Method for promoting expression of gene involved in myotube cell differentiation, method for promoting expression of gene involved in protein constituting muscle, and method for increasing diameter of myotube cell

Abstract

To provide a new and highly safe method having an effect of developing muscle.SOLUTION: A method for promoting expression of a gene involved in myotube cell differentiation, comprising a step of administering a composition containing a pyrimidine nucleotide or a precursor thereof as an active ingredient, wherein the composition is a food or drink, a supplement, a powdered infant formula, an enteral nutrient, a health food or drink, an additive for animal feed, or a medicament for animals other than humans, and the pyrimidine nucleotide or the precursor thereof is one or more selected from the group consisting of cytidylate, uridylate, cytidine, and uridine.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for promoting the expression of genes involved in myotube differentiation, a method for promoting the expression of genes involved in muscle-constituting proteins, and a method for increasing the diameter of myotube cells. [Background technology]

[0002] Muscles are formed by the differentiation of muscle cells. During muscle cell differentiation, myoblasts, which arise from muscle satellite cells, fuse with each other to form multinucleated fused bodies called myotubes. The myotubes then aggregate and align to form muscle fibers, becoming the tissue that generates the great force of muscle.

[0003] It is known that specific transcription factors are expressed and function differentially at each stage of muscle differentiation, such as Pax7 in muscle satellite cells, Myf5 and MyoD in myoblasts, and Myogenin during myotube formation. To promote myogenesis, it is important to increase factors involved in muscle differentiation.

[0004] Muscles are broadly classified into slow-twitch fibers (type I fibers) and fast-twitch fibers (type II fibers), and fast-twitch fibers are further divided into subtypes such as type IIa fibers, type IIb fibers, and type IIx fibers. Type I fibers have a slow contraction rate but are excellent in endurance, have high levels of mitochondria and antioxidants, and have many capillaries adjacent to the muscle fibers. In contrast, type II fibers have a fast contraction rate but are poor in endurance, have few mitochondria and antioxidants, and have few capillaries adjacent to the muscle fibers.

[0005] Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a factor that plays a central role in regulating muscle fiber type. PGC-1α was discovered as a transcriptional cofactor that activates transcription by the nuclear receptor PPARγ in brown adipocytes. PGC-1α is expressed not only in brown adipocytes but also in many other tissues, such as skeletal muscle, heart, kidney, and brain, and is known to regulate mitochondrial biogenesis and energy production.

[0006] Based on these functions of PGC-1α, it is known that PGC-1α overexpression improves various age-related symptoms such as decreased muscle mass, motor function, bone density, and mitochondrial function, as well as insulin resistance, worsening systemic inflammatory responses, and obesity, and extends lifespan in mice (Non-patent document 1).

[0007] On the other hand, it is known that PGC-1α knockout mice develop neurological disorders in addition to muscle dysfunction and obesity (Non-Patent Document 2). Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease are known to be associated with abnormalities and decreased expression of the PGC-1α gene. Therefore, activation of PGC-1α is emerging as a new method for treating various neurodegenerative diseases.

[0008] Since enhanced expression of PGC-1α is expected to be effective against the above-mentioned diseases or symptoms, there are prior arts related to the PGC-1α expression enhancing effect. For example, Patent Document 1 describes that Helipyron A promotes PGC-1α production in nerve cells and has the effect of improving various neurotransmitter disorders and short-term memory disorders in which PGC-1α is involved. Furthermore, Patent Document 2 describes that combining β-hydroxy-β-methylbutyric acid (HMB) with a secondary ingredient enhances the expression of myogenin, a myotube cell differentiation marker, and exhibits a significant muscle-building effect. Furthermore, Patent Document 3 describes that pyrroloquinoline quinone exhibits mitochondrial activation activity.

[0009] Cytidylic acid and uridylic acid are types of nucleotides, and are substances that are widely found in living organisms and in food, making them highly safe materials.

[0010] Patent Document 4 describes an agent for improving affective disorders that contains nucleotides. Patent Document 4 also discloses an immunostimulatory agent that contains nucleotides. Non-Patent Document 3 describes that rats administered a mixture of cytidylic acid and uridylic acid were able to withstand prolonged treadmill exercise.

[0011] However, the physical fitness improvement described in Non-Patent Document 3 only measures the improvement in the ability to continue a certain level of exercise, such as on a treadmill, and the amount of biochemical parameters related to fatigue, such as glycogen and lactic acid in the liver and muscle, but does not analyze the expression of transcription factors such as PGC-1α, which correlate with an increase in muscle mass. Therefore, Non-Patent Document 3 does not consider at all muscle-building effects, such as promoting the differentiation of myotubes.

[0012] Therefore, whether or not nucleic acid-related substances such as cytidylic acid and uridylic acid have the effect of promoting PGC-1α expression, promoting myotube differentiation, and / or mitochondrial activation has not been evaluated so far. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] Japanese Patent Application Publication No. 2017-043566 [Patent Document 2] Japanese Patent Application Publication No. 2018-090504 [Patent Document 3] International Publication No. 2006 / 025247 [Patent Document 4] Japanese Patent Application Publication No. 10-203989 [Patent Document 5] Japanese Patent Application Laid-Open No. 2001-314172 [Non-patent literature]

[0014] [Non-Patent Document 1] Tina Wenz and 4 others, "Increased muscle PGC-1α expression protects from sarcopenia and metabolic disease during aging", PNAS December 1,2009,106 (48) 20405-20410 [Non-patent document 2] Teresa C Leone and 17 others, "PGC-1α Deficiency Causes Multi-System Energy Metabolic Derangements: Muscle Dysfunction, Abnormal Weight Control and Hepatic Steatosis", PLoS Biol 3(4): e101. [Non-patent document 3] Gella, A and 3 others, "Effect of the nucleotides CMP and UMP on exhaustion in exercise rats", J Physiol Biochem, 64(1), 9-18, 2008 Summary of the Invention [Problem to be solved by the invention]

[0015] An object of the present invention is to provide a novel and highly safe method that has the effect of enhancing muscle strength. [Means for solving the problem]

[0016] As a result of intensive research conducted by the present inventors to achieve the above-mentioned object, they discovered for the first time that pyrimidine nucleotides or precursors thereof have the effect of significantly promoting muscle building, which led to the creation of the present invention. That is, the present invention is a method for promoting the expression of genes involved in myotube cell differentiation, a method for promoting the expression of genes involved in muscle-constituting proteins, or a method for increasing the diameter of myotube cells, which comprises the step of administering a composition containing a muscle-enhancing agent containing a pyrimidine nucleotide or a precursor thereof as an active ingredient, wherein the composition is a food or drink, a supplement, an infant formula, an enteral nutrient, a health food or drink, an additive for animal feed, or a pharmaceutical for animals other than humans, and the pyrimidine nucleotide or precursor thereof is one or more selected from the group consisting of cytidylic acid, uridylic acid, cytidine, and uridine. [Effects of the Invention]

[0017] The method of the present invention is novel and highly safe, and has the effect of enhancing muscle mass. Achieving muscle mass enhancement provides a new means of improving exercise performance and the quality of life (QOL) of patients and elderly people suffering from muscle weakness. [Brief explanation of the drawings]

[0018] [Figure 1] 1 shows the PGC-1α expression promoting effect of cytidylic acid in C2C12 cells in Example 1. In the figure, CMP: disodium cytidylic acid, error bars: standard error, *: p<0.05. [Figure 2] 2 shows the PGC-1α expression promoting effect of cytidylic acid and uridylic acid in C2C12 cells in Example 2. In the figure, CMP: disodium cytidylic acid, UMP: disodium uridylic acid, error bars: standard error, *: p<0.05. [Figure 3] 3 shows the myogenin expression promoting effect of cytidylic acid in C2C12 cells in Example 3. In the figure, CMP: disodium cytidylic acid, error bars: standard error, *: p<0.05. [Figure 4]4 shows the myogenin expression promoting effect of cytidylic acid and uridylic acid in C2C12 cells in Example 4. In the figure, CMP: disodium cytidylic acid, UMP: disodium uridylic acid, error bars: standard error, *: p<0.05. [Figure 5] 5 shows the myogenin expression promoting effect of cytidine in C2C12 cells in Example 4. In the figure, error bars indicate standard error, and * indicates p<0.05. [Figure 6] 6 shows the Myh7 (slow-twitch myosin heavy chain) expression promoting effect of cytidylic acid in C2C12 cells in Example 5. In the figure, CMP: disodium cytidylic acid, error bars: standard error, *: p<0.05. [Figure 7] 7 shows the Myh7 (slow-twitch myosin heavy chain) expression promoting effect of cytidylic acid and uridylic acid in C2C12 cells in Example 6. In the figure, CMP: disodium cytidylic acid, UMP: disodium uridylic acid, error bars: standard error, *: p<0.05. [Figure 8] 8 shows the Myh7 (slow-twitch myosin heavy chain) expression promoting effect of cytidine in C2C12 cells in Example 6. In the figure, error bars indicate standard error, and * indicates p<0.05. [Figure 9] 9 shows the effect of cytidylic acid on increasing the copy number of mitochondrial DNA in C2C12 cells in Example 7. In the figure, CMP: disodium cytidylic acid, error bars: standard error, *: p<0.05. [Figure 10] 10 shows the effect of cytidylic acid and cytidylic acid on increasing mitochondrial DNA numbers in C2C12 cells in Example 8. In the figure, CMP: disodium cytidylate, UMP: disodium uridylate, error bars: standard error, *: p<0.05. [Figure 11] 11 is a photograph at 100x magnification showing the effect of cytidylic acid on increasing the diameter of myotube cells in C2C12 cells in Example 9. In the figure, CMP means disodium cytidylic acid. [Figure 12]12 shows the effect of cytidylic acid on increasing the diameter of myotubes in C2C12 cells in Example 9. In the figure, CMP: disodium cytidylic acid, error bars: standard error, *: p<0.05. [Figure 13] 13 shows the myotube diameter-increasing effect of C2C12 cells by cytidylic acid, uridylic acid, cytidine, and uridine in Example 10. In the figure, CMP means disodium cytidylic acid, and UMP means disodium uridylic acid. [Figure 14] 14 shows the effect of cytidylic acid on increasing the diameter of myotubes in C2C12 cells in Example 10. In the figure, CMP: disodium cytidylic acid, error bars: standard error, *: p<0.05. [Figure 15] 15 shows the effect of uridylic acid on increasing the diameter of myotubes in C2C12 cells in Example 10. In the figure, UMP: disodium uridylic acid, error bars: standard error, *: p<0.05. [Figure 16] 16 shows the effect of cytidine on increasing the diameter of myotubes in C2C12 cells in Example 10. In the figure, error bars indicate standard error, and * indicates p<0.05. [Figure 17] 17 shows the effect of uridine in increasing the diameter of myotubes in C2C12 cells in Example 10. In the figure, error bars indicate standard error, and * indicates p<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention relates to a PGC-1α expression promoter, muscle-building agent, or mitochondrial activator containing a pyrimidine nucleotide or its precursor as an active ingredient. Hereinafter, when simply referred to as "agent," it refers collectively to the PGC-1α expression promoter, muscle-building agent, and mitochondrial activator. Furthermore, in the agent of the present invention, the PGC-1α expression promoting effect, muscle-building effect, and mitochondrial activating effect may be correlated with each other and occur simultaneously.

[0020] In the present invention, promotion of PGC-1α expression means that when the PGC-1α expression promoter of the present invention is administered, the measured amount of mRNA increases statistically significantly compared to the control.

[0021] Since PGC-1α is known to be associated with various diseases or symptoms such as muscle atrophy, diabetes, dyslipidemia, obesity, decreased brain function, neurodegenerative diseases, and aging, the PGC-1α expression promoter of the present invention may contribute to the alleviation, prevention, prophylaxis, etc. of these diseases or symptoms.

[0022] In the present invention, muscle building refers to the fact that when the muscle-building agent of the present invention is administered, at least one of the following effects (1) to (3) occurs, resulting in thicker muscle fibers and muscle building. (1) The expression of genes involved in myotube differentiation, such as myogenin, is promoted, promoting myotube differentiation. (2) The expression of genes related to muscle-forming proteins, such as Myh7, is promoted. (3) The diameter of myotubes increases significantly.

[0023] In the present invention, mitochondrial activation refers to an event such as an increase in mitochondrial DNA copy number, and when evaluating this, measurement by a known method such as real-time PCR can be used as an index of the degree of activation.

[0024] As used herein, pyrimidine nucleotides refer to cytidylic acid and uridylic acid.

[0025] Cytidylic acid (cytidine 5'-phosphate, CMP) is a compound represented by CAS Registry Number 63-37-6. When cytidylic acid is mentioned in this specification, salts of cytidylic acid are also included.

[0026] In this specification, when the mass of cytidylic acid is mentioned, it refers to the mass converted into cytidylic acid disodium salt (CMP, 2Na). When the concentration (%) of cytidylic acid is mentioned, unless otherwise specified, it is assumed to be a mass-volume percent concentration (w / v%), and the mass of cytidylic acid is the mass converted into CMP, 2Na. When a salt other than the disodium salt is selected, or in the case of a free acid that does not form a salt, the mass is taken as the mass converted into CMP, 2Na based on the amount of substance of cytidylic acid.

[0027] Uridylic acid (uridine 5'-phosphate, UMP) is a compound designated by CAS Registry Number 58-97-9. When referring to uridylic acid in this specification, this concept also encompasses salts of uridylic acid.

[0028] In this specification, when the mass of uridylic acid is mentioned, it refers to the mass converted into uridylic acid disodium salt (UMP, 2Na). When the concentration (%) of uridylic acid is mentioned, unless otherwise specified, it is assumed to be a mass-volume percent concentration (w / v%), and the mass of uridylic acid is the mass converted into UMP, 2Na. When a salt other than the disodium salt is selected, or in the case of a free acid that does not form a salt, the mass is taken as the mass converted into UMP, 2Na based on the amount of substance of uridylic acid.

[0029] As used herein, "pyrimidine nucleotide precursor" refers to a compound that can be metabolized to a pyrimidine nucleotide, i.e., cytidylic acid and / or uridylic acid. Whether a compound is included in the pyrimidine nucleotide precursor category is determined by whether or not it is known that the compound can be converted into a pyrimidine nucleotide. Specifically, examples of pyrimidine nucleotide precursors in the present specification include cytidine diphosphate, cytidine triphosphate, uridine diphosphate, and uridine triphosphate, which are known to be decomposed into cytidylic acid and / or uridylic acid by the action of ectonucleotidases and the like (Isao Matsuoka, "Ectonucleotidase in the Nervous System," Clinical Chemistry 33:11-18, 2004), and cytidine, cytosine, uridine, and uracil, which are known to be phosphorylated into cytidylic acid and / or uridylic acid by the action of kinases (A. Orengo, "Regulation of enzymic activity by metabolites. I. Uridine-cytidine kinase of Novikoff ascites rat tumor," J. Biol. Chem. 1969 Apr 25;244(8):2204-9).

[0030] As described above, examples of pyrimidine nucleotides or precursors thereof in the present invention include cytidine, cytosine, cytidylic acid, cytidine diphosphate, cytidine triphosphate, uridine, uracil, uridylic acid, uridylic diphosphate, and uridylic triphosphate. Of these, cytidylic acid, uridylic acid, cytidine, and uridine are preferred.

[0031] As mentioned above, the concept of cytidylic acid in the present invention encompasses salts. Examples of cytidylic acid salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt, and barium salt; basic amino acid salts such as arginine and lysine; ammonium salts such as ammonium salt and tricyclohexylammonium salt; and various alkanolamine salts such as monoethanolamine salt, diethanolamine salt, triethanolamine salt, monoisopropanolamine salt, diisopropanolamine salt, and triisopropanolamine salt. Alkali metal salts such as sodium salt are preferred. Specific examples of such alkali metal salts include monosodium cytidylic acid and disodium cytidylic acid, with disodium cytidylic acid being preferred from the standpoint of ease of handling.

[0032] As mentioned above, the concept of uridylic acid in the present invention encompasses salts. Examples of salts of uridylic acid include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt, magnesium salt, and barium salt; basic amino acid salts such as arginine and lysine; ammonium salts such as ammonium salt and tricyclohexylammonium salt; and various alkanolamine salts such as monoethanolamine salt, diethanolamine salt, triethanolamine salt, monoisopropanolamine salt, diisopropanolamine salt, and triisopropanolamine salt. Alkali metal salts such as sodium salt are preferred. Specific examples of such alkali metal salts include monosodium uridylate and disodium uridylate, with disodium uridylate being preferred from the standpoint of ease of handling.

[0033] The active ingredients may be used alone or in combination of two or more.

[0034] There are no particular limitations on the origin of the active ingredient, and those derived from natural products such as yeast, bacteria, seafood, animals, and plants are suitable.

[0035] The agent of the present invention can be put to practical use as a composition for food and beverages, supplements, infant formula, enteral nutrients, health foods and beverages (including foods for specified health uses and foods with functional claims), additives for animal feed, pharmaceuticals for humans or non-human animals, etc.

[0036] When the agent of the present invention is provided as a food or drink, a health food or drink, or an infant formula, the active ingredient can be appropriately added to a known food or drink to produce a food or drink having a PGC-1α expression promoting effect, a muscle-building effect, or a mitochondrial activation effect. Examples of the target food or drink include milk and dairy products, seasonings, beverages, confectioneries, bread, noodles, oils and fats, processed meat products, processed seafood products, processed agricultural products, frozen foods, and instant foods.

[0037] Furthermore, the active ingredient can be mixed into food and beverage ingredients to produce new foods and beverages that have the effect of promoting PGC-1α expression, muscle-building effects, or mitochondrial activation effects. The target food and beverage forms can be selected from various forms, such as tablets, granules, capsules, powders, solutions, syrups, milks, and pastes. In producing these foods and beverages, various excipients, seasoning ingredients, and the like that can be used in foods can be appropriately added in addition to the active ingredient of the present invention.

[0038] The food and drink may be provided or sold as a food or drink labeled with health uses such as PGC-1α expression promoting effects, muscle-building effects, or mitochondrial activation effects. "Labeling" includes all acts intended to inform consumers of the uses, and any expression that can recall or infer the uses falls under the "labeling" act of this technology, regardless of the purpose of the labeling, the content of the labeling, the object or medium on which it is displayed, etc.

[0039] The "labeling" is preferably done in a way that allows consumers to directly recognize the intended use. Specifically, this includes acts of transferring, delivering, displaying for the purpose of transferring or delivering, or importing food and beverage products or product packaging that lists the intended use, displaying or distributing advertisements, price lists, or transaction documents that list the intended use, or providing information containing the above-mentioned uses by electromagnetic means (such as the Internet).

[0040] The content of the labeling is preferably one approved by the government, etc. (for example, a labeling approved based on various systems established by the government and made in a manner based on such approval.) It is also preferable that such content of the labeling be attached to promotional materials at the point of sale, such as packaging, containers, catalogs, pamphlets, and POP displays, as well as other documents.

[0041] Furthermore, when the agent of the present invention is put to practical use as a pharmaceutical, supplement, enteral nutrient, etc., the active ingredient can be formulated alone or in combination with formulation adjuvants, etc. The method of administration of the formulation may be oral or parenteral, but oral or enteral administration is preferred.

[0042] The formulation may be in the form of tablets, granules, capsules, granules, powders, solutions, syrups, emulsions, etc. for oral administration, and in the form of injections, sprays, ointments, patches, etc. for parenteral administration.

[0043] In the formulation, in addition to the active ingredient of the present invention, any formulation auxiliary such as an excipient, a binder, a disintegrant, a lubricant, a flavoring agent, a solubilizing agent, a suspending agent, a coating agent, etc. may be used in appropriate combination according to the respective delivery form.

[0044] The amount of the active ingredient in the agent of the present invention may be appropriately selected from the range of 0.1 to 30% (W / W) depending on the purpose of use (prevention, health care, symptom relief, etc.), the age of the subject, the method of administration or ingestion, the dosage form, etc.

[0045] The amount of administration or intake of the agent of the present invention may vary depending on the subject's age, body weight, severity of symptoms, administration or intake method, etc., but may be appropriately selected from the range of approximately 1 mg to 800 g per day. [Example]

[0046] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention should not be construed as being limited by these examples.

[0047] Example 1: Effect of promoting expression of PGC-1α gene (I) Mouse myoblast cell line C2C12 cells (RIKEN BRC, RCB0987) were suspended in growth medium (Dulbecco's modified Eagle's medium containing 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin) and seeded into 24-well plates. They were cultured at 37°C in a 5% CO2 incubator until cell density reached 70-90%. The growth medium was removed and replaced with differentiation-inducing medium (Dulbecco's modified Eagle's medium containing 2% adult bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin) containing 1 mM disodium cytidylate or 5 mM disodium cytidylate, or differentiation-inducing medium without disodium cytidylate. After 6 days of culture, with medium changes every 2 days, the medium was removed, and total RNA was extracted from the cells using the RNeasy Mini kit (QIAGEN). Unless otherwise specified, including in the following examples, the study was carried out using six samples for each test.

[0048] Using this total RNA as a template, a reverse transcription reaction mixture was prepared using the ReverTra Ace® qPCR RT Kit (Toyobo). The mRNA expression levels of PGC-1α and β-actin as an internal control were measured using the reverse transcription reaction mixture, GoTaq® qPCR Master Mix (Promega), and the Thermal Cycler Dice Real Time System (Takara Bio). Analysis was performed by relative quantification, with β-actin mRNA expression normalized against the endogenous control. Statistical analysis was performed using Dunnett's multiple comparison test. The threshold for statistical significance was set at 5%. The results are shown in Figure 1.

[0049] As shown in Figure 1, the mRNA expression level of PGC-1α was significantly increased by cytidylic acid in a concentration-dependent manner. These results demonstrate that cytidylic acid has an excellent effect of promoting the expression of PGC-1α.

[0050] (Example 2) Effect of promoting expression of PGC-1α gene (II) Mouse myoblast cell line C2C12 cells (RIKEN BRC, RCB0987) were suspended in growth medium (Dulbecco's modified Eagle's medium containing 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin) and seeded into 24-well plates. Cultures were grown in a 37°C, 5% CO2 incubator until cell density reached 70-90%. The growth medium was then removed and replaced with differentiation-inducing medium containing the test substance (Dulbecco's modified Eagle's medium containing 2% adult bovine serum or 2% horse serum, and the antibiotics penicillin 100 units / ml and streptomycin 100 μg / ml) or without the test substance. After culturing for 4-6 days with medium changes every two days, the medium was removed and total RNA was extracted from the cells using an RNeasy Mini kit (QIAGEN) or NucleoSpin RNA (Takara Bio).

[0051] Using this total RNA as a template, a reverse transcription reaction mixture was prepared using the ReverTra Ace® qPCR RT Kit (Toyobo). The mRNA expression levels of PGC-1α and β-actin as an internal control were measured using the reverse transcription reaction mixture, GoTaq® qPCR Master Mix (Promega), and the Thermal Cycler Dice Real Time System (Takara Bio). Analysis was performed by relative quantification, with β-actin mRNA expression normalized as an endogenous control. Statistical analysis was performed using a t-test or Dunnett's multiple comparison test with a group treated with differentiation-inducing medium containing no test substance as a control. The threshold for statistical significance was set at 5%. The results are shown in Figure 2.

[0052] As shown in Figure 2, the expression level of PGC-1α mRNA was significantly and concentration-dependently increased by cytidylic acid and uridylic acid treatment. These results demonstrate that cytidylic acid and uridylic acid have an excellent effect of promoting PGC-1α expression.

[0053] (Example 3) Myogenin gene expression promoting effect (I) The expression level of myogenin mRNA, a marker gene for myotube differentiation, was evaluated by the method described below. Mouse myoblast cell line C2C12 cells were suspended in growth medium, seeded into 24-well plates, and cultured in an incubator at 37°C with 5% CO2 until cell density reached 70-90%. The growth medium was removed and replaced with a differentiation-inducing medium containing 0.1 mM disodium cytidylate, 1 mM disodium cytidylate, or no cytidylic acid. The cells were cultured for 6 days, with the medium replaced every two days. Total RNA extraction and reverse transcription were performed as in Example 1. The mRNA expression levels of myogenin and β-actin as an internal standard were measured by real-time PCR analysis. Statistical analysis was performed as in Example 1. The results are shown in Figure 3.

[0054] As shown in Figure 3, the expression level of myogenin mRNA was significantly increased by cytidylic acid in a concentration-dependent manner. These results demonstrate that cytidylic acid has an excellent effect of promoting myotube differentiation.

[0055] (Example 4) Myogenin gene expression promoting effect (II) The expression level of myogenin mRNA, a marker gene for myotube differentiation, was evaluated by the method described below. Cell culture, test substance treatment, total RNA extraction, and reverse transcription were performed in the same manner as in Example 2. The mRNA expression levels of myogenin and β-actin as an internal standard were measured by real-time PCR analysis. Statistical analysis was also performed in the same manner as in Example 1. The results are shown in Figures 4 and 5.

[0056] As shown in Figure 4, the expression level of myogenin mRNA was significantly increased by treatment with cytidylic acid and uridylic acid. As shown in Figure 5, the expression level of myogenin mRNA was significantly increased by treatment with cytidine. These results demonstrate that cytidylic acid, uridylic acid, and cytidine have an excellent effect of promoting myotube differentiation.

[0057] (Example 5) Effect of promoting Myh7 gene expression (I) The expression level of Myh7 mRNA, which encodes a slow-muscle myosin protein, was evaluated by the method described below. Mouse myoblast cell line C2C12 cells were suspended in growth medium, seeded into 24-well plates, and cultured in an incubator at 37°C with 5% CO2 until cell density reached 70-90%. The growth medium was removed, and the medium was replaced with differentiation-inducing medium containing 5 mM disodium cytidylate or differentiation-inducing medium without cytidylic acid. After culturing for 4 days with medium changes every two days, the medium was removed, and total RNA was extracted and reverse-transcription was performed as in Example 1. The mRNA expression levels of Myh7 and β-actin as an internal control were measured by real-time PCR analysis. Statistical analysis was performed using Welch's t-test. The threshold for statistical significance was set at 5%. The results are shown in Figure 6.

[0058] As a result, the expression level of Myh7 mRNA was significantly increased by cytidylic acid, as shown in Figure 6. These results demonstrate that cytidylic acid has an excellent effect of promoting the expression of slow-twitch myosin protein.

[0059] (Example 6) Effect of promoting Myh7 gene expression (II) The expression level of Myh7 mRNA, which encodes a slow-muscle myosin protein, was evaluated by the method described below. Cell culture, test substance treatment, total RNA extraction, and reverse transcription were performed in the same manner as in Example 2. The mRNA expression levels of Myh7 and β-actin as an internal standard were measured by real-time PCR analysis. Statistical analysis was also performed in the same manner as in Example 1. The results are shown in Figures 7 and 8.

[0060] As shown in Figure 7, Myh7 mRNA expression was significantly increased by cytidylic acid treatment and uridylic acid treatment. As shown in Figure 8, Myh7 mRNA expression was significantly increased by cytidine treatment. These results demonstrate that cytidylic acid, uridylic acid, and cytidine have an excellent effect on promoting the expression of slow-twitch myosin proteins.

[0061] (Example 7) Effect of increasing mitochondrial DNA copy number (I) Cell culture and addition of the test substance were carried out in the same manner as in Example 1. After culturing for 6 days while changing the differentiation-inducing medium containing the test substance every other day, the medium was removed and total DNA was extracted from the cells using a DNeasy Blood & Tissue kit (QIAGEN). Using the extracted DNA, GoTaq® qPCR Master Mix (Promega), and the real-time PCR device Thermal Cycler Dice Real Time System (Takara Bio), COX2 (Cytochrome c oxidase subunit 2), a gene encoded by mitochondrial DNA, and PPIA (Cyclophilin A), a gene encoded by mouse DNA as an internal standard, were measured. The abundance of COX2 relative to the abundance of PPIA was evaluated as mitochondrial DNA copy number. Statistical analysis was performed in the same manner as in Example 1. In this example, the study was performed with n=3. The results are shown in Figure 9.

[0062] As shown in Figure 9, mitochondrial DNA copy number was significantly increased by cytidylic acid in a concentration-dependent manner. These results demonstrate that cytidylic acid has an extremely effective effect on activating mitochondria.

[0063] (Example 8) Effect of increasing mitochondrial DNA copy number (II) Cell culture and test substance addition were carried out in the same manner as in Example 2. After culturing for 6 days, the medium was removed and total DNA was extracted from the cells using a DNeasy Blood & Tissue kit (QIAGEN). Using the extracted DNA, GoTaq® qPCR Master Mix (Promega), and the real-time PCR device Thermal Cycler Dice Real Time System (Takara Bio), COX2 (Cytochrome c oxidase subunit 2), a gene encoded by mitochondrial DNA, and PPIA (Cyclophilin A), a gene encoded by mouse DNA as an internal standard, were measured. The abundance of COX2 relative to the abundance of PPIA was evaluated as mitochondrial DNA copy number. Statistical analysis was performed in the same manner as in Example 1. In this example, the study was performed with n=3. The results are shown in Figure 10.

[0064] As shown in Figure 10, mitochondrial DNA copy number was significantly increased in a concentration-dependent manner by treatment with cytidylic acid and uridylic acid. These results demonstrate that cytidylic acid and uridylic acid have an excellent effect on mitochondrial activation.

[0065] (Example 9) Increasing effect on myotube cell diameter (I) Cell culture and test substance addition were performed in the same manner as in Example 1. After 6 days of culture, with the differentiation-inducing medium containing the test substance being replaced every two days, five locations near the center of the well were photographed at 100x magnification. Using the image analysis software ImageJ, the diameters of the 10 largest myotube cells per photograph were measured, and the average of the 50 was used as the myotube cell diameter for each well. Statistical analysis was performed in the same manner as in Example 1. Representative photographs of each group are shown in Figure 11, and the results of measuring the average myotube cell diameter are shown in Table 1 and Figure 12.

[0066] [Table 1]

[0067] 11 and 12, myotube diameter was significantly increased by cytidylic acid in a concentration-dependent manner. These results demonstrate that cytidylic acid has an excellent effect of increasing myotube diameter.

[0068] (Example 10) Increasing effect on myotube cell diameter (II) Cell culture and test substance addition were carried out in the same manner as in Example 2. After culturing for 5-6 days while changing the differentiation-inducing medium containing the test substance every other day, five points near the center of the well were photographed at 100x magnification. Representative photographs of each group are shown in Figure 13. In addition, using the image analysis software ImageJ, the diameters of the 10 largest myotubes per photograph were measured, and the average of the 50 was used as the myotube diameter for each well. Statistical analysis was performed in the same manner as in Example 2. The results are shown in Figures 14 to 17.

[0069] As shown in FIG. 13, myotube cell diameter was significantly increased by treatment with cytidylic acid, uridylic acid, cytidine, and uridine. As shown in Figure 14, cytidylic acid treatment significantly increased myotube cell diameter in a concentration-dependent manner. As shown in Figure 15, uridylic acid treatment significantly increased myotube cell diameter. As shown in Figure 16, cytidine treatment significantly increased myotube cell diameter in a concentration-dependent manner. As shown in Figure 17, uridine treatment significantly increased myotube cell diameter in a concentration-dependent manner. These results demonstrate that cytidylic acid, uridylic acid, cytidine, and uridine have excellent effects on increasing myotube cell diameter.

Claims

1. The method comprises administering a composition containing a muscle-building agent containing a pyrimidine nucleotide or a precursor thereof as an active ingredient, the composition is a food or drink, a supplement, an infant formula, an enteral nutrient, a health food or drink, an animal feed additive, or a pharmaceutical product for animals other than humans, A method for promoting expression of a gene involved in myotube cell differentiation, wherein the pyrimidine nucleotide or a precursor thereof is one or more selected from the group consisting of cytidylic acid, uridylic acid, cytidine, and uridine.

2. The method comprises administering a composition containing a muscle-building agent containing a pyrimidine nucleotide or a precursor thereof as an active ingredient, the composition is a food or drink, a supplement, an infant formula, an enteral nutrient, a health food or drink, an animal feed additive, or a pharmaceutical product for animals other than humans, A method for promoting the expression of a gene related to a protein that constitutes muscle, wherein the pyrimidine nucleotide or a precursor thereof is one or more selected from the group consisting of cytidylic acid, uridylic acid, cytidine, and uridine.

3. The method comprises administering a composition containing a muscle-building agent containing a pyrimidine nucleotide or a precursor thereof as an active ingredient, the composition is a food or drink, a supplement, an infant formula, an enteral nutrient, a health food or drink, an animal feed additive, or a pharmaceutical product for animals other than humans, A method for increasing the diameter of myotube cells, wherein the pyrimidine nucleotide or precursor thereof is one or more selected from the group consisting of cytidylic acid, uridylic acid, cytidine, and uridine.

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