Novel peptides with muscle-building, anti-obesity, and anti-diabetic activities and their uses

Abstract

The peptides of the present invention exhibit muscle-building activity in myoblasts. The peptides of the present invention exhibit activity in hepatocytes and adipocytes that inhibit fat accumulation and promote lipolysis. The peptides of the present invention inhibit the expression of insulin resistance factor and increase the expression of insulin sensitivity factor in hepatocytes and adipocytes. Therefore, the peptides of the present invention can be used as active substances for treating, preventing, or improving muscle diseases, obesity, fatty liver, and diabetes.

Description

[0001] Cross-reference to related applications

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

[0003] This invention relates to a novel peptide with muscle-building activity, anti-obesity activity and anti-diabetic activity, and its uses. Background Technology

[0004] Age-related loss of skeletal muscle mass leads to a decline in muscle strength and various physical functions. In particular, frailty, skeletal muscle atrophy, and sarcopenia are common in older adults and are chronic problems threatening healthy life expectancy in various countries; therefore, prevention, treatment, and rehabilitation have become major concerns in the field of healthcare today. While muscle mass and strength naturally decline with age, sarcopenia involves an excessive reduction in muscle mass and strength even after taking age and sex factors into account, leading to decreased physical function and increased health risks or mortality. Recently, diagnostic criteria for sarcopenia have been established, and with its inclusion in the International Classification of Diseases (ICD) coding, it is classified as a disease, not merely a result of aging.

[0005] Obesity refers to a condition characterized by an excess of body fat, resulting from an imbalance between energy intake and expenditure. According to the World Health Organization (WHO), over one billion adults worldwide are overweight, with at least three million clinically obese; this number is significantly increasing in the United States and Europe. Overweight and obesity raise blood pressure and cholesterol levels, potentially leading to various diseases such as heart disease, diabetes, and arthritis, and increasing the incidence of various adult-onset diseases. Furthermore, overweight and obesity are contributing factors to increased rates of various adult-onset diseases such as arteriosclerosis, hypertension, hyperlipidemia, and heart disease, affecting not only adults but also children and adolescents.

[0006] Currently, representative obesity treatments approved and widely prescribed by the US FDA include a group of drugs that act on the central nervous system to suppress appetite, and orlistat (Xenical), an inhibitor of the pancreatic digestive enzyme lipase. Regarding drugs acting on the central nervous system, the approval of many (such as sibutramine) has been withdrawn due to cardiovascular and psychiatric side effects, while orlistat is limited by its variability in efficacy with fat intake and various side effects. Meanwhile, liraglutide (a glucagon-like peptide-1 (GLP-1) receptor agonist), an endocrine peptide-targeting drug, has been approved for use, but it carries a risk of thyroid cancer.

[0007] Diabetes mellitus is a metabolic disease characterized by hyperglycemia, or elevated blood glucose levels, caused by insufficient or impaired insulin secretion. Hyperglycemia leads to various symptoms and signs, and results in the excretion of glucose in the urine. In recent years, the incidence of diabetes has exploded due to rising obesity rates, particularly abdominal obesity. Diabetes can be broadly classified into insulin-dependent type 1 diabetes and non-insulin-dependent type 2 diabetes. Type 2 diabetes is characterized by hyperglycemia, insulin resistance, and relative impaired insulin secretion.

[0008] When food is ingested, the digestive tract absorbs glucose from the food, stimulating pancreatic beta cells to secrete insulin. This insulin promotes glucose uptake by muscles. Furthermore, while insulin is partially involved in glucose uptake by the liver, it primarily inhibits glucose production in the liver. Insulin lowers blood glucose levels by inhibiting glucose production in the liver and promoting glucose uptake by peripheral tissues, including muscles. Insulin resistance refers to a condition where, at a given insulin concentration, blood glucose responds to insulin less than normally. Insulin regulates blood glucose by promoting glucose uptake by muscles or inhibiting glucose production in the liver, while insulin resistance refers to a state where these insulin effects are diminished even in the absence of insulin deficiency. Insulin receptors on cell membranes are involved in the process of glucose uptake by peripheral tissue cells; insulin resistance occurs when the number of insulin receptors is reduced or when there are intracellular defects after receptor binding. Although insulin receptor defects have been found in type 2 diabetes, intracellular defects after receptor binding, particularly impaired phosphorylation / dephosphorylation regulated by insulin, are known to play a greater role. Among these mechanisms, impaired PI3K (phosphoinositol 3-kinase) signaling is known to reduce the movement of the glucose transporter GLUT-4 (glucose type 4) across the cell membrane.

[0009] Currently, blood sugar is controlled through lifestyle interventions (diet therapy, exercise therapy) and medication. However, strict management and implementation of diet therapy and exercise therapy are difficult, and their effectiveness is limited. Therefore, most diabetic patients rely on medications such as insulin, insulin secretagogues, insulin sensitivity enhancers, and hypoglycemic agents, combined with lifestyle interventions, to control their blood sugar.

[0010] [Existing Technical Documents]

[0011] [Patent Literature]

[0012] Korean Patent No. 10-2064387

[0013] Korean Patent No. 10-2486996

[0014] Korean Patent No. 10-2507392

[0015] International Patent Application No. PCT-KR2021-011278 Summary of the Invention

[0016] [Technical Issues]

[0017] The inventors have diligently researched and developed a small molecule with excellent therapeutic activity against diseases such as sarcopenia, diabetes, and obesity, while ensuring safety and high drug delivery efficiency. As a result, experiments have confirmed that the peptide developed by the inventors possesses activity in muscle cells that promotes the expression of factors involved in muscle formation and factors involved in muscle protein synthesis. Furthermore, this invention was achieved by confirming that, in addition to these activities, the peptide of this invention also possesses activities that inhibit fat accumulation in adipocytes and hepatocytes, promote lipolysis, inhibit insulin resistance, and improve insulin sensitivity.

[0018] Therefore, one object of the present invention is to provide a novel peptide having muscle-building, 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-diabetes, comprising peptides having the above-mentioned activities as active ingredients.

[0020] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of muscle diseases, comprising 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 the prevention or improvement of muscle diseases, comprising peptides having the above-mentioned activity as active ingredients.

[0022] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of obesity or fatty liver, comprising a peptide having the above-mentioned 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, comprising peptides having the above-mentioned activity as active ingredients.

[0024] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of diabetes, comprising 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 the prevention or improvement of diabetes, comprising peptides having the above-mentioned activity as active ingredients.

[0026] [Technical Solution]

[0027] To achieve the above-mentioned objectives of the present invention, one aspect of the present invention provides a peptide comprising the amino acid sequence shown in SEQ ID NO:1.

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

[0029] Another aspect of the invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases, comprising the peptide as an active ingredient.

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

[0031] Another aspect of the invention provides a pharmaceutical composition for the prevention or treatment of 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 invention provides a pharmaceutical composition for the prevention or treatment of diabetes, comprising the peptide as an active ingredient.

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

[0035] The present invention will now be described in detail.

[0036] 1. Peptides and their activities

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

[0038] [SEQ ID NO:1]

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

[0040] As used in this article, the term "peptide" refers to a linear molecule formed by amino acid residues linked together by peptide bonds.

[0041] The peptide containing the amino acid sequence of SEQ ID NO:1 of the present invention can be used as is, but can also be used to obtain amino acid variants or fragments with different sequences by means of the deletion, insertion or substitution of amino acid residues or combinations thereof, without affecting the original activity of the peptide (e.g., skin condition improvement activity).

[0042] The peptides of the present invention can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc., without changing their activity.

[0043] The peptides of the present invention include peptides and variants thereof having substantially the same amino acid sequence as the peptide comprising the amino acid sequence of SEQ ID NO:1, or their active fragments. Substantially the same amino acid sequence is defined as an amino acid sequence having at least 75%, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 98% sequence identity with the amino acid sequence of SEQ ID NO:1. Furthermore, the peptide may additionally comprise a targeting sequence, a tag, labeled residues, or an amino acid sequence prepared for a specific purpose to increase half-life or peptide stability.

[0044] The peptides of the present invention may have N-terminal and / or C-terminal modifications induced to select a portion of the amino acid sequence and increase its activity. Such N-terminal and / or C-terminal modifications can significantly improve the stability of the peptides of the present invention, for example, by extending the half-life of the peptide after in vivo administration. The term "stability" includes not only in vivo stability that protects the peptides of the present invention from attack by proteolytic enzymes in vivo, but also storage stability (e.g., room temperature storage stability).

[0045] N-terminal modification can be a protecting group attached to the N-terminus of the peptide, selected from the group consisting of acetyl, fluorenemethyloxycarbonyl, formyl, palmitoyl, myristyl, stearoyl, and polyethylene glycol (PEG). C-terminal modification can be, but is not limited to, the formation of hydroxyl (-OH), amino (-NH2), hydrazide (-NHNH2), etc., attached to the C-terminus of the peptide.

[0046] The peptides of the present invention can be prepared by a variety of methods widely known in the art to which this invention pertains. For example, the peptides of the present invention can be prepared according to chemical synthesis methods known in the industry, particularly solid-phase synthesis (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 (US Patent No. 5,516,891).

[0047] The peptides of this invention have muscle-building activity.

[0048] In one embodiment, the peptide of the present invention has the following activity in promoting muscle formation: (i) Promotes the expression of gene expression of cell proliferation-related factors PCNA (proliferating cell nuclear antigen), NDRG2 (N-myc downstream regulatory gene 2), or CDK4 (cyclin-dependent kinase 4) in myoblasts; (ii) Promotes the expression of protein of cell proliferation-related factors SIRT1 (Sirtuin 1), Ki67, Pax7 or pAKT in myoblasts; (iii) Promotes the expression of genes of differentiation-related factors MyoD, Myf6, mTOR, Myf5 or Myf4 in myoblasts; (iv) Promotes the expression of differentiation-related factors α-actin, Myf6, or MyoG in myoblasts; or (v) Promotes the expression of protein signals involved in muscle protein synthesis in myoblasts, such as SIRT1, pAMPKα, pAKT, pmTOR, or p70S6K.

[0049] The peptides of this invention have the activity of inhibiting obesity or fatty liver.

[0050] In one embodiment, the peptide of the present invention has the following activities in inhibiting obesity or fatty liver: (i) Inhibits the activity of lipid accumulation in hepatocytes; (ii) Inhibit the protein expression of adipogenic factors SREBP1 or FAS and promote the protein expression of lipolytic factors pACC, pHSL, ATGL or PLIN in hepatocytes with induced lipid accumulation. (iii) Activities that promote gene expression of fatty acid oxidation-related factors CPT1, PGC1α or PPARα and protein expression of fatty acid oxidation-related factors CPT1 or PGC1α in hepatocytes with induced lipid accumulation. (iv) Inhibit the gene expression of adipogenesis-related factors ACCα or PPAR-γ in adipocytes; (v) Inhibits the protein expression of adipogenesis-related factors SREBP1, FAS, or C / EBPα in adipocytes and promotes the protein expression of fatty acid degradation-related factor pACCα. (vi) The activity of promoting the expression of proteins of lipolysis-related factors PLIN, pHSL or PGC1α in adipocytes. (vii) The activity of promoting gene expression of fatty acid oxidation-related factors PGC1α or CPT1 in adipocytes induced with insulin resistance. (viii) Inhibit the gene expression of fatty acid synthesis factor FAS and promote the gene expression of lipolysis-related factors PLIN, HSL, or ATGL in induced insulin-resistant adipocytes; or (ix) Induced insulin resistance in adipocytes, inhibited the protein expression of fatty acid synthesis factors FAS or SREBP1 and promoted the protein expression of lipolysis-related factor PLIN.

[0051] The peptides of this invention have anti-diabetic activity.

[0052] In one embodiment, the peptide of the present invention has the following activities in terms of antidiabetic activity: (i) Promotes the protein expression of insulin-sensitive factors pIRS (Tyr612) or GLUT4 in adipocytes and inhibits the protein expression of insulin-resistant factors pIRS (Ser302) or p70S6K. (ii) Inhibit the gene expression of the insulin resistance factor P70S6K and promote the gene expression of the insulin sensitivity factors AMPKα, GLUT4 or SIRT1 in induced insulin-resistant adipocytes. (iii) Promotes the protein expression of insulin sensitivity factor pAMPKα and inhibits the protein expression of insulin resistance factor pIRS (Ser302) in induced insulin resistance adipocytes. (iv) Inhibiting the gene expression of insulin resistance factors mTOR or P70S6K and promoting the gene expression of insulin sensitivity factors AKT, SIRT1, or AMPKα in induced insulin-resistant hepatocytes; or (v) Inhibits the protein expression of insulin resistance factors pmTOR or pJNK and promotes the protein expression of insulin sensitivity factors pAMPKα, GLUT4 or SIRT1 in induced insulin-resistant hepatocytes.

[0053] 2. Compositions for promoting muscle formation

[0054] According to another aspect of the present invention, a composition for promoting muscle formation is provided, comprising a peptide containing the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0055] Pharmaceutical Composition

[0056] According to another aspect of the present invention, a pharmaceutical composition for the prevention or treatment of muscle diseases is provided, comprising a peptide containing the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0057] The peptide containing the amino acid sequence of SEQ ID NO:1 is the same as the entry above. Peptides and their activities The peptides described in this section are the same, therefore the description of their activity in preventing or treating muscle diseases is quoted here and will not be repeated.

[0058] The peptides of the present invention, which possess the above-mentioned activities, exhibit excellent efficacy in inhibiting muscle loss and increasing muscle mass, and can be used to prevent or treat muscle diseases.

[0059] Meanwhile, although conventional functional peptides possess effective biological activity, they still exhibit some drawbacks, such as being unable to be effectively absorbed into target tissues or cells due to their size, or being eliminated from the body in a short time due to their short half-life. On the other hand, the pharmaceutical composition of the present invention for the prevention or treatment of muscle diseases comprises a peptide consisting of about 10 or fewer amino acids as the active ingredient. Therefore, the active ingredient has excellent cell penetration, and, for example, when applied topically, can achieve effective prevention or treatment of muscle diseases.

[0060] In this article, the term "muscle disease" refers to a muscle disease, condition, or state associated with a decrease in the proliferation or differentiation of myoblasts, and can be collectively referred to as a muscle disease, such as a muscle disease resulting from muscle dysfunction, muscle atrophy, or muscle degeneration. Muscle diseases can be selected from, but are not limited to, one or more of the following groups: muscle atrophy, sarcopenia, muscular dystrophy, disuse atrophy, spinal muscular atrophy, myotonia, hypotonia, weakness, decreased muscle endurance, amyotrophic lateral sclerosis, spinal-bulbar muscular atrophy, myasthenia gravis, muscle weakness, muscle degeneration, and cachexia.

[0061] In one embodiment, the peptides of the present invention can promote the proliferation and differentiation of myoblasts to inhibit muscle loss and increase muscle mass. Specifically, the peptides of the present invention can promote the proliferation of myoblasts and significantly increase the expression of early, intermediate, and late differentiation markers of myoblasts, thereby promoting the differentiation of myoblasts into myocytes or myofibrils. In addition, the peptides of the present invention can significantly increase the expression of myoblast markers and muscle protein synthesis markers, improve the myoblast angiogenesis capacity, and improve muscle mass recovery / regeneration when myoblasts are damaged by exogenous factors.

[0062] In this document, the term "prevention" refers to any action that suppresses or delays the onset of disease by applying the above-described composition.

[0063] In this article, the term "treatment" refers to any form of therapy that provides therapeutic effects to an individual who has a disease or is at risk of developing it, including improving an individual's condition (e.g., one or more symptoms), slowing disease progression, delaying the onset of symptoms, or alleviating symptom progression. Therefore, "treatment" and "prevention" do not refer to a cure or complete elimination of symptoms.

[0064] In this document, the term "administration" means introducing a particular substance into an individual by any suitable method, and the pharmaceutical compositions of the present invention can be administered via any general route capable of reaching the target in the body. For example, there are no particular limitations on the route of administration of the pharmaceutical compositions of the present invention; they can be administered orally or parenterally. In the case of parenterally administration, they can be administered via intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, local application, percutaneous application, etc., but are not limited thereto.

[0065] The pharmaceutical compositions of the present invention may contain a therapeutically effective amount of the above-described peptides and a pharmaceutically acceptable carrier.

[0066] The term "therapeutic effective amount" refers to an amount sufficient to achieve the activity or efficacy of a peptide (which is the active ingredient of the pharmaceutical composition of the present invention), for example, an amount sufficient to achieve the preventive or therapeutic efficacy of diabetes or obesity.

[0067] Pharmaceutically acceptable carriers are those commonly used in formulations, including but not limited to lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0068] In addition to the components described above, the pharmaceutical compositions of the present invention may further include, but are not limited to, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0069] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy (19th ed., 1995, Williams & Wilkins).

[0070] The pharmaceutical compositions of the present invention can be formulated into unit dosage forms or contained in multi-dose containers using pharmaceutically acceptable carriers and / or excipients, according to methods readily practiced by those skilled in the art. The formulations may be in the form of solutions, suspensions, or emulsions in oily or aqueous media, or in the form of extracts, powders, granules, tablets, or capsules, and may additionally contain dispersants or stabilizers.

[0071] The pharmaceutical composition of the present invention can be administered via any route suitable for treating muscle diseases, such as oral or parenteral administration, and in the case of parenteral administration, it can be administered via intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, local administration, transdermal administration, etc.

[0072] The pharmaceutical composition of the present invention may comprise the peptide of the present invention at a concentration of 0.01 μM to 1000 μM, and specifically, the concentration of the peptide of the present invention may be 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; 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, but not limited to these.

[0073] Food composition

[0074] According to another aspect of the present invention, a food composition for preventing or improving muscle diseases is provided, comprising a peptide containing the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0075] In one embodiment, the peptides of the present invention can promote the proliferation and differentiation of myoblasts, thereby inhibiting muscle loss and increasing muscle mass. Therefore, the peptides of the present invention can be effectively used as active ingredients in food compositions for the prevention or improvement of muscle diseases, and the food compositions of the present invention can be effectively used for the prevention or improvement of muscle diseases.

[0076] In this specification, the term “improvement” may refer to any behavior that at least reduces parameters (such as symptom severity) related to the relief or treatment of the condition.

[0077] The activity of the peptides of the present invention in the food composition of the present invention related to the prevention or improvement of muscle diseases is the same as that described in the above-mentioned pharmaceutical compositions, and therefore is cited herein without further elaboration.

[0078] In the functional food composition of the present invention, the appropriate amount of peptides contained as active ingredients may be selected from 0.000001% by weight to 10% by weight, specifically less than 10% by weight, of the total weight of the composition.

[0079] The food composition of the present invention may contain a food-effective amount of the peptide and a food-acceptable carrier.

[0080] The functional food composition of the present invention not only contains peptides as active ingredients, but also ingredients commonly added during food manufacturing, and may include, for example, proteins, carbohydrates, fats, nutrients, flavoring agents, and taste agents. Examples of carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose, sucrose, and oligosaccharides; and polysaccharides such as dextrin and cyclodextrin, as well as sugar alcohols such as xylitol, sorbitol, and erythritol. As taste agents, natural taste agents (such as sematrandrine and stevia extracts, such as rebaudioside A and glycyrrhizin) and synthetic taste agents (such as saccharin and aspartame) can be used.

[0081] In addition to the above-mentioned ingredients, the food composition of the present invention may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents (such as synthetic and natural flavoring agents), coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the food composition of the present invention may contain fruit pulp used in the manufacture of natural fruit juices, fruit juice beverages, and vegetable beverages. For example, when the functional food composition of the present invention is manufactured into a beverage, in addition to peptides as the active ingredient of the present invention, it may also additionally contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia ulmoides extract, jujube extract, licorice extract, etc.

[0082] There are no particular restrictions on the type of food composition. Examples of food or food compositions include meat, sausage, bread, chocolate, confectionery, snacks, pastries, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea beverages, alcoholic beverages and vitamin complexes, dairy products, fermented milk, etc., and may include all functional foods or foods in the conventional sense.

[0083] Furthermore, food compositions may include health functional foods or dietary compositions. In this document, the term "health functional food" refers to food manufactured and processed using raw materials or ingredients that have functional properties beneficial to the human body, in the form of tablets, capsules, powders, granules, liquid preparations, pills, etc. In this document, "functional" refers to, for example, achieving health-beneficial effects through the regulation of nutrients or physiological functions related to human structure and function.

[0084] Health functional foods or dietary foods can be manufactured using methods commonly used in the art, and commonly used raw materials and ingredients can be added during such manufacturing processes. Furthermore, formulations of health functional foods can be manufactured without restriction, as long as they are considered to be formulations of health functional foods. Food compositions can be manufactured in various formulation forms. Unlike general pharmaceuticals, its advantage lies in being made from food raw materials, thus avoiding the side effects that may occur with long-term drug use, and it is highly portable. Therefore, a health functional food according to one embodiment can be used as an adjuvant to enhance the therapeutic effect of muscle diseases.

[0085] According to another aspect of the present invention, a method for treating a muscle disease in a subject in need is provided, the method comprising administering to the subject in need of treatment an effective amount of a peptide comprising the amino acid sequence of SEQ ID NO:1 or a pharmaceutical composition comprising said peptide as an active ingredient.

[0086] According to another aspect of the invention, the use of a peptide comprising the amino acid sequence of SEQ ID NO:1 in the treatment or prevention of muscle diseases is provided.

[0087] According to another aspect of the invention, the use of a peptide comprising the amino acid sequence of SEQ ID NO:1 in the preparation of a pharmaceutical composition for treating or preventing muscle diseases is provided.

[0088] In one embodiment, regarding the composition of the present invention for promoting muscle formation, the pharmaceutical composition or food composition for preventing, treating or improving muscle diseases, the peptide of the present invention has the following activities: (i) Activities promoting the expression of cell proliferation-related factors PCNA (proliferating cell nuclear antigen), NDRG2 (N-myc downstream regulatory gene 2), or CDK4 (cyclin-dependent kinase 4) in myoblasts; (ii) Activities promoting the expression of cell proliferation-related factors SIRT1 (Sirtuin 1), Ki67, Pax7, or pAKT in myoblasts; (iii) Activities promoting the expression of differentiation-related factors MyoD, Myf6, mTOR, Myf5, or Myf4 in myoblasts; (iv) Activities promoting the expression of differentiation-related factors α-actin, Myf6, or MyoG in myoblasts; or (v) Activities promoting the expression of signal transduction factors involved in muscle protein synthesis SIRT1, pAMPKα, pAKT, pmTOR, or p70S6K in myoblasts.

[0089] 3. Compositions for anti-obesity, anti-fatty liver, and anti-diabetic purposes

[0090] According to another aspect of the present invention, a composition for anti-obesity, anti-fatty liver or anti-diabetes is provided, comprising a peptide containing the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0091] Pharmaceutical Composition

[0092] According to another aspect of the present invention, a pharmaceutical composition for the prevention or treatment of obesity or fatty liver is provided, comprising a peptide containing the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0093] As described above, the peptides of the present invention have the activity of inhibiting fat accumulation in hepatocytes and adipocytes and promoting fat breakdown, and therefore can be used as excellent preventive or therapeutic agents for obesity or fatty liver.

[0094] Peptides containing the amino acid sequence of SEQ ID NO:1 and their activities related to the treatment and prevention of obesity or fatty liver are consistent with the above entry. Peptides and their activities The peptides described in this section are the same, so the specific description is quoted here and will not be repeated.

[0095] According to another aspect of the present invention, a pharmaceutical composition for the prevention or treatment of diabetes is provided, comprising a peptide containing the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0096] In this invention, diabetes can be type 1 diabetes or type 2 diabetes, and more specifically type 2 diabetes.

[0097] As described above, the peptides of the present invention can be used as excellent preventive or therapeutic agents for diabetes by inhibiting the expression of insulin resistance factors and promoting the expression of insulin sensitivity factors in hepatocytes and adipocytes.

[0098] The peptide containing the amino acid sequence of SEQ ID NO:1 and its activities related to the treatment and prevention of diabetes are consistent with the above entry. Peptides and their activities The peptides described in this section are the same, so the specific description is quoted here and will not be repeated.

[0099] The pharmaceutical compositions of the present invention may contain a therapeutically effective amount of the above-described peptides and a pharmaceutically acceptable carrier.

[0100] The term "therapeutic effective amount" refers to an amount sufficient to achieve the activity or efficacy of a peptide (which is the active ingredient of the pharmaceutical composition of the present invention), for example, an amount sufficient to achieve the therapeutic or preventive efficacy of diabetes or obesity.

[0101] Pharmaceutically acceptable carriers are those commonly used in formulations, including but not limited to lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0102] In addition to the components described above, the pharmaceutical compositions of the present invention may further include, but are not limited to, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0103] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington: The Science and Practice of Pharmacy (19th ed., 1995, Williams & Wilkins).

[0104] The pharmaceutical compositions of the present invention can be administered via any route suitable for treating diabetes, fatty liver, or obesity, such as oral or parenteral administration, and in the case of parenteral administration, via intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, local administration, transdermal administration, etc.

[0105] The dosage of the pharmaceutical composition may be from 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, but is not limited thereto, and may be administered in various ways depending on factors such as preparation method, administration method, patient age, weight, sex, pathological condition, diet, administration time, route of administration, excretion rate and responsiveness.

[0106] The pharmaceutical compositions of the present invention can be formulated into unit volume forms or contained in multi-dose containers using pharmaceutically acceptable carriers and / or excipients, according to methods readily practiced by those skilled in the art. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oily or aqueous medium, or in the form of an extract, powder, granules, tablet, or capsule, and may additionally contain dispersants or stabilizers.

[0107] Food composition

[0108] According to another aspect of the present invention, a food composition for preventing or improving obesity or fatty liver is provided, comprising a peptide containing the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0109] As described above, the peptides of the present invention have the activity of inhibiting fat accumulation in hepatocytes and adipocytes and promoting fat breakdown, and therefore can be used as active ingredients in food compositions for preventing or improving obesity or fatty liver.

[0110] The peptide containing the amino acid sequence of SEQ ID NO:1 and its activities related to the prevention or improvement of obesity or fatty liver are consistent with the above-mentioned items. Peptides and their activities The peptides and their activities described in the previous section are the same, so the specific descriptions are quoted from there and will not be repeated here.

[0111] According to another aspect of the present invention, a food composition for the prevention or improvement of diabetes is provided, comprising a peptide containing the amino acid sequence of SEQ ID NO:1 as an active ingredient.

[0112] In this invention, diabetes can be type 1 diabetes or type 2 diabetes, and more specifically type 2 diabetes.

[0113] As described above, the peptides of the present invention can be used as active ingredients in food compositions for the prevention or improvement of diabetes by inhibiting the expression of insulin resistance factors and promoting the expression of insulin sensitivity factors in hepatocytes and adipocytes.

[0114] The food composition of the present invention for the prevention or improvement of diabetes can be a functional food composition for regulating blood sugar levels.

[0115] In the functional food composition of the present invention, regulating blood glucose levels can be achieved by regulating blood glucose levels in diabetic patients or patients at high risk of prediabetes.

[0116] In the functional food composition of the present invention, regulating blood sugar levels can be achieved by lowering blood sugar levels.

[0117] In the food composition of the present invention described above, the appropriate amount of the peptide may be selected from a range of 0.0001% by weight to 10% by weight relative to the total weight of the composition.

[0118] In one embodiment, the functional food composition of the present invention may contain a food-effective amount of peptides and a food-acceptable carrier.

[0119] The food composition of the present invention not only contains peptides as active ingredients, but also ingredients commonly added during food manufacturing, and may include, for example, proteins, carbohydrates, fats, nutrients, flavoring agents, and taste agents. Examples of carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose, sucrose, and oligosaccharides; and polysaccharides such as dextrin and cyclodextrin, as well as sugar alcohols such as xylitol, sorbitol, and erythritol. As taste agents, natural taste agents (such as sematriol and stevia extract (e.g., rebaudioside A and glycyrrhizin)) and synthetic taste agents (such as saccharin and aspartame) can be used. The proportion of carbohydrates is typically from about 1g to 20g per 100g of the food composition of the present invention, preferably from about 5g to 12g, but is not limited thereto.

[0120] In addition to the above-mentioned ingredients, the food compositions of the present invention may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents (such as synthetic and natural flavoring agents), coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, etc. Furthermore, the food compositions of the present invention may contain fruit pulp used in the manufacture of natural fruit juices, fruit juice beverages, and vegetable beverages. For example, when the functional food compositions of the present invention are manufactured into beverages, in addition to peptides as active ingredients of the present invention, they may also contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia ulmoides extract, jujube extract, licorice extract, etc.

[0121] In another aspect of the invention, a method is provided for treating, preventing or improving obesity or fatty liver in a subject in need, the method comprising administering to the subject an effective amount of a peptide comprising the amino acid sequence of SEQ ID NO:1 as described above.

[0122] In another aspect of the invention, the use of the peptide comprising the amino acid sequence SEQ ID NO:1 as described above in the treatment, prevention or improvement of obesity or fatty liver is provided.

[0123] In another aspect of the invention, the use of the peptide comprising the amino acid sequence SEQ ID NO:1 as described above in the preparation of compositions for treating, preventing or improving obesity or fatty liver is provided.

[0124] In another aspect of the invention, a method for treating, preventing or improving diabetes in a subject in need is provided, the method comprising administering to the subject an effective amount of a peptide comprising the amino acid sequence of SEQ ID NO:1 as described above.

[0125] In another aspect of the invention, the use of the peptide comprising the amino acid sequence SEQ ID NO:1 as described above in the treatment, prevention or improvement of diabetes is provided.

[0126] In another aspect of the invention, the use of the peptide comprising the amino acid sequence SEQ ID NO:1 as described above in the preparation of compositions for treating, preventing or improving diabetes is provided.

[0127] In one embodiment, regarding the compositions of the present invention for anti-obesity or anti-fatty liver, and pharmaceutical or food compositions for the prevention, treatment, or improvement of obesity or fatty liver, the peptides of the present invention have the following activities: (i) activity of inhibiting lipid accumulation in hepatocytes; (ii) activity of inhibiting the protein expression of adipogenic factors SREBP1 or FAS and promoting the protein expression of lipolysis factors pACC, pHSL, ATGL, or PLIN in hepatocytes induced to accumulate lipids; (iii) activity of promoting the gene expression of fatty acid oxidation-related factors CPT1, PGC1α, or PPARα and promoting the protein expression of fatty acid oxidation-related factors CPT1 or PGC1α in hepatocytes induced to accumulate lipids; (iv) activity of inhibiting the gene expression of adipogenic factors ACCα or PPAR-γ in adipocytes; (v) activity of inhibiting the gene expression of adipogenic factors ACCα or PPAR-γ in adipocytes; The activities of inhibiting the protein expression of adipogenesis-related factors SREBP1, FAS, or C / EBPα and promoting the protein expression of fatty acid degradation-related factor pACCα in cells; (vi) the activities of promoting the protein expression of lipolysis-related factors PLIN, pHSL, or PGC1α in adipocytes; (vii) the activities of promoting the gene expression of fatty acid oxidation-related factors PGC1α or CPT1 in adipocytes induced with insulin resistance; (viii) the activities of inhibiting the gene expression of fatty acid synthesis factor FAS and promoting the gene expression of lipolysis-related factors PLIN, HSL, or ATGL in adipocytes induced with insulin resistance; or (ix) the activities of inhibiting the protein expression of fatty acid synthesis factor FAS or SREBP1 and promoting the protein expression of lipolysis-related factor PLIN in adipocytes induced with insulin resistance.

[0128] In one embodiment, regarding compositions for antidiabetic purposes and pharmaceutical or food compositions for the prevention, treatment, or improvement of diabetes, the peptides of the present invention have the following activities: (i) the activity of promoting protein expression of insulin-sensitive factors pIRS (Tyr612) or GLUT4 and inhibiting protein expression of insulin-resistant factors pIRS (Ser302) or p70S6K in adipocytes; (ii) the activity of inhibiting gene expression of insulin-resistant factor p70S6K and promoting gene expression of insulin-sensitive factors AMPKα, GLUT4, or SIRT1 in adipocytes induced with insulin resistance; (iii) the activity of inhibiting gene expression of insulin-resistant factor p70S6K and promoting gene expression of insulin-sensitive factors AMPKα, GLUT4, or SIRT1 in adipocytes induced with insulin resistance. (iv) In insulin-resistant adipocytes, the activity of promoting the protein expression of insulin-sensitive factor pAMPKα and inhibiting the protein expression of insulin-resistant factor pIRS (Ser302) is used to inhibit the gene expression of insulin-resistant factor mTOR or p70S6K and promote the gene expression of insulin-sensitive factors AKT, SIRT1 or AMPKα; or (v) In insulin-resistant hepatocytes, the activity of inhibiting the protein expression of insulin-resistant factors pmTOR or pJNK and promoting the protein expression of insulin-sensitive factors pAMPKα, GLUT4 or SIRT1 is used to inhibit the protein expression of insulin-resistant factors pmTOR or pJNK and promote the protein expression of insulin-sensitive factors pAMPKα, GLUT4 or SIRT1 is used to inhibit the protein expression of insulin-resistant factors pmTOR or pJNK and promote the protein expression of insulin-sensitive factors pAMPKα, GLUT4 or SIRT1 is used to inhibit the protein expression of insulin-sensitive ... pmTOR, GLUT1 or SIRT1 is used to inhibit the protein expression of insulin-sensitive factors pmTOR or pJNK and promote the protein expression of insulin-sensitive factors pmTOR, GLUT1 or SIRT1 is used to inhibit the protein expression of insulin-sensitive factors pmTOR, p70S6K and pIRS (Ser302) is used to inhibit the protein expression of insulin-sensitive factors AKT, SIRT1 or AMPKα.

[0129] [Beneficial Effects]

[0130] The peptides of the present invention exhibit muscle-building activity in myoblasts. They also exhibit activity in hepatocytes and adipocytes that inhibit fat accumulation and promote lipolysis. Furthermore, the peptides of the present invention inhibit the expression of insulin resistance factor and increase the expression of insulin sensitivity factor in hepatocytes and adipocytes. Therefore, the peptides of the present invention can be used as active substances for treating, preventing, or improving muscle diseases, obesity, fatty liver, and diabetes.

[0131] However, the effects of the present invention are not limited to those described above, and those skilled in the art will clearly understand from the following description other effects not mentioned. Attached Figure Description

[0132] Figure 1 The figure illustrates the cell proliferation-promoting activity of the peptides of the present invention in C2C12 cells.

[0133] Figure 2a and Figure 2b The results show the effects of the peptides of the present invention on promoting the gene expression of cell proliferation-related factors PCNA (proliferating cell nuclear antigen), NDRG2 (N-myc downstream regulatory gene 2), and CDK4 (cyclin-dependent kinase 4) in C2C12 cells. Non indicates the group not treated with the peptide, wherein the differentiation-induced C2C12 cells were not treated with the peptide.

[0134] Figure 3a and Figure 3b The results show that the peptides of the present invention increased the expression levels of cell proliferation-related factors SIRT1 (Sirtuin 1), Ki67, Pax7, and pAKT proteins in C2C12 cells. Non represents the group untreated with the peptides, where differentiation-induced C2C12 cells were not treated with the peptides.

[0135] Figure 4a and Figure 4b The results show that the peptide of the present invention increased the expression levels of genes MyoD, Myf6, mTOR, Myf5, and Myf4, which are involved in intermediate and late differentiation, in C2C12 cells. Non represents the group untreated with the peptide, in which differentiation-induced C2C12 cells were not treated with the peptide.

[0136] Figure 5a , Figure 5b , Figure 5c and Figure 5d The results show that the peptides of the present invention increased the expression of differentiation marker proteins α-actin, Myf6, and MyoG in C2C12 cells, and increased the expression of signaling proteins involved in muscle protein synthesis, such as SIRT1, pAMPKα, pAKT, pmTOR, and p70S6K. Non represents the group untreated with the peptides, in which differentiation-induced C2C12 cells were not treated with the peptides.

[0137] Figure 6a and Figure 6b The results show that the peptides of the present invention promote C2C12 cell differentiation and increase myotube formation. Non is the group without peptide treatment, in which differentiation-induced C2C12 cells were not treated with the peptide.

[0138] Figure 7a , Figure 7b and Figure 7c The results of the peptides of the present invention inhibiting lipid accumulation in HepG2 hepatocytes are shown. Non represents the untreated group of HepG2 hepatocytes not treated with oleic acid and the peptides. OA represents the oleic acid-treated group.

[0139] Figure 8a and 8b The results show that the peptide of the present invention inhibited the expression of adipogenic factors SREBP1 and FAS and promoted the expression of lipolytic factors ATGL and pHSL in HepG2 hepatocytes induced by oleic acid treatment. Non represents the untreated group of HepG2 hepatocytes without oleic acid and peptide treatment. OA represents the oleic acid-treated group.

[0140] Figure 9a , Figure 9b and Figure 9cThe peptide of the present invention is shown to have inhibitory activity against oleic acid-induced increase in FAS expression in HepG2 cells. Non represents the untreated group of HepG2 hepatocytes without oleic acid and peptide treatment. OA represents the oleic acid-treated group.

[0141] Figure 9d , Figure 9e and Figure 9f The peptide of the present invention is shown to have inhibitory activity against oleic acid-induced increase in SREBP1 expression in HepG2 cells. Non represents the untreated group of HepG2 hepatocytes without oleic acid and peptide treatment. OA represents the oleic acid-treated group.

[0142] Figure 9g , Figure 9h and Figure 9i The peptide of the present invention is shown to have the activity of reducing and then increasing oleic acid-induced pACC expression in HepG2 cells. Non represents the untreated group of HepG2 hepatocytes without oleic acid and peptide treatment. OA represents the oleic acid-treated group.

[0143] Figure 9j , Figure 9k and Figure 9l The peptide of the present invention is shown to have the activity of re-increasing oleic acid-induced pHSL expression in HepG2 cells after a decrease. Non represents the untreated group of HepG2 hepatocytes not treated with oleic acid and the peptide. OA represents the oleic acid-treated group.

[0144] Figure 9m , Figure 9n and Figure 9o The peptide of the present invention is shown to have the activity of reducing and then increasing ATGL expression induced by oleic acid in HepG2 cells. Non represents the untreated group of HepG2 hepatocytes without oleic acid and peptide treatment. OA represents the oleic acid-treated group.

[0145] Figure 9p , Figure 9q and Figure 9r The peptide of the present invention is shown to have the activity of reducing and then increasing oleic acid-induced PLIN expression in HepG2 cells. Non represents the untreated group of HepG2 hepatocytes without oleic acid and peptide treatment. OA represents the oleic acid-treated group.

[0146] Figure 10a and Figure 10b The results show that the peptide of the present invention reduced the expression levels of the insulin resistance factor mTOR and P70S6K genes and increased the expression levels of the insulin sensitivity factors AKT, SIRT1, and AMPKα genes in HepG2 cells treated with oleic acid and exhibiting insulin resistance. Non represents the untreated group of HepG2 hepatocytes without oleic acid and peptide treatment. OA represents the oleic acid-treated group.

[0147] Figure 11a and Figure 11b The results show that the peptide of the present invention reduced the expression levels of insulin resistance factors pmTOR and pJNK proteins and increased the expression levels of insulin sensitivity factors pAMPKα, GLUT4, and SIRT1 proteins in HepG2 cells treated with oleic acid and exhibiting insulin resistance. Non represents the untreated HepG2 hepatocytes that were not treated with oleic acid or the peptide. OA represents the oleic acid-treated group.

[0148] Figure 12a and Figure 12b The invention demonstrates that the peptides of this invention increase the expression of fatty acid oxidation-related factors CPT1, PGC1α, and PPARα genes in HepG2 cells treated with oleic acid and exhibiting insulin resistance. Non represents the untreated HepG2 hepatocytes, untreated with either oleic acid or the peptides. OA represents the oleic acid-treated group.

[0149] Figure 13 The invention demonstrates that the peptides of this invention increase the expression of fatty acid oxidation-related factors CPT1 and PGC1α proteins in HepG2 cells treated with oleic acid and subjected to conditions of fat accumulation. Non represents the untreated HepG2 hepatocytes not treated with oleic acid or the peptides. OA represents the oleic acid-treated group.

[0150] Figure 14a and Figure 14b This demonstrates that the peptides of the present invention increase the protein expression levels of insulin sensitivity factors pIRS (Tyr612) and GLUT4, and decrease the protein expression levels of insulin resistance factors pIRS (Ser302) and p70S6K in 3T3-L1 cells. Non represents the untreated 3T3-L1 cells that were not treated with the peptides.

[0151] Figure 15a and Figure 15b This demonstrates that the peptide of the present invention reduces the expression level of the insulin resistance factor P70S6K gene and increases the expression levels of insulin sensitivity factors AMPKα, GLUT4, and SIRT1 genes in oleic acid-treated 3T3-L1 cells. Non represents the untreated 3T3-L1 cell group without oleic acid and peptide treatment. OA represents the oleic acid-treated group.

[0152] Figure 16 The peptides of the present invention were shown to increase the expression level of the insulin sensitivity factor pAMPKα and decrease the expression level of the insulin resistance factor pIRS (Ser302) in oleic acid-treated 3T3-L1 cells. Figure 16 Non represents the untreated 3T3-L1 cells that were not treated with oleic acid and peptides. OA represents the oleic acid-treated group.

[0153] Figure 17The peptides of the present invention are shown to reduce the gene expression of adipogenesis-related factors ACCα and PPAR-γ in 3T3-L1 cells. Non represents the untreated 3T3-L1 cells that were not treated with the peptides.

[0154] Figure 18a and Figure 18b The peptides of the present invention are shown to reduce the expression of adipogenesis-related factors SREBP1, FAS, and C / EBPα and increase the expression of pACCα, which promotes fatty acid degradation, in 3T3-L1 cells. Non represents the untreated 3T3-L1 cells that were not treated with the peptides.

[0155] Figure 19 The peptides of the present invention are shown to increase the expression of lipolysis-related factors PLIN, pHSL, and PGC1α in 3T3-L1 cells. Non represents the untreated 3T3-L1 cells that were not treated with the peptides.

[0156] Figure 20 The invention demonstrates that the peptides of this invention increase the gene expression of fatty acid oxidation-related factors PGC1α and CPT1 in oleic acid-treated 3T3-L1 cells. Non represents the untreated 3T3-L1 cell group, which was not treated with oleic acid or the peptides. OA represents the oleic acid-treated group.

[0157] Figure 21a and Figure 21b This demonstrates that the peptide of the present invention reduces the expression of the fatty acid synthesis-related factor FAS gene and increases the expression of the lipolysis-related factors PLIN, HSL, and ATGL genes in oleic acid-treated 3T3-L1 cells. Non represents the untreated 3T3-L1 cell group, which was not treated with oleic acid or the peptide. OA represents the oleic acid-treated group.

[0158] Figure 22a and Figure 22b This demonstrates that the peptides of the present invention reduce the expression of fatty acid synthesis-related factor FAS protein and increase the expression of lipolysis-related factors SREBP1 and PLIN protein in oleic acid-treated 3T3-L1 cells. Non represents the untreated 3T3-L1 cell group without oleic acid and peptide treatment. OA represents the oleic acid-treated group. Detailed Implementation

[0159] The present invention will now be described in detail through embodiments. However, the following embodiments are intended to specifically illustrate the present invention, and the scope of the present invention is not limited to the following embodiments.

[0160] Preparation Example 1: Preparation of Peptides

[0161] Peptides with the amino acid sequence listed in Table 1 below, SEQ ID NO:1, were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA) and purified using C18 reversed-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). An ACQUITY UPLC BEH300 C18 column (2.1 mm × 100 mm, 1.7 μm, WatersCo, USA) was used.

[0162] [Table 1]

[0163] The efficacy of the peptide prepared in SEQ ID NO:1 was evaluated through the following experiments.

[0164] [Experimental Examples 1-6] Experimental Examples of Myoblasts

[0165] Experimental Example 1: Analysis of the cell proliferation-promoting activity of myoblasts

[0166] The effects of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the proliferative capacity and cytotoxicity of myoblasts were evaluated.

[0167] C2C12 cells (derived from mouse myoblasts) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluence reached 70% to 80%, cells were seeded into 96-well plates at a concentration of 5 × 10³ cells / 200 μl. The next day, the medium was replaced with serum-free DMEM (1% P / S), and 4 hours later, it was replaced with DMEM medium containing 0.2% FBS and different concentrations of peptides (0 μM, 3.9 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM). At 48 and 72 hours post-treatment, reactions were performed using EZ-cytox (DoGenBio Ltd., Seoul, South Korea) until the absorbance of the non-peptide treatment reached 1.0, and absorbance was measured at 450 nm using a spectrophotometer (SPECTRAMAX M2e). When C2C12 cells were treated with different concentrations of peptide for 24 and 48 hours, it was confirmed that there was no significant difference in concentration between treatments at 24 hours, but at 48 hours, cell proliferation increased in a peptide concentration-dependent manner. Figure 1 ).

[0168] Experimental Example 2: Expression Analysis of Proliferation-Related Marker Genes in Myoblasts

[0169] The effect of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the proliferative capacity of myoblasts was evaluated by analyzing the expression of proliferation-related marker genes.

[0170] C2C12 cells (derived from mouse myoblasts) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluence reached 70% to 80%, the cells were sputtered at a rate of 5 × 10⁶ cells / year. 5 Cells were seeded at a concentration of 1 cell / 2 ml in 6-well culture plates. The next day, the medium was replaced with serum-free DMEM (1% P / S), and after approximately 4 hours, cells were treated with peptides (5 μM, 50 μM). One hour after peptide treatment, cells were collected and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). DNA complementary to the extracted RNA was synthesized using TOPScript™ RT DryMIX (Enzynomics, Korea), and then polymerase chain reaction (PCR) was performed on genes involved in cell proliferation factors (markers) using TOPsimple™ DryMIX-nTaq (Enzynomics, Korea). The reaction products were then electrophoresed on a 1.5% agarose gel to compare the mRNA expression levels of the aforementioned factors (markers) in each sample. The gene primer sequences used in the PCR are listed in Table 2 below, with the GAPDH gene used as a control group.

[0171] [Table 2]

[0172] Experimental results confirmed that the expression of PCNA (proliferating cell nuclear antigen), NDRG2 (N-myc downstream regulatory gene 2), and CDK4 (cyclin-dependent kinase 4) genes related to cell proliferation in C2C12 cells was significantly increased after peptide treatment. Figure 2a and Figure 2b ).

[0173] Experimental Example 3: Expression Analysis of Cell Proliferation-Related Marker Proteins in Myoblasts

[0174] The effect of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the proliferative capacity of myoblasts was evaluated by analyzing the expression of proliferation-related marker proteins.

[0175] C2C12 cells (derived from mouse myoblasts) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluence reached 70% to 80%, the cells were sputtered at a rate of 5 × 10⁶ cells / year. 5Cells were seeded at a concentration of 1 cell / 2 ml in 6-well plates. The next day, the medium was replaced with serum-free DMEM (1% P / S), and after approximately 4 hours, the cells were treated with peptides (5 μM, 50 μM). One hour after peptide treatment, cell lysis buffer was added to lyse the cells, followed by centrifugation at 12,000 rpm for 30 minutes at 4°C to obtain protein. Proteins were quantified using a BCA kit. The proteins were electrotransferred to membranes after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The protein-coated membranes were blocked with 5% skim milk, then treated with primary antibodies and incubated overnight at 4°C. 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) were used as primary antibodies. After washing the reaction mixture with PBS-T, the secondary antibodies (peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L), Jackson Immuno Research, USA; peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L), Jackson Immuno Research, USA) were reacted at room temperature for 1 hour, washed again with PBS-T, and then treated with Western spectroscopy assay kit (Elpis Biotech, Daejeon, Korea) and developed using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0176] Experimental results showed that treatment with 5 μM and 50 μM peptides for 1 hour increased the expression levels of SIRT1 (Sirtuin 1), Ki67, Pax7, and pAKT proteins in a peptide concentration-dependent manner. Specifically, compared to the control group, the expression level of Pax7 protein increased by 2.6-fold and 3.8-fold, respectively, showing a significant increase. Figure 3a and Figure 3b ).

[0177] Example 4: Expression analysis of genes marking early / medium differentiation in myoblasts and genes related to muscle protein synthesis signal transduction.

[0178] The effects of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of early and mid-stage myoblast differentiation marker genes and signal transduction factors involved in muscle protein synthesis were analyzed.

[0179] C2C12 cells (derived from mouse myoblasts) were cultured in DMEM medium containing 1% P / S and 2% BCS (fetal bovine serum). When cell confluence reached 70% to 80%, the cells were cultured at a rate of 5 × 10⁶ cells / year. 5 Cells were seeded at a concentration of 100% per 2 ml in 6-well culture plates. When the cells reached 100% confluence, the medium was replaced with differentiation medium (1% P / S, 2% horse serum) and peptides (5 μM, 50 μM) were added. The differentiation medium and peptides were then replaced every two days. Three days after the first replacement, cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was used to synthesize complementary DNA using TOPScript™ RT DryMIX (Enzynomics, Korea). Polymerase chain reaction (PCR) was then performed on early and mid-stage differentiation markers and marker genes involved in muscle protein synthesis signaling using TOPsimple™ DryMIX-nTaq (Enzynomics, Korea). The reaction products were then electrophoresed on a 1.5% agarose gel to compare the mRNA expression levels of each marker in each sample. The gene primer sequences used in PCR are listed in Table 3 below, with the GAPDH gene used as a control group.

[0180] [Table 3]

[0181] Following the experimental method described above, differentiation was induced in C2C12 myoblasts using 2% horse serum, followed by peptide treatment for 3 days to confirm the effect of peptides on differentiation. RT-PCR results showed that the expression levels of MyoD and Myf6, factors involved in intermediate and late-stage differentiation, increased after peptide treatment. Specifically, Myf6 showed a significant increase, with expression increasing 1.8-fold compared to the control group after treatment with 50 μM peptide. Furthermore, the expression levels of mTOR, Myf5, and Myf4, factors involved in early and intermediate-stage differentiation, were also confirmed to increase in a peptide concentration-dependent manner. Figure 4a and Figure 4b ).

[0182] Example 5: Expression analysis of mid-to-late stage differentiation markers and muscle protein synthesis-related signaling proteins in myoblasts.

[0183] The effects of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on late differentiation markers in myoblasts and the expression of signal transduction proteins involved in muscle protein synthesis were analyzed.

[0184] C2C12 cells (derived from mouse myoblasts) were cultured in DMEM medium containing 1% P / S and 2% BCS (fetal bovine serum). When cell confluence reached 70% to 80%, the cells were cultured at a rate of 5 × 10⁶ cells / year. 5 Cells were seeded at a concentration of 100% per 2 ml in 6-well culture plates. When the cells reached 100% confluence, the medium was replaced with differentiation medium (1% P / S, 2% horse serum) and peptides (5 μM, 50 μM) were added. The differentiation medium (1% P / S, 2% horse serum) and peptides (5 μM, 50 μM) were then replaced every two days. Three days after the first replacement, lysis buffer was added to lyse the cells. The cell lysates were then centrifuged at 12,000 rpm for 30 minutes at 4°C to obtain proteins. Proteins were quantified using a BCA kit. Proteins were electrotransferred to membranes after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The protein-coated membranes were blocked with 5% skim milk, treated with primary antibody, and incubated overnight at 4°C. The primary antibodies used are as follows: anti-α-actin 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); and anti-α-tubulin antibody (Santa Cruz, USA). The reaction mixture was washed with PBS-T, then reacted with secondary antibodies (peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L), Jackson Immuno Research, USA; peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L), Jackson Immuno Research, USA) at room temperature for 1 hour, washed again with PBS-T, then treated with Western spectroscopy assay kit (Elpis Biotech, Daejeon, Korea) and developed with GelDoc (Bio-Rad, Hercules, CA, USA).

[0185] The experimental results showed that the expression of differentiation markers α-actin, Myf6, and MyoG proteins increased in a peptide concentration-dependent manner after peptide treatment (Figures 5A and 5B). Furthermore, the expression of signaling proteins involved in muscle protein synthesis, such as SIRT1, pAMPKα, pAKT, pmTOR, and p70S6K, also increased in a peptide concentration-dependent manner after peptide treatment (Figures 5C and 5D).

[0186] Experimental Example 6: Microscopic Analysis of Differentiation-Induced Myoblast Morphology

[0187] The effect of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on myotube synthesis in myoblasts was analyzed by morphological observation under a microscope.

[0188] C2C12 cells (derived from mouse myoblasts) were cultured in DMEM medium containing 1% P / S and 2% BCS (fetal bovine serum). When the cells reached approximately 80% confluence, they were cultured at a rate of 5 × 10⁶ cells / year. 5 Cells were seeded at a concentration of 100 cells / 2 ml into 6-well culture plates. Subsequently, when the cell confluence in the 6-well plates reached 100%, the medium was replaced with differentiation medium (1% P / S, 2% horse serum) and peptides (5 μM, 50 μM) were added. Thereafter, the differentiation medium (1% P / S, 2% horse serum) and peptides (5 μM, 50 μM) were changed every two days. Cell images were taken three days after differentiation induction using an optical microscope (FLEXACAM C1, Leica, Germany).

[0189] The above experiment aimed to determine whether peptide treatment promoted cell differentiation in C2C12 myoblasts during differentiation induction. Cell morphology was examined under a microscope, and the thickness of the formed myotubes was measured. The results showed that, compared to the control group (differentiation induction, Non), peptide treatment further promoted myoblast differentiation, and high-concentration peptide treatment resulted in the formation of numerous long and thick myotubes. Figure 6a and Figure 6b ).

[0190] [Experimental Examples 7 to 13] Experimental Examples of Hepatocytes

[0191] Experimental Example 7: Analysis of Fat Accumulation in Hepatocytes

[0192] The effect of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on fat accumulation in hepatocytes was analyzed.

[0193] HepG2 cells (human hepatocytes) were cultured in DMEM medium containing 1% P / S and 10% FBS. When the cell confluence reached approximately 70% to 80%, the cells were cultured at a rate of 1 × 10⁻⁶ cells / mL. 5Cells were seeded at a concentration of 10 cells / ml into 12-well culture plates. Subsequently, when cell confluence in the 12-well plates reached approximately 70% to 80%, the medium was replaced with serum-free DMEM. Four hours later, lipid accumulation was induced by replacing the medium with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA). Twenty-four hours after inducing lipid accumulation, 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 adipogenesis, cells were washed twice with PBS and fixed with 10% formaldehyde. Oil-Red O solution (Sigma, USA) was added to the fixed cells to stain lipid globules for 2 hours, followed by washing with distilled water (DW) and observation under a microscope. Subsequently, the distilled water was removed, the cells were dried, washed with isopropanol, and the absorbance was measured at OD 540 nm using a spectrophotometer (SPECTRAMAX M2e).

[0194] Experimental results showed that oleic acid treatment increased lipogenesis in HepG2 hepatocytes, while peptide treatment inhibited lipogenesis. Figure 7a , Figure 7b and Figure 7c ).

[0195] Experimental Example 8: Expression Analysis of Proteins Related to Lipogenesis and Lipolysis in Hepatocytes

[0196] The effects of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of proteins related to adipogenesis and lipolysis in hepatocytes were analyzed.

[0197] HepG2 cells (human hepatocytes) were cultured in DMEM medium containing 1% P / S and 10% FBS. When the cell confluence reached approximately 70% to 80%, the cells were cultured at a rate of 1 × 10⁻⁶ cells / mL. 5Cells were seeded at a concentration of 10 cells / ml into 12-well culture plates. Subsequently, when cell confluence in the 12-well plates reached approximately 70% to 80%, the medium was replaced with serum-free DMEM. Four hours later, lipid accumulation was induced by replacing the medium with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA). Twenty-four hours after inducing lipid accumulation, cells were cultured for 48 hours in serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM). Following culture, lysis buffer was added to lyse the cells, and the cell lysates were centrifuged at 12,000 rpm for 30 minutes at 4°C to obtain proteins. Proteins were quantified using a BCA kit. Proteins were electrotransferred to a membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The protein-coated membranes were blocked with 5% skim milk, treated with primary antibodies, and reacted overnight at 4°C. The primary antibodies used were: 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 reactants were washed with PBS-T, 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, treated with Western spectroscopy reagent (Elpis Biotech, Daejeon, Korea), and developed using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0198] Experimental results showed that in HepG2 hepatocytes, oleic acid treatment increased the expression of SREBP1 (sterol regulatory element-binding protein-1) and FAS (fatty acid synthase), proteins involved in lipogenesis, while peptide treatment reduced the expression of the elevated SREBP1 and FAS again. Figure 8aFurthermore, in HepG2 hepatocytes, oleic acid treatment was confirmed to inhibit the expression of ATGL (triglyceride lipase) and pHSL (hormone-sensitive lipase), proteins involved in lipolysis. However, upon peptide treatment, the decreased expression levels of ATGL and pHSL were restored. Figure 8b ).

[0199] Experimental Example 9: Immunofluorescence analysis of the expression of markers related to adipogenesis and lipolysis in hepatocytes

[0200] The effect of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of markers related to adipogenesis and lipolysis in hepatocytes was analyzed by immunofluorescence.

[0201] HepG2 cells (human hepatocytes) were cultured in DMEM medium containing 1% P / S and 10% FBS. When the cell confluence reached approximately 70% to 80%, the cells were cultured at a rate of 3 × 10⁻⁶ cells / mL. 5Cells were seeded at a concentration of 10 cells / 2 ml into 6-well culture plates. Subsequently, when cell confluence in the 6-well plates reached approximately 70% to 80%, the medium was replaced with serum-free DMEM. Four hours later, lipid accumulation was induced by replacing the medium with serum-free DMEM (1% BSA) supplemented with 350 μM oleic acid (Sigma, USA). Twenty-four hours after inducing lipid accumulation, the cell culture 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 culture, cells were fixed with 4% paraformaldehyde (PFA) at room temperature (RT) for 10 minutes 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 at room temperature for 1 hour, then treated with primary antibody and incubated at room temperature for 1 hour. The primary antibodies used were: 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); and anti-PLIN antibody (Affinity Biosciences, USA). After incubation, the cells were washed three times and incubated with the secondary antibody (goat anti-rabbit IgG H&L, Abcam, USA) for 30 minutes at room temperature. The samples were washed three times, and the cell nuclei were counterstained with DAPI (Santa Cruz, USA). Images were acquired using a LEICA fluorescence microscope (TCS SP8, LEICA, Germany).

[0202] Regarding the expression level of FAS (fatty acid synthase), an enzyme involved in fatty acid synthesis, it was confirmed that FAS expression increased in HepG2 cells when lipid accumulation was induced by oleic acid, and that the increased FAS expression level decreased again in a peptide concentration-dependent manner when cells were treated with peptides. Figure 9a , Figure 9b and Figure 9c ).

[0203] Regarding the expression level of SREBP1 (sterol regulatory element-binding protein-1), which induces lipid and cholesterol synthesis in the liver, it was confirmed that SREBP1 expression increased in HepG2 cells when lipid accumulation was induced by oleic acid, and that the elevated SREBP1 expression level decreased again in a peptide concentration-dependent manner when cells were treated with peptides. Figure 9d , Figure 9e and Figure 9f ).

[0204] Acetyl-CoA carboxylase (ACC) is known to be phosphorylated by AMP-activated protein kinase (AMPK), thereby promoting fatty acid oxidation (lipolysis). Regarding the expression level of phosphorylated acetyl-CoA carboxylase (pACC), it was confirmed that pACC expression decreased in HepG2 cells induced by oleic acid-induced lipid accumulation, while the inhibited pACC expression level re-increased in a peptide concentration-dependent manner upon cell treatment with peptides. Figure 9g , Figure 9h and Figure 9i ).

[0205] HSL (hormone-sensitive lipase) is a lipid hydrolase that breaks down triglycerides into free fatty acids and diglycerides, or diglycerides into free fatty acids and monoglycerides, in adipose tissue. HSL is activated by phosphorylation of protein kinase A (PKA), which is activated during hormone-induced signaling. Regarding the expression level of pHSL (phosphorylated hormone-sensitive lipase), it was confirmed that pHSL expression decreased in HepG2 cells induced by oleic acid-induced lipid accumulation, while the inhibited pHSL expression level re-increased in a peptide concentration-dependent manner upon peptide treatment. Figure 9j , Figure 9k and Figure 9l ).

[0206] ATGL (triglyceride lipase) possesses the activity of breaking down triglycerides and releasing fatty acids during intracellular lipolysis, enabling the released fatty acids to be used for energy. Regarding the expression level of ATGL, it was confirmed that ATGL expression decreased in HepG2 cells induced by oleic acid-induced lipid accumulation, while the inhibited ATGL expression level was restored in a peptide concentration-dependent manner upon cell treatment with peptides. Figure 9m , Figure 9n and Figure 9o ).

[0207] Lipid droplet coating protein (PLIN) is one of the proteins that constitute lipid droplet structure and is known to promote the breakdown of triglycerides by lipases. When the lipolytic hormone signaling pathway is activated, intracellular cAMP levels increase, and after protein kinase PKA is activated, the activated PKA phosphorylates PLIN, thereby activating PLIN. Regarding the expression level of PLIN, it was confirmed that PLIN expression was decreased in HepG2 cells when lipid accumulation was induced by oleic acid, and that when cells were treated with peptides, the inhibited PLIN expression level was restored in a peptide concentration-dependent manner. Figure 9p , 9q and 9r).

[0208] The above experimental results show that the peptide of SEQ ID NO:1 of the present invention has the activity of inhibiting lipid synthesis in hepatocytes and promoting fat breakdown.

[0209] Experimental Example 10: Analysis of the expression of genes related to insulin resistance and sensitivity in hepatocytes

[0210] The effects of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of genes related to insulin resistance and sensitivity in hepatocytes were analyzed.

[0211] HepG2 cells (human hepatocytes) were cultured in DMEM medium containing 1% P / S and 10% FBS. When the cell confluence reached approximately 70% to 80%, the cells were cultured at a rate of 2 × 10⁻⁶ cells / mL. 5 Cells were seeded at a concentration of [number] cells / ml into 12-well culture plates. Subsequently, when cell confluence in the 12-well plates reached approximately 70% to 80%, the medium was replaced with serum-free DMEM. Four hours later, an insulin-resistant environment was induced by replacing the medium with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, USA). After 24 hours of induction, the cell 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, cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was synthesized using TOPScript™ RTDryMIX (Enzynomics, Korea) to obtain DNA complementary to the RNA, and then polymerase chain reaction (PCR) was performed on genes related to insulin resistance and sensitivity using TOPsimple™ DryMIX-nTaq (Enzynomics, Korea). The reaction products were then electrophoresed on a 1.5% agarose gel to compare the mRNA expression levels of each biomarker. The gene primer sequences used in the PCR are listed in Table 4 below, with the GAPDH gene used as a control group.

[0212] [Table 4]

[0213] The above experiments aimed to determine whether peptide treatment of HepG2 cells induced with oleic acid in a state of insulin resistance induced insulin sensitivity. Insulin sensitivity was confirmed by analyzing the expression levels of factors related to insulin resistance or sensitivity using RT-PCR. The results showed that oleic acid treatment increased the expression levels of insulin resistance-related factors mTOR and P70S6K in HepG2 cells, but when cells were treated with peptides, the increased mTOR and P70S6K expression levels decreased again in a peptide concentration-dependent manner. Furthermore, oleic acid treatment decreased the expression levels of insulin sensitivity-related factors AKT, SIRT1, and AMPKα in HepG2 cells, but when cells were treated with peptides, the decreased AKT, SIRT1, and AMPKα expression levels increased again. Figure 10a and Figure 10b ).

[0214] Example 11: Analysis of the expression of proteins related to insulin resistance and sensitivity in hepatocytes

[0215] The effects of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of insulin resistance and sensitivity-related proteins in hepatocytes were analyzed.

[0216] HepG2 cells (human hepatocytes) were cultured in DMEM medium containing 1% P / S and 10% FBS. When the cell confluence reached approximately 70% to 80%, the cells were cultured at a rate of 2 × 10⁻⁶ cells / mL. 5Cells were seeded at a concentration of [number] cells / ml into 12-well culture plates. Subsequently, when cell confluence in the 12-well plates reached approximately 70% to 80%, the medium was replaced with serum-free DMEM. Four hours later, 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). Twenty-four hours after induction, the cell 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, lysis buffer was added to lyse the cells, and the cell lysates were centrifuged at 12,000 rpm at 4°C for 30 minutes to obtain proteins. The obtained proteins were quantified using a BCA kit. Proteins were electrotransferred to a membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The membranes containing the electrotransfer proteins were blocked with 5% skim milk, then treated with primary antibodies and incubated overnight at 4°C. The primary antibodies used were: 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); and anti-β-actin antibody (Santa Cruz, USA). The reaction mixture was washed with PBS-T, then reacted with secondary antibodies (peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L), Jackson Immuno Research, USA; peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L), Jackson Immuno Research, USA) at room temperature for 1 hour, washed again with PBS-T, then treated with Western spectroscopy assay kit (Elpis Biotech, Daejeon, Korea) and developed with GelDoc (Bio-Rad, Hercules, CA, USA).

[0217] The above experiments aimed to determine whether peptide treatment of HepG2 cells induced with oleic acid in a state of insulin resistance induced insulin sensitivity. Western blot analysis of the expression levels of insulin resistance or sensitivity-related factors was used to confirm whether insulin sensitivity was induced. The results showed that oleic acid treatment increased the expression levels of insulin resistance-related factors pmTOR and pJNK in HepG2 cells; however, when cells were treated with peptides, the increased expression levels of pmTOR and pJNK decreased again. Furthermore, oleic acid treatment decreased the expression levels of insulin sensitivity-related factors pAMPKα, GLUT4, and SIRT1 in HepG2 cells; however, when cells were treated with peptides, the decreased expression levels of pAMPKα, GLUT4, and SIRT1 increased again. Figure 11a and Figure 11b ).

[0218] Example 12: Analysis of the expression of fatty acid oxidation-related genes in hepatocytes

[0219] The effects of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of fatty acid oxidation-related genes in hepatocytes were analyzed.

[0220] HepG2 cells (human hepatocytes) were cultured in DMEM medium containing 1% P / S and 10% FBS. When the cell confluence reached approximately 70% to 80%, the cells were cultured at a rate of 2 × 10⁻⁶ cells / mL. 5Cells were seeded at a concentration of [number] cells / ml into 12-well culture plates. Subsequently, when cell confluence in the 12-well plates reached approximately 70% to 80%, the medium was replaced with serum-free DMEM. Four hours later, lipid accumulation was induced by replacing the medium with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, USA). Twenty-four hours after inducing lipid accumulation, 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. Following culture, cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was synthesized using TOPScript™ RTDryMIX (Enzynomics, Korea) to obtain DNA complementary to the RNA, and then polymerase chain reaction (PCR) was performed on fatty acid oxidation-related biomarker genes using TOPsimple™ DryMIX-nTaq (Enzynomics, Korea). The reaction products were then electrophoresed on a 1.5% agarose gel to compare the mRNA expression levels of the above markers for each sample. The gene primer sequences used in the PCR are listed in Table 5 below, with the GAPDH gene used as a control.

[0221] [Table 5]

[0222] Experimental results showed that oleic acid treatment in HepG2 cells reduced the expression of fatty acid oxidation-related factors, namely CPT1 (carnitine palmitoyltransferase I), PGC1α (peroxisome proliferator-activated receptor gamma coactivator 1-α), and PPARα (peroxisome proliferator-activated receptor α). When cells were treated with peptides, the reduced expression levels of CPT1, PGC1α, and PPARα increased again. Figure 12a and Figure 12b ).

[0223] Experimental Example 13: Analysis of the expression of fatty acid oxidation-related proteins in hepatocytes

[0224] The effect of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of fatty acid oxidation-related proteins in hepatocytes was analyzed.

[0225] HepG2 cells (human hepatocytes) were cultured in DMEM medium containing 1% P / S and 10% FBS. When the cell confluence reached approximately 70% to 80%, the cells were cultured at a rate of 2 × 10⁻⁶ cells / mL. 5Cells were seeded at a concentration of [number] cells / ml into 12-well culture plates. Subsequently, when cell confluence in the 12-well plates reached approximately 70% to 80%, the medium was replaced with serum-free DMEM. Four hours later, lipid accumulation was induced by replacing the medium with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, USA). Twenty-four hours after inducing lipid accumulation, 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, lysis buffer was added to lyse the cells, and the cell lysates were centrifuged at 12,000 rpm for 30 minutes at 4°C to obtain proteins. The obtained proteins were quantified using a BCA kit. Proteins were electrotransferred to a membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The protein-coated membranes were blocked with 5% skim milk, treated with primary antibodies, and incubated overnight at 4°C. The primary antibodies used were: 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, 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, treated with Western blotting reagent (Elpis Biotech, Daejeon, Korea), and developed using GelDoc (Bio-Rad, Hercules, CA, USA).

[0226] Experimental results showed that in HepG2 cells, oleic acid treatment reduced the expression of fatty acid oxidation-related factors CPT1 (carnitine palmitoyltransferase I) and PGC1α (peroxisome proliferator-activated receptor gamma coactivator 1-α) proteins; however, when peptides were administered, the reduced CPT1 and PGC1α protein expression levels increased again. Figure 13 ).

[0227] [Experimental Examples 14 to 22] Experimental Examples of Adipocytes

[0228] Example 14: Analysis of the expression of proteins related to insulin resistance and sensitivity in adipocytes

[0229] The effects of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of insulin resistance and sensitivity-related proteins in adipocytes were analyzed.

[0230] 3T3-L1 cells (preadipocytes, USA Type Culture Collection) were cultured in DMEM medium containing 2% BCS (fetal bovine serum) (Welgene, South Korea). To differentiate 3T3-L1 cells into adipocytes, they were cultured at a concentration of 1 × 10⁻⁶ cells / cells. 5Cells were seeded at a concentration of [number] cells / well into 12-well plates. When the cells reached confluence (day 2), the medium was replaced and the cells were cultured for an additional 48 hours. After 48 hours (day 0), the medium was replaced with DMEM containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutyl methylxanthine (IBMX) (Sigma, USA), and 1 μM dexamethasone (Sigma, USA) and the cells were cultured. After 2 days (day 2), the medium was replaced with DMEM containing 10% FBS, 1% P / S, and 1 μg / ml insulin and the cells were cultured. After 3 days (day 5), the medium was replaced with medium containing only 10% FBS and 1% P / S, and peptides (5 μM, 50 μM) were added and the cells were cultured for 48 hours. After culture, lysis buffer was added to lyse the cells, and proteins were obtained by centrifugation at 12,000 rpm for 30 minutes at 4°C. The obtained proteins were then quantified using a BCA kit. Subsequently, the proteins were electrotransferred to the membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The protein-coated membrane was blocked with 5% skim milk and then incubated with primary antibodies overnight at 4°C. 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); 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 Immuno Research, USA; peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L), Jackson Immuno Research, USA) at room temperature for 1 hour. After washing again with PBS-T, the mixture was treated with Western spectroscopy assay kit (Elpis Biotech, Daejeon, Korea) and developed using GelDoc (Bio-Rad, Hercules, CA, USA).

[0231] In the above experiment, 3T3-L1 preadipocytes were induced to differentiate for 5 days using an MDI mixture. The insulin sensitivity-promoting effect was then confirmed by measuring the expression levels of insulin sensitivity- or insulin resistance proteins after peptide treatment. The results showed that in 3T3-L1 cells, peptide treatment increased the expression levels of insulin sensitivity-related factors pIRS (Tyr612) and GLUT4, while decreasing the expression levels of insulin resistance-related factors pIRS (Ser302) and p70S6K. Figure 14a and Figure 14b ).

[0232] Example 15: Analysis of the expression of genes related to insulin resistance and sensitivity in adipocytes

[0233] The effects of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of genes related to insulin resistance and sensitivity in adipocytes were analyzed.

[0234] 3T3-L1 cells (preadipocytes, USA Type Culture Collection) were cultured in DMEM medium containing 2% BCS (fetal bovine serum) (Welgene, South Korea). To differentiate 3T3-L1 cells into adipocytes, they were cultured at a concentration of 1 × 10⁻⁶ cells / cells. 5Cells were seeded at a concentration of [number] cells / well into 12-well plates. When the cells reached confluence (day 2), the medium was changed again and the cells were cultured for an additional 48 hours. After 48 hours (day 0), the medium was changed to DMEM containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutyl methyl xanthine (IBMX) (Sigma, USA), and 1 μM dexamethasone (Sigma, USA) and cultured. After 2 days (day 2), the medium was changed to DMEM containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. After 3 days (day 5), the medium was changed to medium containing only 10% FBS and 1% P / S, and an insulin-resistant environment was induced by adding 350 μM oleic acid (Sigma, St. Louis, MO, USA) for 24 hours. After 24 hours of induction, the culture 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. Following culture, cells were harvested, and RNA was extracted using Trizol (ThermoFisher Scientific, USA). The extracted RNA was synthesized using TOPScript™ RT DryMIX (Enzynomics, Korea) to obtain complementary DNA, which was then used to perform polymerase chain reaction (PCR) on genes associated with insulin resistance or sensitivity using TOPsimple™ DryMIX-nTaq (Enzynomics, Korea). The reaction products were then electrophoresed on a 1.5% agarose gel to compare the mRNA expression levels of these markers for each sample. The gene primer sequences used in the PCR are listed in Table 6 below, with the GAPDH gene used as a control.

[0235] [Table 6]

[0236] The above experiments aimed to determine whether insulin resistance was inhibited and insulin sensitivity was induced in 3T3-L1 adipocytes induced by oleic acid treatment with peptides. Western blot analysis was used to confirm the expression levels of factors related to the inhibition of insulin resistance or the induction of insulin sensitivity. The results showed that oleic acid treatment increased the expression level of the insulin resistance-related factor p70S6K in 3T3-L1 cells, while peptide treatment reduced the elevated p70S6K expression level. Furthermore, oleic acid treatment in 3T3-L1 cells decreased the expression levels of insulin sensitivity-related factors AMPKα, GLUT4, and SIRT1, while peptide treatment increased the expression levels of these factors again. Figure 15a and Figure 15b ).

[0237] Experimental Example 16: Analysis of the expression of proteins related to insulin resistance and sensitivity in adipocytes

[0238] The effects of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of insulin resistance and sensitivity-related proteins in adipocytes were analyzed.

[0239] 3T3-L1 cells (preadipocytes, USA Type Culture Collection) were cultured in DMEM medium containing 2% BCS (fetal bovine serum) (Welgene, South Korea). To differentiate 3T3-L1 cells into adipocytes, the cells were cultured at a density of 1 × 10⁶ cells / year. 5Cells were seeded at a concentration of [number] cells / well into 12-well plates. When the cells reached confluence (day 2), the medium was changed again and the cells were cultured for an additional 48 hours. After 48 hours (day 0), the medium was changed to DMEM containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutyl methyl xanthine (IBMX) (Sigma, USA), and 1 μM dexamethasone (Sigma, USA) and cultured. After 2 days (day 2), the medium was changed to DMEM containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. After 3 days (day 5), the medium was changed to medium containing only 10% FBS and 1% P / S, and an insulin-resistant environment was induced by adding 350 μM oleic acid (Sigma, St. Louis, MO, USA) for 24 hours. After 24 hours of induction, the culture 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. Following culture, lysis buffer was added to lyse the cells, and proteins were obtained by centrifugation at 12,000 rpm at 4°C for 30 minutes. The obtained proteins were then quantified using a BCA kit. Subsequently, the proteins were electrotransferred to a membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The protein-coated membrane was blocked with 5% skim milk, and then incubated overnight at 4°C with primary antibodies. The primary antibodies used were: 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, then reacted with secondary antibodies (peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L), Jackson Immuno Research, USA; peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L), Jackson Immuno Research, USA) at room temperature for 1 hour, washed again with PBS-T, then treated with Western spectroscopy assay kit (Elpis Biotech, Daejeon, Korea) and developed using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0240] Experimental results showed that in 3T3-L1 cells, oleic acid treatment reduced the expression level of the insulin sensitivity-related factor pAMPKα, while the reduced pAMPKα expression level increased again upon the addition of peptides. Furthermore, in 3T3-L1 cells, oleic acid treatment increased the expression level of the insulin resistance-related factor pIRS (Ser302), while the increased pIRS (Ser302) expression level decreased again upon the addition of peptides. Figure 16 ).

[0241] Experimental Example 17: Analysis of the expression of genes related to adipogenesis in adipocytes

[0242] The effects of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of adipogenesis-related genes in adipocytes were analyzed.

[0243] 3T3-L1 cells (preadipocytes, USA Type Culture Collection) were cultured in DMEM medium containing 2% BCS (fetal bovine serum) (Welgene, South Korea). To differentiate 3T3-L1 cells into adipocytes, the cells were cultured at a density of 1 × 10⁶ cells / year. 5 Cells were seeded at a concentration of [number] cells / well into 12-well plates. When the cells reached confluence (day 2), the medium was changed again and cultured for an additional 48 hours. After 48 hours (day 0), the medium was changed to DMEM containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutyl methylxanthine (IBMX) (Sigma, USA), and 1 μM dexamethasone (Sigma, USA) and cultured. After 2 days (day 2), the medium was changed to DMEM containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. After 3 days (day 5), the medium was changed to 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 (ThermoFisher Scientific, USA). RNA-complementary DNA was synthesized and extracted using TOPScript™ RT DryMIX (Enzynomics, Korea), and then polymerase chain reaction (PCR) was performed on adipogenesis-related biomarker genes using TOPsimple™ DryMIX-nTaq (Enzynomics, Korea). The reaction products were then electrophoresed on a 1.5% agarose gel to compare the mRNA expression levels of biomarkers for each sample. The gene primer sequences used in the PCR are listed in Table 7 below, with the GAPDH gene used as a control group.

[0244] [Table 7]

[0245] ACCα (acetyl-CoA carboxylase α) is an enzyme gene involved in lipogenesis, catalyzing the carboxylation of acetyl-CoA to malonyl-CoA, which is the rate-limiting step in fatty acid synthesis. PPAR-γ (peroxisome proliferator-activated receptor γ) is known to regulate fatty acid storage and glucose metabolism, and is known to enhance the ability of adipocytes to store fatty acids. Experimental results confirmed that peptide treatment of 3T3-L1 cells reduced the expression of ACCα and PPAR-γ. Figure 17 ).

[0246] Experimental Example 18: Analysis of the expression of adipogenesis-related proteins in adipocytes

[0247] The effect of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of adipogenesis-related proteins in adipocytes was analyzed.

[0248] 3T3-L1 cells (preadipocytes, USA Type Culture Collection) were cultured in DMEM medium containing 2% BCS (fetal bovine serum) (Welgene, South Korea). To differentiate 3T3-L1 cells into adipocytes, the cells were cultured at a density of 1 × 10⁶ cells / year. 5Cells were seeded at a concentration of [number] cells / well into 12-well plates. When the cells reached confluence (day 2), the medium was replaced and the cells were cultured for an additional 48 hours. After 48 hours (day 0), the medium was replaced with DMEM containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutyl methylxanthine (IBMX) (Sigma, USA), and 1 μM dexamethasone (Sigma, USA) and cultured. After 2 days (day 2), the medium was replaced with DMEM 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, lysis buffer was added to lyse the cells, and then protein was obtained by centrifugation at 12,000 rpm at 4°C for 30 minutes. The obtained proteins were then quantified using a BCA kit. Subsequently, the proteins were electrotransferred to a membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The protein-coated membrane was blocked with 5% skim milk and then incubated overnight at 4°C with primary antibodies. The primary antibodies used were as follows: anti-SREBP1 antibody (Abcam, USA); anti-FAS antibody (Cell Signaling Technology, USA); anti-C / EBPα antibody (Cell Signaling Technology, USA); anti-pACCα antibody (Cell Signaling Technology, USA); and anti-β-actin antibody (Santa Cruz, USA). The reaction mixture was washed with PBS-T, then reacted with secondary antibodies (peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L), Jackson Immuno Research, USA; peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L), Jackson Immuno Research, USA) at room temperature for 1 hour, washed again with PBS-T, then treated with Western spectroscopy assay kit (Elpis Biotech, Daejeon, Korea) and developed using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0249] In the above experiment, 3T3-L1 adipocytes were differentiated using an MDI mixture, and the inhibitory effect of peptide treatment on adipogenesis was confirmed by measuring the expression levels of related factors. SREBP1 (sterol regulatory element-binding protein-1) is known to induce lipid and cholesterol synthesis, FAS is a fatty acid synthase, and C / EBPα (CCAAT / enhancer-binding protein-1) is known to induce adipogenesis via PPAR-γ (Genes Dev. 2002 Jan 1; 16(1): 22-26). In addition, ACCα (acetyl-CoA carboxylase-1) is known to be phosphorylated by AMPK (AMP-activated protein kinase) to promote fatty acid oxidation (degradation). The experimental results showed that in 3T3-L1 adipocytes, peptide treatment decreased the expression levels of adipogenesis-related factors SREBP1, FAS, and C / EBPα, while conversely, the expression level of pACCα, which promotes fatty acid degradation, increased. Figure 18a and Figure 18b ).

[0250] Experimental Example 19: Analysis of the expression of proteins related to lipolysis in adipocytes

[0251] The effect of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of lipolysis-related proteins in adipocytes was analyzed.

[0252] 3T3-L1 cells (preadipocytes, USA Type Culture Collection) were cultured in DMEM medium containing 2% BCS (fetal bovine serum) (Welgene, South Korea). To differentiate 3T3-L1 cells into adipocytes, the cells were cultured at a density of 1 × 10⁶ cells / year. 5Cells were seeded at a concentration of [number] cells / well into 12-well plates. When the cells reached confluence (day 2), the medium was replaced and the cells were cultured for an additional 48 hours. After 48 hours (day 0), the medium was replaced with DMEM containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutyl methylxanthine (IBMX) (Sigma, USA), and 1 μM dexamethasone (Sigma, USA) and cultured. After 2 days (day 2), the medium was replaced with DMEM 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, lysis buffer was added to lyse the cells, and protein was obtained by centrifugation at 12,000 rpm at 4°C for 30 minutes. The obtained protein was then quantified using a BCA kit. Subsequently, the proteins were electrotransferred to the membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The protein-coated membrane was blocked with 5% skim milk and then incubated with primary antibodies 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); 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 Immuno Research, USA; peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L), Jackson Immuno Research, USA; peroxidase-conjugated AffiniPure rabbit anti-goat IgG (H+L), Jackson Immuno Research, USA) at room temperature for 1 hour. After washing with PBS-T again, the mixture was treated with Western spectroscopy assay kit (Elpis Biotech, Daejeon, Korea) and developed using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0253] Through the above experiments, 3T3-L1 adipocytes were induced to differentiate using MDI mixture, and the expression levels of related factors were measured to confirm the promoting effect of peptide treatment on lipolysis. PLIN (lipid-coated protein) is phosphorylated by activated PKA and promotes the degradation of triglycerides by lipase. HSL (hormone-sensitive lipase) is a lipid-hydrolyzing enzyme that is activated by phosphorylation by activated kinase PKA (protein kinase A). PGC1α (peroxisome proliferator-activated receptor gamma coactivator 1-α) is a transcriptional coactivator that regulates the expression of energy metabolism-related genes (Adv Physiol Educ. 2006 Dec;30(4):145-51). The experimental results confirmed that when 3T3-L1 adipocytes were treated with peptides, the expression levels of lipolysis-related factors PLIN, pHSL and PGC1α increased. Figure 19 ).

[0254] Example 20: Analysis of fatty acid oxidation-related gene expression in adipocytes after inducing an insulin-resistant environment.

[0255] The effects of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of fatty acid oxidation-related genes in adipocytes induced by insulin resistance were analyzed.

[0256] 3T3-L1 cells (preadipocytes, USA Type Culture Collection) were cultured in DMEM medium containing 2% BCS (fetal bovine serum) (Welgene, South Korea). To differentiate 3T3-L1 cells into adipocytes, the cells were cultured at a density of 1 × 10⁶ cells / year. 5Cells were seeded at a concentration of [number] cells / well into 12-well plates. When the cells reached confluence (day 2), the medium was changed again and the cells were cultured for an additional 48 hours. After 48 hours (day 0), the medium was changed to DMEM containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutyl methylxanthine (IBMX) (Sigma, USA), and 1 μM dexamethasone (Sigma, USA) and cultured. After 2 days (day 2), the medium was changed to DMEM containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. After 3 days (day 5), the medium was changed to medium containing only 10% FBS and 1% P / S, and an insulin-resistant environment was induced by treatment with 350 μM oleic acid (Sigma, St. Louis, MO, USA) for 24 hours. After 24 hours of induction, the medium was replaced with DMEM (10% FBS, 1% BSA) supplemented with 350 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM), and the cells were cultured for 48 hours. Following culture, cells were harvested, and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was synthesized using TOPScript™ RT DryMIX (Enzynomics, Korea) to obtain complementary DNA, which was then used to perform polymerase chain reaction (PCR) on fatty acid oxidation-related biomarkers using TOPsimple™ DryMIX-nTaq (Enzynomics, Korea). The reaction products were then electrophoresed on a 1.5% agarose gel to compare the mRNA expression levels of each biomarker. The gene primer sequences used in the PCR are listed in Table 8 below, with the GAPDH gene used as a control group.

[0257] [Table 8]

[0258] Experimental results showed that in 3T3-L1 cells, oleic acid treatment reduced the expression of fatty acid oxidation-related genes PGC1α (peroxisome proliferator-activated receptor gamma coactivator 1-α) and CPT1 (carnitine palmitoyltransferase I), but when cells were treated with peptides, the reduced expression of PGC1α and CPT1 increased again. Figure 20 ).

[0259] Example 21: Analysis of the expression of genes related to adipogenesis and lipolysis in adipocytes after inducing an insulin-resistant environment.

[0260] The effects of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of genes related to adipogenesis and lipolysis in insulin resistance-induced adipocytes were analyzed.

[0261] 3T3-L1 cells (preadipocytes, USA Type Culture Collection) were cultured in DMEM medium containing 2% BCS (fetal bovine serum) (Welgene, South Korea). To differentiate 3T3-L1 cells into adipocytes, the cells were cultured at a density of 1 × 10⁶ cells / year. 5 Cells were seeded at a concentration of [number] cells / well into 12-well plates. When the cells reached confluence (day 2), the medium was changed again and the cells were cultured for an additional 48 hours. After 48 hours (day 0), the medium was changed to DMEM containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutyl methyl xanthine (IBMX) (Sigma, USA), and 1 μM dexamethasone (Sigma, USA) and cultured. After 2 days (day 2), the medium was changed to DMEM containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. After 3 days (day 5), the medium was changed to medium containing only 10% FBS and 1% P / S, and an insulin-resistant environment was induced by treatment with 350 μM oleic acid (Sigma, USA) for 24 hours. After 24 hours of induction, the medium was replaced with DMEM (10% FBS, 1% BSA) supplemented with 350 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM), and the cells were cultured for 48 hours. Following culture, cells were harvested, and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was used to synthesize complementary DNA using TOPScript™ RT DryMIX (Enzynomics, Korea), followed by polymerase chain reaction (PCR) of adipogenesis and lipolysis-related biomarkers using TOPsimple™ DryMIX-nTaq (Enzynomics, Korea). The reaction products were then electrophoresed on a 1.5% agarose gel to compare the mRNA expression levels of each biomarker. The primer sequences used in the PCR are listed in Table 9 below, with the GAPDH gene used as a control group.

[0262] [Table 9]

[0263] Through the above experiments, after inducing an insulin-resistant environment in 3T3-L1 adipocytes, the expression levels of related factors were measured to confirm the inhibitory effect of peptide treatment on adipogenesis or the promoting effect on lipolysis. The results showed that oleic acid treatment increased the expression level of the adipogenesis-related factor FAS gene in 3T3-L1 adipocytes; however, upon the addition of peptides, the increased FAS gene expression level decreased again. Furthermore, oleic acid treatment decreased the expression levels of the lipolysis-related factors PLIN, HSL, and ATGL genes in 3T3-L1 adipocytes; however, upon the addition of peptides, the decreased PLIN, HSL, and ATGL gene expression levels increased again. Figure 21a and Figure 21b ).

[0264] Example 22: Analysis of the expression of proteins related to adipogenesis and lipolysis in adipocytes after inducing an insulin-resistant environment.

[0265] The effects of the peptide of SEQ ID NO:1 prepared in Preparation Example 1 on the expression of proteins related to adipogenesis and lipolysis in insulin resistance-induced adipocytes were analyzed.

[0266] 3T3-L1 cells (preadipocytes, USA Type Culture Collection) were cultured in DMEM medium containing 2% BCS (fetal bovine serum) (Welgene, South Korea). To differentiate 3T3-L1 cells into adipocytes, the cells were cultured at a density of 1 × 10⁶ cells / year. 5Cells were seeded at a concentration of [number] cells / well into 12-well plates. When the cells reached confluence (day 2), the medium was changed again and the cells were cultured for an additional 48 hours. After 48 hours (day 0), the medium was changed to DMEM containing 10% FBS, 1% P / S, 1 μg / ml insulin (Sigma, USA), 0.5 mM isobutyl methylxanthine (IBMX) (Sigma, USA), and 1 μM dexamethasone (Sigma, USA) and cultured. After 2 days (day 2), the medium was changed to DMEM containing 10% FBS, 1% P / S, and 1 μg / ml insulin and cultured. After 3 days (day 5), the medium was changed to medium containing only 10% FBS and 1% P / S, and an insulin-resistant environment was induced by treatment with 350 μM oleic acid (Sigma, St. Louis, MO, USA) for 24 hours. After 24 hours of induction, the medium was replaced with DMEM (10% FBS, 1% BSA) supplemented with 350 μM oleic acid (Sigma, St. Louis, MO, USA) and peptides (5 μM, 50 μM), and the cells were cultured for 48 hours. Following culture, lysis buffer was added to lyse the cells, and proteins were obtained by centrifugation at 12,000 rpm at 4°C for 30 minutes. The obtained proteins were then quantified using a BCA kit. Subsequently, the proteins were electrotransferred to a membrane after SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The protein-coated membrane was blocked with 5% skim milk, and then incubated overnight at 4°C with primary antibodies. The primary antibodies used were: anti-SREBP1 antibody (Abcam, US); anti-FAS antibody (Cell Signaling Technology, USA); anti-PLIN antibody (Abcam, US); 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 Immuno Research, USA; peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L), Jackson Immuno Research, USA; peroxidase-conjugated AffiniPure rabbit anti-goat IgG (H+L), Jackson Immuno Research, USA) at room temperature for 1 hour. The mixture was washed again with PBS-T, then treated with Western spectroscopy assay kit (Elpis Biotech, Daejeon, Korea) and developed using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0267] Experimental results showed that oleic acid treatment increased the expression levels of adipogenesis-related factors FAS and SREBP1 proteins in 3T3-L1 adipocytes; however, the increased expression levels of FAS and SREBP1 proteins decreased again upon the addition of peptides. Furthermore, oleic acid treatment decreased the expression level of lipolysis-related factor PLIN protein in 3T3-L1 adipocytes; however, the decreased PLIN protein expression level increased again upon the addition of peptides. Figure 22a and Figure 22b ).

[0268] Although representative embodiments of this application have been described above by way of example, the scope of this application is not limited to these specific embodiments, and those skilled in the art will be able to make appropriate modifications within the scope described in the claims of this application.

Claims

1. A peptide comprising the amino acid sequence of SEQ ID NO:

1.

2. The peptide according to claim 1, wherein the peptide has the following activities: (i) Promotes the expression of gene expression of cell proliferation-related factors PCNA (proliferating cell nuclear antigen), NDRG2 (N-myc downstream regulatory gene 2), or CDK4 (cyclin-dependent kinase 4) in myoblasts; (ii) Promotes the expression of protein of cell proliferation-related factors SIRT1 (Sirtuin 1), Ki67, Pax7 or pAKT in myoblasts; (iii) Promotes the expression of genes of differentiation-related factors MyoD, Myf6, mTOR, Myf5 or Myf4 in myoblasts; (iv) Promotes the expression of differentiation-related factors α-actin, Myf6, or MyoG in myoblasts; or (v) Promotes the expression of protein signals involved in muscle protein synthesis in myoblasts, such as SIRT1, pAMPKα, pAKT, pmTOR, or p70S6K.

3. The peptide according to claim 1, wherein the peptide has the following activity: (i) Inhibits the activity of lipid accumulation in hepatocytes; (ii) In hepatocytes induced to accumulate lipids, inhibit the protein expression of adipogenic factors SREBP1 or FAS and promote the protein expression of lipolytic factors pACC, pHSL, ATGL or PLIN. (iii) Activities that promote gene expression of fatty acid oxidation-related factors CPT1, PGC1α or PPARα and protein expression of fatty acid oxidation-related factors CPT1 or PGC1α in hepatocytes with induced lipid accumulation. (iv) Inhibit the gene expression of adipogenesis-related factors ACCα or PPAR-γ in adipocytes; (v) Inhibits the protein expression of adipogenesis-related factors SREBP1, FAS, or C / EBPα in adipocytes and promotes the protein expression of fatty acid degradation-related factor pACCα. (vi) The activity of promoting the expression of proteins of lipolysis-related factors PLIN, pHSL or PGC1α in adipocytes. (vii) The activity of promoting gene expression of fatty acid oxidation-related factors PGC1α or CPT1 in adipocytes induced with insulin resistance. (viii) Inhibit the gene expression of fatty acid synthesis factor FAS and promote the gene expression of lipolysis-related factors PLIN, HSL, or ATGL in induced insulin-resistant adipocytes; or (ix) Induced insulin resistance in adipocytes, inhibited the protein expression of fatty acid synthesis factors FAS or SREBP1 and promoted the protein expression of lipolysis-related factor PLIN.

4. The peptide according to claim 1, wherein the peptide has the following activity: (i) Promotes the protein expression of insulin-sensitive factors pIRS (Tyr612) or GLUT4 in adipocytes and inhibits the protein expression of insulin-resistant factors pIRS (Ser302) or p70S6K. (ii) Inhibit the gene expression of the insulin resistance factor P70S6K and promote the gene expression of the insulin sensitivity factors AMPKα, GLUT4 or SIRT1 in induced insulin-resistant adipocytes. (iii) Promotes the protein expression of insulin sensitivity factor pAMPKα and inhibits the protein expression of insulin resistance factor pIRS (Ser302) in induced insulin resistance adipocytes. (iv) Inhibiting the gene expression of insulin resistance factors mTOR or P70S6K and promoting the gene expression of insulin sensitivity factors AKT, SIRT1, or AMPKα in induced insulin-resistant hepatocytes; or (v) Inhibits the protein expression of insulin resistance factors pmTOR or pJNK and promotes the protein expression of insulin sensitivity factors pAMPKα, GLUT4 or SIRT1 in induced insulin-resistant hepatocytes.

5. A composition for promoting muscle formation, combating obesity, combating fatty liver, or combating diabetes, comprising a peptide as an active ingredient according to any one of claims 1 to 4.

6. A pharmaceutical composition for the prevention or treatment of muscle diseases, comprising the peptide according to claim 1 or 2 as an active ingredient.

7. The pharmaceutical composition for the prevention or treatment of muscle diseases according to claim 6, wherein the muscle disease is selected from one or more diseases in the group consisting of muscular atrophy, sarcopenia, muscular dystrophy, disuse atrophy, spinal muscular atrophy, myotonia, hypotonia, muscle weakness, muscle weakness, amyotrophic lateral sclerosis, spinal-bulbar muscular atrophy, myasthenia gravis, myasthenia, muscle degeneration, and cachexia.

8. A food composition for the prevention or improvement of muscle diseases, comprising the peptide according to claim 1 or 2 as an active ingredient.

9. The food composition for preventing or improving muscle diseases according to claim 8, wherein the muscle disease is selected from one or more diseases in the group consisting of muscular atrophy, sarcopenia, muscular dystrophy, disuse atrophy, spinal muscular atrophy, myotonia, hypotonia, muscle weakness, muscle endurance, amyotrophic lateral sclerosis, spinal-bulbar muscular atrophy, myasthenia gravis, myasthenia, muscle degeneration, and cachexia.

10. A pharmaceutical composition for the prevention or treatment of obesity or fatty liver, comprising the peptide according to claim 1 or 3 as an active ingredient.

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

12. A pharmaceutical composition for the prevention or treatment of diabetes, comprising the peptide according to claim 1 or 4 as an active ingredient.

13. A food composition for the prevention or improvement of diabetes, comprising the peptide according to claim 1 or 4 as an active ingredient.

Images