Novel peptide having muscle formation-promoting activity, Anti-obesity activity and Anti-diabetic activity and uses thereof

Abstract

A peptide of the present invention has muscle formation-promoting activity in myoblasts. The peptide of the present invention has the activities of inhibiting fat accumulation and promoting lipolysis in hepatocytes and adipocytes. The peptide of the present invention inhibits the expression of insulin resistance factors and increases the expression of insulin sensitivity factors in hepatocytes and adipocytes. Therefore, the peptide of the present invention can be used as an active substance for the treatment, prevention or amelioration of muscle diseases, obesity, fatty liver, and diabetes.

Description

Novel peptides having muscle-building promoting activity, anti-obesity and anti-diabetic activity and their uses

[0001] [Cross-citation with related applications]

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2024-0008399, filed January 18, 2024, the entire contents of which are incorporated herein by reference.

[0003] [Technical Field]

[0004] The present invention relates to a novel peptide having muscle building promoting activity, anti-obesity activity and anti-diabetic activity and its use.

[0005] Aging-related declines in skeletal muscle mass lead to a decline in muscle strength and various bodily functions. Frailty, skeletal muscle atrophy, and sarcopenia are particularly common in the elderly and are chronic problems that threaten the healthy life expectancy of many countries. Their prevention, treatment, and rehabilitation have become major concerns in today's healthcare. While muscle mass and strength naturally decline with age, sarcopenia is a condition in which muscle mass and strength are excessively reduced, even after accounting for factors such as age and gender, leading to a decline in physical function and increased health risks and mortality. Recently, diagnostic criteria for sarcopenia have been established and it has been added to the International Classification of Diseases (ICD), classifying it as a disease rather than a simple consequence of aging.

[0006] Obesity is a condition in which excess energy is stored as body fat, resulting in excessive adipose tissue in the body when the energy intake and expenditure are not balanced. According to the World Health Organization (WHO), more than 1 billion adults worldwide are overweight, and at least 3 million of them are clinically obese, with the number increasing significantly in the United States and Europe. Overweight and obesity increase blood pressure and cholesterol levels, which can lead to various diseases such as heart disease, diabetes, and arthritis, and increase the incidence of various adult diseases. Furthermore, overweight and obesity are factors that increase the incidence of various adult diseases such as arteriosclerosis, hypertension, hyperlipidemia, and heart disease not only in adults but also in children and adolescents.

[0007] Currently, the representative obesity treatment drugs that are widely prescribed and approved by the US FDA include a group of drugs that act on the central nervous system and act as appetite suppressants, and orlistat (Xenical), an inhibitor of the digestive enzyme lipase secreted by the pancreas. Many drugs that act on the central nervous system, such as sibutramine, have had their approval revoked due to cardiovascular and psychiatric side effects, and orlistat has the limitation of having variable drug effects depending on fat intake in addition to various side effects. Meanwhile, liraglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist, has been approved and used as an endocrine peptide targeting drug, but the risk of thyroid cancer is emerging.

[0008] Diabetes mellitus is a metabolic disease characterized by insufficient insulin secretion or abnormal insulin function. It is characterized by hyperglycemia, which is an increase in blood glucose concentration. Hyperglycemia causes various symptoms and signs and causes glucose to be excreted in the urine. Recently, the incidence of diabetes has been explosively increasing due to the increase in obesity, especially abdominal obesity. Diabetes can be broadly divided into type 1 diabetes, which is insulin-dependent diabetes, and type 2 diabetes, which is non-insulin-dependent diabetes. Type 2 diabetes is characterized by hyperglycemia, insulin resistance, and relative insulin secretion impairment.

[0009] When you eat food, glucose is absorbed from the digestive tract, stimulating insulin secretion from pancreatic beta cells. This insulin then promotes glucose uptake into muscles. While insulin also plays a role in hepatic glucose uptake, it primarily inhibits hepatic glucose production. Insulin lowers blood glucose levels by inhibiting hepatic glucose production and promoting glucose uptake into peripheral tissues, including muscles. Insulin resistance refers to a condition in which blood glucose levels are lower than normal in response to insulin under given insulin concentrations. Insulin regulates blood glucose levels by promoting glucose uptake into muscles or inhibiting glucose production in the liver. Insulin resistance refers to a condition in which this insulin action is reduced even in the absence of insulin deficiency. Insulin receptors on the cell membrane are involved in the process of glucose uptake into cells in peripheral tissues. Insulin resistance occurs when the number of insulin receptors decreases or intracellular defects occur after receptor binding. Although insulin receptor defects are found in type 2 diabetes, post-receptor intracellular defects, namely insulin-regulated phosphorylation / dephosphorylation disorders, are known to play a far greater role. Among these mechanisms, impaired PI3K (Phosphoinositide 3-kinase) signaling is known to reduce the translocation of the glucose transporter type 4 (GLUT-4) to the cell membrane.

[0010] Current methods for controlling blood sugar include lifestyle modifications (diet and exercise) and medication. However, these methods are difficult to strictly manage and implement, and their effectiveness is limited. Therefore, most diabetic patients rely on lifestyle modifications and medications such as insulin, insulin secretagogues, insulin sensitizers, and hypoglycemic agents to control their blood sugar.

[0011] [Prior Art Literature]

[0012] [Patent Document]

[0013] Republic of Korea Patent No. 10-2064387

[0014] Republic of Korea Patent No. 10-2486996

[0015] Republic of Korea Patent No. 10-2507392

[0016] International Patent Application No. PCT-KR2021-011278

[0017] The present inventors have conducted research efforts to develop small molecule substances that exhibit excellent therapeutic activity for diseases such as sarcopenia, diabetes, and obesity, while also ensuring safety and high drug delivery efficiency. As a result, the inventors experimentally confirmed that the peptide developed by the present inventors has the activity of promoting the expression of factors involved in myogenesis and muscle protein synthesis in muscle cells. Furthermore, the peptide of the present invention has the activity of suppressing fat accumulation and promoting fat breakdown in adipocytes and hepatocytes, suppressing insulin resistance, and enhancing insulin sensitivity, thereby completing the present invention.

[0018] Accordingly, the object of the present invention is to provide a novel peptide having muscle building promotion, anti-obesity, or anti-diabetic activity.

[0019] Another object of the present invention is to provide a composition for promoting muscle formation, anti-obesity, anti-fatty liver, or anti-diabetic activity, which comprises a peptide having the above-described activity as an active ingredient.

[0020] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating muscle diseases, which comprises a peptide having the above-described activity as an active ingredient.

[0021] Another object of the present invention is to provide a food composition for preventing or improving muscle disease, which comprises a peptide having the above-described activity as an active ingredient.

[0022] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity or fatty liver, comprising a peptide having the above-described activity as an active ingredient.

[0023] Another object of the present invention is to provide a food composition for preventing or improving obesity or fatty liver, which comprises a peptide having the above-described activity as an active ingredient.

[0024] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes, which comprises a peptide having the above-described activity as an active ingredient.

[0025] Another object of the present invention is to provide a food composition for preventing or improving diabetes, which comprises a peptide having the above-described activity as an active ingredient.

[0026] In order to achieve the purpose of the present invention described above,

[0027] One aspect of the present invention provides a peptide comprising the amino acid sequence disclosed in SEQ ID NO: 1.

[0028] Another aspect of the present invention provides a composition for promoting muscle formation, anti-obesity, anti-fatty liver or anti-diabetic, comprising the peptide as an active ingredient.

[0029] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating muscle disease, comprising the peptide as an active ingredient.

[0030] Another aspect of the present invention provides a food composition for preventing or improving muscle disease, comprising the peptide as an active ingredient.

[0031] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating obesity or fatty liver, comprising the peptide as an active ingredient.

[0032] Another aspect of the present invention provides a food composition for preventing or improving obesity or fatty liver, comprising the peptide as an active ingredient.

[0033] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising the peptide as an active ingredient.

[0034] Another aspect of the present invention provides a food composition for preventing or improving diabetes, comprising the peptide as an active ingredient.

[0035] The present invention is described in detail below.

[0036] 1. Peptides and their activities

[0037] According to one aspect of the present invention, a peptide comprising an amino acid sequence disclosed in SEQ ID NO: 1 is provided.

[0038] [Sequence number 1]

[0039] HGTY (His-Gly-Thr-Tyr)

[0040] The term "peptide" as used herein means a linear molecule formed by amino acid residues being linked to each other by peptide bonds.

[0041] The peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention may be used without modification, but a variant or fragment of an amino acid having a different sequence by deletion, insertion, substitution, or a combination thereof of amino acid residues may be used within a range that does not affect the original activity of the peptide, for example, the activity of improving skin condition.

[0042] The peptide of the present invention can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc., within a range that does not change its activity.

[0043] The peptide of the present invention includes a peptide comprising an amino acid sequence substantially identical to a peptide comprising an amino acid sequence of SEQ ID NO: 1, and a variant or active fragment thereof. The substantially identical amino acid sequence refers to an amino acid sequence having a sequence identity of 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, or 98% or more, with the amino acid sequence of SEQ ID NO: 1. In addition, the peptide may additionally include a targeting sequence, a tag, a labeled residue, an amino acid sequence manufactured for a specific purpose to increase half-life or peptide stability.

[0044] The peptide of the present invention may be modified at the N-terminus and / or C-terminus to select a portion of the amino acid sequence and increase its activity. Such N-terminus and / or C-terminus modifications can significantly improve the stability of the peptide of the present invention, for example, increasing the half-life of the peptide when administered in vivo. The term "stability" encompasses not only in vivo stability, which protects the peptide of the present invention from attack by in vivo protein-cleaving enzymes, but also storage stability (e.g., room temperature storage stability).

[0045] The above N-terminal modification may be a modification in which a protecting group selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG) is bonded to the N-terminus of the peptide. The above C-terminal modification may be a modification in which a hydroxyl group (-OH), an amino group (-NH2), a hydrazino group (-NHNH2), or the like is bonded to the C-terminus of the peptide, but is not limited thereto.

[0046] The peptide of the present invention can be prepared by various methods widely known in the art to which the present invention pertains. For example, the peptide of the present invention can be prepared by chemical synthesis methods known in the art, particularly solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co.: Rockford, 111 (1984)) or liquid-phase synthesis techniques (US Patent No. 5,516,891).

[0047] The peptide of the present invention has an activity that promotes muscle formation.

[0048] In one embodiment, the peptide of the present invention has the following activities with respect to muscle building promoting activity:

[0049] (i) Activity that promotes the expression of genes related to cell proliferation, such as PCNA (Proliferating cell nuclear antigen), NDRG2 (N-myc Downstream-Regulated Gene 2), or CDK4 (Cyclin-dependent kinase 4), in myoblasts;

[0050] (ii) activity that promotes the expression of cell proliferation-related factors SIRT1 (Sirtuin1), Ki67, Pax7, or pAKT protein in myogenic cells;

[0051] (iii) activity that promotes the expression of genes of differentiation-related factors MyoD, Myf6, mTOR, Myf5, or Myf4 in myogenic cells;

[0052] (iv) activity that promotes the expression of differentiation-related factors α-actinin, Myf6 or MyoG proteins in myogenic cells; or

[0053] (v) Activity that promotes protein expression of signal transduction factors SIRT1, pAMPKα, pAKT, pmTOR or p70S6K involved in muscle protein synthesis in myoblasts.

[0054] The peptide of the present invention has an activity of suppressing obesity or fatty liver.

[0055] In one embodiment, the peptide of the present invention has the following activities with respect to its activity in suppressing obesity or fatty liver:

[0056] (i) Activity that inhibits fat accumulation in hepatocytes;

[0057] (ii) activity of inhibiting protein expression of fat synthesis factors SREBP1 or FAS and promoting protein expression of fat decomposition factors pACC, pHSL, ATGL, or PLIN in hepatocytes where fat accumulation is induced;

[0058] (iii) Activity that promotes the expression of the gene of fatty acid oxidation-related factor CPT1, PGC1α or PPARα in hepatocytes where fat accumulation is induced, and promotes the expression of the protein of fatty acid oxidation-related factor CPT1 or PGC1α.

[0059] (iv) Activity that inhibits gene expression of fat synthesis-related factors ACCα or PPAR-γ in adipocytes;

[0060] (v) Activity that inhibits protein expression of fat synthesis-related factors SREBP1, FAS, or C / EBPα in adipocytes and promotes protein expression of fatty acid degradation-related factor pACCα;

[0061] (vi) activity that promotes protein expression of lipolysis-related factors PLIN, pHSL, or PGC1α in adipocytes;

[0062] (vii) Activity that promotes gene expression of fatty acid oxidation-related factors PGC1α or CPT1 in adipocytes where insulin resistance is induced;

[0063] (viii) activity that inhibits the gene expression of fatty acid synthesis factor FAS and promotes the gene expression of lipolysis-related factors PLIN, HSL, or ATGL in adipocytes where insulin resistance is induced; or

[0064] (ix) Activity that inhibits the protein expression of fatty acid synthesis factors FAS and SREBP1 in fat cells where insulin resistance is induced, and promotes the protein expression of fat decomposition-related factor PLIN.

[0065] The peptide of the present invention has antidiabetic activity.

[0066] In one embodiment, the peptide of the present invention has the following activities with respect to antidiabetic activity:

[0067] (i) Activity of promoting protein expression of insulin-sensitive factor pIRS (Tyr612) or GLUT4 in adipocytes and inhibiting protein expression of insulin-resistant factor pIRS (Ser302) or p70S6K;

[0068] (ii) Activity of suppressing the expression of the gene of insulin resistance factor P70S6K and promoting the expression of the gene of insulin sensitivity factor AMPKα, GLUT4 or SIRT1 in fat cells where insulin resistance is induced;

[0069] (iii) Activity that promotes protein expression of insulin-sensitive factor pAMPKα and inhibits protein expression of insulin resistance factor pIRS (Ser302) in fat cells where insulin resistance is induced.

[0070] (iv) activity that inhibits the expression of genes for insulin resistance factors mTOR or P70S6K and promotes the expression of genes for insulin sensitivity factors AKT, SIRT1, or AMPKα in hepatocytes where insulin resistance is induced; or

[0071] (v) Activity that inhibits the expression of proteins of insulin resistance factors pmTOR or pJNK in hepatocytes where insulin resistance is induced, and promotes the expression of proteins of insulin sensitivity factors pAMPKα, GLUT4, or SIRT1.

[0072] 2. Composition for promoting muscle formation

[0073] According to another aspect of the present invention, a composition for promoting muscle formation is provided, which comprises a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0074] pharmaceutical composition

[0075] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating muscle disease is provided, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0076] The peptide containing the amino acid sequence of the above sequence number 1 is identical to the peptide described in the above “Peptide and its activity” section, and the description of the activity related to the use for preventing or treating muscle diseases is based on the above and is not described in duplicate.

[0077] The peptide of the present invention having the above-described activity exhibits excellent efficacy in suppressing muscle loss and increasing muscle mass, and can be used for the prevention or treatment of muscle diseases.

[0078] Meanwhile, conventional functional peptides, despite their effective biological activity, have shown the disadvantage of not being able to effectively enter target tissues or cells due to the size of the peptide itself, or being eliminated from the body in a short period of time due to a short half-life. On the other hand, the pharmaceutical composition for preventing or treating muscle diseases of the present invention comprises a peptide composed of about 10 amino acids or less as an active ingredient, and accordingly, the cell penetration rate of the active ingredient is very excellent, and for example, when administered topically, an effective preventive or therapeutic effect for muscle diseases can be obtained.

[0079] As used herein, the term "muscle disease" may collectively refer to a muscle disease, illness or condition associated with a decrease in the proliferation or differentiation of myoblasts, for example, muscle diseases resulting from muscle dysfunction, muscle wasting or muscle degeneration. The muscle disease may be, but is not limited to, one or more selected from the group consisting of muscular atrophy, sarcopenia, muscular dystrophy, disuse atrophy, spinal muscular amyotrophy, muscle rigidity, muscular hypotonia, muscle weakness, muscle endurance weakness, amyotrophic lateral sclerosis, spinal muscular atrophy, myasthenia gravis, myasthenia, muscle degeneration and cachexia.

[0080] In one embodiment, the peptide of the present invention can promote the proliferation and differentiation of myogenic cells, thereby inhibiting muscle loss and increasing muscle mass. Specifically, the peptide of the present invention can promote the proliferation of myogenic cells and significantly increase the expression of early differentiation markers, mid-differentiation markers, and late differentiation markers of myogenic cells, thereby promoting differentiation into muscle cells or muscle fibers. In addition, the peptide of the present invention can significantly increase the expression of myogenic markers and muscle protein synthesis markers, and can also increase the myotube synthesis capacity of myogenic cells, and can enhance muscle mass recovery / regeneration when myogenic cells are damaged by exogenous factors.

[0081] As used herein, the term “prevention” means any act of inhibiting or delaying the onset of a disease by administering the composition.

[0082] As used herein, the term "treatment" refers to any form of treatment that provides a benefit to a subject suffering from a disease or at risk of developing a disease, including improving the condition of the subject (e.g., one or more symptoms), delaying the progression of the disease, delaying the onset of symptoms, or slowing the progression of symptoms. Accordingly, the terms "treatment" and "prevention" are not intended to imply a cure or complete elimination of symptoms.

[0083] As used herein, the term "administration" means introducing a given substance into an individual by any appropriate method, and the pharmaceutical composition of the present invention may be administered via any general route capable of reaching an in vivo target. For example, the route of administration of the pharmaceutical composition of the present invention is not particularly limited, but may be administered orally or parenterally, and in the case of parenteral administration, may be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc., but is not limited thereto.

[0084] The pharmaceutical composition of the present invention may comprise a therapeutically effective amount of the above-described peptide and a pharmaceutically acceptable carrier.

[0085] The term "therapeutically effective amount" above means an amount sufficient for the peptide, which is an active ingredient of the pharmaceutical composition of the present invention, to achieve its activity or efficacy, for example, an amount sufficient to achieve efficacy in treating or preventing diabetes or obesity.

[0086] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0087] The pharmaceutical composition of the present invention may additionally include, in addition to the above ingredients, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc., but is not limited thereto.

[0088] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy, (19th ed., 1995, Williams & Wilkins).

[0089] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the method can be performed. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0090] The pharmaceutical composition of the present invention can be administered by any suitable route for treating muscle diseases, for example, it can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, transdermal administration, etc.

[0091] The pharmaceutical composition of the present invention may contain the peptide of the present invention at a concentration of 0.01 μM to 1000 μM, specifically, the peptide of the present invention may contain the peptide of the present invention at a concentration of 0.01 μM to 1000 μM; 0.05 μM to 800 μM, 0.05 μM to 700 μM, 0.05 μM to 600 μM, 0.05 μM to 500 μM, 0.05 μM to 300 μM, 0.05 μM to 200 μM; 0.1 μM to 800 μM, 0.1 μM to 700 μM, 0.1 μM to 600 μM, 0.1 μM to 500 μM, 0.1 μM to 300 μM, 0.1 μM to 200 μM; It may be included at a concentration of, but is not limited to, 1 μM to 800 μM, 1 μM to 700 μM, 1 μM to 600 μM, 1 μM to 500 μM, 1 μM to 300 μM, 1 μM to 200 μM; 5 μM to 800 μM, 5 μM to 700 μM, 5 μM to 600 μM, 5 μM to 500 μM, 5 μM to 300 μM, or 5 μM to 200 μM.

[0092] Food composition

[0093] According to another aspect of the present invention, a food composition for preventing or improving muscle disease is provided, which comprises a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0094] In one embodiment, the peptide of the present invention can promote the proliferation and differentiation of myogenic cells, thereby inhibiting muscle loss and increasing muscle mass. Therefore, the peptide of the present invention can be effectively utilized as an active ingredient in a food composition for preventing or ameliorating muscle diseases, and the food composition of the present invention can be effectively utilized for the prevention or amelioration of muscle diseases.

[0095] As used herein, the term "improvement" may mean any action that at least reduces the severity of a symptom, for example, a parameter associated with alleviating or treating a condition.

[0096] The activity of the peptide of the present invention related to the prevention or improvement of muscle disease in the food composition of the present invention is the same as that described in the pharmaceutical composition described above, and therefore is cited and not described in duplicate.

[0097] In the functional food composition of the present invention, the active ingredient, peptide, may be included in an appropriate amount selected within a range of 10 wt% or less, specifically 0.000001 wt% to 10 wt%, based on the total composition weight.

[0098] The food composition of the present invention may contain a food-effective amount of a peptide and a food-acceptable carrier.

[0099] The functional food composition of the present invention comprises not only the peptide as the effective ingredient, but also components commonly added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the carbohydrates mentioned above include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavoring agents, thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.) can be used.

[0100] In addition to the above-described ingredients, 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 and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain fruit pulp for the production of natural fruit juices and fruit juice drinks and vegetable drinks. For example, when the functional food composition of the present invention is produced as a drink, in addition to the peptide, which is an effective ingredient of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia extract, jujube extract, licorice extract, etc. may be additionally contained.

[0101] There is no particular limitation on the type of the above food composition. Examples of the above food or food composition include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea drinks, alcoholic beverages, vitamin complexes, dairy products, fermented milk, etc., and may include all functional foods or foods in the conventional sense.

[0102] Meanwhile, the food composition may include a health functional food. As used herein, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients with beneficial functional properties for the human body, in the form of tablets, capsules, powders, granules, liquids, pills, etc. "Functionality" here refers to achieving beneficial health effects, such as regulating nutrients or physiological effects on the structure and function of the human body.

[0103] The above health functional food can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art during the manufacturing process. In addition, the formulation of the health functional food can be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition can be manufactured in various forms of formulation, and unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs because it uses food as a raw material, and has excellent portability, and the health functional food according to one embodiment can be consumed as a supplement to enhance the therapeutic effect of muscle disease.

[0104] According to another aspect of the present invention, a method for treating a muscle disease is provided, comprising administering to a subject in need of treatment of a muscle disease a peptide comprising an amino acid sequence of SEQ ID NO: 1 or a pharmaceutical composition comprising the peptide as an active ingredient.

[0105] According to another aspect of the present invention, there is provided a use of a peptide comprising an amino acid sequence of SEQ ID NO: 1 for treating or preventing muscle disease.

[0106] According to another aspect of the present invention, there is provided a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for the manufacture of a pharmaceutical composition for treating or preventing muscle disease.

[0107] In one embodiment, the peptide of the present invention, in relation to the composition for promoting muscle formation, the pharmaceutical composition for preventing, treating or improving muscle disease or the food composition of the present invention, has the following activities:

[0108] (i) Activity of promoting the expression of the gene of PCNA (Proliferating cell nuclear antigen), NDRG2 (N-myc Downstream-Regulated Gene 2), or CDK4 (Cyclin-dependent kinase 4), which are cell proliferation-related factors in myoblasts; (ii) Activity of promoting the expression of SIRT1 (Sirtuin1), Ki67, Pax7, or pAKT protein, which are cell proliferation-related factors in myoblasts; (iii) Activity of promoting the expression of the gene of MyoD, Myf6, mTOR, Myf5, or Myf4, which are differentiation-related factors in myoblasts; (iv) Activity of promoting the expression of α-actinin, Myf6, or MyoG protein, which are differentiation-related factors in myoblasts; or (v) Activity of promoting the protein expression of SIRT1, pAMPKα, pAKT, pmTOR, or p70S6K, which are signaling factors related to muscle protein synthesis in myoblasts.

[0109] 3. Composition for anti-obesity, anti-fatty liver, and anti-diabetic use

[0110] According to another aspect of the present invention, there is provided an anti-obesity, anti-fatty liver or anti-diabetic composition comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0111] pharmaceutical composition

[0112] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating obesity or fatty liver is provided, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0113] As described above, the peptide of the present invention has the activity of inhibiting fat accumulation in hepatocytes and adipocytes and promoting fat decomposition, and therefore can be used as an excellent agent for preventing or treating obesity or fatty liver.

[0114] The peptide comprising the amino acid sequence of the above sequence number 1 and its activity related to the treatment and prevention of obesity or fatty liver are identical to the peptide and its activity described in the above “Peptide and its activity” section, and the specific description thereof is cited and not described repeatedly.

[0115] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating diabetes is provided, which comprises a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0116] In the present invention, the diabetes may be type 1 diabetes or type 2 diabetes, and specifically, type 2 diabetes.

[0117] As described above, the peptide of the present invention can be used as an excellent agent for preventing or treating diabetes by suppressing the expression of insulin resistance factors and promoting the expression of insulin sensitivity factors in hepatocytes and adipocytes.

[0118] The peptide comprising the amino acid sequence of the above sequence number 1 and its activity related to the treatment and prevention of diabetes are identical to the peptide and its activity described in the above “Peptide and its activity” section, and a specific description thereof is cited and not described repeatedly.

[0119] The pharmaceutical composition of the present invention may comprise a therapeutically effective amount of the above-described peptide and a pharmaceutically acceptable carrier.

[0120] The term "therapeutically effective amount" above means an amount sufficient for the peptide, which is an active ingredient of the pharmaceutical composition of the present invention, to achieve its activity or efficacy, for example, an amount sufficient to achieve efficacy in treating or preventing diabetes or obesity.

[0121] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0122] The pharmaceutical composition of the present invention may additionally include, in addition to the above ingredients, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc., but is not limited thereto.

[0123] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy, (19th ed., 1995, Williams & Wilkins).

[0124] The pharmaceutical composition of the present invention can be administered by any suitable route for treating diabetes, fatty liver, or obesity, for example, can be administered orally or parenterally, and in the case of parenteral administration, can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, transdermal administration, etc.

[0125] The dosage of the pharmaceutical composition may be, but is not limited to, 0.0001 μg to 100 mg, 0.001 μg to 100 mg, 0.01 μg to 100 mg, 0.1 μg to 100 mg, or 1.0 μg to 1000 mg per day, and may be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity.

[0126] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the method can be performed. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0127] Food composition

[0128] According to another aspect of the present invention, a food composition for preventing or improving obesity or fatty liver is provided, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0129] As described above, the peptide of the present invention has the activity of inhibiting fat accumulation in hepatocytes and adipocytes and promoting fat decomposition, and therefore can be used as an active ingredient of an excellent food composition for preventing or improving obesity or fatty liver.

[0130] The peptide comprising the amino acid sequence of the above sequence number 1 and its activity related to the prevention or improvement of obesity or fatty liver are identical to the peptide and its activity described in the above “peptide and its activity” section, and the specific description thereof is cited and not described repeatedly.

[0131] According to another aspect of the present invention, a food composition for preventing or improving diabetes is provided, which comprises a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0132] In the present invention, the diabetes may be type 1 diabetes or type 2 diabetes, and specifically, type 2 diabetes.

[0133] As described above, the peptide of the present invention can be used as an active ingredient of a food composition for preventing or improving diabetes by suppressing the expression of insulin resistance factors and promoting the expression of insulin sensitivity factors in hepatocytes and adipocytes.

[0134] The food composition for preventing or improving diabetes of the present invention may be a functional food composition for controlling blood sugar levels.

[0135] In the functional food composition of the present invention, the control of blood sugar level may be the control of blood sugar level of a diabetic patient or a high-risk patient in the pre-diabetic stage.

[0136] In the functional food composition of the present invention, the control of blood sugar level may be a lowering of blood sugar level.

[0137] In the food composition of the present invention described above, the peptide may be included in an appropriate amount selected within the range of 0.0001 wt% to 10 wt% based on the total composition weight.

[0138] In one embodiment, the functional food composition of the present invention may comprise a food-effective amount of the peptide and a food-acceptable carrier.

[0139] The food composition of the present invention comprises not only the peptide as the effective ingredient but also components commonly added during food manufacturing, and may include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the carbohydrates mentioned above include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents, thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. The proportion of the carbohydrates may generally be about 1 to 20 g, preferably about 5 to 12 g, per 100 g of the food composition of the present invention, but is not limited thereto.

[0140] In addition to the above-described ingredients, 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 and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain fruit pulp for the production of natural fruit juice and fruit juice drinks and vegetable drinks. For example, when the functional food composition of the present invention is produced as a drink, in addition to the peptide, which is an effective ingredient of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia extract, jujube extract, licorice extract, etc. may be additionally contained.

[0141] In another aspect of the present invention, a method for treating, preventing, or improving obesity or fatty liver disease is provided, comprising administering to a patient suffering from obesity or fatty liver disease a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 described above.

[0142] In another aspect of the present invention, a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 described above for treating, preventing or improving obesity or fatty liver is provided.

[0143] In another aspect of the present invention, there is provided a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 described above for the preparation of a composition for treating, preventing or improving obesity or fatty liver.

[0144] In another aspect of the present invention, a method for treating, preventing, or improving diabetes is provided, comprising administering to a diabetic patient a therapeutically effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 described above.

[0145] In another aspect of the present invention, a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 described above for treating, preventing or improving diabetes is provided.

[0146] In another aspect of the present invention, there is provided a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 described above for the preparation of a composition for treating, preventing or improving diabetes.

[0147] In one embodiment, the peptide of the present invention, in relation to the anti-obesity or anti-fatty liver composition of the present invention and the pharmaceutical composition or food composition for preventing, treating or improving obesity or fatty liver, has the following activities: (i) activity of inhibiting fat accumulation in hepatocytes, (ii) activity of inhibiting protein expression of fat synthesis factors SREBP1 or FAS in hepatocytes in which fat accumulation is induced, and promoting protein expression of fat decomposition factors pACC, pHSL, ATGL, or PLIN; (iii) activity of promoting gene expression of fatty acid oxidation-related factors CPT1, PGC1α or PPARα in hepatocytes in which fat accumulation is induced, and promoting protein expression of fatty acid oxidation-related factors CPT1 or PGC1α; (iv) activity of inhibiting gene expression of fat synthesis-related factors ACCα or PPAR-γ in adipocytes; (v) activity of inhibiting protein expression of lipogenesis-related factors SREBP1, FAS, or C / EBPα in adipocytes and promoting protein expression of fatty acid degradation-related factor pACCα; (vi) activity of promoting protein expression of lipolysis-related factors PLIN, pHSL, or PGC1α in adipocytes; (vii) activity of promoting gene expression of fatty acid oxidation-related factors PGC1α or CPT1 in adipocytes in which insulin resistance is induced; (viii) activity of inhibiting gene expression of fatty acid synthesis factor FAS and promoting gene expression of lipolysis-related factors PLIN, HSL, or ATGL in adipocytes in which insulin resistance is induced; or (ix) activity of inhibiting protein expression of fatty acid synthesis factors FAS and SREBP1 and promoting protein expression of lipolysis-related factor PLIN in adipocytes in which insulin resistance is induced.

[0148] In one embodiment, the peptide of the present invention, in relation to the antidiabetic composition of the present invention and the pharmaceutical composition or food composition for preventing, treating or improving diabetes, has the following activities: (i) activity of promoting protein expression of insulin sensitive factor pIRS (Tyr612) or GLUT4 in adipocytes and inhibiting protein expression of insulin resistance factor pIRS (Ser302) or p70S6K; (ii) activity of suppressing gene expression of insulin resistance factor P70S6K in adipocytes in which insulin resistance is induced and promoting gene expression of insulin sensitive factor AMPKα, GLUT4 or SIRT1; (iii) activity of promoting protein expression of insulin sensitive factor pAMPKα in adipocytes in which insulin resistance is induced and inhibiting protein expression of insulin resistance factor pIRS (Ser302); (iv) activity of inhibiting the expression of the gene of the insulin resistance factor mTOR or P70S6K in hepatocytes in which insulin resistance is induced, and promoting the expression of the gene of the insulin sensitivity factor AKT, SIRT1, or AMPKα; or (v) activity of inhibiting the expression of the protein of the insulin resistance factor pmTOR or pJNK in hepatocytes in which insulin resistance is induced, and promoting the expression of the protein of the insulin sensitivity factor pAMPKα, GLUT4, or SIRT1.

[0149] The peptide of the present invention has the activity of promoting muscle formation in myogenic cells. Furthermore, the peptide of the present invention has the activity of inhibiting fat accumulation and promoting fat breakdown in hepatocytes and adipocytes. Furthermore, the peptide of the present invention suppresses the expression of insulin resistance factors and increases the expression of insulin sensitivity factors in hepatocytes and adipocytes. Therefore, the peptide of the present invention can be used as an active substance for the treatment, prevention, or improvement of muscle diseases, obesity, fatty liver, and diabetes.

[0150] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0151] Figure 1 is a drawing showing that the peptide of the present invention has cell proliferation promoting activity in C2C12 cells.

[0152] Figures 2a and 2b show the results of the peptide of the present invention promoting the expression of genes related to cell proliferation, such as PCNA (Proliferating cell nuclear antigen), NDRG2 (N-myc Downstream-Regulated Gene 2), and CDK4 (Cyclin-dependent kinase 4), in C2C12 cells. Non is a group not treated with peptide in which differentiation-induced C2C12 cells were not treated with peptide.

[0153] Figures 3a and 3b show the results of the peptide of the present invention increasing the expression levels of cell proliferation-related factors SIRT1 (Sirtuin1), Ki67, Pax7, and pAKT proteins in C2C12 cells. Non is the non-peptide-treated group in which differentiation-induced C2C12 cells were not treated with the peptide.

[0154] Figures 4a and 4b show the results of the peptide of the present invention increasing the expression levels of the genes MyoD, Myf6, mTOR, Myf5, and Myf4, which are factors involved in mid- and late-stage differentiation in C2C12 cells. Non represents a group not treated with the peptide in which differentiation-induced C2C12 cells were not treated with the peptide.

[0155] Figures 5a, 5b, 5c, and 5d show that the peptide of the present invention increases the expression of differentiation markers α-actinin, Myf6, and MyoG proteins in C2C12 cells, and increases the expression of signaling proteins involved in muscle protein synthesis, SIRT1, pAMPKα, pAKT, pmTOR, and p70S6K. Non is a peptide-untreated group in which differentiation-induced C2C12 cells were not treated with the peptide.

[0156] Figures 6a and 6b show the results of the peptide of the present invention promoting differentiation of C2C12 cells and increasing myotube formation. Non is a non-peptide-treated group in which differentiation-induced C2C12 cells were not treated with the peptide.

[0157] Figures 7a, 7b, and 7c show the results of the peptide of the present invention inhibiting fat accumulation in HepG2 hepatocytes. Non is the untreated group in which HepG2 hepatocytes were not treated with oleic acid or the peptide. OA is the oleic acid-treated group.

[0158] Figures 8a and 8b show that the peptide of the present invention inhibits the expression of fat synthesis factors SREBP1 and FAS, and promotes the expression of fat decomposition factors ATGL and pHSL in HepG2 hepatocytes in which fat accumulation was induced by oleic acid treatment. Non is the untreated group in which HepG2 hepatocytes were not treated with oleic acid or peptide. OA is the oleic acid-treated group.

[0159] Figures 9a, 9b, and 9c demonstrate that the peptides of the present invention have the activity of re-suppressing the increase in FAS expression induced by oleic acid in HepG2 cells. Non represents the untreated group in which HepG2 hepatocytes were not treated with oleic acid or peptide. OA represents the oleic acid-treated group.

[0160] Figures 9d, 9e, and 9f demonstrate that the peptide of the present invention has the activity of re-suppressing the increase in SREBP1 expression induced by oleic acid in HepG2 cells. Non is the untreated group in which HepG2 hepatocytes were not treated with oleic acid or the peptide. OA is the oleic acid-treated group.

[0161] Figures 9g, 9h, and 9i demonstrate that the peptide of the present invention has the activity of re-increasing the decreased expression of pACC induced by oleic acid in HepG2 cells. Non is the untreated group in which HepG2 hepatocytes were not treated with oleic acid or peptide. OA is the oleic acid-treated group.

[0162] Figures 9j, 9k, and 9l demonstrate that the peptides of the present invention have the activity of re-increasing the decreased expression of pHSL induced by oleic acid in HepG2 cells. Non is the untreated group in which HepG2 hepatocytes were not treated with oleic acid or peptide. OA is the oleic acid-treated group.

[0163] Figures 9m, 9n, and 9o demonstrate that the peptide of the present invention has the activity of re-increasing the decreased expression of ATGL induced by oleic acid in HepG2 cells. Non is the untreated group in which HepG2 hepatocytes were not treated with oleic acid or peptide. OA is the oleic acid-treated group.

[0164] Figures 9p, 9q, and 9r demonstrate that the peptide of the present invention has the activity of re-increasing the decreased expression of PLIN induced by oleic acid in HepG2 cells. Non is the untreated group in which HepG2 hepatocytes were not treated with oleic acid or the peptide. OA is the oleic acid-treated group.

[0165] Figures 10a and 10b show that the peptide of the present invention reduces the expression levels of genes for insulin resistance factors mTOR and P70S6K and increases the expression levels of genes for insulin sensitivity factors AKT, SIRT1, and AMPKα in HepG2 cells treated with oleic acid and insulin resistance conditions. Non is the untreated group in which HepG2 hepatocytes were not treated with oleic acid or peptide. OA is the oleic acid-treated group.

[0166] Figures 11a and 11b show that the peptide of the present invention reduces the protein expression levels of insulin resistance factors pmTOR and pJNK, and increases the protein expression levels of insulin sensitivity factors pAMPKα, GLUT4, and SIRT1 in oleic acid-treated insulin-resistant HepG2 cells. Non is the untreated group in which HepG2 hepatocytes were not treated with oleic acid or peptide. OA is the oleic acid-treated group.

[0167] Figures 12a and 12b show that the peptide of the present invention increases the expression of genes for CPT1, PGC1α, and PPARα, which are factors involved in fatty acid oxidation, in HepG2 cells treated with oleic acid and under fat accumulation conditions. Non is the untreated group in which HepG2 hepatocytes were not treated with oleic acid or peptide. OA is the oleic acid-treated group.

[0168] Figure 13 shows that the peptide of the present invention increases the expression of CPT1 and PGC1α proteins, which are fatty acid oxidation-related factors, in HepG2 cells treated with oleic acid and under fat accumulation conditions. Non represents the untreated group in which HepG2 hepatocytes were not treated with oleic acid or peptide. OA represents the oleic acid-treated group.

[0169] Figures 14a and 14b show that the peptide of the present invention increases the protein expression levels of insulin-sensitive factors pIRS (Tyr612) and GLUT4, and decreases the protein expression levels of insulin-resistant factors pIRS (Ser302) and p70S6K in 3T3-L1 cells. Non represents an untreated group in which 3T3-L1 cells were not treated with the peptide.

[0170] Figures 15a and 15b show that the peptide of the present invention reduces the expression level of the gene of P70S6K, an insulin resistance factor, and increases the expression levels of the genes of AMPKα, GLUT4, and SIRT1, which are insulin sensitivity factors, in 3T3-L1 cells treated with oleic acid. Non is the untreated group in which 3T3-L1 cells were not treated with oleic acid or the peptide. OA is the oleic acid-treated group.

[0171] Figure 16 shows that the peptide of the present invention increases the expression level of pAMPKα, an insulin-sensitive factor, and decreases the expression level of pIRS (Ser302), an insulin-resistant factor, in oleic acid-treated 3T3-L1 cells (Figure 16). Non represents the untreated group in which 3T3-L1 cells were not treated with oleic acid or the peptide. OA represents the oleic acid-treated group.

[0172] Figure 17 shows that the peptide of the present invention reduces the gene expression of adipogenesis-related factors, ACCα and PPAR-γ, in 3T3-L1 cells. Non is an untreated group in which 3T3-L1 cells were not treated with the peptide.

[0173] Figures 18a and 18b show that the peptide of the present invention reduces the expression of SREBP1, FAS, and C / EBPα, which are lipogenesis-related factors, and increases the expression of pACCα, which promotes fatty acid breakdown, in 3T3-L1 cells. Non is an untreated group in which 3T3-L1 cells were not treated with the peptide.

[0174] Figure 19 shows that the peptide of the present invention increases the expression of PLIN, pHSL, and PGC1α, which are lipolysis-related factors, in 3T3-L1 cells. Non is an untreated group in which 3T3-L1 cells were not treated with the peptide.

[0175] Figure 20 shows that the peptide of the present invention increases the gene expression of fatty acid oxidation-related factors PGC1α and CPT1 in oleic acid-treated 3T3-L1 cells. Non is the untreated group in which 3T3-L1 cells were not treated with oleic acid or peptide. OA is the oleic acid-treated group.

[0176] Figures 21a and 21b show that the peptide of the present invention reduces the expression of the FAS gene, a fatty acid synthesis-related factor, and increases the expression of the PLIN, HSL, and ATGL genes, which are lipolysis-related factors, in oleic acid-treated 3T3-L1 cells. Non represents the untreated group in which 3T3-L1 cells were not treated with oleic acid or the peptide. OA represents the oleic acid-treated group.

[0177] Figures 22a and 22b show that the peptide of the present invention reduces the expression of the fatty acid synthesis-related factor FAS protein and increases the expression of the lipolysis-related factors SREBP1 and PLIN protein in oleic acid-treated 3T3-L1 cells. Non is the untreated group in which 3T3-L1 cells were not treated with oleic acid or the peptide. OA is the oleic acid-treated group.

[0178] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0179] Manufacturing Example 1: Manufacturing of peptides

[0180] A peptide having the amino acid sequence of SEQ ID NO: 1 shown in Table 1 below was synthesized using an automatic peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptide was purified using C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was ACQUITY UPLC BEH300 C18 (2.1 mm Х 100 mm, 1.7 μm, Waters Co, USA).

[0181] Sequence number Amino acid sequence (N-terminal → C-terminal) 1HGTY (His-Gly-Thr-Tyr)

[0182] The efficacy of the peptide of sequence number 1 manufactured above was evaluated through the following experiment.

[0183] [Experimental Example 1-6] Experimental Example on Myoblasts

[0184] Experimental Example 1: Analysis of cell proliferation-promoting activity in myoblasts

[0185] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the proliferation ability and cytotoxicity of myoblasts was evaluated.

[0186] C2C12 cells (mouse-derived myoblasts) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached 70-80%, 5 x 10 cells were seeded in 96-well culture plates. 3Cells were seeded at a concentration of 200 μl. The following day, the medium was replaced with serum-free DMEM (1% P / S), and 4 hours later, the medium was replaced with DMEM containing 0.2% FBS and peptides at different concentrations (0 μM, 3.9 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500 μM). At 48 and 72 hours after replacement, the medium was reacted using EZ-cytox (DoGenBio Co., Ltd, Seoul, Korea) until the absorbance of Non (no peptide added) reached 1.0, and the absorbance was measured at 450 nm using a spectrophotometer (SPECTRAMAX M2e). When C2C12 cells were treated with peptides at different concentrations for 24 and 48 hours, there was no significant difference in the concentrations in the case of 24-hour treatment, but in the case of 48-hour treatment, it was confirmed that cell proliferation increased in a peptide concentration-dependent manner (Fig. 1).

[0187] Experimental Example 2: Expression Analysis of Proliferation-Related Marker Genes in Myoblasts

[0188] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the proliferation capacity of myoblasts was evaluated by analyzing the expression of proliferation-related marker genes.

[0189] C2C12 cells (mouse-derived myoblasts) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached 70-80%, 5 x 10 cells were seeded in 6-well culture plates. 5Cells were seeded at a concentration of 2 ml / cell. The following day, the medium was replaced with serum-free DMEM (1% P / S), and approximately 4 hours later, peptides (5 μM, 50 μM) were treated. One hour after peptide treatment, cells were harvested and RNA was extracted with Trizol (Thermo Fisher Scientific, USA). The extracted RNA was analyzed using TOPScript TM After synthesizing DNA complementary to RNA using RT DryMIX (enzynomics, Korea), TOPsimple TM Polymerase chain reaction (PCR) was performed on genes of factors (markers) involved in cell proliferation using DryMIX-nTaq (enzynomics, Korea). The reaction products were then run on a 1.5% agarose gel, and the mRNA expression levels of the factors (markers) mentioned above were compared for each sample. Table 2 below lists the primer sequences for the genes used in the PCR, and the GAPDH gene was used as a control.

[0190] Primer name sequence (5'-> 3') SEQ ID NO: PCNA ForwardAGC GGA GAA GGT GCT GCA G2PCNA ReverseATA GCG GCG GTA TGT GTC GG3NDRG2 ForwardCAC ACA GAC CTC GTT CCT CA4NDRG2 ReverseGAT GCT GCA CTG GTC AGA GA5CDK4 ForwardAGT TTC TAA GCG GCC TGG AT6CDK4 ReverseAAC TTC AGG AGC TCG GTA CC7GAPDH ForwardGTG ATG GCA TGG ACT GTG GT8GAPDH ReverseGGA GCC AAA AGG GTC ATC AT9

[0191] As a result of the experiment, it was confirmed that the expression of PCNA (Proliferating cell nuclear antigen), NDRG2 (N-myc Downstream-Regulated Gene 2), and CDK4 (Cyclin-dependent kinase 4) genes related to cell proliferation in C2C12 cells was significantly increased by peptide treatment (Fig. 2a, Fig. 2b).

[0192] Experimental Example 3: Analysis of expression of cell proliferation-related marker proteins in myoblasts.

[0193] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the proliferation capacity of myoblasts was evaluated by analyzing the expression of proliferation-related marker proteins.

[0194] C2C12 cells (mouse-derived myoblasts) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached 70-80%, 5 x 10 cells were seeded in 6-well culture plates. 5Cells were seeded at a concentration of 2 ml. The following day, the medium was replaced with serum-free DMEM (1% P / S), and approximately 4 hours later, peptides (5 μM, 50 μM) were treated. One hour after peptide treatment, cell lysis buffer was added to lyse the cells, and the cells were centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins. The obtained proteins were quantified using a BCA kit. The proteins were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then electrotransferred to a membrane. The membrane with the protein attached was blocked with 5% skim milk, and then treated with primary antibodies and reacted overnight at 4°C. The primary antibodies used were anti-SIRT1 antibody (Cell signaling Technology, USA), anti-Ki67 antibody (Santa Cruz, USA), anti-Pax7 antibody (Santa Cruz, USA), anti-pAKT antibody (Cell signaling Technology, USA), and anti-β-actin antibody (Santa Cruz, USA). The reaction mixture was washed with PBS-T, and then reacted with secondary antibodies (Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson immunoResearch, USA; Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L), Jackson immunoResearch, USA) at room temperature for 1 hour, washed again with PBS-T, and visualized by Western detection reagent (Elpis Biotech, Daejeon, Korea) treatment using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0195] As a result of the experiment, when the peptide was treated at concentrations of 5 μM and 50 μM for 1 hour, the expression levels of SIRT1 (Sirtuin1), Ki67, Pax7, and pAKT proteins all increased in a peptide concentration-dependent manner. In particular, the expression level of Pax7 protein was significantly increased by 2.6- and 3.8-fold, respectively, compared to the control group (Fig. 3a and Fig. 3b).

[0196] Experimental Example 4: Expression analysis of genes for early / mid-stage differentiation markers and signaling genes related to muscle protein synthesis in myoblasts.

[0197] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of genes of early and mid-stage differentiation markers of myogenic cells and signaling factors involved in muscle protein synthesis was analyzed.

[0198] C2C12 cells (mouse-derived myoblasts) were cultured in DMEM medium containing 1% P / S and 2% BCS (bovine calf serum). When cell confluency reached 70-80%, 5 x 10 cells were seeded in 6-well culture plates. 5 Cells were seeded at a concentration of 2 ml per well. After reaching 100% confluency in a 6-well culture plate, the differentiation medium (1% P / S, 2% horse serum) and peptides (5 μM, 50 μM) were replaced. Afterwards, the differentiation medium (1% P / S, 2% horse serum) and peptides (5 μM, 50 μM) were replaced every other day, and 3 days after the first replacement, cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was TOPScript TM After synthesizing DNA complementary to RNA using RT DryMIX (enzynomics, Korea), TOPsimple TMPolymerase chain reaction (PCR) was performed using DryMIX-nTaq (enzynomics, Korea) for genes involved in early and mid-stage differentiation markers and signal transduction involved in muscle protein synthesis. The reaction products were then run on a 1.5% agarose gel, and the mRNA expression levels of the markers were compared for each sample. Table 3 lists the primer sequences for the genes used in the PCR, and the GAPDH gene was used as a control.

[0199] Primer Name Sequence (5'-> 3') Sequence Number mTOR ForwardTTG AGG TCG CTA TGA CCA GAG AGA A10mTOR ReverseTTA CCA GAA GGG ACA CCA GCC AAT G11Myf6 ForwardATC AGC TAC ATT GAG CGT CTA CA12Myf6 ReverseCCT GGA ATG ATC CGA AAC ACT TG13Myf5 ForwardTAT GAA GGC TCC TGT CC14Myf5 ReverseACG TGC TCC TCA TCG TCT G15Myf4 ForwardATC AGC TAC ATT GAG CGT CTA CA16Myf4 ReverseCCT GGA ATG ATC CGA AAC ACT TG17MyoD ForwardAGT GAA TGA GGC CTT CGA GA18MyoD ReverseCTG GGT TCC CTG TTC TGT GT19GAPDH ForwardGTG ATG GCA TGG ACT GTG GT8GAPDH ReverseGGA GCC AAA AGG GTC ATC AT9

[0200] According to the above experimental method, differentiation was induced in C2C12 myoblasts with 2% horse serum, and the peptide was treated for 3 days to determine the effect of the peptide on differentiation. As a result of RT-PCR, the expression levels of MyoD and Myf6, which are factors involved in mid- and late differentiation, increased when the peptide was treated. In particular, the expression of Myf6 showed a significant increase, increasing 1.8-fold compared to the control group when treated with 50 μM of the peptide. In addition, the expression levels of mTOR, Myf5, and Myf4, which are factors involved in early and mid-differentiation, were also confirmed to increase in a concentration-dependent manner (Fig. 4a and Fig. 4b).

[0201] Experimental Example 5: Expression Analysis of Mid- and Late Differentiation Markers and Signaling Proteins Involved in Muscle Protein Synthesis in Myogenic Cells

[0202] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of mid- and late-stage differentiation markers of myogenic cells and signaling proteins involved in muscle protein synthesis was analyzed.

[0203] C2C12 cells (mouse-derived myoblasts) were cultured in DMEM medium containing 1% P / S and 2% BCS (bovine calf serum). When cell confluency reached 70-80%, 5 x 10 cells were seeded in 6-well culture plates. 5Cells were seeded at a concentration of 2 ml per well. After reaching 100% confluency in a 6-well culture plate, the differentiation medium (1% P / S, 2% horse serum) and peptides (5 μM, 50 μM) were replaced. Thereafter, the differentiation medium (1% P / S, 2% horse serum) and peptides (5 μM, 50 μM) were replaced every other day, and lysis buffer was added 3 days after the first replacement to lyse the cells. The cell lysate was then centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins. The obtained proteins were quantified using a BCA kit. The proteins were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then electrotransferred to a membrane. The membranes with protein attachment were blocked with 5% skim milk, and then treated with primary antibodies and incubated overnight at 4°C. The primary antibodies used were as follows: anti-α-actinin antibody (Santa Cruz, USA); anti-MyoG antibody (Santa Cruz, USA); anti-Myf6 antibody (Abcam, UK); anti-SIRT1 antibody (Cell signaling Technology, USA); anti-pAMPKα antibody (Cell signaling Technology, USA); anti-pAKT antibody (Cell signaling Technology, USA); anti-pmTOR antibody (Cell signaling Technology, USA); anti-p70S6 Kinase antibody (Cell signaling Technology, USA); anti-α-tubulin antibody (Santa Cruz, USA).The reaction mixture was washed with PBS-T, reacted with secondary antibodies (Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson immunoResearch, USA; Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L), Jackson immunoResearch, USA) for 1 hour at room temperature, washed again with PBS-T, and visualized by Western detection reagent (Elpis Biotech, Daejeon, Korea) treatment using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0204] As a result of the experiment, the expression of differentiation markers α-actinin, Myf6, and MyoG proteins increased in a concentration-dependent manner by peptide treatment (Fig. 5a, Fig. 5b). In addition, the expression of signaling proteins involved in muscle protein synthesis, SIRT1, pAMPKα, pAKT, pmTOR, and p70S6K, also increased in a concentration-dependent manner by peptide treatment (Fig. 5c, Fig. 5d).

[0205] Experimental Example 6: Microscopic Analysis of the Morphology of Differentiated Myoblasts

[0206] Through morphological observation using a microscope, the effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the synthesis of myotubes from myoblasts was analyzed.

[0207] C2C12 cells (mouse-derived myoblasts) were cultured in DMEM medium containing 1% P / S and 2% BCS (bovine calf serum). When cell confluency reached approximately 80%, 5 x 10 cells were seeded in 6-well culture plates. 5Cells were seeded at a concentration of 2 ml per well. After reaching 100% confluency in a 6-well culture plate, the differentiation medium (1% P / S, 2% horse serum) and peptides (5 μM, 50 μM) were replaced. Thereafter, the differentiation medium (1% P / S, 2% horse serum) and peptides (5 μM, 50 μM) were replaced every other day, and images of the cells were taken using an optical microscope (FLEXACAM C1, Leica, Germany) 3 days after differentiation induction.

[0208] Through the above experiment, we aimed to determine whether peptide treatment simultaneously with differentiation induction in C2C12 myoblasts promotes cell differentiation. Cell morphology was observed under a microscope, and the thickness of the formed myotubes was measured. As a result of the experiment, it was confirmed that differentiation of myoblasts was further promoted when treated with peptides compared to the control group (differentiation-inducing, Non), and it was confirmed that a large number of long and thick myotubes were formed when treated with high concentrations of peptides (Fig. 6a, Fig. 6b).

[0209] [Experimental Example 7-13] Experimental Example on Hepatocytes

[0210] Experimental Example 7: Analysis of Fat Accumulation in Hepatocytes

[0211] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on fat accumulation in hepatocytes was analyzed.

[0212] HepG2 cells (human hepatocytes) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70-80%, 1 x 10 cells were seeded in 12-well culture plates. 5Cells were seeded at a concentration of 1 ml per well. After reaching approximately 70-80% confluency in a 12-well culture plate, the medium was replaced with serum-free DMEM. After 4 hours, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA) to induce lipid accumulation. After 24 hours of lipid accumulation induction, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM), and cultured for 48 hours. To compare the degree of lipid synthesis, the cells were washed twice with PBS and fixed with 10% formaldehyde. Fixed cells were stained with Oil-Red O solution (sigma, USA) for 2 hours to stain lipid droplets, then washed with distilled water (DW) and observed under a microscope. The DW was then removed, dried, and washed with isopropanol. The absorbance was measured at an OD of 540 nm using a spectrophotometer (SPECTRAMAX M2e).

[0213] As a result of the experiment, it was observed that fat formation was increased by oleic acid treatment in HepG2 hepatocytes, and fat formation was inhibited by peptide treatment (Fig. 7a, Fig. 7b, Fig. 7c).

[0214] Experimental Example 8: Analysis of the expression of proteins related to lipogenesis and lipolysis in hepatocytes.

[0215] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of proteins related to fat synthesis and fat decomposition in hepatocytes was analyzed.

[0216] HepG2 cells (human hepatocytes) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70-80%, 1 x 10 cells were seeded in 12-well culture plates. 5Cells were seeded at a concentration of 1 ml per well. After reaching approximately 70-80% confluency in a 12-well culture plate, the medium was replaced with serum-free DMEM. After 4 hours, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA) to induce lipid accumulation. After 24 hours of lipid accumulation induction, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM), and incubated for 48 hours. After incubation, the cells were lysed by adding lysis buffer, and the cell lysate was centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins. The obtained proteins were quantified using a BCA kit. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred onto a membrane. The membrane with the protein attached was blocked with 5% skim milk, and then treated with primary antibodies and incubated overnight at 4°C. The primary antibodies used were as follows: anti-SREBP1 antibody (Abcam, UK); anti-FAS antibody (Cell signaling Technology, USA); anti-ATGL antibody (Cell signaling Technology, USA); anti-pHSL antibody (Cell signaling Technology, USA); and anti-β-actin antibody (Santa Cruz, USA).The reaction mixture was washed with PBS-T, reacted with secondary antibodies (Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson immunoResearch, USA; Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L), Jackson immunoResearch, USA) for 1 hour at room temperature, washed again with PBS-T, and visualized by Western detection reagent (Elpis Biotech, Daejeon, Korea) treatment using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0217] As a result of the experiment, it was confirmed that the expression of SREBP1 (Sterol Regulatory Element Binding Protein-1) and FAS (Fatty acid synthase), which are proteins involved in fat synthesis, increased by oleic acid treatment in HepG2 hepatocytes, and when peptides were treated thereto, the increased expression of SREBP1 and FAS decreased again (Fig. 8a). In addition, the expression of ATGL (Adipose triglyceride lipase) and pHSL (Hormone-sensitive lipase), which are proteins involved in fat decomposition, was suppressed by oleic acid treatment in HepG2 hepatocytes, but when peptides were treated thereto, the decreased expression levels of ATGL and pHSL increased again (Fig. 8b).

[0218] Experimental Example 9: Immunofluorescence analysis of the expression of markers related to lipogenesis and lipolysis in hepatocytes.

[0219] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of markers related to fat synthesis and fat decomposition in hepatocytes was analyzed through immunofluorescence.

[0220] HepG2 cells (human hepatocytes) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70-80%, 3 x 10 cells were seeded in 6-well culture plates. 5Cells were seeded at a concentration of 2 ml per well. After reaching approximately 70-80% confluency in a 6-well culture plate, the medium was replaced with serum-free DMEM. After 4 hours, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 350 μM oleic acid (Sigma, St. Louis, MO, USA) to induce lipid accumulation. After 24 hours of lipid accumulation induction, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 350 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM), and cultured for 48 hours. After incubation, cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature (RT) and permeabilized with 0.1% Triton X-100 for 5 minutes. Subsequently, cells were blocked with PBS, 10% FBS, and 0.1% Triton X-100 for 1 hour at RT, followed by treatment with primary antibodies and incubation for 1 hour at RT. The primary antibodies used were as follows: anti-FAS antibody (Cell signaling Technology, USA); anti-SREBP1 antibody (Abcam, UK); anti-pACC antibody (Cell signaling Technology, USA); anti-pHSL antibody (Cell signaling Technology, USA); anti-pATGL antibody (Cell signaling Technology, USA); anti-PLIN antibody (Affinity biosciences, USA). After incubation, cells were washed three times and incubated with secondary antibody (goat anti-Rabbit IgG H&L, Abcam, USA) at room temperature for 30 minutes. Samples were washed three times, and nuclei were counterstained with DAPI (Santa Cruz, USA). Images were acquired using a LEICA fluorescence microscope (TCS SP8, LEICA, Germany).

[0221] Regarding the expression level of FAS (Fatty acid synthase), an enzyme involved in fatty acid synthesis, when fat accumulation was induced with oleic acid in HepG2 cells, the expression of FAS increased, and when peptides were treated here, the increased expression level of FAS decreased again in a concentration-dependent manner (Fig. 9a, Fig. 9b, Fig. 9c).

[0222] Regarding the expression level of SREBP1 (Sterol Regulatory Element Binding Protein-1), which induces the synthesis of fat and cholesterol in the liver, when fat accumulation was induced with oleic acid in HepG2 cells, the expression of SREBP1 increased, and when peptides were treated here, the increased expression level of SREBP1 decreased again in a concentration-dependent manner (Fig. 9d, Fig. 9e, Fig. 9f).

[0223] ACC (Acetyl CoA carboxylase) is known to be phosphorylated by AMPK (AMP-activating protein kinase) and promote fatty acid oxidation (lipolysis). Regarding the expression level of pACC (phospho-Acetyl CoA carboxylase), when fat accumulation was induced with oleic acid in HepG2 cells, the expression of pACC decreased, and when peptide was treated here, the expression level of pACC, which had been suppressed, increased again in a concentration-dependent manner (Fig. 9g, Fig. 9h, Fig. 9i).

[0224] Hormone-sensitive lipase (HSL) is an enzyme that hydrolyzes fat, and has the activity of breaking down triacylglycerol into free fatty acids and diacylglycerol in adipose tissue, or breaking down diacylglycerol into free fatty acids and monoglycerides. HSL is activated through phosphorylation by protein kinase A (PKA), which is activated during a hormone-induced signal transduction process. Regarding the expression level of pHSL (phospho-Hormone-sensitive lipase), when fat accumulation was induced with oleic acid in HepG2 cells, the expression of pHSL decreased, and when peptide was treated here, the expression level of pHSL, which had been suppressed, increased again in a concentration-dependent manner (Fig. 9j, Fig. 9k, Fig. 9l).

[0225] ATGL (Adipose triglyceride lipase) has the activity of decomposing and liberating fatty acids from triacylglycerol during the intracellular lipolysis process, thereby allowing the free fatty acids to be used for energy. Regarding the expression level of ATGL (Adipose triglyceride lipase), when fat accumulation was induced with oleic acid in HepG2 cells, ATGL expression was reduced, and when peptide was treated here, the expression level of ATG, which had been suppressed, was confirmed to increase again in a concentration-dependent manner (Fig. 9m, Fig. 9n, Fig. 9o).

[0226] Perilipin (PLIN) is a protein that forms the structure of lipid droplets and is known to promote the triacylglycerol breakdown of lipolytic enzymes. When lipolytic hormone signaling is activated, the intracellular cAMP level increases, and the protein kinase PKA (Protein Kinase A) is activated, and the activated PKA phosphorylates PLIN to activate it. Regarding the expression level of Perilipin (PLIN), when fat accumulation was induced with oleic acid in HepG2 cells, the expression of PLIN decreased, and when treated with peptide, the expression level of PLIN, which had been suppressed, was confirmed to increase again in a concentration-dependent manner (Fig. 9p, 9q, 9r).

[0227] The above experimental results prove that the peptide of sequence number 1 of the present invention has the activity of inhibiting fat synthesis in hepatocytes and promoting fat decomposition.

[0228] Experimental Example 10: Expression Analysis of Genes Related to Insulin Resistance and Sensitivity in Hepatocytes

[0229] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of genes related to insulin resistance and sensitivity in hepatocytes was analyzed.

[0230] HepG2 cells (human hepatocytes) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70-80%, 2 x 10 cells were seeded in 12-well culture plates. 5Cells were seeded at a concentration of 10 cells / ml. After reaching approximately 70-80% cell confluency in a 12-well culture plate, the medium was replaced with serum-free DMEM. After 4 hours, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA) to induce an insulin resistance environment. After 24 hours of induction, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM) and cultured for 48 hours. After culture, the cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was TOPScript TM After synthesizing DNA complementary to RNA using RT DryMIX (enzynomics, Korea), TOPsimple TM Polymerase chain reaction (PCR) was performed on genes associated with insulin resistance and sensitivity markers using DryMIX-nTaq (enzynomics, Korea). The reaction products were then run on a 1.5% agarose gel to compare the mRNA expression levels of the markers for each sample. Table 4 below lists the primer sequences for the genes used in the PCR, with the GAPDH gene serving as a control.

[0231] <h2 style=";text-align:left;direction:ltr">5'->3' mTOR ForwardCTG GGA CTC AAA TGT GTG CAG TTC mTOR ReverseGAA CAA TAG GGT GAA TGA TCC GGG21P70S6k ForwardACT TCT GGC TCG AAA GGT GG22P70S6k ReverseTTG AGT CAT CTG GGC TGT CG23AKT ForwardTTG TCA TGG AGT ACG CCA ACG24AKT ReverseACA GCC CGA AGT CTG TGA TCT T25SIRT1 ForwardTTG GCA CAG ATC CTC GAA CAA26SIRT1 ReverseAAG TCT ACA GCA AGG CGA GC27AMPKα ForwardGTC ATG ATA GCT TGC ATA AAT GGT G28AMPKα ReverseAGT TGA ATA GAA CAA GCC CTG GAC29GAPDH ForwardGAG TCA ACG GAT TTG GTC GT30GAPDH ReverseGAC AAG CTT CCC GTT CTC AG31<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0232] Through the above experiment, we tried to confirm whether insulin sensitivity is induced by peptide treatment in HepG2 cells in which an insulin-resistant environment is induced by oleic acid treatment. Induction of insulin sensitivity was confirmed by examining the expression levels of insulin resistance or sensitivity-related factors through RT-PCR. As a result of the experiment, the expression levels of mTOR and P70S6K, which are factors related to insulin resistance, increased by oleic acid treatment in HepG2 cells. However, when peptides were treated thereto, the increased expression levels of mTOR and P70S6K decreased again in a peptide concentration-dependent manner. In addition, the expression levels of AKT, SIRT1, and AMPKα, which are factors related to insulin sensitivity, decreased by oleic acid treatment in HepG2 cells. However, when peptides were treated thereto, the decreased expression levels of AKT, SIRT1, and AMPKα increased again (Fig. 10a and Fig. 10b).

[0233] Experimental Example 11: Analysis of the expression of proteins related to insulin resistance and sensitivity in hepatocytes.

[0234] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of proteins related to insulin resistance and sensitivity in hepatocytes was analyzed.

[0235] HepG2 cells (human hepatocytes) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70-80%, 2 x 10 cells were seeded in 12-well culture plates. 5Cells were seeded at a concentration of 10 cells / ml. After reaching approximately 70-80% confluency in a 12-well culture plate, the medium was replaced with serum-free DMEM. After 4 hours, insulin resistance was induced by replacing the medium with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA). After 24 hours of induction, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM), and incubated for 48 hours. After incubation, the cells were lysed by adding lysis buffer, and the cell lysate was centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins. The obtained proteins were quantified using a BCA kit. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred onto a membrane. The electrotransferred membrane was blocked with 5% skim milk, and then treated with primary antibodies and incubated overnight at 4°C. The primary antibodies used were as follows: anti-pmTOR antibody (Cell signaling Technology, USA); anti-pJNK antibody (Santa Cruz, USA); anti-GLUT4 antibody (Invitrogen, USA); anti-SIRT1 antibody (Cell signaling Technology, USA); anti-pAMPKα antibody (Cell signaling Technology, USA); anti-β-actin antibody (Santa Cruz, USA).The reaction mixture was washed with PBS-T, reacted with secondary antibodies (Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson immunoResearch, USA; Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L), Jackson immunoResearch, USA) for 1 hour at room temperature, washed again with PBS-T, and visualized by Western detection reagent (Elpis Biotech, Daejeon, Korea) treatment using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0236] Through the above experiment, we tried to determine whether insulin sensitivity is induced by peptide treatment in HepG2 cells in which an insulin-resistant environment is induced by oleic acid treatment. Induction of insulin sensitivity was confirmed by examining the expression levels of insulin resistance or sensitivity-related factors through Western blotting. As a result of the experiment, the expression levels of pmTOR and pJNK, factors related to insulin resistance, increased by oleic acid treatment in HepG2 cells, but when peptides were treated thereto, the increased expression levels of pmTOR and pJNK decreased again. In addition, the expression levels of pAMPKα, GLUT4, and SIRT1, factors related to insulin sensitivity, decreased by oleic acid treatment in HepG2 cells, but when peptides were treated thereto, the decreased expression levels of pAMPKα, GLUT4, and SIRT1 increased again (Fig. 11a and Fig. 11b).

[0237] Experimental Example 12: Expression Analysis of Genes Related to Fatty Acid Oxidation in Hepatocytes

[0238] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of fatty acid oxidation-related genes in hepatocytes was analyzed.

[0239] HepG2 cells (human hepatocytes) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70-80%, 2 x 10 cells were seeded in 12-well culture plates. 5 Cells were seeded at a concentration of 10 cells / ml. After reaching approximately 70-80% cell confluency in a 12-well culture plate, the medium was replaced with serum-free DMEM. After 4 hours, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA) to induce lipid accumulation. After 24 hours of lipid accumulation induction, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM) and cultured for 48 hours. After culture, the cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was TOPScript TM After synthesizing DNA complementary to RNA using RT DryMIX (enzynomics, Korea), TOPsimple TM Polymerase chain reaction (PCR) was performed on genes of fatty acid oxidation-related markers using DryMIX-nTaq (enzynomics, Korea). The reaction products were then run on a 1.5% agarose gel, and the mRNA expression levels of the markers were compared for each sample. Table 5 below lists the primer sequences for the genes used in the PCR, and the GAPDH gene was used as a control.

[0240] Primer name sequence (5'-> 3') SEQ ID NO: CPT1 ForwardCCT CCA GTT GGC TTA TCG TG32CPT1 ReverseTTC TTC GTC TGG CTG GAC AT33PGC1α ForwardAGT CTG TAT GGA GTG ACA TCG AG34PGC1α ReverseGGC AAT CCG TCT TCA TCC AC35PPARα ForwardAAG GGC TTC TTT CGG CGA AC36PPARα ReverseTGA CCT TGT TCA TGT TGA AGT TCT TCA37GAPDH ForwardGAG TCA ACG GAT TTG GTC GT30GAPDH ReverseGAC AAG CTT CCC GTT CTC AG31

[0241] As a result of the experiment, the expression of CPT1 (Carnitine Palmitoyltransferase I), PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha), and PPARα (Peroxisome Proliferator-Activated Receptor α), which are factors related to fatty acid oxidation, decreased by oleic acid treatment in HepG2 cells, and when peptide was treated here, the expression levels of CPT1, PGC1α, and PPARα, which had been decreased, increased again (Fig. 12a and Fig. 12b).

[0242] Experimental Example 13: Analysis of the expression of fatty acid oxidation-related proteins in hepatocytes.

[0243] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of fatty acid oxidation-related proteins in hepatocytes was analyzed.

[0244] HepG2 cells (human hepatocytes) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70-80%, 2 x 10 cells were seeded in 12-well culture plates. 5Cells were seeded at a concentration of 10 cells / ml. After reaching approximately 70-80% confluency in a 12-well culture plate, the medium was replaced with serum-free DMEM. After 4 hours, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA) to induce lipid accumulation. After 24 hours of lipid accumulation induction, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM), and incubated for 48 hours. After incubation, the cells were lysed by adding lysis buffer, and the cell lysate was centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins. The obtained proteins were quantified using a BCA kit. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred onto a membrane. The membrane with the attached proteins was blocked with 5% skim milk, and then treated with primary antibodies and reacted overnight at 4°C. The primary antibodies used were as follows: anti-CPT1 antibody (Abcam, UK), anti-PGC1α antibody (Abcam, UK), and anti-β-actin antibody (Santa Cruz, USA).The reaction mixture was washed with PBS-T, reacted with secondary antibodies (Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson immunoResearch, USA; Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L), Jackson immunoResearch, USA) for 1 hour at room temperature, washed again with PBS-T, and visualized by Western detection reagent (Elpis Biotech, Daejeon, Korea) treatment using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0245] As a result of the experiment, the expression of CPT1 (Carnitine Palmitoyltransferase I) and PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) proteins, which are factors related to fatty acid oxidation, decreased by oleic acid treatment in HepG2 cells. However, when peptides were treated thereto, the expression levels of CPT1 and PGC-1α proteins, which had been decreased, increased again (Fig. 13).

[0246] [Experimental Example 14-22] Experimental Example on Adipocytes

[0247] Experimental Example 14: Analysis of the expression of proteins related to insulin resistance and sensitivity in adipocytes.

[0248] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of proteins related to insulin resistance and sensitivity in adipocytes was analyzed.

[0249] 3T3-L1 cells (adipocyte precursor cells, American Type Culture Collection) were cultured in DMEM medium containing 2% bovine calf serum (BCS) (Welgene, Korea). To differentiate 3T3-L1 cells into adipocytes, 1 x 10 5Cells were seeded at a concentration of 10 cells / well, and when the culture reached confluence (Day=2), the medium was replaced once more and cultured for an additional 48 hours. After 48 hours (Day=0), the culture medium was replaced with DMEM medium containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 μM Dexamethasone (Sigma, USA) and cultured. Two days later (Day=2), the medium was replaced with DMEM medium containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. Three days later (Day=5), the medium was replaced with medium containing only 10% FBS and 1% P / S, and peptides (5 μM, 50 μM) were treated and cultured for 48 hours. After incubation, the cells were lysed by adding lysis buffer, and the cells were centrifuged at 4℃ and 12,000 rpm for 30 minutes to obtain proteins, which were then quantified using a BCA kit. Subsequently, the proteins were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and electrotransferred to a membrane. The membrane with the attached proteins was blocked by treating it with 5% skim milk, and then reacted with the primary antibody overnight at 4℃. The primary antibodies used were as follows: anti-pIRS (Tyr612) antibody (Invitrogen, USA); anti-GLUT4 antibody (Invitrogen, USA); anti-pIRS (Ser302) antibody (Cell signaling Technology, USA); anti-P70S6K antibody (Cell signaling Technology, USA); Anti-β-actin antibody (Santa Cruz, USA).The reaction mixture was washed with PBS-T, reacted with secondary antibodies (Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson immunoResearch, USA; Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L), Jackson immunoResearch, USA) for 1 hour at room temperature, washed again with PBS-T, and visualized by Western detection reagent (Elpis Biotech, Daejeon, Korea) treatment using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0250] Through the above experiment, after inducing differentiation in 3T3-L1 preadipocytes with an MDI cocktail for 5 days, the effect of promoting insulin sensitivity when treated with peptides was confirmed by measuring the expression levels of insulin-sensitive or -resistant proteins. As a result of the experiment, the expression levels of pIRS (Tyr612) and GLUT4, which are factors related to insulin sensitivity, increased by peptide treatment in 3T3-L1 cells, and the expression levels of pIRS (Ser302) and p70S6K, which are factors related to insulin resistance, decreased (Fig. 14a and Fig. 14b).

[0251] Experimental Example 15: Expression Analysis of Genes Related to Insulin Resistance and Sensitivity in Adipocytes

[0252] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of genes related to insulin resistance and sensitivity in adipocytes was analyzed.

[0253] 3T3-L1 cells (adipocyte precursor cells, American Type Culture Collection) were cultured in DMEM medium containing 2% bovine calf serum (BCS) (Welgene, Korea). To differentiate 3T3-L1 cells into adipocytes, 1 x 10 5 Cells were seeded at a concentration of 10 cells / well, and the medium was replaced once more at the confluent culture point (Day=2) and cultured for an additional 48 hours. After 48 hours (Day=0), the culture medium was replaced with DMEM medium containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 μM Dexamethasone (Sigma, USA) and cultured. Two days later (Day=2), the medium was replaced with DMEM medium containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. After 3 days (Day = 5), the medium was replaced with only 10% FBS and 1% P / S, and 350 μM oleic acid (Sigma, St. Louis, MO, USA) was treated for 24 hours to induce an insulin-resistant environment. After 24 hours of induction, the medium was replaced with DMEM medium (10% FBS, 1% BSA) supplemented with 350 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM), and cultured for 48 hours. After culture, cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was TOPScript TM After synthesizing DNA complementary to RNA using RT DryMIX (enzynomics, Korea), TOPsimple TMPolymerase chain reaction (PCR) was performed on genes associated with insulin resistance or sensitivity markers using DryMIX-nTaq (enzynomics, Korea). The reaction products were then run on a 1.5% agarose gel, and the mRNA expression levels of the markers were compared for each sample. The primer sequences for the genes used in the PCR are listed in Table 6 below, and the GAPDH gene was used as a control.

[0254] Primer name sequence (5'-> 3') SEQ ID NO: P70S6K ForwardGGA GCC TGG GAG CCC TGA TGT38P70S6K ReverseGAA GCC CTC TTT GAT GCT GTC C39GLUT4 ForwardACT AAG AGC ACC GAG ACC AA40GLUT4 ReverseCTG CCC GAA AGA GTC TAA AG41SIRT1 ForwardGAT CCT TTG GAT TCC TGC AA42SIRT1 ReverseAGT TCC AGC CGT CTC TGT GT43AMPKα ForwardTCA CCG GAC ATA AAG TGG CT44AMPKα ReverseTGA TGA TGT GAG GGT GCC TG45GAPDH ForwardGTG ATG GCA TGG ACT GTG GT8GAPDH ReverseGGA GCC AAA AGG GTC ATC AT9

[0255] Through the above experiment, we tried to confirm whether insulin resistance was suppressed and insulin sensitivity was induced by treating peptides in 3T3-L1 adipocytes in which an insulin-resistant environment was induced by treatment with oleic acid. The expression levels of related factors were confirmed through Western blotting to determine whether insulin resistance was suppressed or insulin sensitivity was induced. As a result of the experiment, the expression level of p70S6K, a factor related to insulin resistance, increased in 3T3-L1 cells by oleic acid treatment, and when peptides were treated thereto, the increased expression level of p70S6K decreased again. In addition, the expression levels of AMPKα, GLUT4, and SIRT1, factors related to insulin sensitivity, decreased in 3T3-L1 cells by oleic acid treatment, and when peptides were treated thereto, the decreased expression levels of AMPKα, GLUT4, and SIRT1 increased again. (Figures 15a and 15b)

[0256] Experimental Example 16: Analysis of the expression of proteins related to insulin resistance and sensitivity in adipocytes.

[0257] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of proteins related to insulin resistance and sensitivity in adipocytes was analyzed.

[0258] 3T3-L1 cells (adipocyte precursor cells, American Type Culture Collection) were cultured in DMEM medium containing 2% bovine calf serum (BCS) (Welgene, Korea). To differentiate 3T3-L1 cells into adipocytes, 1 x 10 5Cells were seeded at a concentration of 10 cells / well, and the medium was replaced once more at the confluent culture point (Day=2) and cultured for an additional 48 hours. After 48 hours (Day=0), the culture medium was replaced with DMEM medium containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 μM Dexamethasone (Sigma, USA) and cultured. Two days later (Day=2), the medium was replaced with DMEM medium containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. After 3 days (Day=5), the medium was replaced with only 10% FBS and 1% P / S, and 350 μM oleic acid (Sigma, St. Louis, MO, USA) was treated for 24 hours to induce an insulin-resistant environment. After 24 hours of induction, the medium was replaced with DMEM medium (10% FBS, 1% BSA) supplemented with 350 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM), and cultured for 48 hours. After culture, the cells were lysed by adding lysis buffer, and the protein was obtained by centrifugation at 4°C and 12,000 rpm for 30 minutes, and the obtained protein was quantified using a BCA kit. Next, the proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred onto a membrane. The membrane with the proteins attached was blocked with 5% skim milk, and then reacted with the primary antibody overnight at 4°C.The primary antibodies used were as follows: anti-pAMPKα antibody (Cell signaling Technology, USA), anti-pIRS (Ser302) antibody (Cell signaling Technology, USA), and anti-β-actin antibody (Santa Cruz, USA). The reaction mixture was washed with PBS-T, and then incubated with secondary antibodies (Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson immunoResearch, USA; Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L), Jackson immunoResearch, USA) at room temperature for 1 hour. The mixture was washed again with PBS-T, and visualized by Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0259] As a result of the experiment, the expression level of pAMPKα, a factor related to insulin sensitivity, was reduced by oleic acid treatment in 3T3-L1 cells, and when peptide was treated thereto, the decreased expression level of pAMPKα increased again. In addition, the expression level of pIRS (Ser302), a factor related to insulin resistance, was increased by oleic acid treatment in 3T3-L1 cells, and when peptide was treated thereto, the increased expression level of pIRS (Ser302) decreased again (Fig. 16).

[0260] Experimental Example 17: Expression Analysis of Fat Synthesis-Related Genes in Adipocytes

[0261] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of genes related to fat synthesis in adipocytes was analyzed.

[0262] 3T3-L1 cells (adipocyte precursor cells, American Type Culture Collection) were cultured in DMEM medium containing 2% bovine calf serum (BCS) (Welgene, Korea). To differentiate 3T3-L1 cells into adipocytes, 1 x 10 5 Cells were seeded at a concentration of 10 cells / well, and when the culture reached confluence (Day=2), the medium was replaced once more and cultured for an additional 48 hours. After 48 hours (Day=0), the culture medium was replaced with DMEM medium containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 μM Dexamethasone (Sigma, USA) and cultured. After 2 days (Day=2), the medium was replaced with DMEM medium containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. After 3 days (Day=5), the medium was replaced with medium containing only 10% FBS and 1% P / S, treated with peptides (5 μM, 50 μM), and cultured for 48 hours. After culture, cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was used in TOPScript TM After synthesizing DNA complementary to RNA using RT DryMIX (enzynomics, Korea), TOPsimple TMPolymerase chain reaction (PCR) was performed on genes of markers related to fat synthesis using DryMIX-nTaq (enzynomics, Korea). The reaction products were then run on a 1.5% agarose gel, and the mRNA expression levels of the markers were compared for each sample. Table 7 below lists the primer sequences for the genes used in the PCR, and the GAPDH gene was used as a control.

[0263] Primer name sequence (5'-> 3') Sequence number ACCα Forward ACC TTA CTG CCA TCC CAT GTG CTA 46 ACCα Reverse GTG CCT GAT GAT CGC ACG AAC AAA 47 PPARγ Forward TCC GCT GAT GCA CTG CCT AT 48 PPARγ Reverse ACA GAC TCG GCA CTC AAT GG 49 GAPDH Forward GTG ATG GCA TGG ACT GTG GT 8 GAPDH Reverse GGA GCC AAA AGG GTC ATC AT 9

[0264] ACCα (Acetyl-CoA Carboxylase-alpha) is a gene encoding an enzyme involved in lipogenesis, catalyzing the carboxylation of acetyl-CoA to malonyl-CoA, a rate-limiting step in fatty acid synthesis. PPAR-γ (Peroxisome proliferator-activated receptor gamma) is known to regulate fatty acid storage and glucose metabolism, and is known to have the effect of increasing the ability of adipocytes to store fatty acids. Experimental results confirmed that when peptides were treated in 3T3-L1 cells, the expression of ACCα and PPAR-γ was reduced (Fig. 17).

[0265] Experimental Example 18: Analysis of the expression of proteins related to fat synthesis in adipocytes.

[0266] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of proteins related to fat synthesis in adipocytes was analyzed.

[0267] 3T3-L1 cells (adipocyte precursor cells, American Type Culture Collection) were cultured in DMEM medium containing 2% bovine calf serum (BCS) (Welgene, Korea). To differentiate 3T3-L1 cells into adipocytes, 1 x 10 5Cells were seeded at a concentration of 10 cells / well, and when the culture reached confluence (Day=2), the medium was replaced once more and cultured for an additional 48 hours. After 48 hours (Day=0), the culture medium was replaced with DMEM medium containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 μM Dexamethasone (Sigma, USA) and cultured. After 2 days (Day=2), the medium was replaced with DMEM medium containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. After 3 days (Day=5), the medium was replaced with medium containing only 10% FBS and 1% P / S, treated with peptides (5 μM, 50 μM), and cultured for 48 hours. After incubation, the cells were lysed by adding lysis buffer, and the cells were centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins, which were quantified using a BCA kit. Subsequently, the proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred to a membrane. The membrane with the attached proteins was blocked by treating it with 5% skim milk, and then reacted with the primary antibody overnight at 4°C. The primary antibodies used were as follows: anti-SREBP1 antibody (Abcam, US); anti-FAS antibody (Cell signaling Technology, USA); anti-C / EBPα antibody (Cell signaling Technology, USA); anti-pACCα antibody (Cell signaling Technology, USA); anti-β-actin antibody (Santa Cruz, USA).The reaction mixture was washed with PBS-T, reacted with secondary antibodies (Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson immunoResearch, USA; Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L), Jackson immunoResearch, USA) for 1 hour at room temperature, washed again with PBS-T, and visualized by Western detection reagent (Elpis Biotech, Daejeon, Korea) treatment using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0268] Through the above experiment, we confirmed the inhibitory effect of peptide treatment on adipogenesis by measuring the expression levels of related factors after inducing differentiation of 3T3-L1 adipocytes with MDI cocktail. SREBP1 (Sterol Regulatory Element Binding Protein-1) is known to induce the synthesis of fat and cholesterol, FAS (Fatty acid synthase) is a fatty acid synthesis enzyme, and C / EBPα (CCAAT / enhancer-binding protein α) is known to induce adipogenesis through PPAR-γ (Genes Dev. 2002 Jan 1; 16(1): 22-26). In addition, ACCα (Acetyl CoA carboxylase α) is known to be phosphorylated by AMPK (AMP-activating protein kinase) and promote the oxidation (decomposition) of fatty acids. As a result of the experiment, it was confirmed that the expression levels of SREBP1, FAS, and C / EBPα, which are fat synthesis-related factors, decreased by peptide treatment in 3T3-L1 adipocytes, and conversely, the expression level of pACCα, which promotes fatty acid decomposition, increased (Fig. 18a and Fig. 18b).

[0269] Experimental Example 19: Analysis of the expression of proteins related to lipolysis in adipocytes.

[0270] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of lipolysis-related proteins in adipocytes was analyzed. 3T3-L1 cells (adipocyte precursor cells, American Type Culture Collection) were cultured in DMEM medium containing 2% Bovine Calf Serum (BCS) (Welgene, Korea). To differentiate 3T3-L1 cells into adipocytes, 1 x 10 5Cells were seeded at a concentration of 10 cells / well, and when the culture reached confluence (Day=2), the medium was replaced once more and cultured for an additional 48 hours. After 48 hours (Day=0), the culture medium was replaced with DMEM medium containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 μM Dexamethasone (Sigma, USA) and cultured. After 2 days (Day=2), the medium was replaced with DMEM medium containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. After 3 days (Day=5), the medium was replaced with medium containing only 10% FBS and 1% P / S, treated with peptides (5 μM, 50 μM), and cultured for 48 hours. After incubation, the cells were lysed by adding lysis buffer, and the cells were centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins, which were quantified using a BCA kit. Subsequently, the proteins were subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then electrotransferred to a membrane. The membrane with the attached proteins was blocked by treating it with 5% skim milk, and then reacted with the primary antibody overnight at 4°C. The primary antibodies used were as follows: anti-PLIN antibody (Abcam, US); anti-pHSL antibody (Cell signaling Technology, USA); anti-PGC1α antibody (Cell signaling Technology, USA); anti-β-actin antibody (Santa Cruz, USA).The reaction mixture was washed with PBS-T, and then reacted with secondary antibodies (Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson immunoResearch, USA; Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L), Jackson immunoResearch, USA; Peroxidase-conjugated AffiniPure Rabbit Anti-Goat IgG (H+L), Jackson immunoResearch, USA) at room temperature for 1 hour. After washing again with PBS-T, the mixture was visualized by Western detection reagent (Elpis Biotech, Daejeon, Korea) treatment using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0271] Through the above experiment, we confirmed the effect of promoting lipolysis by peptide treatment after inducing differentiation of 3T3-L1 adipocytes with MDI cocktail by measuring the expression levels of related factors. PLIN (Perilipin) is phosphorylated by activated PKA and promotes the triacylglycerol decomposition action of lipolytic enzymes. HSL (Hormone-sensitive lipase) is an enzyme that hydrolyzes fat and is activated by phosphorylation by activated kinase PKA (Protein Kinase A). PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) is a transcriptional coactivator that regulates the expression of genes related to energy metabolism (Adv Physiol Educ. 2006 Dec;30(4):145-51). As a result of the experiment, it was confirmed that when peptides were treated in 3T3-L1 adipocytes, the expression levels of PLIN, pHSL, and PGC1α, which are factors related to fat decomposition, increased (Fig. 19).

[0272] Experimental Example 20: Analysis of the expression of genes related to fatty acid oxidation after inducing an insulin-resistant environment in adipocytes.

[0273] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of genes related to fatty acid oxidation in insulin-resistant adipocytes was analyzed.

[0274] 3T3-L1 cells (adipocyte precursor cells, American Type Culture Collection) were cultured in DMEM medium containing 2% bovine calf serum (BCS) (Welgene, Korea). To differentiate 3T3-L1 cells into adipocytes, 1 x 10 5Cells were seeded at a concentration of 10 cells / well, and the medium was replaced once more at the confluent culture point (Day=2) and cultured for an additional 48 hours. After 48 hours (Day=0), the culture medium was replaced with DMEM medium containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 μM Dexamethasone (Sigma, USA) and cultured. Two days later (Day=2), the medium was replaced with DMEM medium containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. After 3 days (Day = 5), the medium was replaced with only 10% FBS and 1% P / S, and 350 μM oleic acid (Sigma, St. Louis, MO, USA) was treated for 24 hours to induce an insulin-resistant environment. After 24 hours of induction, the medium was replaced with DMEM medium (10% FBS, 1% BSA) supplemented with 350 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM), and cultured for 48 hours. After culture, cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was TOPScript TM After synthesizing DNA complementary to RNA using RT DryMIX (enzynomics, Korea), TOPsimple TM Polymerase chain reaction (PCR) was performed on genes of fatty acid oxidation-related markers using DryMIX-nTaq (enzynomics, Korea). The reaction products were then run on a 1.5% agarose gel, and the mRNA expression levels of the markers were compared for each sample. Table 8 below lists the primer sequences for the genes used in the PCR, with the GAPDH gene used as a control.

[0275] Primer name sequence (5'-> 3') Sequence number PGC1α Forward ATG TGC AGC CAA GAC TCT GTA 50 PGC1α Reverse CGC TAC ACC ACT TCA ATC CAC 51 CPT1 Forward CGT ACC AAG TAG CCA AGG CA 52 CPT1 Reverse CAG GAA CGC ACA GTC TCA GT 53 GAPDH Forward GTG ATG GCA TGG ACT GTG GT 8 GAPDH Reverse GGA GCC AAA AGG GTC ATC AT 9

[0276] As a result of the experiment, the expression of PGC1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) and CPT1 (Carnitine Palmitoyltransferase I), genes related to fatty acid oxidation, decreased by oleic acid treatment in 3T3-L1 cells, but when peptide was treated thereto, the expression of PGC1α and CPT1, which had been decreased, increased again (Fig. 20).

[0277] Experimental Example 21: Analysis of expression of genes related to fat synthesis and lipolysis after inducing an insulin-resistant environment in adipocytes.

[0278] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of genes related to fat synthesis and fat decomposition in insulin-resistant adipocytes was analyzed.

[0279] 3T3-L1 cells (adipocyte precursor cells, American Type Culture Collection) were cultured in DMEM medium containing 2% bovine calf serum (BCS) (Welgene, Korea). To differentiate 3T3-L1 cells into adipocytes, 1 x 10 5Cells were seeded at a concentration of 10 cells / well, and the medium was replaced once more at the confluent culture point (Day=2) and cultured for an additional 48 hours. After 48 hours (Day=0), the culture medium was replaced with DMEM medium containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 μM Dexamethasone (Sigma, USA) and cultured. Two days later (Day=2), the medium was replaced with DMEM medium containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. After 3 days (Day = 5), the medium was replaced with only 10% FBS and 1% P / S, and 350 μM oleic acid (Sigma, St. Louis, MO, USA) was treated for 24 hours to induce an insulin-resistant environment. After 24 hours of induction, the medium was replaced with DMEM medium (10% FBS, 1% BSA) supplemented with 350 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM), and cultured for 48 hours. After culture, cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was TOPScript TM After synthesizing DNA complementary to RNA using RT DryMIX (enzynomics, Korea), TOPsimple TM Polymerase chain reaction (PCR) was performed on genes of markers related to fat synthesis and lipolysis using DryMIX-nTaq (enzynomics, Korea). The reaction products were then run on a 1.5% agarose gel, and the mRNA expression levels of the markers were compared for each sample. Table 9 below lists the primer sequences for the genes used in the PCR, and the GAPDH gene was used as a control.

[0280] Primer name sequence (5'-> 3') SEQ ID NO: FAS ForwardTGC TGG CAC TAC AGA ATG C54FAS ReverseAAC AGC CTC AGA GCG ACA AT55HSL ForwardGGA CAC ACA CAC ACC TG56HSL ReverseCCC TTT CGC AGC AAC TTT AG57ATGL ForwardTCG TGG ATG TTG GTG GAG CT58ATGL ReverseTGT GGC CTC ATT CCT CCT A59PLIN ForwardAAG GAT CCT GCA CCT CAC AC60PLIN ReverseCCT CTG CTG AAG GGT TAT CG61GAPDH ForwardGTG ATG GCA TGG ACT GTG GT8GAPDH ReverseGGA GCC AAA AGG GTC ATC AT9

[0281] Through the above experiment, after inducing an insulin-resistant environment in 3T3-L1 adipocytes, we confirmed whether peptide treatment had an inhibitory effect on lipogenesis or an activating effect on lipolysis by measuring the expression levels of related factors. As a result of the experiment, it was confirmed that the expression level of the FAS gene, a lipogenesis-related factor, increased by oleic acid treatment in 3T3-L1 adipocytes, but when peptides were treated thereto, the increased expression level of the FAS gene decreased again. In addition, the expression levels of the PLIN, HSL, and ATGL genes, which are lipolysis-related factors, decreased by oleic acid treatment in 3T3-L1 adipocytes, but when peptides were treated thereto, the decreased expression levels of the PLIN, HSL, and ATGL genes increased (Fig. 21a, Fig. 21b).

[0282] Experimental Example 22: Analysis of the expression of proteins related to fat synthesis and fat decomposition after inducing an insulin-resistant environment in adipocytes.

[0283] The effect of the peptide of sequence number 1 manufactured in Manufacturing Example 1 on the expression of proteins related to fat synthesis and fat decomposition in insulin-resistant adipocytes was analyzed.

[0284] 3T3-L1 cells (adipocyte precursor cells, American Type Culture Collection) were cultured in DMEM medium containing 2% bovine calf serum (BCS) (Welgene, Korea). To differentiate 3T3-L1 cells into adipocytes, 1 x 10 5Cells were seeded at a concentration of 10 cells / well, and the medium was replaced once more at the confluent culture point (Day=2) and cultured for an additional 48 hours. After 48 hours (Day=0), the culture medium was replaced with DMEM medium containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 μM Dexamethasone (Sigma, USA) and cultured. Two days later (Day=2), the medium was replaced with DMEM medium containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. After 3 days (Day=5), the medium was replaced with only 10% FBS and 1% P / S, and 350 μM oleic acid (Sigma, St. Louis, MO, USA) was treated for 24 hours to induce an insulin-resistant environment. After 24 hours of induction, the medium was replaced with DMEM medium (10% FBS, 1% BSA) supplemented with 350 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM), and cultured for 48 hours. After culture, the cells were lysed by adding lysis buffer, and the protein was obtained by centrifugation at 4°C and 12,000 rpm for 30 minutes, and the obtained protein was quantified using a BCA kit. Next, the proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred onto a membrane. The membrane with the proteins attached was blocked with 5% skim milk, and then reacted with the primary antibody overnight at 4°C.The primary antibodies used were as follows: anti-SREBP1 antibody (Abcam, US); anti-FAS antibody (Cell signaling Technology, USA); anti-PLIN antibody (Abcam, US); anti-β-actin antibody (Santa Cruz, USA). The reaction mixture was washed with PBS-T, and then incubated with secondary antibodies (Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L), Jackson immunoResearch, USA; Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L), Jackson immunoResearch, USA; Peroxidase-conjugated AffiniPure Rabbit Anti-Goat IgG (H+L), Jackson immunoResearch, USA) at room temperature for 1 hour. After washing again with PBS-T, the mixture was visualized by Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0285] As a result of the experiment, it was confirmed that the expression level of FAS protein, a fat synthesis-related factor, increased by oleic acid treatment in 3T3-L1 adipocytes, but when peptide was treated thereto, the increased expression level of FAS protein decreased again. In addition, the expression levels of SREBP1 and PLIN proteins, which are fat decomposition-related factors, decreased by oleic acid treatment in 3T3-L1 adipocytes, but when peptide was treated thereto, the decreased expression levels of SREBP1 and PLIN proteins increased (Fig. 22a and Fig. 22b).

[0286] Although representative embodiments of the present application have been described above as examples, the scope of the present application is not limited to the specific embodiments described above, and a person with ordinary knowledge in the relevant field will be able to make appropriate changes within the scope described in the claims of the present application.

Claims

1. A peptide comprising the amino acid sequence of sequence number 1.

2. In claim 1, The above peptide is a peptide having the following activities: (i) Activity that promotes the expression of genes related to cell proliferation in myoblasts, including PCNA (Proliferating cell nuclear antigen), NDRG2 (N-myc Downstream-Regulated Gene 2), or CDK4 (Cyclin-dependent kinase 4); (ii) activity that promotes the expression of cell proliferation-related factors SIRT1 (Sirtuin1), Ki67, Pax7, or pAKT proteins in myogenic cells; (iii) activity that promotes the expression of genes for differentiation-related factors MyoD, Myf6, mTOR, Myf5, or Myf4 in myogenic cells; (iv) activity that promotes the expression of differentiation-related factors α-actinin, Myf6 or MyoG protein in myogenic cells; or (v) Activity that promotes protein expression of signal transduction factors SIRT1, pAMPKα, pAKT, pmTOR or p70S6K involved in muscle protein synthesis in myogenic cells.

3. In claim 1, The above peptide is a peptide having the following activities: (i) Activity that inhibits fat accumulation in hepatocytes; (ii) activity of inhibiting protein expression of lipogenic factors SREBP1 or FAS and promoting protein expression of lipolytic factors pACC, pHSL, ATGL, or PLIN in hepatocytes where fat accumulation is induced; (iii) Activity of promoting the expression of the gene of fatty acid oxidation-related factor CPT1, PGC1α or PPARα in hepatocytes where fat accumulation is induced, and promoting the expression of the protein of fatty acid oxidation-related factor CPT1 or PGC1α. (iv) Activity that inhibits gene expression of fat synthesis-related factors ACCα or PPAR-γ in adipocytes; (v) activity of inhibiting protein expression of fat synthesis-related factors SREBP1, FAS, or C / EBPα in adipocytes and promoting protein expression of fatty acid degradation-related factor pACCα; (vi) activity that promotes protein expression of lipolysis-related factors PLIN, pHSL, or PGC1α in adipocytes; (vii) Activity that promotes gene expression of fatty acid oxidation-related factors PGC1α or CPT1 in adipocytes where insulin resistance is induced; (viii) activity that inhibits the gene expression of fatty acid synthesis factor FAS and promotes the gene expression of lipolysis-related factors PLIN, HSL, or ATGL in adipocytes where insulin resistance is induced; or (ix) Activity that inhibits protein expression of fatty acid synthesis factors FAS and SREBP1 and promotes protein expression of lipolysis-related factor PLIN in adipocytes where insulin resistance is induced.

4. In claim 1, The above peptide is a peptide having the following activities: (i) Activity of promoting protein expression of insulin-sensitive factor pIRS (Tyr612) or GLUT4 in adipocytes and inhibiting protein expression of insulin resistance factor pIRS (Ser302) or p70S6K; (ii) activity of inhibiting the expression of the gene of insulin resistance factor P70S6K and promoting the expression of the gene of insulin sensitivity factor AMPKα, GLUT4 or SIRT1 in adipocytes where insulin resistance is induced; (iii) Activity that promotes protein expression of insulin-sensitive factor pAMPKα and inhibits protein expression of insulin resistance factor pIRS (Ser302) in adipocytes where insulin resistance is induced. (iv) activity of inhibiting the expression of genes for insulin resistance factors mTOR or P70S6K and promoting the expression of genes for insulin sensitivity factors AKT, SIRT1, or AMPKα in hepatocytes where insulin resistance is induced; or (v) Activity of inhibiting the expression of proteins of insulin resistance factors pmTOR or pJNK and promoting the expression of proteins of insulin sensitivity factors pAMPKα, GLUT4 or SIRT1 in hepatocytes where insulin resistance is induced.

5. A composition for promoting muscle formation, anti-obesity, anti-fatty liver or anti-diabetic use, comprising the peptide of any one of claims 1 to 4 as an active ingredient.

6. A pharmaceutical composition for preventing or treating muscle disease, comprising the peptide of claim 1 or 2 as an active ingredient.

7. In claim 6, A pharmaceutical composition for preventing or treating a muscle disease, wherein the muscle disease is at least one selected from the group consisting of muscular atrophy, sarcopenia, muscular dystrophy, disuse atrophy, spinal muscular amyotrophy, muscle stiffness, muscular hypotonia, muscle weakness, muscle endurance weakness, amyotrophic lateral sclerosis, spinal muscular atrophy, myasthenia gravis, myasthenia, muscle degeneration, and cachexia.

8. A food composition for preventing or improving muscle disease, comprising the peptide of claim 1 or 2 as an active ingredient.

9. In claim 8, A food composition for preventing or improving a muscle disease, wherein the muscle disease is at least one selected from the group consisting of muscular atrophy, sarcopenia, muscular dystrophy, disuse atrophy, spinal muscular amyotrophy, muscle stiffness, muscular hypotonia, weakened muscle strength, weakened muscle endurance, amyotrophic lateral sclerosis, spinal muscular atrophy, myasthenia gravis, myasthenia, muscle degeneration, and cachexia.

10. A pharmaceutical composition for preventing or treating obesity or fatty liver, comprising the peptide of claim 1 or claim 3 as an active ingredient.

11. A food composition for preventing or improving obesity or fatty liver, comprising the peptide of claim 1 or claim 3 as an active ingredient.

12. A pharmaceutical composition for preventing or treating diabetes, comprising the peptide of claim 1 or claim 4 as an active ingredient.

13. A food composition for preventing or improving diabetes, comprising the peptide of claim 1 or claim 4 as an active ingredient.

Images