Peptide having muscle formation-promoting, Anti-obesity, fatty liver-inhibiting, and Anti-diabetic activities, and use thereof

Abstract

The present invention relates to a peptide having muscle formation-promoting activity, anti-obesity activity, fatty liver-inhibiting activity, or anti-diabetic activity, and to a use thereof. Specifically, the peptide of the present invention exhibits activities of inhibiting muscle loss, increasing muscle mass, promoting lipid degradation in hepatocytes or adipocytes, suppressing insulin resistance signaling, or enhancing insulin sensitivity signaling. The peptide of the present invention can be effectively used as an active ingredient in a pharmaceutical composition or a food composition for treating, preventing, or alleviating muscle diseases, obesity, fatty liver, or diabetes.

Description

Peptides having muscle building promoting, anti-obesity, fatty liver inhibition and anti-diabetic activities and their uses

[0001] [Cross-reference with related applications]

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2024-0090400, filed July 9, 2024, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to a peptide having muscle building promotion, anti-obesity, fatty liver inhibition and anti-diabetic activities and its use.

[0004]

[0005] Sarcopenia, caused by degeneration of spinal nerves, motor nerves, or skeletal muscle fibers associated with muscle disease, is a representative intractable disease with unknown causes. Previous research has shown that the motor nerves that drive skeletal muscle contraction degenerate, preventing skeletal muscle contraction. Alternatively, the expression of proteins involved in muscle contraction within skeletal muscle is reduced or altered, preventing normal skeletal muscle contraction. Over the long term, the motor nerves or skeletal muscle are known to transform into fibrous tissue. Because the fundamental cause of sarcopenia remains unknown, and methods to prevent or reverse the degeneration of motor nerves or skeletal muscles have yet to be developed, active research is currently underway to develop methods to slow the progression of sarcopenia. While exercise, protein, and calorie supplementation are known to be helpful for sarcopenia, these treatments are not particularly effective in the elderly, who account for the majority of patients. Therefore, there is a pressing need for treatments for sarcopenia.

[0006] Meanwhile, muscle size or mass is regulated by intracellular signaling processes that induce anabolic or catabolic reactions within the muscle. Specifically, when the signaling response that induces muscle protein synthesis is superior to that of muscle protein breakdown, muscle protein synthesis increases, resulting in increased muscle size (hypertrophy) or muscle fiber number (hyperplasia). Furthermore, muscle cell differentiation and muscle formation are regulated by various muscle regulatory factors. MyoD initiates the expression of muscle-specific genes and induces the differentiation of muscle satellite cells into myoblasts. The induction of myogenin expression by MyoD activation is a crucial factor in the fusion of myoblasts, contributing to the formation of myotubes. Muscle fibers formed through this process form bundles that ultimately form muscles.

[0007] Under this technological background, various studies are being conducted to inhibit muscle loss caused by external or internal factors and to promote muscle protein synthesis (Korean Patent No. 10-2064387), but the results are still insufficient.

[0008] Obesity also refers to a condition in which excess fat tissue exists in the body, as a result of an imbalance between the energy intake and expenditure of food, leading to the accumulation of excess energy as body fat. According to the World Health Organization (WHO), more than 1 billion adults worldwide are overweight, and at least 3 million of them are clinically obese, with the number increasing significantly in the United States and Europe. Overweight and obesity increase blood pressure and cholesterol levels, which can lead to various diseases such as heart disease, diabetes, and arthritis, and increase the incidence of various adult diseases. Furthermore, overweight and obesity are factors that increase the incidence of various adult diseases such as arteriosclerosis, hypertension, hyperlipidemia, and heart disease not only in adults but also in children and adolescents.

[0009] Currently, the representative obesity treatment drugs that are widely prescribed and approved by the US FDA include a group of drugs that act on the central nervous system and act as appetite suppressants, and orlistat (Xenical), an inhibitor of the digestive enzyme lipase secreted by the pancreas. Many drugs that act on the central nervous system, such as sibutramine, have had their approval revoked due to cardiovascular and psychiatric side effects, and orlistat has the limitation of having variable drug effects depending on fat intake in addition to various side effects. Meanwhile, liraglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist, has been approved and used as an endocrine peptide targeting drug, but the risk of thyroid cancer is emerging.

[0010] Diabetes mellitus is a metabolic disease characterized by insufficient insulin secretion or abnormal insulin function. It is characterized by hyperglycemia, which is an increase in blood glucose concentration. This hyperglycemia causes various symptoms and signs and causes glucose to be excreted in the urine. The incidence of diabetes has been rapidly increasing due to the recent increase in obesity, especially abdominal obesity. Diabetes can be broadly divided into type 1 diabetes, which is insulin-dependent, and type 2 diabetes, which is non-insulin-dependent. Type 2 diabetes is characterized by hyperglycemia, insulin resistance, and relative insulin secretion impairment.

[0011] When you eat food, glucose is absorbed from the digestive tract, stimulating insulin secretion from pancreatic beta cells. This insulin then promotes glucose uptake into muscles. While insulin also plays a role in hepatic glucose uptake, it primarily inhibits hepatic glucose production. Insulin lowers blood glucose levels by inhibiting hepatic glucose production and promoting glucose uptake into peripheral tissues, including muscles. Insulin resistance refers to a condition in which blood glucose levels are lower than normal in response to insulin under given insulin concentrations. Insulin regulates blood glucose levels by promoting glucose uptake into muscles or inhibiting glucose production in the liver. Insulin resistance refers to a condition in which this insulin action is reduced even in the absence of insulin deficiency. Insulin receptors on the cell membrane are involved in the process of glucose uptake into cells in peripheral tissues. Insulin resistance occurs when the number of insulin receptors decreases or intracellular defects occur after receptor binding. Although insulin receptor defects are found in type 2 diabetes, post-receptor intracellular defects, namely insulin-regulated phosphorylation or dephosphorylation disorders, are known to play a far greater role. Among these mechanisms, impaired PI3K (Phosphoinositide 3-kinase) signaling is known to reduce the translocation of the glucose transporter type 4 (GLUT-4) to the cell membrane.

[0012] Current methods for controlling blood sugar include lifestyle modifications (diet and exercise) and medication. However, these methods are difficult to strictly manage and implement, and their effectiveness is limited. Therefore, most diabetic patients rely on lifestyle modifications and medications such as insulin, insulin secretagogues, insulin sensitizers, and hypoglycemic agents to control their blood sugar.

[0013]

[0014] [Prior Art Literature]

[0015] [Patent Document]

[0016] Korean Patent No. 10-2064387

[0017]

[0018] The present inventors have conducted research to develop an effective active substance that can be used to prevent, improve, or treat muscle diseases by suppressing sarcopenia and promoting muscle growth. As a result, the inventors experimentally confirmed that the peptide they synthesized exhibited excellent sarcopenia-suppressing and muscle growth-stimulating activity, thereby completing the present invention.

[0019] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating muscle diseases, which comprises a peptide having the physiological activity of inhibiting muscle loss and increasing muscle mass as an active ingredient.

[0020] In addition, another object of the present invention is to provide a food composition for preventing or improving muscle disease, which contains the above-described peptide as an active ingredient.

[0021] In addition, the inventors of the present invention have made efforts to find an active substance with improved efficacy and secured safety for the treatment of obesity, fatty liver, and diabetes, from the viewpoint of promoting lipolysis in hepatocytes and adipocytes, and promoting glucose uptake by suppressing insulin resistance and increasing insulin signal sensitivity.

[0022] As a result, the inventors experimentally proved that the peptide they synthesized had the above-mentioned activity, thereby completing the present invention.

[0023] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating obesity, which comprises a peptide having the above-described activity as an active ingredient.

[0024] In addition, another object of the present invention is to provide a food composition for preventing or improving obesity, which contains a peptide having the above-described activity as an active ingredient.

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

[0026] Another object of the present invention is to provide a food composition for preventing or improving fatty liver, which comprises a peptide having the above-described activity as an active ingredient.

[0027] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes, which comprises a peptide having the above-described activity as an active ingredient.

[0028] Another object of the present invention is to provide a food composition for controlling blood sugar levels, which comprises a peptide having the above-described activity as an active ingredient.

[0029]

[0030] To achieve the above purpose,

[0031] One aspect of the present invention provides a pharmaceutical composition for preventing or treating muscle disease, comprising a peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0032] Another aspect of the present invention provides a food composition for preventing or improving muscle disease, comprising a peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0033] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating obesity, comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0034] Another aspect of the present invention provides a food composition for preventing or improving obesity, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0035] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating fatty liver, comprising a peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0036] Another aspect of the present invention provides a food composition for preventing or improving fatty liver, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0037] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising a peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0038] Another aspect of the present invention provides a food composition for controlling blood sugar levels, comprising a peptide comprising an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0039]

[0040] The present invention is described in detail below.

[0041]

[0042] 1. Peptide of the present invention

[0043] According to one aspect of the present invention, a peptide comprising an amino acid sequence disclosed in SEQ ID NO: 1 is provided.

[0044] [Amino acid sequence of sequence number 1: GVGR]

[0045] The term "peptide" as used herein means a linear molecule formed by amino acid residues being linked to each other by peptide bonds.

[0046] The peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention can be used without modification, but a variant or fragment of an amino acid having a different sequence by deletion, insertion, substitution, or a combination thereof of amino acid residues can be used within a range that does not affect the original activity of the peptide, such as muscle formation promotion activity, anti-diabetic activity, anti-obesity activity, and fatty liver inhibition activity.

[0047] The peptide of the present invention can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc., within a range that does not change its activity.

[0048] The peptide of the present invention includes a peptide comprising an amino acid sequence substantially identical to a peptide comprising the amino acid sequence of SEQ ID NO: 1, and a variant or active fragment thereof. The substantially identical amino acid sequence refers to an amino acid sequence having a sequence identity of 75% or more, for example, 80% or more, 85% or more, 90% or more, 95% or more, or 97% or more, with the amino acid sequence of SEQ ID NO: 1. In addition, the peptide may additionally include a targeting sequence, a tag, a labeled residue, an amino acid sequence manufactured for a specific purpose to increase half-life or peptide stability.

[0049] The peptide of the present invention may be modified at the N-terminus and / or C-terminus to select a portion of the amino acid sequence and increase its activity. Such N-terminus and / or C-terminus modifications can significantly improve the stability of the peptide of the present invention, for example, increasing the half-life of the peptide when administered in vivo. The term "stability" encompasses not only in vivo stability, which protects the peptide of the present invention from attack by in vivo protein-cleaving enzymes, but also storage stability (e.g., room temperature storage stability).

[0050] The above N-terminal modification may be a modification in which a protecting group selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG) is bonded to the N-terminus of the peptide. The above C-terminal modification may be a modification in which a hydroxyl group (-OH), an amino group (-NH2), a hydrazino group (-NHNH2), or the like is bonded to the C-terminus of the peptide, but is not limited thereto.

[0051] The peptide of the present invention can be manufactured by various methods widely known in the technical field to which the present invention pertains, and specifically, can be manufactured according to a chemical synthesis method known in the art, particularly a solid-phase synthesis technique or a liquid-phase synthesis technique. For example, the peptide of the present invention can be manufactured according to a chemical synthesis method known in the art, particularly a solid-phase synthesis technique (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 a liquid-phase synthesis technique (US Patent No. 5,516,891).

[0052] In addition, the peptide of the present invention is artificially synthesized, or non-naturally occurring or engineered, wherein "non-naturally occurring or engineered" means a state that is not a state that occurs in nature, but is created by artificial modification. Here, the artificial modification may include artificially synthesizing an amino acid sequence by mimicking a plurality of amino acid structures, or engineering to obtain chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity.

[0053]

[0054] 2. Activity of the peptide of the present invention

[0055] The peptide of the present invention has an activity that promotes muscle formation.

[0056] In one embodiment, the peptide of the present invention has muscle loss inhibition and muscle mass increasing activity.

[0057] Therefore, the peptide of the present invention can be used for the prevention, improvement, or treatment of muscle diseases.

[0058] In one embodiment, the peptide of the present invention has one or more of the following activities:

[0059] (i) Activity that promotes proliferation and differentiation of myoblasts;

[0060] (ii) activity of increasing the mRNA expression level of a gene involved in differentiation into muscle cells or muscle protein synthesis, selected from the group consisting of Myf5 (Myogenic factor 5), MyoD (myoblast determination protein 1), AKT (Protein kinase B), and P70S6K (Ribosomal protein S6 kinase beta-1, p70S6 kinase);

[0061] (ii) activity of increasing the expression level of a marker or protein involved in differentiation into muscle cells or muscle protein synthesis, selected from the group consisting of MyoD, Myf6 (Myogenic factor 6), MyoG (Myogenin), MyHC (Myosin heavy chain), α-actinin, SIRT1 (silent mating type information regulation 2 homologue 1), pmTOR (mammalian target of rapamycin), and p70S6K;

[0062] (iii) activity that increases myotube synthesis capacity;

[0063] (iv) activity that increases mRNA levels of mTOR, P70S6K, MyoG, or IGF-1 (Insulin-like growth factor-1) genes in a muscular dystrophy environment;

[0064] (v) activity that reduces the mRNA level of the MuRF1 (Muscle-specific RING finger protein 1) gene in a muscular dystrophy environment;

[0065] (vi) activity that increases the expression level of a marker or protein selected from the group consisting of MyHC, α-actinin, Myf6, MyoG, IGF-1, pAKT and p70S6K in a muscular atrophy environment;

[0066] (vii) In a muscular atrophy environment, activity that increases the expression level of a marker or protein selected from the group consisting of MSTN (Myostatin), MAFbx (muscle atrophy F-box), MuRF1, Cleaved-PARP (Poly (ADP-ribose) polymerase), Cleaved caspase-3 and BAX (Bcl-2-associated X protein).

[0067]

[0068] The peptide of the present invention has anti-obesity activity.

[0069] In one embodiment, the peptide of the present invention has the activity of promoting the breakdown of fat in adipocytes or promoting the oxidation of fatty acids.

[0070] In one embodiment, the peptide of the present invention has an activity of reducing the mRNA expression level of a fat synthesis-related gene FAS (Fatty acid synthase) or ACCα (Acetyl-CoA carboxylase alpha) in adipocytes; or an activity of reducing the protein expression level of a fat synthesis-related marker FAS or SREBP1 (Sterol-regulatory element binding protein 1).

[0071] In one embodiment, the peptide of the present invention has an activity of increasing the mRNA expression level of a lipolysis-related gene, HSL (Hormone-Sensitive Lipase) or PLIN (Perilipin, lipid droplet-associated protein), in adipocytes; or an activity of increasing the protein expression level of a lipogenesis-related marker, ATGL (Adipose triglyceride lipase) or PLIN.

[0072] In one embodiment, the peptide of the present invention has an activity of increasing the expression level of mRNA or protein of fatty acid oxidation-related markers, AMPKα (AMP-activated Protein Kinase α), SIRT1 (silent mating type information regulation 2 homologue 1), PGC1α (Peroxisome proliferator-activated receptor-gamma coactivator), or CPT1 (Carnitine palmitoyl transferase I), in adipocytes.

[0073]

[0074] The peptide of the present invention has activity in treating, preventing, or improving fatty liver.

[0075] In one embodiment, the peptide of the present invention has the activity of promoting the breakdown of fat or promoting the oxidation of fatty acids in hepatocytes.

[0076] In one embodiment, the peptide of the present invention has an activity of reducing the expression level of mRNA of a fat synthesis-related gene, FAS (Fatty Acid Synthase), or ACCα (Acetyl-CoA carboxylase alpha), in hepatocytes; or an activity of reducing the expression level of a protein of a fat synthesis-related marker, FAS, or SREBP1 (Sterol-regulatory element binding protein 1).

[0077] In one embodiment, the peptide of the present invention has an activity of increasing the expression level of mRNA of a fat-decomposition-related gene, ATGL (Adipose triglyceride lipase), or HSL (Hormone-Sensitive Lipase), in hepatocytes; or an activity of increasing the expression level of protein of a fat-decomposition-related marker, ATGL, pHSL (phosphorylated Hormone-Sensitive Lipase), or pACCα (phosphorylated Acetyl-CoA carboxylase alpha).

[0078] In one embodiment, the peptide of the present invention has an activity of increasing the expression level of mRNA or protein of fatty acid oxidation-related markers AMPKα (AMP-activated Protein Kinase α), SIRT1 (silent mating type information regulation 2 homologue 1), PGC1α (Peroxisome proliferator-activated receptor-gamma coactivator 1-alpha), or CPT1 (Carnitine palmitoyl transferase I) in hepatocytes.

[0079]

[0080] The peptide of the present invention has antidiabetic activity.

[0081] In one embodiment, the peptide of the present invention has the activity of inhibiting insulin resistance signaling or promoting insulin sensitivity signaling.

[0082] In one embodiment, the peptide of the present invention has one or more of the following activities: (i) decreasing the expression level of the P70S6K (p70S6 kinase) gene, or increasing the expression level of the AKT gene; (ii) increasing the expression level of the insulin sensitivity-related protein pPI3K, pAKT, or pIRS(Tyr); or (iii) decreasing the expression level of the insulin resistance-related protein P70S6K or pJNK.

[0083]

[0084] Since the peptide of the present invention described above has the above-described activity, it can exhibit excellent efficacy in treating, preventing, or improving muscle disease, obesity, fatty liver, or diabetes.

[0085]

[0086] 3. Composition for preventing, improving or treating muscle disease, obesity, fatty liver or diabetes

[0087] pharmaceutical composition

[0088] According to another aspect of the present invention, a composition for treating, preventing or improving muscle disease is provided, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0089] In one embodiment, the composition is a pharmaceutical composition.

[0090] In the pharmaceutical composition of the present invention, the peptide comprising the amino acid sequence of SEQ ID NO. 1, which is an active ingredient, and its activity are the same as the peptide and its activity described in the above “1. Peptide and 2. Activity of Peptide” section, and a detailed description thereof is cited and is not described repeatedly.

[0091] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating muscle disease is provided, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0092] The peptide of the present invention exhibits excellent efficacy in suppressing muscle loss or increasing muscle mass, and can be used for the prevention or treatment of muscle diseases.

[0093] As used herein, the term "muscle disease" may collectively refer to a muscle disease, illness or condition associated with a decrease in the proliferation or differentiation of myoblasts, for example, muscle diseases resulting from muscle dysfunction, muscle wasting or muscle degeneration.

[0094] In one embodiment, the muscle disease may be at least one selected from the group consisting of muscular atrophy, sarcopenia, muscular dystrophy, disuse atrophy, spinal muscular amyotrophy, muscle rigidity, muscular hypotonia, muscle weakness, muscle endurance weakness, amyotrophic lateral sclerosis, spinal muscular atrophy, myasthenia gravis, myasthenia, muscle degeneration and cachexia, but is not limited thereto.

[0095] In one embodiment, the peptide of the present invention can inhibit muscle loss and increase muscle mass by promoting proliferation and differentiation of myogenic cells.

[0096] In one embodiment, the peptide of the present invention can promote proliferation of myogenic cells and significantly increase the expression of early differentiation markers, mid-differentiation markers, and late differentiation markers of myogenic cells, thereby promoting differentiation into muscle cells or muscle fibers.

[0097] In one embodiment, the peptide of the present invention can significantly increase the expression of myogenic markers and muscle protein synthesis markers, and can also increase the myotube synthesis ability of myoblasts, and can enhance muscle mass recovery or regeneration when myoblasts are damaged by exogenous factors.

[0098] In one embodiment, the peptide, which is an active ingredient of the pharmaceutical composition, has one or more of the following activities:

[0099] (i) Activity that promotes proliferation and differentiation of myoblasts;

[0100] (ii) activity of increasing the mRNA expression level of a gene involved in differentiation into muscle cells or muscle protein synthesis, selected from the group consisting of Myf5 (Myogenic factor 5), MyoD (myoblast determination protein 1), AKT (Protein kinase B), and P70S6K (Ribosomal protein S6 kinase beta-1, p70S6 kinase);

[0101] (ii) activity of increasing the expression level of a marker or protein involved in differentiation into muscle cells or muscle protein synthesis, selected from the group consisting of MyoD, Myf6 (Myogenic factor 6), MyoG (Myogenin), MyHC (Myosin heavy chain), α-actinin, SIRT1 (silent mating type information regulation 2 homologue 1), pmTOR (mammalian target of rapamycin), and p70S6K;

[0102] (iii) activity that increases myotube synthesis capacity;

[0103] (iv) activity that increases mRNA levels of mTOR, P70S6K, MyoG, or IGF-1 (Insulin-like growth factor-1) genes in a muscular dystrophy environment;

[0104] (v) activity that reduces the mRNA level of the MuRF1 (Muscle-specific RING finger protein 1) gene in a muscular dystrophy environment;

[0105] (vi) activity that increases the expression level of a marker or protein selected from the group consisting of MyHC, α-actinin, Myf6, MyoG, IGF-1, pAKT and p70S6K in a muscular atrophy environment;

[0106] (vii) In a muscular atrophy environment, activity that increases the expression level of a marker or protein selected from the group consisting of MSTN (Myostatin), MAFbx (muscle atrophy F-box), MuRF1, Cleaved-PARP (Poly (ADP-ribose) polymerase), Cleaved caspase-3 and BAX (Bcl-2-associated X protein).

[0107]

[0108] According to another aspect of the present invention, a composition for preventing or treating obesity is provided, which comprises a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0109] In one embodiment, the composition is a pharmaceutical composition.

[0110] The peptide of the present invention exhibits excellent efficacy in promoting fat decomposition and fatty acid oxidation in fat cells, and thus can be used for the prevention or treatment of obesity.

[0111] In one embodiment, in a pharmaceutical composition for preventing or treating obesity, the peptide can promote the breakdown of fat in adipocytes or promote the oxidation of fatty acids.

[0112] In one embodiment, the peptide, which is an active ingredient of the pharmaceutical composition, has an activity of reducing the mRNA expression level of a fat synthesis-related gene, FAS (Fatty acid synthase) or ACCα (Acetyl-CoA carboxylase alpha), in adipocytes; or an activity of reducing the protein expression level of a fat synthesis-related marker, FAS, or SREBP1 (Sterol-regulatory element binding protein 1).

[0113] In one embodiment, the peptide, which is an active ingredient of the pharmaceutical composition, has an activity of increasing the mRNA expression level of a fat-decomposition-related gene, HSL (Hormone-Sensitive Lipase) or PLIN (Perilipin, lipid droplet-associated protein), in adipocytes; or an activity of increasing the protein expression level of a fat synthesis-related marker, ATGL (Adipose triglyceride lipase) or PLIN.

[0114] In one embodiment, the peptide, which is an active ingredient of the pharmaceutical composition, has an activity of increasing the expression level of mRNA or protein of fatty acid oxidation-related markers, AMPKα (AMP-activated Protein Kinase α), SIRT1 (silent mating type information regulation 2 homologue 1), PGC1α (Peroxisome proliferator-activated receptor-gamma coactivator), or CPT1 (Carnitine palmitoyl transferase I), in adipocytes.

[0115]

[0116] According to another aspect of the present invention, a composition for preventing or treating fatty liver is provided, which comprises a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0117] In one embodiment, the composition is a pharmaceutical composition.

[0118] The peptide of the present invention exhibits excellent efficacy in promoting fat decomposition and fatty acid oxidation in hepatocytes, and thus can be used for the prevention or treatment of fatty liver.

[0119] In one embodiment, in a pharmaceutical composition for preventing or treating fatty liver, the peptide can promote the breakdown of fat in hepatocytes or promote the oxidation of fatty acids.

[0120] In one embodiment, the fatty liver may be alcoholic fatty liver or non-alcoholic fatty liver.

[0121] In one embodiment, the peptide, which is an active ingredient of the pharmaceutical composition, has an activity of reducing the expression level of mRNA of a fat synthesis-related gene, FAS (Fatty Acid Synthase), or ACCα (Acetyl-CoA carboxylase alpha), in hepatocytes; or an activity of reducing the expression level of a protein of a fat synthesis-related marker, FAS, or SREBP1 (Sterol-regulatory element binding protein 1).

[0122] In one embodiment, the peptide, which is an active ingredient of the pharmaceutical composition, has an activity of increasing the expression level of mRNA of a fat-decomposition-related gene, ATGL (Adipose triglyceride lipase) or HSL (Hormone-Sensitive Lipase), in hepatocytes; or an activity of increasing the expression level of protein of a fat-decomposition-related marker, ATGL, pHSL (phosphorylated Hormone-Sensitive Lipase), or pACCα (phosphorylated Acetyl-CoA carboxylase alpha).

[0123] In one embodiment, the peptide, which is an active ingredient of the pharmaceutical composition, has an activity of increasing the expression level of mRNA or protein of fatty acid oxidation-related markers AMPKα (AMP-activated Protein Kinase α), SIRT1 (silent mating type information regulation 2 homologue 1), PGC1α (Peroxisome proliferator-activated receptor-gamma coactivator 1-alpha), or CPT1 (Carnitine palmitoyl transferase I) in hepatocytes.

[0124]

[0125] According to another aspect of the present invention, a composition for preventing or treating diabetes is provided, which comprises a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0126] In one embodiment, the composition is a pharmaceutical composition.

[0127] The peptide of the present invention exhibits excellent efficacy in suppressing insulin resistance and promoting insulin sensitivity, and thus can be used for the prevention or treatment of diabetes.

[0128] In one embodiment, the diabetes may be type 1 diabetes or type 2 diabetes, and specifically type 2 diabetes.

[0129] In one embodiment, in the pharmaceutical composition for preventing or treating diabetes, the peptide can inhibit insulin resistance signaling or promote insulin sensitivity signaling.

[0130] In one embodiment, the peptide, which is an active ingredient of the pharmaceutical composition, has one or more of the following activities: (i) decreasing the expression level of the P70S6K (p70S6 kinase) gene, or increasing the expression level of the AKT gene; (ii) increasing the expression level of the insulin sensitivity-related protein pPI3K, pAKT, or pIRS (Tyr); or (iii) decreasing the expression level of the insulin resistance-related protein P70S6K or pJNK.

[0131] Conventional functional peptides, despite their effective biological activity, have shown problems such as not being able to effectively enter target tissues or cells due to their large size, or being rapidly eliminated from the body due to their short half-life. In contrast, the pharmaceutical composition of the present invention comprises a peptide composed of 10 or fewer amino acids as its active ingredient. Accordingly, the composition exhibits excellent cellular penetration of the active ingredient, and even when administered topically, it can exhibit excellent effects in the prevention or treatment of muscle disease, obesity, fatty liver, and diabetes.

[0132] As used herein, the term “prevention” means any act of inhibiting or delaying the onset of a disease by administering the composition.

[0133] As used herein, the term "treatment" means any form of treatment that provides a benefit to a subject suffering from a disease or at risk of developing a disease, including improving the condition of the subject (e.g., one or more symptoms), delaying the progression of the disease, delaying the onset of symptoms, or slowing the progression of symptoms.

[0134] Therefore, the above “treatment” and “prevention” are not intended to mean cure or complete elimination of symptoms.

[0135] As used herein, the term "administration" means introducing a given substance into an individual by any appropriate method, and the pharmaceutical composition of the present invention may be administered via any general route capable of reaching an in vivo target. For example, the route of administration of the pharmaceutical composition of the present invention is not particularly limited, but may be administered orally or parenterally, and in the case of parenteral administration, may be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc., but is not limited thereto.

[0136] The dosage of the pharmaceutical composition may be, but is not limited to, 0.0001 to 1000 μg, 0.001 to 1000 μg, 0.01 to 1000 μg, 0.1 to 1000 μg, or 1.0 to 1000 μg per day, and may be administered in various amounts depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. In addition, the frequency of administration of the pharmaceutical composition may be 1 to 4 times, 2 to 3 times, or 2 times per day, and the administration period may be, but is not limited to, 4 weeks or more, 8 weeks or more, 4 to 12 weeks, or 8 to 12 weeks.

[0137] The pharmaceutical composition of the present invention may additionally comprise a therapeutically effective amount of the above-described peptide and / or an appropriate carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions.

[0138] The term "therapeutically effective amount" above means an amount sufficient for the peptide, which is an active ingredient of the pharmaceutical composition of the present invention, to achieve its activity or efficacy, and for example, an amount sufficient to achieve the efficacy of preventing or treating muscle disease, obesity, fatty liver, or diabetes.

[0139] Examples of carriers, excipients or diluents usable in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

[0140] The pharmaceutical composition of the present invention can be formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods, and can be manufactured in the form of unit doses or by being placed in multi-dose containers.

[0141] When formulating the pharmaceutical composition of the present invention, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

[0142] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium, styrax, and talc can also be used.

[0143] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives.

[0144] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include withepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0145] Preparations for transdermal administration may include dusting powders, emulsions, suspensions, oils, sprays, ointments, cream pastes, gels, foams, or solutions. The pharmaceutical compositions of the present invention may be an anhydrous ointment, containing paraffin, especially low viscosity paraffin, which is suitable for topical use and is liquid at body temperature, or may contain natural or partially synthetic fats, for example coconut fatty acid triglycerides, hydrogenated oils, for example hydrogenated peanut oil or castor oil, partial fatty acid esters of glycerol, for example glycerol monostearate and distearate, silicones, for example polymethylsiloxanes, such as hexamethyldisiloxane or octamethyltrisiloxane, fatty alcohols, for example those associated with aqueous creams and which increase the water absorption capacity, and sterols, wool wax, other emulsifiers and / or other additives.

[0146] Suitable pharmaceutically acceptable carriers and agents in the pharmaceutical composition of the present invention are described in detail in Remington: The Science and Practice of Pharmacy, (21st ed., 2005, Lippincott Williams & Wilkins).

[0147] The dosage of the peptide comprising the amino acid sequence of SEQ ID NO: 1 contained in the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the severity of the disease, the drug form, the route and period of administration, and may be appropriately selected depending on the case. For example, the peptide comprising the amino acid sequence may be administered at a dosage of 0.000001 to 1000 mg / kg per day, specifically 0.001 to 1000 mg / kg, and the application may be applied once a day or divided into several times. The dosage of the peptide comprising the peptide comprising the amino acid sequence of the present invention may be increased or decreased depending on the route of administration, the severity of the disease, gender, weight, age, etc. Therefore, the dosage does not limit the scope of the present invention in any way.

[0148] The pharmaceutical composition of the present invention may contain a peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention at a concentration of 0.001 μM to 1000 μM, specifically, the peptide is present at a concentration of 0.01 μM to 1000 μM; 0.05 μM to 800 μM, 0.05 μM to 700 μM, 0.05 μM to 600 μM, 0.05 μM to 500 μM, 0.05 μM to 300 μM, 0.05 μM to 200 μM; 0.1 μM to 800 μM, 0.1 μM to 700 μM, 0.1 μM to 600 μM, 0.1 μM to 500 μM, 0.1 μM to 300 μM, 0.1 μM to 200 μM; It may be included at a concentration of, but is not limited to, 1 μM to 800 μM, 1 μM to 700 μM, 1 μM to 600 μM, 1 μM to 500 μM, 1 μM to 300 μM, 1 μM to 200 μM; 5 μM to 800 μM, 5 μM to 700 μM, 5 μM to 600 μM, 5 μM to 500 μM, 5 μM to 300 μM, or 5 μM to 200 μM.

[0149] The weight ratio between the peptide and the pharmaceutically acceptable carrier may be, for example, 500:1 to 1:500, and as an example, the weight ratio may be 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to It can be, but is not limited to, 1:4, or 2:1 or 1:2.

[0150]

[0151] Food composition

[0152] According to another aspect of the present invention, a composition for treating, preventing or improving muscle disease is provided, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0153] In one embodiment, the composition is a food composition.

[0154] According to another aspect of the present invention, a food composition is provided comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0155] The peptide containing the amino acid sequence of SEQ ID NO: 1, which is an active ingredient in the food composition of the present invention, and its activity are the same as the peptide and its activity described in the above "1. Peptide, 2. Activity of peptide and pharmaceutical composition" items, and the specific description thereof is cited and is not described repeatedly.

[0156] In addition, terms or technical configurations that can be equally applied to the food composition of the present invention in the description of the pharmaceutical composition described above are cited and not described repeatedly.

[0157] According to another aspect of the present invention, a food composition for preventing or improving muscle disease is provided, which comprises a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0158] The peptide of the present invention exhibits excellent efficacy in suppressing muscle loss and increasing muscle mass, and thus can be used for the prevention or improvement of muscle diseases.

[0159] In one embodiment, the peptide of the present invention can promote proliferation and differentiation of myogenic cells, thereby inhibiting muscle loss and increasing muscle mass.

[0160] The above muscle disease is a muscle disease, illness or condition associated with a decrease in the proliferation or differentiation of myoblasts, and may be a general term for muscle diseases resulting from, for example, muscle dysfunction, muscle loss or muscle degeneration.

[0161] In one embodiment, the muscle disease may be at least one selected from the group consisting of muscular atrophy, sarcopenia, muscular dystrophy, disuse atrophy, spinal muscular amyotrophy, muscle rigidity, muscular hypotonia, muscle weakness, muscle endurance weakness, amyotrophic lateral sclerosis, spinal muscular atrophy, myasthenia gravis, myasthenia, muscle degeneration and cachexia, but is not limited thereto.

[0162]

[0163] According to another aspect of the present invention, a composition for preventing or improving obesity is provided, which comprises a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0164] In one embodiment, the composition is a food composition.

[0165] Since the peptide of the present invention exhibits excellent efficacy in promoting fat decomposition or fatty acid oxidation in fat cells, it can be used for the prevention or improvement of obesity.

[0166] In one embodiment, the peptide of the present invention can promote lipolysis and fatty acid oxidation in adipocytes.

[0167]

[0168] According to another aspect of the present invention, a composition for preventing or improving fatty liver is provided, which comprises a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0169] In one embodiment, the composition is a food composition.

[0170] The peptide of the present invention exhibits excellent efficacy in promoting fat decomposition or fatty acid oxidation in hepatocytes, and thus can be used for the prevention or improvement of fatty liver.

[0171] In one embodiment, the peptide of the present invention can promote lipolysis and fatty acid oxidation in hepatocytes.

[0172] In one embodiment, the fatty liver may be alcoholic fatty liver or non-alcoholic fatty liver.

[0173]

[0174] According to another aspect of the present invention, a composition for preventing or improving diabetes is disclosed, which comprises a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

[0175] In one embodiment, the composition is a food composition.

[0176] According to another aspect of the present invention, a composition for controlling blood sugar levels is provided, comprising a peptide comprising an amino acid sequence of sequence number 1 as an active ingredient.

[0177] In one embodiment, the composition is a food composition.

[0178] The peptide of the present invention exhibits excellent efficacy in suppressing insulin resistance and promoting insulin sensitivity, and thus can be used for preventing or improving diabetes or for controlling blood sugar levels.

[0179] In one embodiment, the peptide of the present invention can significantly reduce the expression of factors associated with insulin resistance and increase the expression of factors promoting insulin sensitivity in hepatocytes and adipocytes.

[0180] The above diabetes may be type 1 diabetes or type 2 diabetes, and specifically type 2 diabetes.

[0181] In one embodiment, the control of blood sugar levels may be control of blood sugar levels in a diabetic patient or a high-risk patient with pre-diabetes.

[0182] In one embodiment, the control of blood sugar levels may be a lowering of blood sugar levels.

[0183] In one embodiment, the peptide may inhibit insulin resistance signaling or promote insulin sensitivity signaling.

[0184] As used herein, the term "improvement" may mean any action that at least reduces the severity of a symptom, for example, a parameter associated with alleviating or treating a condition.

[0185] In the food composition of the present invention, the peptide may be included in an appropriate amount selected within the range of 0.0001 wt% to 30 wt% based on the total composition weight.

[0186] The food composition described above can be used by adding the peptide as is or in combination with other foods or food ingredients, and can be used appropriately according to a conventional method. For example, it can contain various flavoring agents or natural carbohydrates as additional ingredients, like a conventional beverage. Examples of the natural carbohydrates described above include conventional sugars such as monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc.; and sugar alcohols such as xylitol, sorbitol, erythritol, etc. In addition to the flavoring agents described above, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrates described above can be appropriately determined by a person skilled in the art.

[0187] The food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These components may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

[0188] Meanwhile, the food composition may include a health functional food.

[0189] As used herein, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients that have functionality useful to the human body. Here, "functionality" means obtaining a useful effect for health purposes, such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food can be manufactured by a method commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art can be added. In addition, the formulation of the health functional food can also be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition can be manufactured in various forms of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs because it uses food as a raw material, and is highly portable. According to one embodiment, the health functional food can be consumed as a supplement to enhance the therapeutic effect of muscle disease.

[0190] In one embodiment, a health functional food to which the food composition of the present invention can be applied can be manufactured by mixing other appropriate auxiliary ingredients and known additives according to the selection of a person skilled in the art. Examples of foods to which the composition can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and in addition, it can be manufactured by adding it to juice, tea, jelly, and juice.

[0191]

[0192] Another aspect of the present invention provides a method for treating, preventing, or improving a muscle disease, comprising the step of administering to a subject in need of treatment of a muscle disease an effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a composition comprising the peptide as an active ingredient.

[0193] Another aspect of the present invention provides a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for treating or preventing muscle disease.

[0194] Another aspect of the present invention provides a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 in the manufacture of a pharmaceutical composition or food composition for treating, preventing, or improving a muscle disease.

[0195]

[0196] Another aspect of the present invention provides a method for treating, improving, or preventing obesity, comprising administering to a subject in need of treatment for obesity an effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a composition comprising the peptide as an active ingredient.

[0197] Another aspect of the present invention provides a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for treating or preventing obesity.

[0198] Another aspect of the present invention provides a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 in the manufacture of a pharmaceutical composition or food composition for treating, preventing or improving obesity.

[0199]

[0200] Another aspect of the present invention provides a method for treating, improving, or preventing fatty liver, comprising administering to a subject in need of treatment of fatty liver an effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a composition comprising the peptide as an active ingredient.

[0201] Another aspect of the present invention provides a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for the treatment or prevention of fatty liver.

[0202] Another aspect of the present invention provides a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 in the manufacture of a pharmaceutical composition or food composition for treating, preventing or improving fatty liver.

[0203]

[0204] In another aspect of the present invention, a method for treating, preventing, or improving diabetes is provided, comprising administering to a diabetic patient an effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a composition comprising the peptide as an active ingredient.

[0205] Another aspect of the present invention provides a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 for treating or preventing diabetes.

[0206] Another aspect of the present invention provides a use of a peptide comprising the amino acid sequence of SEQ ID NO: 1 in the manufacture of a pharmaceutical composition or food composition for treating, preventing or improving diabetes.

[0207] In another aspect of the present invention, a method for controlling blood sugar levels is provided, comprising administering to a subject in need of blood sugar level control an effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1 or a composition comprising the peptide as an active ingredient.

[0208] The term "subject" above refers to a human or non-human animal, such as humans, primates, mammals and vertebrates.

[0209]

[0210] The peptide of the present invention has activity that promotes muscle formation, anti-obesity activity, fatty liver inhibition activity, or anti-diabetic activity. Specifically, the peptide of the present invention has activity that inhibits muscle loss, activity that increases muscle mass, activity that promotes fat breakdown in hepatocytes or adipocytes, or activity that inhibits insulin resistance signaling or promotes insulin sensitivity signaling. Therefore, the peptide of the present invention can be utilized as an active ingredient in a pharmaceutical composition or food composition for the treatment, prevention, or improvement of muscle disease, obesity, fatty liver, or diabetes.

[0211] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0212]

[0213] Figure 1 shows the effect of the peptide of the present invention on the proliferation of myoblasts.

[0214] Figure 2 shows that when the peptide of the present invention is treated to muscle cells, the expression of genes related to muscle cell proliferation increases.

[0215] Figures 3a and 3b show that when the peptide of the present invention is treated on muscle cells, the expression of proteins related to signaling of myogenesis and muscle cell proliferation increases.

[0216] Figures 4a and 4b show that when the peptide of the present invention is treated on muscle cells, the expression of early and mid-stage differentiation marker genes of muscle cells and genes involved in muscle protein synthesis increases.

[0217] Figures 5a, 5b and 5c show that when the peptide of the present invention is treated on muscle cells, the expression of early, middle and late differentiation marker proteins of muscle cells and signaling proteins involved in muscle protein synthesis increases.

[0218] Figure 6 shows that the peptide of the present invention increases myotube synthesis ability in C2C12 cells.

[0219] Figures 7a, 7b and 7c show that the peptide of the present invention restores the expression levels of differentiation-related genes, genes of signaling proteins involved in muscle protein synthesis and genes of factors involved in muscle growth in C2C12 cells in which an atrophy environment is induced, and reduces the expression levels of genes of factors involved in muscle atrophy.

[0220] Figures 8a to 8f show that the peptide of the present invention restores the expression levels of differentiation marker proteins, signaling proteins involved in muscle protein synthesis, and factors involved in muscle growth in C2C12 cells in which an atrophy environment is induced, and reduces the expression levels of factors involved in muscle atrophy and cell death.

[0221] Figures 9a and 9b show that the peptide of the present invention increases the expression of fat synthesis-related genes and decreases the expression of fat decomposition-related genes in hepatocytes.

[0222] Figures 9c and 9d show that the peptide of the present invention increases the expression of fat synthesis-related genes and decreases the expression of fat decomposition-related genes in adipocytes.

[0223] Figures 10a and 10b show that the peptide of the present invention increases the expression of fat synthesis-related proteins and decreases the expression of fat decomposition-related proteins in hepatocytes.

[0224] Figures 10c and 10d show that the peptide of the present invention increases the expression of fat synthesis-related proteins and decreases the expression of fat decomposition-related proteins in adipocytes.

[0225] Figures 11a and 11b show that the peptide of the present invention increases the expression of genes related to the promotion of fatty acid oxidation in hepatocytes.

[0226] Figures 11c and 11d show that the peptide of the present invention increases the expression of genes related to the promotion of fatty acid oxidation in adipocytes.

[0227] Figures 12a and 12b show that the peptide of the present invention increases the expression of proteins associated with the promotion of fatty acid oxidation in hepatocytes.

[0228] Figure 12c shows that the peptide of the present invention increases the expression of proteins associated with the promotion of fatty acid oxidation in adipocytes.

[0229] Figure 13a shows that the peptide of the present invention reduces the expression of genes related to insulin resistance and increases the expression of genes related to insulin sensitivity in hepatocytes.

[0230] Figure 13b shows that the peptide of the present invention reduces the expression of genes related to insulin resistance and increases the expression of genes related to insulin sensitivity in adipocytes.

[0231] Figure 14a shows that the peptide of the present invention reduces the expression of insulin resistance-related proteins in hepatocytes.

[0232] Figures 14b and 14c show that the peptide of the present invention reduces the expression of insulin resistance-related proteins and increases the expression of insulin sensitivity-related proteins in adipocytes.

[0233]

[0234] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0235]

[0236] Example

[0237]

[0238] Manufacturing Example 1: Manufacturing of peptides

[0239] A peptide having the amino acid sequence of SEQ ID NO: 1 shown in Table 1 below was synthesized using an automatic peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptide was purified using C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was ACQUITY UPLC BEH300 C18 (2.1 mm Х 100 mm, 1.7 μm, Waters Co, USA).

[0240] Amino acid sequence of the peptide sequence number 1GVGR

[0241] Next, the efficacy of the peptide of sequence number 1 manufactured above was evaluated.

[0242] Experimental Example 1: Cytotoxicity Analysis of Peptide on Myoblasts

[0243] To analyze the effect of the peptide manufactured in Manufacturing Example 1 on the proliferation of myoblasts, a cytotoxicity test was performed.

[0244] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, cells were seeded at 5 x 10 3Cells were seeded in each well of a 96-well culture plate at 200 μL each. After 1 day, the medium was replaced with serum-free DMEM medium (1% P / S), and 4 hours later, the medium was replaced with DMEM containing 0.2% FBS, and the peptide of SEQ ID NO: 1 was added at concentrations of 3.9, 7.81, 15.63, 31.25, 62.5, 125, 250, or 500 μM. At this time, the group without the peptide of SEQ ID NO: 1 was used as a control group. 24 and 48 hours after the addition, the reaction was performed using EZ-cytox (DoGenBio Co., Ltd, Seoul, Korea) until the absorbance of Non became 1.0, and the absorbance at 450 nm was measured using a spectrophotometer (SPECTRAMAX M2e).

[0245] As a result of the experiment, when the peptide of sequence number 1 was treated at various concentrations in C2C12 myoblast cells, the difference in cytotoxicity compared to the control group was not significant at all concentrations, confirming that the peptide of sequence number 1 of the present invention is not cytotoxic to myoblast cells (Fig. 1).

[0246]

[0247] Experimental Example 2: Analysis of gene expression related to muscle cell proliferation

[0248] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the proliferation of myogenic cells, the expression levels of CCND1 (Cyclin D1) and PCNA (Proliferating Cell Nuclear Antigen), which are cell proliferation-related gene markers, were evaluated through RT-PCR analysis.

[0249] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, cells were seeded at 5 x 10 5Cells were seeded into each well of a 6-well culture plate at 2 mL / well. After that, when the cell confluency reached 100%, the medium was replaced with serum-free DMEM medium (1% P / S), and after about 4 hours, the peptide of sequence number 1 was added at a concentration of 50 μM or 100 μM. After 1 hour of treatment with the peptide, the cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was TOPScript TM After obtaining DNA complementary to RNA using RT DryMIX (enzynomics, Korea), TOPsimple TM Polymerase chain reaction (PCR) was performed on marker genes involved in cell proliferation using DryMIX-nTaq (enzynomics, Korea), and then run on a 1.5% agarose gel to compare the mRNA expression levels of the markers under each sample treatment condition. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 2.

[0250] Primer sequence (5'-> 3')SEQ ID NO:CCND1 Forward(5') GCC ACG CCC TCC GTA TCT (3')2CCND1 Reverse(5') GTA ACC AGC GGC TCT TCT TCA (3')3PCNA Forward(5') AGC GGA GAA GGT GCT GGA G (3')4PCNA Reverse(5') ATA GCG GCG GTA TGT GTC G (3')5GAPDH Forward(5') GTG ATG GCA TGG ACT GTG GT (3')6GAPDH Reverse(5') GGA GCC AAA AGG GTC ATC AT (3')7

[0251] As a result of the experiment, it was confirmed that the mRNA expression levels of CCND1 and PCNA genes, which are gene markers related to muscle cell proliferation, were significantly increased in a concentration-dependent manner by the peptide of sequence number 1 in C2C12 myoblasts (Fig. 2).

[0252]

[0253] Experimental Example 3: Analysis of the expression of proteins related to muscle cell proliferation.

[0254] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on myogenesis, the expression levels of SIRT1 (Sirtuin 1), Ki67 (Antigen Kiel 67), CCND1 (Cyclin D1), pERK (phospho-ERK), and pAKT (phospho-Akt), which are protein markers involved in cell proliferation in myogenic cells, were evaluated through Western blot analysis.

[0255] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, cells were seeded at 5 x 10 5Cells were seeded in each well of a 6-well culture plate at 2 mL each. After 1 day, the medium was replaced with serum-free DMEM medium (1% P / S), and 4 hours later, the peptide of SEQ ID NO: 1 was added at a concentration of 50 μM or 100 μM. After 1 hour, the solution was dissolved by adding lysis buffer, and the protein was obtained by centrifugation at 4°C and 12,000 rpm for 30 minutes, which was quantified using a BCA kit. The protein was subjected to SDS-PAGE and electrotransferred to a membrane. After blocking the membrane on which the protein was electrophoresed with 5% skim milk, the membrane was reacted overnight at 4°C with anti-SIRT1 primary antibody (Cell signaling Technology, USA), anti-Ki67 primary antibody (Santa Cruz, USA), anti-Cyclin D1 primary antibody (Cell signaling Technology, USA), anti-pERK primary antibody (Cell signaling Technology, USA), and anti-pAKT primary antibody (Cell signaling Technology, USA), and anti-β-actin primary antibody (Santa Cruz, USA) as a loading control. Next, the membrane was washed with PBS-T and incubated with peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibodies (Jackson immunoResearch, USA) and peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibodies (Jackson immunoResearch, USA) at room temperature for 1 h. The membrane was washed again with PBS-T and visualized using a Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0256] As a result of the experiment, it was confirmed that after treatment with the peptide of sequence number 1 in C2C12 myoblasts, the expression of SIRT1, Ki67, and Cyclin D1, which are cell proliferation-related proteins, increased in a peptide concentration-dependent manner, and the expression of pERK and pAKT, which are cell proliferation signaling-related proteins, increased in a peptide concentration-dependent manner (Fig. 3a and Fig. 3b).

[0257]

[0258] Experimental Example 4: Analysis of expression of early and mid-stage differentiation markers and genes involved in muscle protein synthesis in myoblasts.

[0259] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of genes related to muscle protein synthesis, the expression levels of early and mid-stage differentiation-related gene markers and genes involved in muscle protein synthesis (Myf5, MyoD, AKT, and P70S6K) in myogenic cells were evaluated through RT-PCR analysis.

[0260] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 2% BCS. When cell confluency reached approximately 70 to 80%, cells were seeded at 5 x 10 5 Cells were seeded in each well of a 6-well culture plate at 2 mL each. Afterwards, when the cell confluency reached 100%, the medium was replaced with differentiation medium (1% P / S, 2% horse serum), and the peptide of SEQ ID NO: 1 was added at a concentration of 50 μM or 100 μM. Afterwards, the differentiation medium and the peptide of SEQ ID NO: 1 were replaced every other day, and 3 days after the first replacement, the cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was TOPScript TM After obtaining DNA complementary to RNA using RT DryMIX (enzynomics, Korea), TOPsimple TMPolymerase chain reaction (PCR) was performed on signal transduction genes related to muscle protein synthesis using DryMIX-nTaq (enzynomics, Korea), and the results were run on a 1.5% agarose gel to compare the mRNA expression levels of the markers under each sample treatment condition. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 3.

[0261] Primer sequence (5'-> 3') SEQ ID NO: Myf5 Forward(5') TAT GAA GGC TCC TGT CC (3')8Myf5 1 Reverse(5') ACG TGC TCC TCA TCG TCT G (3')9MyoD Forward(5') AGT GAA TGA GGC CTT CGA GA (3')10MyoD Reverse(5') CTG GGT TCC CTG TTC TGT GT (3')11AKT Forward(5') GGT AGC GAT AAT GGA GGT CT (3')12AKT Reverse(5') GCT TCT GTC CTC TTC TCC TT (3')13P70S6K Forward(5') GAA GCC CTC TTT GAT GCT GTC C (3')14P70S6K Reverse(5') GGA GCC TGG GAG CCC TGA TGT A (3')15GAPDH Forward(5') GTG ATG GCA TGG ACT GTG GT (3')6GAPDH Reverse(5') GGA GCC AAA AGG GTC ATC AT (3')7

[0262] As a result of the experiment, it was confirmed that the mRNA expression levels of Myf5 and MyoD genes, which are gene markers related to muscle protein synthesis or muscle differentiation, were significantly increased in a peptide concentration-dependent manner by the peptide of sequence number 1 in C2C12 myogenic cells, and that the mRNA expression levels of AKT and P70S6K, which are signal transduction factors related to muscle protein synthesis, were increased in a peptide concentration-dependent manner (Fig. 4a and Fig. 4b).

[0263]

[0264] Experimental Example 5: Expression Analysis of Early, Mid, and Late Differentiation-Related Markers and Muscle Protein Synthesis-Involved Signaling Proteins in Myogenic Cells

[0265] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the differentiation of myoblasts into muscle cells or muscle fibers, the expression levels of early, middle, and late differentiation-related protein markers MyoD, Myf6, MyoG, MyHC, and α-actinin and muscle protein synthesis-related signaling proteins SIRT1, pmTOR, and p70S6K in myoblasts were evaluated through Western blot analysis.

[0266] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 2% BCS. When cell confluency reached approximately 70 to 80%, cells were seeded at 5 x 10 5Cells were seeded in each well of a 6-well culture plate at 2 mL each. When the cell confluency reached 100%, the medium was replaced with differentiation medium (1% P / S, 2% horse serum), and the peptide of SEQ ID NO: 1 was added at a concentration of 50 μM or 100 μM. Thereafter, the differentiation medium and the peptide of SEQ ID NO: 1 were replaced every other day, and 3 days after the first replacement, the cells were lysed by adding lysis buffer, and the proteins were obtained by centrifugation at 4°C and 12,000 rpm for 30 minutes, which were quantified using a BCA kit. SDS-PAGE was performed on the proteins and electrotransferred to a membrane. After blocking the protein-electrophoretic membrane with 5% skim milk, the membrane was incubated overnight at 4°C with anti-MyoD primary antibody (Santa Cruz, USA), anti-Myf6 primary antibody (Santa Cruz, USA), anti-MyoG primary antibody (Santa Cruz, USA), anti-MyHC primary antibody (Santa Cruz, USA), anti-α-actinin primary antibody (Santa Cruz, USA), anti-SIRT1 primary antibody (Cell signaling Technology, USA), anti-pmTOR primary antibody (Cell signaling Technology, USA), and anti-p70S6K primary antibody (Cell signaling Technology, USA), and anti-α-tubulin primary antibody (Santa Cruz, USA) as a loading control. The membrane was washed with PBS-T and incubated with Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibody (Jackson immunoResearch, USA) and Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Jackson immunoResearch, USA) for 1 hour at room temperature.After washing the membrane again with PBS-T, it was visualized using Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0267] As a result of the experiment, the expression of MyoD, Myf6, and MyoG, which are early and mid-stage differentiation-related protein markers of C2C12 myoblasts, was increased in a peptide concentration-dependent manner by the peptide of sequence number 1 in the peptide, and in particular, the expression of MyHC and α-actinin, which are late differentiation-related protein markers, was significantly increased in a peptide concentration-dependent manner (Fig. 5a and Fig. 5b). In addition, in the case of SIRT1, pmTOR, and p70S6K, which are signaling proteins involved in muscle protein synthesis, it was confirmed that the protein expression was increased in a peptide concentration-dependent manner by the peptide of sequence number 1 (Fig. 5c).

[0268]

[0269] Experimental Example 6: Analysis of myotube synthesis ability

[0270] To analyze the effect of the peptide manufactured in Manufacturing Example 1 on the myotube synthesis ability in myoblasts, differentiation-induced myoblasts were observed under a microscope and the thickness of the myotubes was compared.

[0271] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 2% BCS. When cell confluency reached approximately 80%, 5 x 10 5Cells were seeded into each well of a 6-well culture plate at 2 mL each. When the cell confluency reached 100%, the medium was replaced with differentiation medium (1% P / S, 2% horse serum), and the peptide of SEQ ID NO: 1 was added at a concentration of 50 μM or 100 μM. Thereafter, the differentiation medium and the peptide of SEQ ID NO: 1 were replaced every other day, and 3 days after differentiation induction, images were taken with an optical microscope (FLEXACAM C1, Leica, Germany) to compare the thickness of the myotubes.

[0272] Experimental results showed that myotubes formed by the peptide of sequence number 1 in C2C12 myotubes fused with surrounding myotubes, resulting in thicker myotubes. This occurred in a peptide concentration-dependent manner (Fig. 6). Therefore, it was confirmed that the peptide of sequence number 1 promoted differentiation in myotubes.

[0273]

[0274] Experimental Example 7: Analysis of Marker Gene Expression in Muscle Atrophy-Induced Myogenic Cells

[0275] The expression of the following genes in myogenic cells in which the peptide manufactured in Manufacturing Example 1 induced muscle atrophy was evaluated through RT-PCR analysis: differentiation-related genes (MyoG), genes of signaling proteins involved in muscle protein synthesis (mTOR, P70S6K), genes related to muscle proliferation (IGF-1), and genes involved in muscle atrophy (MuRF1).

[0276] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 2% BCS. When cell confluency reached approximately 70 to 80%, cells were seeded at 5 x 10 5Cells were seeded into each well of a 6-well culture plate at 2 mL / well. Afterwards, when the cell confluency reached 100%, the medium was replaced with differentiation medium (1% P / S, 2% horse serum) to induce differentiation. Thereafter, the differentiation medium (1% P / S, 2% horse serum) was replaced every other day. On the fourth day after differentiation induction, the peptide of SEQ ID NO: 1 was added at a concentration of 10 μM or 100 μM for pretreatment for 30 minutes, and then dexamethasone (Dexamethasone-soluble, Sigma, USA) was added and cultured for 24 hours. Afterwards, the cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was TOPScript TM After obtaining DNA complementary to RNA using RT DryMIX (enzynomics, Korea), TOPsimple TM Polymerase chain reaction (PCR) was performed on signal transduction genes related to muscle protein synthesis using DryMIX-nTaq (enzynomics, Korea), and the results were run on a 1.5% agarose gel to compare the mRNA expression levels of the markers under each sample treatment condition. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 4.

[0277] 프라이머서열 (5'-> 3')서열번호MyoG Forward(5') ACC AGG AGC CCC ACT TCT AT (3')16MyoG Reverse(5') ACG ATG GAC GTA AGG GAG TG (3')17mTOR Forward(5') TTG AGG TCG CTA TGA CCA GAG AGA A (3')18mTOR Reverse(5') TTA CCA GAA GGG ACA CCA GCC AAT G (3')19P70S6K Forward(5') GAA GCC CTC TTT GAT GCT GTC C (3')20P70S6K Reverse(5') GGA GCC TGG GAG CCC TGA TGT A (3')21IGF-1 Forward(5') CTG GGT GTC CAA ATG TAA CT (3')22IGF-1 Reverse(5') GTA TCT TTA TTG GAG GTG CG (3')23MuRF1 Forward(5') CAC CAA GAA GGT CAA ACA GGA (3')24MuRF1 Reverse(5') GCA AGA ACT TTA TTC AAA GTG CAA (3')25GAPDH Forward(5') GTG ATG GCA TGG ACT GTG GT (3')6GAPDH Reverse(5') GGA GCC AAA AGG GTC ATC AT (3')7

[0278] As a result of the experiment, when C2C12 myoblasts were treated with dexamethasone (DEX-soluble), the expression levels of mTOR and P70S6K genes involved in muscle synthesis signaling and MyoG, a muscle cell differentiation-related gene, were significantly reduced. However, it was confirmed that the reduced gene expression levels (mRNA) were restored in a peptide concentration-dependent manner by treatment with the peptide of sequence number 1 (Fig. 7a and Fig. 7b). In addition, when C2C12 myoblasts were treated with dexamethasone, the expression of IGF-1 involved in muscle proliferation decreased and the expression of MuRF1 involved in muscle atrophy increased. However, it was confirmed that the expression level of IGF-1 was restored and the expression level of MuRF1 was reduced by treatment with the peptide of sequence number 1 (Fig. 7c).

[0279]

[0280] Experimental Example 8: Analysis of Marker Protein Expression in Muscle Atrophy-Induced Myogenic Cells

[0281] The effect of the peptide manufactured in Manufacturing Example 1 on the expression of the following proteins in myoblasts that induced muscle atrophy was evaluated through Western blotting: differentiation-related marker proteins (MyHC, α-actinin, Myf6, and MyoG), muscle proliferation-related proteins (IGF-1), proteins involved in muscle atrophy (MSTN, MAFbx, and MuRF1), signal transduction-related proteins involved in muscle protein synthesis (pAKT, P70S6K), and proteins involved in cell death (apoptosis) (Cleaved-PAPR, Cleaved-caspase-3, and BAX).

[0282] C2C12 cells (mouse myoblast cell line) were cultured in DMEM medium containing 1% P / S and 2% BCS. When cell confluency reached approximately 70 to 80%, cells were seeded at 5 x 10 5Cells were seeded into each well of a 6-well culture plate at 2 mL per well. After reaching 100% cell confluency, the medium was replaced with differentiation medium (1% P / S, 2% horse serum) to induce differentiation. Thereafter, the differentiation medium (1% P / S, 2% horse serum) was replaced every other day. On the 4th day of differentiation induction, the peptide of SEQ ID NO: 1 was added at a concentration of 10 μM or 100 μM and pretreated for 30 minutes, and then dexamethasone (Dexamethasone-soluble, Sigma, USA) was added and incubated for 24 hours. After incubation, the cells were lysed by adding lysis buffer and centrifuged at 4°C, 12,000 rpm for 30 minutes to obtain the protein, which was quantified using a BCA kit. SDS-PAGE was performed on the proteins and electrotransferred onto a membrane. The membrane on which the proteins were electrotransferred was blocked with 5% skim milk, and then incubated overnight at 4°C with anti-MyHC primary antibody (Santa Cruz, USA), anti-α-actinin primary antibody (Santa Cruz, USA), anti-Myf6 primary antibody (Santa Cruz, USA), anti-MyoG primary antibody (Santa Cruz, USA), and anti-β-actin primary antibody (Santa Cruz, USA) as a loading control. The membrane was washed with PBS-T and incubated with peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Jackson immunoResearch, USA) for 1 hour at room temperature.

[0283] Meanwhile, for muscle proliferation-related proteins and muscle atrophy-related proteins, the membrane on which the proteins were electrophoretically transferred was blocked with 5% skim milk, and then reacted overnight at 4°C with anti-IGF-1 primary antibody (Cell signaling Technology, USA), anti-MSTN primary antibody (Abcam, UK), anti-MAFbx primary antibody (Santa Cruz, USA), anti-MuRF1 primary antibody (Santa Cruz, USA), and anti-α-tubulin primary antibody (Santa Cruz, USA) as a loading control.

[0284] Additionally, for signaling proteins involved in muscle protein synthesis and proteins involved in apoptosis, anti-Anti-pAKT primary antibody (Cell signaling Technology, USA), anti-P70S6K primary antibody (Cell signaling Technology, USA), anti-Cleaved-PAPR primary antibody (Cell signaling Technology, USA), anti-Cleaved-caspase-3 primary antibody (Cell signaling Technology, USA), and anti-BAX primary antibody (Santa Cruz, USA) and anti-β-actin primary antibody (Santa Cruz, USA) as a loading control were reacted overnight. Afterwards, the membrane was reacted with peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibodies (Jackson immunoResearch, USA) and peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibodies (Jackson immunoResearch, USA) at room temperature for 1 hour. The membrane was washed again with PBS-T and visualized using a Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0285] As a result of the experiment, it was confirmed that when C2C12 myoblasts were treated with dexamethasone, the expression levels of intermediate and late differentiation-related proteins (MyHC, α-actinin, Myf6, and MyoG) were significantly reduced, but the reduced protein expression levels were restored in a peptide concentration-dependent manner by the peptide of sequence number 1 (Fig. 8a and Fig. 8b).

[0286] In addition, when C2C12 myoblasts were treated with dexamethasone, the IGF-1 protein expression level was significantly reduced, but it was confirmed that the reduced IGF-1 protein expression level was restored in a peptide concentration-dependent manner by the peptide of SEQ ID NO: 1 (Figs. 8c and 8d). In addition, when C2C12 myoblasts were treated with dexamethasone, the expression levels of muscle atrophy-related marker proteins (MSTN, MAFbx, and MuRF1) were significantly increased, but it was confirmed that the increased protein expression levels were all reduced in a peptide concentration-dependent manner by treatment with the peptide of SEQ ID NO: 1 (Figs. 8c and 8d). In addition, when C2C12 myoblasts were treated with dexamethasone, the protein expression levels of pAKT and p70S6K, which are markers related to muscle protein synthesis, were significantly reduced, but the reduced protein expression levels were restored in a peptide concentration-dependent manner by treatment with the peptide of sequence number 1 (Figs. 8e and 8f). In addition, when C2C12 myoblasts were treated with dexamethasone, the expression of Cleaved-PARP, Cleaved caspase-3, and BAX, which are cell death-related proteins, increased, but it was confirmed that the increased cell death-related protein expression levels were reduced again by treatment with the peptide of sequence number 1 (Figs. 8e and 8f).

[0287]

[0288] Through the experiments of the above Experimental Examples 1 to 8, it was confirmed that the peptide of sequence number 1 of the present invention can promote the proliferation of muscle cells, promote the differentiation of muscle cells into muscle fibers, and have a muscle regeneration effect even under muscle atrophy conditions.

[0289]

[0290] Experimental Example 9: Analysis of gene expression related to fat synthesis and lipolysis

[0291] Experimental Example 9-1: Analysis of gene expression related to fat synthesis and lipolysis in hepatocytes.

[0292] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of genes related to fat synthesis and fat decomposition, the expression levels of fat synthesis-related genes (FAS, ACCα) and fat decomposition genes (ATGL, HSL) in hepatocytes (HepG2) were evaluated through RT-PCR analysis.

[0293] HepG2 cells (human hepatocyte cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, cells were seeded at a density of 1 x 10 5Cells were seeded in each well of a 12-well culture plate at 1 mL each. When the cell confluency in the 12-well culture plate reached approximately 70-80%, the medium was replaced with serum-free DMEM medium. After 4 hours, lipid accumulation was induced by replacing the medium with serum-free DMEM medium (1% BSA) supplemented with 500 μM oleic acid (OA; Sigma, USA). After 24 hours of induction, the medium was replaced with serum-free DMEM medium (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA), and the peptide of SEQ ID NO: 1 was treated at a concentration of 5 μM or 50 μM, followed by incubation for 48 hours. After incubation, the cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was TOPScript TM After obtaining DNA complementary to RNA using RT DryMIX (enzynomics, Korea), TOPsimple TM Polymerase chain reaction (PCR) was performed on genes related to fat synthesis and fat decomposition using DryMIX-nTaq (enzynomics, Korea), and the results were run on a 1.5% agarose gel to compare the mRNA expression levels of the markers under each sample treatment condition. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 5.

[0294] Primer sequence (5'-> 3')SEQ ID NO:FAS Forward(5') CGG AAA CTG CAG GAG CTG TC (3')26FAS Reverse(5') CAC GGA GTT GAG CCG CAT (3')27ACCα Forward(5') GAA TGT TTG GGG ATA TTT CAG (3')28ACCα Reverse(5') TTC TGC TAT CAG TCT GTC CAG (3')29ATGL Forward(5') GCG TGT CAG ACG GCG AGA ATG (3')30ATGL Reverse(5') GCA GCT CGT GGA TGT TGG (3')31HSL Forward(5') CAC TAC AAA CGC AAC GAG AC (3')32HSL Reverse(5') CCA GAG ACC GAT AGC ACT TCC (3')33GAPDH Forward(5') GGA GCC AAA AGG GTC ATC AT (3')6GAPDH Reverse(5') GTG ATG GCA TGG ACT GTG GT (3')7

[0295] As a result of the experiment, when oleic acid (OA) was treated to HepG2 hepatocytes, the mRNA expression levels of the FAS and ACCα genes, which are fat synthesis-related genes, increased. However, by treatment with the peptide of SEQ ID NO: 1, the increased mRNA expression levels of the FAS and ACCα genes were decreased again in a peptide concentration-dependent manner. In addition, when oleic acid (OA) was treated to HepG2 hepatocytes, the mRNA expression levels of the ATGL and HSL genes, which are fat decomposition genes, were decreased. However, by treatment with the peptide of SEQ ID NO: 1, the decreased mRNA expression levels of the ATGL and HSL genes were increased again in a peptide concentration-dependent manner. (Figs. 9a and 9b) From the above experimental results, it was found that the peptide of Preparation Example 1 has the activity of inhibiting fat synthesis and promoting fat decomposition in hepatocytes.

[0296]

[0297] Experimental Example 9-2: Analysis of gene expression related to fat synthesis and lipolysis in adipocytes

[0298] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of genes related to fat synthesis and decomposition, the expression levels of fat synthesis-related genes (FAS, ACCα) and fat decomposition genes (HSL, PLIN) in adipocytes were evaluated through RT-PCR analysis.

[0299] 3T3-L1 adipocytes purchased from the American Type Culture Collection (ATCC) were cultured in DMEM medium containing 2% bovine calf serum (BCS). To differentiate 3T3-L1 cells into adipocytes, 1 x 10 5The cells were seeded in a 12-well culture plate at a concentration of 10 cells / well, and the medium was replaced once more at the time of reaching confluence (Day = -2) and cultured for an additional 48 h. After the additional 48 h of culture (Day = 0), the medium was replaced with differentiation induction medium [DMEM medium containing 10% FBS, 1 μg / ml insulin, 0.5 mM isobutylmethylxanthine (IBMX) (Sigma, USA), and 1 μM dexamethasone (Sigma, USA)] and cultured to induce differentiation into adipocytes. Two days after the differentiation was induced (Day=2), the medium was replaced with DMEM containing 10% FBS, 1% P / S, and 1 ug / ml insulin and cultured. Three days after the culture (Day=5), the medium was replaced with only 10% FBS and 1% P / S and 500 μM oleic acid (Sigma, USA) was treated for 24 hours. After the oleic acid treatment, the peptide of SEQ ID NO: 1 was treated at a concentration of 5 μM or 50 uM and cultured for 48 hours. After the culture, the cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was TOPScript TM After obtaining DNA complementary to RNA using RT DryMIX (enzynomics, Korea), TOPsimple TM Polymerase chain reaction (PCR) was performed on genes related to fat synthesis and fat decomposition using DryMIX-nTaq (enzynomics, Korea), and the results were run on a 1.5% agarose gel to compare the mRNA expression levels of the markers under each sample treatment condition. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 6.

[0300] Primer sequence (5'-> 3')SEQ ID NO:FAS Forward(5') TGC TGG CAC TAC AGA ATG C (3')34FAS Reverse(5') AAC AGC CTC AGA GCG ACA AT (3')35ACCα Forward(5') ACC TTA CTG CCA TCC CAT GTG CTA (3')36ACCα Reverse(5') GTG CCT GAT GAT CGC ACG AAC AAA (3')37HSL Forward(5') GGA CAC ACA CAC ACC TG (3')38HSL Reverse(5') CCC TTT CGC AGC AAC TTT AG (3')38PLIN Forward(5') AAG GAT CCT GCA CCT CAC AC (3')40PLIN Reverse(5') CCT CTG CTG AAG GGT TAT CG (3')41GAPDH Forward(5') GGA GCC AAA AGG GTC ATC AT (3')6GAPDH Reverse(5') GTG ATG GCA TGG ACT GTG GT (3')7

[0301] As a result of the experiment, when oleic acid was treated to adipocytes, the mRNA expression levels of the FAS and ACCα genes, which are fat synthesis-related genes, increased. However, by treatment with the peptide of sequence number 1, the increased mRNA expression levels of the FAS and ACCα genes were reduced again, and it was confirmed that the effect of the peptide was particularly great at low concentrations. In addition, when oleic acid was treated to adipocytes, the mRNA expression levels of the HSL and PLIN genes, which are fat decomposition genes, were reduced. However, by treatment with the peptide of sequence number 1, the decreased mRNA expression levels of the HSL and PLIN genes were restored to levels similar to those of the control group (Figs. 9c and 9d). From the above experimental results, it was found that the peptide of Preparation Example 1 has the activity of inhibiting fat synthesis and promoting fat decomposition in adipocytes.

[0302]

[0303] Experimental Example 10: Analysis of protein expression related to fat synthesis and lipolysis.

[0304] Experimental Example 10-1: Analysis of Protein Expression Related to Lipid Synthesis and Lipolysis in Hepatocytes

[0305] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of proteins related to fat synthesis and fat decomposition, the expression levels of fat synthesis-related protein markers (FAS, SREBP1) and fat decomposition-related protein markers (pACCα, pHSL, and ATGL) in hepatocytes were evaluated through Western blot analysis.

[0306] HepG2 liver cells were cultured using the same method described in Experimental Example 9-1. The cultured cells were lysed by adding lysis buffer, and the cells were centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins. The obtained proteins were quantified using a BCA kit. SDS-PAGE was performed on the proteins and electrotransferred to a membrane. The membrane on which the proteins were electrotransferred was blocked with 5% skim milk and then reacted with primary antibodies overnight at 4°C. The membrane was washed with PBS-T and reacted with secondary antibodies for 1 hour at room temperature. Anti-FAS primary antibody (Cell signaling Technology, USA), anti-SREBP1 primary antibody (Abcam, UK), anti-pACCα primary antibody (Cell signaling Technology, USA), anti-pHSL primary antibody (Cell signaling Technology, USA), and anti-ATGL primary antibody (Cell signaling Technology, USA) were used as primary antibodies, anti-β-actin primary antibody (Santa Cruz, USA) was used as a loading control, and Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibody (Jackson immunoResearch, USA) and Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Jackson immunoResearch, USA) were used as secondary antibodies. After washing the membrane again with PBS-T, it was visualized using Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0307] As a result of the experiment, when HepG2 hepatocytes were treated with oleic acid, the expression levels of fat synthesis-related proteins FAS and SREBP1 were significantly increased, but the increased expression levels of the proteins FAS and SREBP1 were decreased by treatment with the peptide of sequence number 1. In addition, when HepG2 hepatocytes were treated with oleic acid, the expression levels of fat decomposition-related proteins pACCα, pHSL, and ATGL were decreased, but it was confirmed that the decreased expression levels of the pACCα, pHSL, and ATGL proteins were increased by treatment with the peptide of sequence number 1 (Fig. 10a and Fig. 10b).

[0308] From the above experimental results, it was found that the peptide of Manufacturing Example 1 has the activity of inhibiting fat synthesis and promoting fat decomposition in liver cells.

[0309]

[0310] Experimental Example 10-2: Analysis of Protein Expression Related to Fat Synthesis and Fat Decomposition in Adipocytes

[0311] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of proteins related to fat synthesis and decomposition, the expression levels of fat synthesis-related protein markers (FAS and SREBP1) and fat decomposition-related protein markers (ATGL and PLIN) in adipocytes were evaluated through Western blot analysis.

[0312] 3T3-L1 adipocytes were cultured using the same method described in Experimental Example 9-2, and the cultured cells were lysed by adding a lysis buffer, and then centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins. The obtained proteins were quantified using a BCA kit. SDS-PAGE was performed on the proteins and electrotransferred onto a membrane. The membrane to which the proteins had been transferred was blocked with 5% skim milk, and then reacted overnight at 4°C with anti-FAS primary antibody (Cell signaling Technology, USA), anti-SREBP1 primary antibody (Abcam, UK), anti-ATGL primary antibody (Cell signaling Technology, USA), and anti-PLIN primary antibody (Abcam, US), and as a loading control, anti-β-actin primary antibody (Santa Cruz, USA). The membrane was washed with PBS-T and incubated with peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibodies (Jackson immunoResearch, USA), peroxidase-conjugated AffiniPure Rabbit Anti-Goat IgG (H+L) secondary antibodies (Jackson immunoResearch, USA), and peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibodies (Jackson immunoResearch, USA) for 1 h at room temperature. The membrane was washed again with PBS-T and visualized using a Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0313] As a result of the experiment, when oleic acid was treated to adipocytes, the expression levels of FAS and SREBP1, which are fat synthesis-related proteins, were significantly increased, but the increased expression levels of FAS and SREBP1 proteins were decreased by treatment with the peptide of sequence number 1. In addition, when oleic acid was treated to adipocytes, the expression levels of ATGL and PLIN, which are fat decomposition-related proteins, were decreased, but it was confirmed that the decreased expression levels of ATGL and PLIN proteins were increased in a peptide concentration-dependent manner by treatment with the peptide of sequence number 1 (Fig. 10c and Fig. 10d).

[0314] From the above experimental results, it was found that the peptide of Manufacturing Example 1 has the activity of inhibiting fat synthesis and promoting fat decomposition in fat cells.

[0315]

[0316] Experimental Example 11: Analysis of Gene Expression Related to Fatty Acid Oxidation

[0317] Experimental Example 11-1: Analysis of Gene Expression Related to Fatty Acid Oxidation in Hepatocytes

[0318] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of genes related to fatty acid oxidation, the expression levels of fatty acid oxidation-related gene markers AMPKα, SIRT1, and PGC1α in liver cells HepG2 were evaluated through RT-PCR analysis.

[0319] HepG2 cells (human hepatocyte cell line) were cultured in DMEM medium containing 1% P / S and 10% FBS. When cell confluency reached approximately 70 to 80%, 2 x 10 5Cells were seeded into each well of a 12-well culture plate at 1 mL each. When the cell confluency in the 12-well culture plate reached approximately 70-80%, the medium was replaced with serum-free DMEM. After 4 hours, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (OA; Sigma, USA) to induce lipid accumulation. After 24 hours of induction, the medium was replaced with serum-free DMEM (1% BSA) supplemented with 500 μM oleic acid (Sigma, St. Louis, MO, USA), and the peptide of SEQ ID NO: 1 was treated at a concentration of 5 μM or 50 μM, followed by incubation for 48 hours. After incubation, the cells were harvested and RNA was extracted using Trizol (Thermo Fisher Scientific, USA). The extracted RNA was used to obtain complementary DNA using TOPScript RT DryMIX (enzynomics, Korea), and then polymerase chain reaction (PCR) was performed on genes related to fat synthesis and fat decomposition using TOPsimple DryMIX-nTaq (enzynomics, Korea). The results were run on a 1.5% agarose gel to compare the mRNA expression levels of the markers under each sample treatment condition. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 5.

[0320] The extracted RNA was used to obtain complementary DNA using TOPScript RT DryMIX (enzynomics, Korea), and then polymerase chain reaction (PCR) was performed on fatty acid oxidation-related genes using TOPsimple DryMIX-nTaq (enzynomics, Korea). The results were run on a 1.5% agarose gel to compare the mRNA expression levels of the markers under each sample treatment condition. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 7.

[0321] Primer sequence (5'-> 3')SEQ ID NO:AMPKα Forward(5') GTC ATG ATA GCT TGC ATA AAT GGT G (3')42AMPKαReverse(5') AGT TGA ATA GAA CAA GCC CTG GCA (3')43SIRT1 Forward(5') TCA GTG GCT GGA ACA GTG AG (3')44SIRT1 Reverse(5') TCT GGC ATG TCC CAC TAT CA (3')45PGC1α Forward(5') AGT CTG TAT GGA GTG ACA TCG AG (3')46PGC1α Reverse(5') GGC AAT CCG TCT TCA TCC AC (3')47GAPDH Forward(5') GGA GCC AAA AGG GTC ATC AT (3')6GAPDH Reverse(5') GTG ATG GCA TGG ACT GTG GT (3')7

[0322] As a result of the experiment, when HepG2 hepatocytes were treated with oleic acid, the expression of fatty acid oxidation-related genes AMPKα, SIRT1, and PGC1α was reduced, but it was confirmed that the expression of the reduced genes was increased again by treatment with the peptide of Preparation Example 1 (Figs. 11a and 11b). From the above experimental results, it was found that the peptide of Preparation Example 1 has the activity of promoting fatty acid oxidation in hepatocytes.

[0323]

[0324] Experimental Example 11-2: Analysis of Gene Expression Related to Fatty Acid Oxidation in Adipocytes

[0325] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of genes related to fatty acid oxidation, the expression levels of fatty acid oxidation-related gene markers AMPKα, SIRT1, CPT1, and PGC1α in adipocytes were evaluated through RT-PCR analysis.

[0326] Using the same method described in Experimental Example 9-2, 3T3-L1 adipocytes were cultured, RNA was extracted from the cultured adipocytes, and the expression of fatty acid oxidation-related gene markers was analyzed. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 8.

[0327] Primer sequence (5'-> 3') SEQ ID NO:PGC1α Forward(5') ATG TGC AGC CAA GAC TCT GTA (3')48PGC1α Reverse(5') CGC TAC ACC ACT TCA ATC CAC (3')49CPT1 Forward(5') CGT ACC AAG TAG CCA AGG CA (3')50CPT1 Reverse(5') CAG GAA CGC ACA GTC TCA GT (3')51SIRT1 Forward(5') GAT CCT TTG GAT TCC TGC AA (3')52SIRT1 Reverse(5') AGT TCC AGC CGT CTC TGT GT (3')53AMPKα Forward(5') TCA CCG GAC ATA AAG TGG CT(3')54AMPKα Reverse(5') TGA TGA TGT GAG GGT GCC TG (3')55GAPDH Forward(5') GTG ATG GCA TGG ACT GTG GT (3')6GAPDH Reverse(5') GGA GCC AAA AGG GTC ATC AT (3')7

[0328] As a result of the experiment, when oleic acid was treated in 3T3-L1 adipocytes, the expression of fatty acid oxidation-related genes AMPKα, SIRT1, CPT1, and PGC1α was reduced, but it was confirmed that the expression of the reduced genes was increased again by treatment with the peptide of sequence number 1 (Figs. 11c and 11d). From the above experimental results, it was found that the peptide of Preparation Example 1 has the activity of promoting fatty acid oxidation in adipocytes.

[0329]

[0330] Experimental Example 12: Analysis of Protein Expression Related to Fatty Acid Oxidation

[0331] Experimental Example 12-1: Analysis of the expression of proteins related to fatty acid oxidation in hepatocytes.

[0332] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of proteins related to fatty acid oxidation, the expression levels of fatty acid oxidation-related protein markers SIRT1, CPT1, and PGC1α in hepatocytes were evaluated through Western blot analysis.

[0333] HepG2 liver cells were cultured using the same method described in Experimental Example 11-1. The cultured cells were lysed by adding lysis buffer, and then centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins. The obtained proteins were quantified using a BCA kit. SDS-PAGE was performed on the proteins and electrotransferred to a membrane. The membrane on which the proteins were electrotransferred was blocked with 5% skim milk and then reacted with primary antibodies overnight at 4°C. The membrane was washed with PBS-T and reacted with secondary antibodies for 1 hour at room temperature. At this time, anti-SIRT1 primary antibody (Cell signaling Technology, USA), anti-CPT1 primary antibody (Abcam, UK), and anti-PGC1α primary antibody (Abcam, UK) were used as primary antibodies, anti-β-actin primary antibody (santa cruz, USA) was used as a control, and Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibody (Jackson immunoResearch, USA) and Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Jackson immunoResearch, USA) were used as secondary antibodies. After washing the membrane again with PBS-T, it was visualized through Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0334] As a result of the experiment, it was confirmed that when hepatocytes were treated with oleic acid, the expression levels of fatty acid oxidation-related proteins SIRT1, CPT1, and PGC1α were significantly reduced, but the peptide of Preparation Example 1 increased the expression levels of the reduced proteins. In particular, it was confirmed that the expression levels of SIRT1 and CPT1 proteins in the high-concentration peptide treatment group were significantly restored to the levels of the oleic acid-untreated group (Figs. 12a and 12b).

[0335] From the above experimental results, it was confirmed that the peptide of Manufacturing Example 1 has the activity of promoting the oxidation of fatty acids in liver cells.

[0336]

[0337] Experimental Example 12-2: Analysis of the expression of proteins related to fatty acid oxidation in adipocytes.

[0338] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of proteins related to fatty acid oxidation, the expression level of fatty acid oxidation-related protein markers (SIRT1, CPT1) in adipocytes was evaluated through Western blot analysis.

[0339] 3T3-L1 adipocytes were cultured using the same method described in Experimental Example 10-2. The cultured cells were lysed by adding lysis buffer, and then centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins. The obtained proteins were quantified using a BCA kit. SDS-PAGE was performed on the proteins and electrotransferred to a membrane. The membrane to which the proteins had been transferred was blocked with 5% skim milk and then reacted with primary antibodies overnight at 4°C. The membrane was washed with PBS-T and reacted with secondary antibodies for 1 hour at room temperature. At this time, anti-SIRT1 primary antibody (Cell signaling Technology, USA) and anti-CPT1 primary antibody (Abcam, UK) were used as primary antibodies, anti-β-actin primary antibody (santa cruz, USA) was used as a control, and Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibody (Jackson immunoResearch, USA) and Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Jackson immunoResearch, USA) were used as secondary antibodies. After washing the membrane again with PBS-T, it was visualized through Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0340] As a result of the experiment, it was confirmed that when oleic acid was treated to adipocytes, the expression levels of fatty acid oxidation-related proteins SIRT1 and CPT1 were significantly reduced, but the expression levels of the reduced proteins were increased again by treatment with the peptide of Manufacturing Example 1 (Fig. 12c).

[0341] From the above experimental results, it was confirmed that the peptide of Manufacturing Example 1 has the activity of promoting oxidation of fatty acids in fat cells.

[0342]

[0343] Experimental Example 13: Analysis of Gene Expression Related to Insulin Resistance and Sensitivity

[0344] Experimental Example 13-1: Analysis of Gene Expression Related to Insulin Resistance and Sensitivity in Hepatocytes

[0345] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of genes related to insulin resistance and sensitivity, the expression levels of the insulin resistance-related gene P70S6K and the insulin sensitivity-related gene AKT in HepG2 liver cells were evaluated through RT-PCR analysis.

[0346] HepG2 hepatocytes were cultured using the same method described in Example 11-1. RNA was extracted from the cultured hepatocytes, and RT-PCR was performed on genes related to insulin resistance and insulin sensitivity. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 9.

[0347] Primer sequence (5'-> 3')SEQ ID NO:AKT Forward(5') TTG TCA TGG AGT ACG CCA ACG (3')56AKT Reverse(5') ACA GCC CGA AGT CTG TGA TCT T (3')57P70S6K Forward(5') ACT TCT GGC TCG AAA GGT GG (3')58P70S6K Reverse(5') TTG AGT CAT CTG GGC TGT CG (3')59GAPDH Forward(5') GGA GCC AAA AGG GTC ATC AT (3')6GAPDH Reverse(5') GTG ATG GCA TGG ACT GTG GT (3')7

[0348] As a result of the experiment, when HepG2 hepatocytes were treated with oleic acid, the expression of AKT, a gene related to insulin sensitivity, was reduced, but the expression of the reduced AKT gene was increased by treatment with the peptide of Preparation Example 1. In addition, when HepG2 hepatocytes were treated with oleic acid, the expression of P70S6K, a gene related to insulin resistance, was increased, but it was confirmed that the increased expression of the P70S6K gene was reduced by treatment with the peptide of Preparation Example 1 (Fig. 13a). From the above experimental results, it was confirmed that the peptide of Preparation Example 1 has the activity of promoting the expression of insulin-sensitive genes and reducing the expression of insulin resistance genes in hepatocytes.

[0349]

[0350] Experimental Example 13-2: Analysis of Gene Expression Related to Insulin Resistance and Sensitivity in Adipocytes

[0351] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of genes related to insulin resistance and sensitivity, the expression levels of the insulin resistance-related gene (P70S6K) and the insulin sensitivity-related gene (AKT) in 3T3-L1 adipocytes were evaluated through RT-PCR analysis.

[0352] 3T3-L1 adipocytes were cultured using the same method described in Example 11-2, RNA was extracted from the cultured adipocytes, and the expression of fatty acid oxidation-related gene markers was analyzed using RT-PCR. The primer sequences of the genes used in the experiment and the primer sequence of the GAPDH gene as a control are shown in Table 10.

[0353] Primer sequence (5'-> 3') SEQ ID NO: AKT Forward(5') GGT AGC GAT AAT GGA GGT CT (3')60AKT Reverse(5') GCT TCT GTC CTC TTC TCC TT (3')61P70S6K Forward(5') GAA GCC CTC TTT GAT GCT GTC C (3')62P70S6K Reverse(5') GGA GCC TGG GAG CCC TGA TGT A (3')63GAPDH Forward(5') GTG ATG GCA TGG ACT GTG GT (3')6GAPDH Reverse(5') GGA GCC AAA AGG GTC ATC AT (3')7

[0354] As a result of the experiment, when oleic acid was treated to 3T3-L1 adipocytes, the expression of the AKT gene related to insulin sensitivity was reduced, but by treatment with the peptide of Preparation Example 1, the decreased expression of the AKT gene was increased in a peptide concentration-dependent manner. In addition, when oleic acid was treated to 3T3-L1 adipocytes, the expression of the P70S6K gene related to insulin resistance was increased, but by treatment with the peptide of Preparation Example 1, the increased expression of the P70S6K gene was confirmed to be reduced (Fig. 13b). From the above experimental results, it was confirmed that the peptide of Preparation Example 1 has the activity of promoting the expression of insulin-sensitive genes and reducing the expression of insulin resistance genes in adipocytes.

[0355]

[0356] Experimental Example 14: Analysis of protein expression related to insulin resistance and sensitivity

[0357] Experimental Example 14-1: Analysis of Insulin Sensitivity-Related Protein Expression in Hepatocytes

[0358] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of insulin sensitivity-related proteins, the expression levels of insulin sensitivity-related protein markers pPI3K and pAKT in hepatocytes were evaluated through Western blot analysis.

[0359] HepG2 liver cells were cultured using the same method described in Example 11-1. The cultured cells were lysed by adding lysis buffer, and then centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins. The obtained proteins were quantified using a BCA kit. SDS-PAGE was performed on the proteins and electrotransferred to a membrane. The membrane on which the proteins were electrotransferred was blocked with 5% skim milk and then reacted with primary antibodies overnight at 4°C. The membrane was washed with PBS-T and reacted with secondary antibodies for 1 hour at room temperature. At this time, anti-pPI3K primary antibody (Cell signaling Technology, USA) and anti-pAKT primary antibody (Cell signaling Technology) were used as primary antibodies, anti-β-actin primary antibody (santa cruz, USA) was used as a control, and Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibody (Jackson immunoResearch, USA) and Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Jackson immunoResearch, USA) were used as secondary antibodies. After washing the membrane again with PBS-T, it was visualized through Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0360] As a result of the experiment, it was confirmed that when hepatocytes were treated with oleic acid (OA), the expression levels of insulin sensitivity-related proteins pPI3K and pAKT were reduced, but when treated with the peptide of Preparation Example 1, the expression levels of the reduced proteins were increased again in a peptide concentration-dependent manner (Fig. 14a).

[0361] From the above experimental results, it was confirmed that the peptide of Manufacturing Example 1 has the activity of promoting the expression of insulin-sensitive protein in hepatocytes.

[0362]

[0363] Experimental Example 14-2: Analysis of Protein Expression Related to Insulin Resistance and Sensitivity in Adipocytes

[0364] To confirm the effect of the peptide manufactured in Manufacturing Example 1 on the expression of proteins related to insulin resistance and sensitivity, the expression levels of p70S6K and pJNK, which are insulin resistance-related proteins, and pIRS (Tyr) and pAKT, which are insulin-sensitive proteins, were evaluated in adipocytes using Western blot analysis.

[0365] 3T3-L1 adipocytes were cultured using the same method described in Experimental Example 10-2. The cultured cells were lysed by adding lysis buffer, and then centrifuged at 4°C and 12,000 rpm for 30 minutes to obtain proteins. The obtained proteins were quantified using a BCA kit. SDS-PAGE was performed on the proteins and electrotransferred to a membrane. The membrane to which the proteins had been transferred was blocked with 5% skim milk and then reacted with primary antibodies overnight at 4°C. The membrane was washed with PBS-T and reacted with secondary antibodies for 1 hour at room temperature. At this time, anti-pIRS (Tyr612) primary antibody (Invitrogen, USA), anti-pAKT primary antibody (Cell signaling Technology, USA), anti-pmTOR primary antibody (Cell signaling Technology, USA), anti-p70S6K primary antibody (Cell signaling Technology, USA), and anti-pJNK primary antibody (santa cruz, USA) were used as primary antibodies, anti-β-actin primary antibody (santa cruz, USA) was used as a control, and Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibody (Jackson immunoResearch, USA) and Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Jackson immunoResearch, USA) were used as secondary antibodies. After washing the membrane again with PBS-T, it was visualized using Western detection reagent (Elpis Biotech, Daejeon, Korea) using Gel Doc (Bio-Rad, Hercules, CA, USA).

[0366] As a result of the experiment, when oleic acid was treated to adipocytes, the expression of proteins related to insulin sensitivity (pIRS (Tyr) and pAKT) was significantly reduced, but it was confirmed that the expression of the reduced pIRS (Tyr) and pAKT proteins increased in a concentration-dependent manner by treatment with the peptide of Preparation Example 1. In addition, when oleic acid was treated to adipocytes, the expression of proteins related to insulin resistance (p70S6K and pJNK) increased, but it was confirmed that the expression of the increased p70S6K and pJNK proteins decreased in a concentration-dependent manner by treatment with the peptide of Preparation Example 1 (Figs. 14b and 14c).

[0367] From the above experimental results, it was confirmed that the peptide of Manufacturing Example 1 has the activity of promoting the expression of insulin-sensitive proteins and suppressing the expression of insulin-resistant proteins in adipocytes.

[0368]

[0369] Manufacturing Example 2: Manufacturing of a pharmaceutical composition

[0370] 2-1. Manufacturing of Sanje

[0371] 2 g of the peptide of the present invention

[0372] 1 g lactose

[0373] The above ingredients were mixed and filled into a sealed bag to prepare a powder.

[0374]

[0375] 2-2. Manufacturing of tablets

[0376] 100 mg of the peptide of the present invention

[0377] 100 mg of corn starch

[0378] 100 mg of lactose

[0379] Magnesium stearate 2 mg

[0380] After mixing the above ingredients, tablets were manufactured by pressing them according to a conventional tablet manufacturing method.

[0381]

[0382] 2-3. Manufacturing of capsules

[0383] 100 mg of the peptide of the present invention

[0384] 100 mg of corn starch

[0385] 100 mg of lactose

[0386] Magnesium stearate 2 mg

[0387] After mixing the above ingredients, the mixture was filled into a gelatin capsule according to a conventional capsule manufacturing method to produce a capsule.

[0388]

[0389] 2-4. Manufacturing of rings

[0390] 1 g of the peptide of the present invention

[0391] 1.5 g lactose

[0392] 1 g of glycerin

[0393] 0.5 g of xylitol

[0394] After mixing the above ingredients, it was manufactured to have 4g per pill according to the usual method.

[0395]

[0396] 2-5. Preparation of granules

[0397] 150 mg of the peptide of the present invention

[0398] 50 mg of soybean extract

[0399] 200 mg of glucose

[0400] 600 mg of starch

[0401] After mixing the above ingredients, 100 mg of 30% ethanol was added, dried at 60°C to form granules, and then filled into a bag.

[0402]

[0403] Manufacturing Example 3: Manufacturing of a functional food composition

[0404] 3-1. Manufacturing of health foods

[0405] 500 μg of the peptide of the present invention

[0406] Vitamin mixture appropriate amount

[0407] Vitamin A acetate 70 mg

[0408] Vitamin E 1.0 mg

[0409] Vitamin D 0.13 mg

[0410] Vitamin B2 0.15 mg

[0411] Vitamin B6 0.5 mg

[0412] Vitamin B12 0.2 mg

[0413] 10 mg of vitamin C

[0414] 10 mg of biotin

[0415] 1.7 mg of nicotinamide

[0416] 50 mg of folic acid

[0417] Calcium pantothenate 0.5 mg

[0418] Appropriate amount of mineral mixture

[0419] 1.75 mg of ferrous sulfate

[0420] 0.82 mg of zinc oxide

[0421] Magnesium carbonate 25.3 mg

[0422] 15 mg of monobasic potassium phosphate

[0423] 55 mg of dibasic calcium phosphate

[0424] 90 mg of potassium citrate

[0425] 100mg of calcium carbonate

[0426] Magnesium chloride 24.8 mg

[0427]

[0428] The composition ratio of the above vitamin and mineral mixture is a preferred example of a mixture of ingredients suitable for relatively healthy foods, but the mixing ratio may be arbitrarily modified, and the above ingredients may be mixed according to a conventional health food manufacturing method, granules may be manufactured, and used to manufacture a health food composition according to a conventional method.

[0429]

[0430] 3-2. Manufacturing of health drinks

[0431] 500 μg of the peptide of the present invention

[0432] 1000mg of citric acid

[0433] 100g of oligosaccharide

[0434] 2g plum concentrate

[0435] 1g of taurine

[0436] Add purified water to make a total of 900ml

[0437]

[0438] The above ingredients were mixed according to the conventional health beverage manufacturing method, then stirred and heated at 85℃ for about 1 hour, the resulting solution was filtered, placed in a sterilized container, sealed and sterilized, and then stored in a refrigerator before being used to manufacture a health beverage composition. The above composition ratio is a preferred example of a mixture of ingredients relatively suitable for a preferred beverage, but the mixing ratio can be arbitrarily modified according to regional and ethnic preferences such as the demand class, demand country, and intended use.

[0439]

[0440] Although representative embodiments of the present application have been described above as examples, the scope of the present application is not limited to the specific embodiments described above, and a person with ordinary knowledge in the relevant field will be able to make appropriate changes within the scope described in the claims of the present application.

Claims

1. A pharmaceutical composition for preventing or treating muscle disease, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

2. In claim 1, A pharmaceutical composition, wherein the muscle disease is at least one selected from the group consisting of muscular atrophy, sarcopenia, muscular dystrophy, disuse atrophy, spinal muscular amyotrophy, muscle rigidity, muscular hypotonia, muscle weakness, muscle endurance weakness, amyotrophic lateral sclerosis, spinal muscular atrophy, myasthenia gravis, myasthenia, muscle degeneration, and cachexia.

3. In claim 1, A pharmaceutical composition wherein the peptide has one or more of the following activities: (i) Activity that promotes proliferation and differentiation of myoblasts; (ii) activity of increasing the mRNA expression level of a gene involved in differentiation into muscle cells or muscle protein synthesis, selected from the group consisting of Myf5, MyoD, AKT, and P70S6K; (ii) activity of increasing the expression level of a marker or protein involved in differentiation into muscle cells or muscle protein synthesis, selected from the group consisting of MyoD, Myf6, MyoG, MyHC, α-actinin, SIRT1, pmTOR and p70S6K; (iii) activity that increases myotube synthesis capacity; (iv) activity that increases mRNA levels of mTOR, P70S6K, MyoG, or IGF-1 genes in a muscular dystrophy setting; (v) activity that reduces the mRNA level of the MuRF1 gene in a muscular dystrophy environment; (vi) activity that increases the expression level of a marker or protein selected from the group consisting of MyHC, α-actinin, Myf6, MyoG, IGF-1, pAKT and p70S6K in a muscular atrophy environment; (vii) Activity that increases the expression level of a marker or protein selected from the group consisting of MSTN, MAFbx, MuRF1, Cleaved-PARP, Cleaved caspase-3 and BAX in a muscular atrophy setting.

4. A food composition for preventing or improving muscle disease, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

5. In claim 4, A food composition, wherein the muscle disease is at least one selected from the group consisting of muscular atrophy, sarcopenia, muscular dystrophy, disuse atrophy, spinal muscular amyotrophy, muscle stiffness, muscular hypotonia, muscle weakness, muscle endurance weakness, amyotrophic lateral sclerosis, spinal muscular atrophy, myasthenia gravis, myasthenia, muscle degeneration, and cachexia.

6. In claim 4, A food composition wherein the peptide has one or more of the following activities: (i) Activity that promotes proliferation and differentiation of myoblasts; (ii) activity of increasing the mRNA expression level of a gene involved in differentiation into muscle cells or muscle protein synthesis, selected from the group consisting of Myf5, MyoD, AKT, and P70S6K; (ii) activity of increasing the expression level of a marker or protein involved in differentiation into muscle cells or muscle protein synthesis, selected from the group consisting of MyoD, Myf6, MyoG, MyHC, α-actinin, SIRT1, pmTOR and p70S6K; (iii) activity that increases myotube synthesis capacity; (iv) activity that increases mRNA levels of mTOR, P70S6K, MyoG, or IGF-1 genes in a muscular dystrophy setting; (v) activity that reduces the mRNA level of the MuRF1 gene in a muscular dystrophy environment; (vi) activity that increases the expression level of a marker or protein selected from the group consisting of MyHC, α-actinin, Myf6, MyoG, IGF-1, pAKT and p70S6K in a muscular atrophy environment; (vii) Activity that increases the expression level of a marker or protein selected from the group consisting of MSTN, MAFbx, MuRF1, Cleaved-PARP, Cleaved caspase-3 and BAX in a muscular atrophy setting.

7. A pharmaceutical composition for preventing or treating obesity, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

8. In claim 7, A pharmaceutical composition wherein the above peptide promotes the decomposition of fat in fat cells or promotes the oxidation of fatty acids.

9. In claim 7, A pharmaceutical composition wherein the peptide has one or more of the following activities: (i) In fat cells, Activity that reduces the mRNA expression level of the fat synthesis-related genes FAS (Fatty acid synthase) or ACCα (Acetyl-CoA carboxylase alpha); or Activity that reduces the expression level of the protein of fat synthesis-related markers FAS or SREBP1 (Sterol-regulatory element binding protein 1); (ii) in fat cells, Activity that increases the mRNA expression level of the lipolysis-related genes HSL (Hormone-Sensitive Lipase) or PLIN (Perilipin, lipid droplet-associated protein); or Activity that increases the expression level of the protein of fat synthesis-related marker ATGL (Adipose triglyceride lipase) or PLIN; or (iii) In fat cells, Activity that increases the expression levels of mRNA or protein of fatty acid oxidation-related markers AMPKα (AMP-activated Protein Kinase α), SIRT1 (silent mating type information regulation 2 homologue 1), PGC1α (Peroxisome proliferator-activated receptor-gamma coactivator), or CPT1 (Carnitine palmitoyl transferase I).

10. A food composition for preventing or improving obesity, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

11. In claim 10, A food composition wherein the above peptide promotes the decomposition of fat in fat cells or promotes the oxidation of fatty acids.

12. In claim 10, A food composition wherein the peptide has one or more of the following activities: (i) In fat cells, Activity that reduces the mRNA expression level of the fat synthesis-related genes FAS (Fatty acid synthase) or ACCα (Acetyl-CoA carboxylase alpha); or Activity that reduces the expression level of the protein of fat synthesis-related markers FAS or SREBP1 (Sterol-regulatory element binding protein 1); (ii) in fat cells, Activity that increases the mRNA expression level of the lipolysis-related genes HSL (Hormone-Sensitive Lipase) or PLIN (Perilipin, lipid droplet-associated protein); or Activity that increases the expression level of the protein of fat synthesis-related marker ATGL (Adipose triglyceride lipase) or PLIN; or (iii) In fat cells, Activity that increases the expression levels of mRNA or protein of fatty acid oxidation-related markers AMPKα (AMP-activated Protein Kinase α), SIRT1 (silent mating type information regulation 2 homologue 1), PGC1α (Peroxisome proliferator-activated receptor-gamma coactivator), or CPT1 (Carnitine palmitoyl transferase I).

13. A pharmaceutical composition for preventing or treating fatty liver, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

14. In claim 13, A pharmaceutical composition wherein the above peptide promotes the decomposition of fat in liver cells or promotes the oxidation of fatty acids.

15. In claim 13, A pharmaceutical composition wherein the peptide has one or more of the following activities: (i) In hepatocytes, Activity that reduces the expression level of mRNA of the fat synthesis-related genes FAS (Fatty Acid Synthase) or ACCα (Acetyl-CoA carboxylase alpha); or Activity that reduces the expression levels of the fat synthesis-related markers FAS and SREBP1 (Sterol-regulatory element binding protein 1); (ii) In hepatocytes, Activity that increases the expression level of mRNA of the fat-decomposition-related genes ATGL (Adipose triglyceride lipase) or HSL (Hormone-Sensitive Lipase); or Activity that increases the protein expression level of lipolysis-related markers ATGL, pHSL (phosphorylated Hormone-Sensitive Lipase), or pACCα (phosphorylated Acetyl-CoA carboxylase alpha); or (iii) In hepatocytes, Activity that increases the expression levels of mRNA or protein of fatty acid oxidation-related markers AMPKα (AMP-activated Protein Kinase α), SIRT1 (silent mating type information regulation 2 homologue 1), PGC1α (Peroxisome proliferator-activated receptor-gamma coactivator 1-alpha), or CPT1 (Carnitine palmitoyl transferase I).

16. A food composition for preventing or improving fatty liver, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

17. In claim 16, A food composition wherein the above peptide promotes the decomposition of fat in liver cells or promotes the oxidation of fatty acids.

18. In claim 16, A food composition wherein the peptide has one or more of the following activities: (i) In hepatocytes, Activity that reduces the expression level of mRNA of the fat synthesis-related genes FAS (Fatty Acid Synthase) or ACCα (Acetyl-CoA carboxylase alpha); or Activity that reduces the expression levels of the fat synthesis-related markers FAS and SREBP1 (Sterol-regulatory element binding protein 1); (ii) In hepatocytes, Activity that increases the expression level of mRNA of the fat-decomposition-related genes ATGL (Adipose triglyceride lipase) or HSL (Hormone-Sensitive Lipase); or Activity that increases the protein expression level of lipolysis-related markers ATGL, pHSL (phosphorylated Hormone-Sensitive Lipase), or pACCα (phosphorylated Acetyl-CoA carboxylase alpha); or (iii) In hepatocytes, Activity that increases the expression levels of mRNA or protein of fatty acid oxidation-related markers AMPKα (AMP-activated Protein Kinase α), SIRT1 (silent mating type information regulation 2 homologue 1), PGC1α (Peroxisome proliferator-activated receptor-gamma coactivator 1-alpha), or CPT1 (Carnitine palmitoyl transferase I).

19. A pharmaceutical composition for preventing or treating diabetes, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

20. In claim 19, A pharmaceutical composition wherein the peptide inhibits insulin resistance signaling or promotes insulin sensitivity signaling.

21. In claim 19, A pharmaceutical composition wherein the peptide has one or more of the following activities: (i) decreased expression of the P70S6K (p70S6 kinase) gene, or increased expression level of the AKT gene; (ii) increased expression levels of insulin sensitivity-related proteins pPI3K, pAKT, or pIRS(Tyr); or (iii) Reduced expression level of insulin resistance-related protein P70S6K or pJNK.

22. A food composition for controlling blood sugar levels, comprising a peptide having an amino acid sequence of sequence number 1 as an active ingredient.

23. In claim 22, A food composition wherein the peptide suppresses insulin resistance signaling or promotes insulin sensitivity signaling.

24. In claim 22, A food composition wherein the peptide has one or more of the following activities: (i) decreased expression of the P70S6K (p70S6 kinase) gene, or increased expression level of the AKT gene; (ii) increased expression levels of insulin sensitivity-related proteins pPI3K, pAKT, or pIRS(Tyr); or (iii) Reduced expression level of insulin resistance-related protein P70S6K or pJNK.

Images