addition, in the present invention, a pharmaceutically acceptable carrier may be additionally administered together with the composition of the present invention during the administration. Here, the pharmaceutically acceptable carrier may include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a fragrance, etc. for oral administration, and may include a mixture of a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, a stabilizer, etc. for injections, and may include a base, an excipient, a lubricant, a preservative, etc. for topical administration. The formulation of the compound of the present invention may be prepared in various ways by mixing it with the pharmaceutically acceptable carrier described above. For example, the formulation may be prepared in the form of a tablet, troche, capsule, elixir, suspension, syrup, wafer, etc. for oral administration, and the formulation may be prepared in the form of a unit dosage ampoule or a multiple dosage form for injections. Others can be formulated as solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0065] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0066] Routes of administration of the composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0067] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0068] As used herein, a "pharmaceutically effective amount" refers to a sufficient amount of an agent to produce a desired biological result. This result may be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. For example, an "effective amount" for therapeutic purposes is the amount of a composition disclosed herein required to produce a clinically significant reduction in a disease. The appropriate "effective" amount in any individual case can be determined by one skilled in the art using routine experimentation. Accordingly, the expression "effective amount" generally refers to an amount of an active agent that has a therapeutic effect. In the present invention, the active agent is a therapeutic agent for preventing, improving, or treating muscle disorders, or for promoting muscle regeneration or muscle growth.

[0069] The composition of the present invention may vary depending on various factors including the activity of the active substance used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the active substance may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 100 mg / kg or 0.001 to 100 mg / kg per day. Administration may be administered once a day or divided into several times. The above dosage does not limit the scope of the present invention in any way. The compound according to the present invention may be formulated as a pill, a dragee, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0070] The active substance of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

[0071] The present invention relates to a composition for promoting muscle regeneration and muscle growth, comprising GFER (Growth Factor, Augmenter of Liver Regeneration) and a GFER protein derivative as active ingredients, and a method using the same. The composition of the present invention can prevent, improve, or treat muscle loss due to aging and muscle-related diseases by enhancing muscle function and regulating metabolism.

[0072] Figure 1a is a diagram showing the correlation between GFER expression by age group in skeletal muscle according to one embodiment of the present invention.

[0073] FIG. 1b is a diagram showing the correlation between GFER expression in healthy individuals or sarcopenic individuals according to one embodiment of the present invention.

[0074] FIG. 2 is a schematic diagram illustrating an experiment for GFER treatment of wild-type C2C12 and Split-C2C12 cells, which are muscle cells, according to one embodiment of the present invention.

[0075] FIG. 3 is a diagram showing the results of quantifying the expression levels of MyoG and MHC, which are myogenic biomarkers, according to changes in GFER capacity in C2C12 cells according to one embodiment of the present invention, using Western blotting.

[0076] FIG. 4a and FIG. 4b are diagrams showing the results of confirming GFP signal intensity to demonstrate the effect of GFER / ALR treatment on muscle formation and muscle fusion in Split-C2C12 cells according to one embodiment of the present invention.

[0077] FIG. 5 is a diagram showing the results of comparing the number of nuclei per root canal, the percentage of root canals, and the number of mature root canals between the GFER / ALR treatment group and the control group according to one embodiment of the present invention.

[0078] Figure 6 is a diagram showing the results of measuring the expression levels of MyoD, MyoG, Myh1, and Myh3 by performing RT-qPCR in wild-type C2C12 cells after GFER / ALR treatment according to one embodiment of the present invention.

[0079] FIG. 7a and FIG. 7b are diagrams showing the results of measuring the expression level of myogenic markers (MyoD, MyoG, MHC) through Western blotting in C2C12 cells after GFER / ALR treatment according to one embodiment of the present invention.

[0080] FIG. 8a and FIG. 8b are diagrams showing the results of protein synthesis of C2C12 cells after GFER treatment through SUnSET analysis according to one embodiment of the present invention and the results of quantifying Western blot signals using ImageJ software.

[0081] Figure 9 is a diagram showing an experimental design for evaluating the effect of in vivo GFER administration according to one embodiment of the present invention.

[0082] Figure 10 is a diagram showing the results of confirming the effect of GFER / ALR injection on the body weight of mice for about two months according to one embodiment of the present invention.

[0083] Figures 11a and 11b are diagrams showing changes in body fat mass and lean body mass by GFER measured by DEXA according to one embodiment of the present invention.

[0084] Figure 12 is a diagram showing the effect of improving muscle function using a hang test after GFER / ALR administration according to one embodiment of the present invention.

[0085] Figures 13a and 13b are diagrams showing the effect of improving muscle endurance using an endurance test after GFER administration according to one embodiment of the present invention.

[0086] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0087]

[0088] [Experimental Method]

[0089] cell culture

[0090] Wild-type C2C12 and Split-C2C12 mouse myoblast cell lines were purchased from the American Type Culture Collection (ATCC, CRL-1772) and Applied Biological Materials (T8003, T8004), respectively, and used in the experiments. Cells were cultured in growth medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S) in high-glucose DMEM (Dulbecco's Modified Eagle Medium). To promote myogenic differentiation, 2% horse serum (HS), inactivated at 56°C for 30 min, was added to high-glucose DMEM.

[0091]

[0092] GFER / ALR preparation and administration

[0093] GFER / ALR (Growth Factor, Augmenter of Liver Regeneration / augmenter of liver regeneration, GFER / ALR) was purchased from Novopro (Cat#503686) and diluted with autoclaved distilled water before use. Experiments were performed using GFER treatment concentrations ranging from 2 ng / mL to 50 ng / mL. 1) Cells were treated with GFER / ALR for 5 days from differentiation day 0, and the medium was replaced every 24 or 48 hours. Cells were harvested after 5 days of differentiation. 2) Cells were treated with GFER / ALR on day 5 after changing to differentiation medium and harvested after an additional 5 days.

[0094]

[0095] Western Blot

[0096] Cells were harvested and protein extracted by adding protease duo inhibitor to RIPA buffer. Protein concentration was measured using BCA assay. 20 μg of protein per sample was loaded onto an SDS-PAGE gel and transferred to a PVDF membrane.

[0097] Primary antibodies against MyoD (MA1-41017, Invitrogen), MyoG (MAB3876, Merck), MHC (MAB4470, R&D system, Bio-Techne brand), and beta-actin (loading control) were used, and HRP-conjugated secondary antibodies were used as secondary antibodies.

[0098]

[0099] RT-qPCR

[0100] RNA was extracted from cells using Trizol reagent, and cDNA was synthesized from the extracted RNA using a reverse transcriptase kit. Specific primers for MyoD, MyoG, Myh1, and Myh3 were used for detection (see Table 1). RT-qPCR was performed under cycling conditions of 40 cycles, and gene expression levels were compared with relative expression levels using beta-actin using ΔΔCt. Bands were visually confirmed using ECL solution and quantified and detected using ImageJ software.

[0101]

[0102] Observation of morphological differentiation

[0103] Observation was performed using a fluorescence microscope. Split-C2C12 cells were seeded with GFP-10 and GFP11 cells at a 2:1 ratio, and GFER / ALR was treated together with the differentiation medium from differentiation day 0. GFP signals were observed using a Bio Tek Cytation 5 Cell Imaging Multimode Reader, and fluorescence intensity was recorded daily for 6 days.

[0104]

[0105] Muscle fusion index: MHC staining

[0106] Fixation and permeabilization: Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and prevented non-specific binding with 1% BSA.

[0107] Staining: Cells were incubated overnight with MHC primary antibody, then incubated with Alexa Fluor-conjugated secondary antibody, and nuclei were stained with DAPI.

[0108] Quantification: Images were captured and the number of root canals, nuclei per root canal, and mature root canals (≥5 nuclei) were quantified using Myocount software.

[0109]

[0110] Protein production rate analysis: SUnSET analysis

[0111] Puromycin labeling: Cells were treated with puromycin (10 ug / mL) for 60, 90, or 120 minutes.

[0112] Western blotting analysis: Newly synthesized proteins were detected and quantified using anti-puromycin antibodies.

[0113]

[0114] In vivo GFER administration and body weight measurement

[0115] Mice were administered GFER at 5 ng / g / day via intraperitoneal injection daily, and the weight of mice was measured every 3 days, and lean body mass and body fat mass were measured using InAlyzer.

[0116]

[0117] Muscle function tests

[0118] Hang Test

[0119] After all four legs of the mouse were suspended, the hanging time was measured. A 10-minute rest period was provided between each test, and three measurements were taken for each mouse, and the average was calculated. Before the first test, the mice were allowed to acclimate to the testing equipment by hanging for approximately one minute the day before.

[0120] Endurance Test

[0121] The day before the first endurance test, mice were given a 5-minute warm-up at 5 m / min to acclimate to the treadmill. The endurance test began at 10.8 m / min and increased by 1.8 m / min every 12 minutes. The endurance test was continued until the mice were exhausted and could no longer run.

[0122]

[0123] Statistical analysis

[0124] All data are expressed as mean ± standard deviation (SD), and statistical significance was assessed using one-way ANOVA and Tukey's post hoc test. A P value less than 0.05 was considered statistically significant.

[0125]

[0126] [Experimental Results]

[0127] Confirmation of the association between GFER expression and age and sarcopenia in skeletal muscle.

[0128] To compare the expression pattern of GFER according to age, the expression level of GFER by age group was confirmed in GTEx skeletal muscle data and is shown in Figure 1a. The number of samples for each age group was 67 in the 20s, 65 in the 30s, 124 in the 40s, 255 in the 50s, 263 in the 60s, and 28 in the 70s. As a result of confirming GFER expression in various age groups, it was confirmed that the expression level of GFER showed a negative correlation with age (Spearman's Rho was -0.094, p-value was 0.0079). This suggests that GFER expression tends to decrease with age.

[0129] To compare GFER expression between healthy controls and sarcopenia patients, the group-specific expression levels of GFER identified from the NCBI GEO (GSE111017) sarcopenia data are shown in Figure 1b. The number of healthy individuals was 86, and the number of sarcopenic individuals was 32. The Student's t-test result showed a p-value of 0.0103, confirming that the GFER expression level in the sarcopenic group was statistically significantly reduced compared to the healthy group.

[0130] Thus, the age-related decrease in GFER expression and the decrease in GFER expression in sarcopenic patients suggest that GFER has an important effect on muscle maintenance.

[0131]

[0132] In vitro confirmation of the effects of GFER on muscle

[0133] To determine the effect of GFER / ALR treatment on mouse wild-type C2C12 cells and Split-C2C12 myoblasts, an overview of the experiments performed to test the effect of GFER on muscle in vitro, including wild-type C2C12 cell and Split-C2C12 cell culture conditions, evaluation methods for myogenesis and protein synthesis, and mitochondrial function, is schematically shown in Fig. 2. Specifically, cells were initially cultured in growth medium containing 10% FBS, and when cells reached 100% confluency, the medium was switched to differentiation medium containing 2% HS to promote myogenic differentiation. During differentiation, cells were treated with 50 ng / ml GFER / ALR, and cellular processes such as myogenesis, myogenic differentiation process, and protein synthesis were evaluated. Cell viability was assessed using the MTT assay, and myogenesis was confirmed by morphology, myogenic biomarkers, and gene expression. Protein turnover was analyzed by measuring the rates of protein synthesis and degradation, and mitochondrial function was evaluated by assessing oxygen consumption rate (OCR), ATP production, mitochondrial membrane potential (MMP), and mtDNA (mitochondrial DNA).

[0134]

[0135] Effects of GFER Dosage on C2C12 Myogenesis

[0136] To investigate the myogenic effects of C2C12 according to the change in GFER dose, mouse myoblast cell line C2C12 was used. To ensure cell stability, cells were thawed, passaged three times, and then cultured in high glucose DMEM supplemented with 10% fetal bovine serum (FBS) for 2 days. Subsequently, the medium was changed to high glucose DMEM containing 2% horse serum (HS) to induce differentiation (day 0). GFER / ALR was purchased from Novopro (Cat#503686), diluted with distilled water sterilized in an autoclave, and treated with GFER according to two protocols for verification.

[0137] (1) Cells were treated with GFER / ALR at concentrations of 2, 10, 20, 30, and 50 ng / ml from day 0 and harvested after 5 days. (2) GFER treatment was started on day 5 after initial differentiation and continued at the same concentration for 5 days. In each protocol, we tested two conditions for the medium replacement frequency, every 24 hours and every 48 hours, using GFER / ALR to determine the optimal treatment schedule. The initial concentration starting from 2 ng / ml was selected based on previous results indicating GFER / ALR concentrations in plasma of humans, mice, and rabbits. Cells were harvested using RIPA buffer containing protease inhibitor, and approximately 20 μg of protein was loaded per well of SDS-PAGE for Western blotting analysis of the extracted proteins. Beta-actin was used to quantify the myogenic biomarkers MyoG and MHC.

[0138] Referring to Figure 3, it was confirmed that the protein amounts of major myogenic markers (MyoG and MHC) were significantly enhanced in a concentration-dependent manner of GFER / ALR depending on the GFER dose, with the highest protein expression observed at 50 ng / ml. This suggests a strong effect on myogenic differentiation of C2C12 cells.

[0139]

[0140] Promotion of myogenic differentiation in Split-C2C12

[0141] The final concentration of GFER / ALR in C2C12 cells was set to 50 ng / ml, and the experiment was performed starting from day 0. To confirm the effect of GFER / ALR treatment on myogenesis and muscle fusion in Split-C2C12 cells composed of GFP1-10 and GFP11 cells, the changes in C2C12 differentiation over time after GFER treatment are shown in Fig. 4a and Fig. 4b. The cells express N-terminal and C-terminal green fluorescent proteins (GFP1-10 and GFP11), respectively, which do not show GFP signals until fusion, and the signal intensity is related to myogenic marker expression and muscle fusion. After thawing the stored cells, the cells were passaged 3 to 4 times to ensure stability, and then seeded in 12-well plates at a density of 0.72 x 10 per well. 5 GFP1-10 cells and 0.36 x 10 5 GFP11 cells were cultured in triplicate, and the cells were seeded in the same well at a ratio of 2:1 due to the growth rate of GFP11 cells. After 2 days in a medium containing 10% FBS, the cells were added together with the differentiation medium GFER / ALR from day 0, and the medium was replaced every 24 hours. From day 2, the differentiation process was monitored by tracking the GFP signal using a BioTek Cytation 5 Cell Imaging Multimode Reader, and DMEM without red phenol was used to minimize the color effect caused by red phenol. GFP signals were recorded every 24 hours until day 6, and GFP intensity was quantified at 25 random points per well.

[0142] The GFP intensity between the GFER / ALR treatment group and the control group was compared and analyzed. Fluorescence microscopy images of the control group and GFER-treated cells on days 2, 3, 4, 5, and 6 are shown in Fig. 4a. Compared to the control group at all time points, the GFP fluorescence intensity significantly increased in the GFER / ALR treatment group, and improved cell morphology was confirmed. The change in fluorescence intensity over time was quantified and shown in Fig. 4b, and it was confirmed that the fluorescence was significantly increased in the GFER treatment group at each time point. In particular, it can be seen that the difference in fluorescence intensity was significantly improved starting from day 2 (*p < 0.05) of GFER treatment and continued to day 3 (**p < 0.01), day 4 (****p < 0.0001), day 5 (****p < 0.0001), and day 6 (***p < 0.001). These results indicate that GFER treatment significantly promotes muscle morphological differentiation by inducing more myogenesis and muscle fusion, as indicated by increased GFP fluorescence intensity, suggesting that GFER treatment is correlated with muscle cell differentiation and muscle growth.

[0143]

[0144] Myofusion enhancement effect of C2C12 muscle

[0145] To evaluate the myogenesis index after GFER / ALR treatment using wild-type C2C12 cells, the medium was replaced every 24 hours after 5 days of GFER / ALR treatment, and the cells were stained for myosin heavy chain (MHC), a myogenic differentiation marker. The staining process involved fixing the cells with 4% paraformaldehyde (PFA) in a darkroom for 20 minutes, washing them twice with PBS, permeabilizing them with 0.1% Triton X in a darkroom for 5 minutes, and washing them twice with PBS. The cells were incubated with MHC antibody (MAB4470) overnight at 4°C, washed three times, incubated with Alexa Fluor 488 secondary antibody for 2 hours at room temperature, washed twice, and stained with DAPI for 5 minutes at room temperature. Fluorescence intensity was measured using a BioTek Cytation 5 Cell Imaging Multimode Reader, and images of GFP signals merged with DAPI were captured at 25 random points per well. The number of myotubes and nuclei within muscle cells was quantified using MATLAB-based open-source Myocount software, which provides information on the number of nuclei per myotube, the percentage of myotubes, and the number of mature myotubes.

[0146] As shown in Fig. 5, the experimental results confirmed that GFER significantly enhanced myofusion and myotube formation in C2C12 cells treated with GFER. Both 10x and 20x magnification microscopic images showed that GFER-treated cells had increased green fluorescence intensity compared to the control group, and exhibited longer and more organized myotubes. These observations are supported by quantitative analysis. The total fluorescence intensity representing MHC protein levels was significantly increased in the GFER-treated group (***p < 0.001), and the percentage of myotubes, the number of nuclei per myotube, and the number of mature myotubes were all significantly increased in the GFER-treated group (**p < 0.01, ***p < 0.001, and **p < 0.01, respectively). These results suggest that GFER / ALR treatment enhances myogenic differentiation and myofusion in C2C12 cells, as evidenced by increased MHC staining and quantitative analysis of myotube formation and maturation.

[0147]

[0148] Increased mRNA expression of muscle building biomarkers (MyoD, MyoG, Myh1, Myh3)

[0149] RT-qPCR was performed to measure the expression levels of MyoD, MyoG, Myh1, and Myh3 in wild-type C2C12 cells after GFER / ALR treatment. Cells were cultured and seeded in 6-well plates, and harvested on days 4 and 7 after GFER / ALR treatment every 24 h. RNA was isolated using Trizol reagent, and cDNA was synthesized from the extracted RNA. RT-qPCR was performed using specific primers for MyoD, MyoG, Myh1, and Myh3, as listed in Table 1 below. RT-qPCR was performed up to 40 cycles, and gene expression levels were normalized to beta-actin using the ΔΔCt method.

[0150] Marker Primer SequenceMyoDForward: 5'-CTACAGCGGCGACTCAGATG-3'Reverse: 5'-GGGGTGACGCGGAGTAGA-3'MyoGForward: 5'-GCACTGGAGTTCGGTCCAGA-3'Reverse: 5'-CGCAGTAAAAGGTGTGAGG-3'Myh1Forward: 5'-TGCAGGAGGACGAGGAAGAG-3'Reverse: 5'-GGGCTTGAGGTTGTCGAGAA-3'Myh3Forward: 5'-TGGACCTTGCTCTCCTGTG-3'Reverse: 5'-GGTGGCTGTGGAAGAGATGG-3'

[0151] The experimental results show that the relative gene expression levels of myogenic markers MyoD, MyoG (myogenin), Myh1, and Myh3 in C2C12 cells treated with GFER compared to the control group on days 4 and 7 are shown in Fig. 6. The bar graph shows that the expression of all four genes was significantly increased at both time points in the GFER-treated group, and on day 4, the expression of MyoD (****p < 0.0001), MyoG (**p < 0.01), Myh1 (****p < 0.0001), and Myh3 (****p < 0.0001) in GFER-treated cells was significantly increased compared to the control group. Similarly, on day 7, we confirmed that the expression of MyoD (****p < 0.0001), MyoG (**p < 0.01), Myh1 (**p < 0.01), and Myh3 (**p < 0.01) was significantly increased in the GFER-treated group. On both days 4 and 7, we confirmed that the expression of MyoD, MyoG, Myh1, and Myh3 was significantly increased compared to the control group. This indicates that GFER / ALR enhances myogenic differentiation in muscle regeneration and repair at the molecular level, supporting its potential.

[0152]

[0153] Increased protein expression levels of myogenic markers (MyoD, MyoG, MHC)

[0154] C2C12 cells were thawed and passaged three times, and then cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) for 2 days. Differentiation was initiated by switching the medium to high-glucose DMEM containing 2% horse serum (HS), and cells were treated with 50 ng / ml GFER / ALR from day 0. To ensure consistent exposure of GFER in the differentiation medium containing GFER / ALR, the medium was replaced with fresh medium every 24 hours, and cells were harvested for analysis 5 days after GFER treatment. This procedure was repeated six times under identical conditions to ensure consistency and reliability of the results. Approximately 20 μg of protein per sample was loaded onto SDS-PAGE gels. Western blotting was performed to detect the expression levels of myogenic biomarkers, including MyoD (45 kDa), MyoG (34 kDa), and MHC (220 kDa). At this time, beta-actin was used to confirm the same loading amount.

[0155] Our experimental results confirmed that GFER treatment significantly increased the protein levels of major myogenic markers, MyoD, MyoG (myogenin), and MHC (myosin heavy chain), in C2C12 myoblast cells 5 days after differentiation (see Fig. 7a). Western blot analysis confirmed that the protein levels of MyoD, MyoG, and MHC were significantly increased in the GFER-treated group compared to the control group, and quantification results confirmed that the expression of MyoD (***p < 0.001), MyoG (****p < 0.0001), and MHC (***p < 0.001) also significantly increased in response to GFER (see Fig. 7b). Quantitative analysis of Western blots showed a substantial increase in these myogenic markers, demonstrating the potent effect of GFER / ALR in promoting myogenic differentiation in C2C12 cells.

[0156]

[0157] Increased protein synthesis in C2C12 cells after GFER treatment confirmed by SUnSET analysis.

[0158] To evaluate the effect of GFER / ALR treatment on protein turnover, particularly on the rate of protein synthesis, wild-type C2C12 cells were cultured in 6-well plates according to the previously described culture and seeding procedure, and exposed to GFER daily for 5 days with fresh medium containing GFER / ALR replaced every 24 h. After that, the cells were treated with puromycin at a final concentration of 10 μg / mL using the SUnSET method to label newly synthesized proteins. Cells were harvested in triplicate at different time points (60, 90, and 120 min), and for Western blot analysis, cells were lysed in RIPA buffer containing protease inhibitors, and 20 μg of protein per sample was loaded onto each well of an SDS-PAGE gel. Puromycin-labeled newly synthesized proteins were detected using an anti-puromycin antibody (MABE343), and the Western blot signals were quantified using ImageJ software.

[0159] SUnSET analysis using puromycin labeling confirmed that the protein synthesis rate of C2C12 cells treated with GFER increased over time compared to the control group (see Fig. 8a). The detection intensity of puromycin labeling increased in the GFER / ALR treatment group across all time points, which was quantified and shown in Fig. 8b. The quantification results confirmed that the GFER / ALR-treated group showed a significantly higher protein synthesis rate than the control group in response to GFER. This higher puromycin labeling increase over time suggests that GFER / ALR promotes protein synthesis in C2C12 cells.

[0160]

[0161] In vivo effects of GFER on muscle

[0162] To examine the effects of GFER administration in aged mice, the experimental design was as shown in Figure 9. Specifically, six 22-month-old mice were administered GFER / ALR via intraperitoneal injection at a dose of 5 ng / g / day for 12 weeks. Body weight, body fat, and lean body mass were measured via DEXA, and muscle function was measured via the hang test and endurance test. Baseline measurements were recorded at week 0, and data were collected at weeks 4, 8, and 12, with repeated assessments of body weight, body composition, and muscle function. The final analysis at week 12 included a comprehensive assessment of the effects of GFER administration on muscle function and metabolic health.

[0163]

[0164] Effects of GFER / ALR injection on body weight in rats

[0165] The body weight changes of six 22-month-old mice during the 57-day GFER / ALR administration period are shown in Figure 10. The weights of the mice before GFER administration were also measured. To observe the weight changes of the mice due to GFER, body weights were measured every three days throughout the treatment period. The results are recorded several times until the 8th week, and the initial body weight was approximately 36 g. During the GFER administration period, the weights of the mice remained mostly around 36 g, and the final weight change at the 57th treatment was almost identical to the initial weight, confirming that GFER administration had no effect on body weight. This suggests that GFER administration maintains metabolic stability without inducing significant weight loss or gain in aged mice. With regard to body weight, the above results suggest that long-term GFER administration in elderly patients can ensure not only metabolic benefits but also stability.

[0166]

[0167] Confirmation of changes in body fat mass and lean mass by GFER

[0168] In order to confirm the relative changes in body composition, especially the ratio of body fat mass to lean mass, due to GFER administration to 22-month-old aged mice, GFER was administered intraperitoneally at a dose of 5 ng / g / day, and the body composition changes were measured using DEXA over 8 weeks. The results are shown in Fig. 11. Fig. 11a shows the relative changes in body fat percentage at weeks 0, 4, and 8, and it was confirmed that the body fat percentage tended to decrease slightly (ns) as the GFER administration time passed, although the difference was not significant. This suggests that it had a minimal effect on fat reduction. Fig. 11b shows the relative changes in lean mass at the same time point, and it was confirmed that the lean mass percentage tended to increase slightly (ns) as the GFER administration period passed. Although the changes in body composition due to GFER administration were not statistically significant for 8 weeks, it suggests that GFER administration may have a subtle effect on body composition by decreasing body fat and increasing lean mass. Although statistical significance was not achieved, the observed trend confirms the potential of GFER to improve body composition in aged mice. This suggests that GFER administration may contribute to improving overall body composition and thus health and physical function in the elderly.

[0169]

[0170] Confirmation of improved muscle function through Hang test

[0171] The changes in hanging time at 0, 4, and 8 weeks after GFER administration to aged mice were relatively measured and are shown in Fig. 12. Compared to week 0, the hanging time significantly increased after GFER administration at week 4 (*p < 0.05) and week 8 (**p < 0.01), indicating that muscle endurance and strength were significantly improved. Although the increase between weeks 4 and 8 after GFER administration was not statistically significant (ns), it suggests that muscle function steadily improved as GFER administration continued. This increase in hanging time demonstrates the effect of GFER on improving muscle function in aged mice.

[0172]

[0173] Results of muscular endurance test using the Endurance test

[0174] The changes in running time and distance after GFER administration to aged mice were measured and shown in Fig. 13. The running times were relatively measured at 0, 4, and 8 weeks and are shown in Fig. 13a. In week 4, the running time increased slightly compared to week 0, but it was not significant (ns). However, when comparing week 8 with week 0 (**p < 0.01), and week 8 with week 4 (*p < 0.05), it was confirmed that the running times all increased significantly. The changes in running distance at weeks 0, 4, and 8 were relatively measured and are shown in Fig. 13b. In week 4, the running distance increased slightly compared to week 0, but it was not significant (ns). However, when comparing week 8 with week 0 (**p < 0.01), and week 8 with week 4 (*p < 0.05), it was confirmed that the running distances all increased significantly. These results suggest that GFER administration can significantly improve endurance in aged mice by increasing the running distance and time. The lack of significant difference between weeks 0 and 4 (ns) indicates that the GFER effect stabilizes after an initial period, suggesting that GFER administration effectively improves physical endurance and physical strength in aged mice. Although week 4 after GFER administration did not show a significant difference compared to week 0, the significant increase between week 8 and week 0, and between week 8 and week 4 suggests that GFER administration has the potential to improve both physical endurance and physical strength in aged humans.

[0175]

[0176] In summary, the above results confirmed that GFER significantly enhanced muscle production, as evidenced by increased expression of muscle production-related biomarkers (MyoD, MyoG, MHC), improved muscle morphological differentiation, and increased muscle fusion index. In addition, GFER enhanced protein synthesis rate as measured by the surface sensing of translation (SUnSET) method, and this effect was observed in myoblasts, particularly in wild-type C2C12 and Split-C2C12 cells. Furthermore, when GFER was administered to aged mice, body weight did not change, but fat mass tended to decrease and lean mass increased. In addition, hanging ability (hang test performance time) increased in proportion to the period of GFER administration, and endurance ability (endurance test time and distance) increased. Furthermore, GFER, a glutathione-dependent formaldehyde dehydrogenase, enhances mitochondrial function by regulating oxidative stress and assisting protein folding, thereby improving muscle cell health and metabolic regulation. These findings confirm the potential of GFER and GFER protein derivatives as novel therapeutics for muscle-related diseases, including age-related muscle loss, muscle dysfunction, and sarcopenia, as well as associated metabolic disorders, and are therefore expected to be actively utilized in clinical practice.

[0177]

[0178] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0179] 서열번호 1 : GRER 단백질, homo sapiens

[0180] MAAPGERGRFHGGNLFFLPGGARSEMMDDLATDARGRGAGRRDAAASASTPAQAPTSDSPVAEDASRRRPCRACVDFKTWMRTQQKRDTKFREDCPPDREELGRHSWAVLHTLAAYYPDLPTPEQQQDMAQFIHLFSKFYPCEECAEDLRKRLCRNHPDTRTRACFTQWLCHLHNEVNRKLGKPDFDCSKVDERWRDGWKDGSCD

[0181] 서열번호 2 : GFER 유전자, homo sapiens

[0182] ttggccccgtgcggctgctggcttctgtagaggctgcccagaggggccaggtggcacaaataagagaggggagatggggggcagccaggagaggaggtgtcccttcctcgcccagacacagcgcgcttctctctggcctttcccgaggcctgtgagtgcctcaggaagcagctgggccctctgggaaggctgtgttcagcttaggaacataccgcctgtatctgctgtccctcccctgcccccctgccccccccaccgccttccctttttccctgtcttccttaaagtttcactcctgaataaaacttcactttgccttagaa

Claims

1. A pharmaceutical composition for promoting muscle regeneration or muscle growth, comprising as an active ingredient at least one selected from the group consisting of GFER (Growth Factor, Augmenter of Liver Regeneration) protein and a gene encoding GFER protein.

2. In paragraph 1, A composition characterized in that the GFER protein is a wild-type GFER protein or a GFER protein derivative.

3. A food composition for promoting muscle regeneration or muscle growth, containing GFER (Growth Factor, Augmenter of Liver Regeneration) protein as an active ingredient.

4. In paragraph 3, A composition characterized in that the GFER protein is a wild-type GFER protein or a GFER protein derivative.

5. A pharmaceutical composition for preventing or treating muscle-related diseases, comprising as an active ingredient at least one selected from the group consisting of GFER (Growth Factor, Augmenter of Liver Regeneration) protein and a gene encoding GFER protein.

6. In paragraph 5, A composition characterized in that the GFER protein is a wild-type GFER protein or a GFER protein derivative.

7. In paragraph 5, A composition characterized in that the muscle-related disease is at least one selected from the group consisting of sarcopenia, muscle damage, muscle atrophy, muscle rigidity, hypotonia, myotonic dystrophy, muscular atrophy, myasthenia gravis, muscular degeneration, muscular dystrophy, and myositis.

8. In paragraph 5, A composition, characterized in that the above muscle-related disease is caused by aging.

9. In paragraph 7, A composition characterized in that the above sarcopenia is senile sarcopenia.

10. A food composition for preventing or improving muscle disease, containing GFER (Growth Factor, Augmenter of Liver Regeneration) protein as an active ingredient.

11. In paragraph 10, A composition characterized in that the GFER protein is a wild-type GFER protein or a GFER protein derivative.

12. In paragraph 10, A composition characterized in that the muscle-related disease is at least one selected from the group consisting of sarcopenia, muscle damage, muscle atrophy, muscle rigidity, hypotonia, myotonic dystrophy, muscular atrophy, myasthenia gravis, muscular degeneration, muscular dystrophy, and myositis.

13. In paragraph 12, A composition, characterized in that the above muscle-related disease is caused by aging.

14. In paragraph 12, A composition characterized in that the above sarcopenia is senile sarcopenia.

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