PGC-1α expression enhancer and mitochondrial activator

Abstract

The present invention provides a PGC-1α expression promoting agent containing a pyrimidine nucleotide or precursor thereof as an active ingredient. Also provided are a muscle-building agent and a mitochondria activating agent.

Description

Technical Field

[0001] The present invention relates to a PGC-1α expression promoter, a muscle enhancer, and a mitochondrial activator.

Background Art

[0002] Muscles are formed by the differentiation of muscle cells. In the differentiation of muscle cells, myoblasts derived from satellite cells fuse with each other to form a multinucleated fusant called a myotube cell. Thereafter, the myotube cells aggregate and align to form muscle fibers, which become a tissue that produces a large force as muscle.

[0003] It is known that specific groups of transcription factors are specifically expressed and function at each stage of the muscle differentiation process. In satellite cells, Pax7 functions, in myoblasts, Myf5, MyoD function, and further, Myogenin functions during the formation of myotube cells. To promote muscle formation, it is important to increase factors involved in muscle differentiation.

[0004] Muscles are roughly classified into slow muscle fibers (type I fibers) and fast muscle fibers (type II fibers). Further, fast muscle fibers are divided into subtypes such as type IIa fibers, type IIb fibers, and type IIx fibers. Type I fibers have a slow contraction speed but excellent endurance, a large number of mitochondria and antioxidants, and a large number of capillaries adjacent to the muscle fibers. In contrast, type II fibers have a fast contraction speed but poor endurance, a small number of mitochondria and antioxidants, and a small number of capillaries adjacent to the muscle fibers.

[0005] As a factor that plays a central role in controlling muscle fiber type, there is PGC-1α (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha). 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 tissues such as skeletal muscle, heart, kidney, and brain, and is known to control mitochondrial biosynthesis and energy production.

[0006] Based on these functions of PGC-1α, it is known that PGC-1α overexpressing mice improve age-related symptoms such as decreased muscle mass, motor function, bone density, and mitochondrial function, worsening insulin resistance and systemic inflammatory response, and obesity, thereby extending lifespan (Non-Patent Literature 1).

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

[0008] Since enhanced PGC-1α expression is expected to have effects on the diseases or symptoms described above, prior art related to PGC-1α expression enhancement exists. For example, Patent Document 1 describes that helipilone A promotes PGC-1α production in nerve cells and has the effect of improving various neurotransmission disorders and short-term memory impairments involving PGC-1α. Furthermore, Patent Document 2 describes that combining β-hydroxy-β-methylbutyrate (HMB) with a secondary component enhances the expression of myogenin, a myotubular differentiation marker, and exhibits a significant muscle-building effect. Furthermore, Patent Document 3 describes that pyrroloquinoline quinone exhibits mitochondrial activating effects.

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

[0010] Patent document 4 describes an agent for improving emotional disorders characterized by containing nucleotides. Patent document 4 also discloses an immunostimulatory agent characterized by containing nucleotides. Non-patent document 3 describes that rats administered a mixture of cytidylic acid and uridylic acid were able to withstand prolonged exercise on a treadmill.

[0011] However, the physical fitness improvement described in Non-Patent Document 3 above only involves measuring the ability to sustain a certain state of exercise, such as on a treadmill, and the levels of biochemical parameters related to fatigue, such as glycogen and lactic acid in the liver and muscles. It does not analyze the expression of transcription factors such as PGC-1α that correlate with increased muscle mass. Therefore, Non-Patent Document 3 does not consider muscle-enhancing effects such as promoting myotube differentiation at all.

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

[0013] [Patent Document 1] Japanese Patent Publication No. 2017-043566 [Patent Document 2] Japanese Patent 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 Publication 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 [Overview of the Initiative] [Problems that the invention aims to solve]

[0015] The object of the present invention is to provide a novel and highly safe PGC-1α expression promoter, muscle enhancer, or mitochondrial activator that, when used as food, animal feed, pharmaceuticals, quasi-drugs, etc., has the effect of promoting PGC-1α expression and exhibiting muscle-enhancing effects. [Means for solving the problem]

[0016] The inventors of this application conducted diligent research to achieve the above objectives and, as a result, discovered for the first time that pyrimidine nucleotides or their precursors have clear effects not only on the conventionally known anti-fatigue and physical strength-enhancing effects, but also on promoting PGC-1α expression, promoting myotubular differentiation, and mitochondrial activation, and that they have a remarkable effect on promoting muscle strengthening, leading to the present invention. That is, the invention of the present application is Cytidylic acid or uridylic acid a PGC-1α expression promoter or a mitochondrial activator for preventing and treating diseases or symptoms caused by low expression of PGC-1α, which contains Cytidylic acid or uridylic acid as an active ingredient 。

Advantages of the Invention

[0017] The PGC-1α expression promoter of the present invention promotes the differentiation of myotube cells and achieves muscle strengthening. The achievement of muscle strengthening provides a new means for improving the exercise performance and the quality of life (QOL) of patients and the elderly caused by muscle strength decline. In addition, it is known that muscle atrophy associated with aging is suppressed in PGC-1α overexpressing mice (Non-Patent Document 1), and an effect of suppressing muscle atrophy can also be expected by promoting the expression of PGC-1α.

[0018] In addition, diseases or symptoms caused by low expression of PGC-1α include diabetes, dyslipidemia, obesity, decline in brain function, neurodegenerative diseases, aging, etc. The PGC-1α expression promoter of the present invention can provide a new means for preventing and treating diseases or symptoms caused by low expression of PGC-1α.

Brief Description of the Drawings

[0019] [Figure 1] Figure 1 shows the effect of promoting PGC-1α expression by cytidylic acid in C2C12 cells of Example 1. In the figure, CMP: disodium cytidylic acid, error bar: standard error, *: means p < 0.05. [Figure 2] Figure 2 shows the effect of promoting PGC-1α expression by cytidylic acid and uridylic acid in C2C12 cells of Example 2. In the figure, CMP: disodium cytidylic acid, UMP: disodium uridylic acid, error bar: standard error, *: means p < 0.05. [Figure 3] Figure 3 shows the effect of promoting Myogenin expression by cytidylic acid in C2C12 cells of Example 3. In the figure, CMP: disodium cytidylic acid, error bar: standard error, *: means p < 0.05. [Figure 4] Figure 4 shows the myogenin expression-promoting effects of cytidylic acid and uridylic acid in C2C12 cells of Example 4. In the figure, CMP: disodium cytidylic acid, UMP: disodium uridylic acid, error bars: standard error, *: p<0.05. [Figure 5] Figure 5 shows the effect of cytidine on promoting myogenin expression in C2C12 cells of Example 4. In the figure, error bars represent the standard error, and * indicates p<0.05. [Figure 6] Figure 6 shows the effect of cytidylic acid on promoting Myh7 (slow-twitch myosin heavy chain) expression in C2C12 cells of Example 5. In the figure, CMP means disodium cytidylate, error bars mean standard error, and * means p<0.05. [Figure 7] Figure 7 shows the effect of cytidylic acid and uridylic acid on promoting Myh7 (slow-twitch myosin heavy chain) expression in C2C12 cells of Example 6. In the figure, CMP: disodium cytidylic acid, UMP: disodium uridylic acid, error bars: standard error, *: p<0.05. [Figure 8] Figure 8 shows the effect of cytidine on promoting Myh7 (slow-twitch myosin heavy chain) expression in C2C12 cells of Example 6. In the figure, error bars represent the standard error, and * indicates p<0.05. [Figure 9] Figure 9 shows the effect of cytidylic acid on increasing the mitochondrial DNA copy number in C2C12 cells of Example 7. In the figure, CMP means disodium cytidylate, error bars mean standard error, and * means p<0.05. [Figure 10] Figure 10 shows the effects of cytidylic acid and cytidylic acid on increasing mitochondrial DNA number in C2C12 cells of Example 8. In the figure, CMP: disodium cytidylate, UMP: disodium uridylate, error bars: standard error, *: p<0.05. [Figure 11] Figure 11 shows a 100x magnification photograph illustrating the effect of cytidylic acid on increasing the myotubular diameter of C2C12 cells in Example 9. In the figure, CMP refers to disodium cytidylate. [Figure 12]Figure 12 shows the effect of cytidylic acid on increasing the myotubular diameter of C2C12 cells in Example 9. In the figure, CMP means disodium cytidylic acid, error bars mean standard error, and * means p<0.05. [Figure 13] Figure 13 shows the effect of cytidylic acid, uridylic acid, cytidine, and uridine on increasing the myotubular diameter of C2C12 cells in Example 10. In the figure, CMP refers to disodium cytidylic acid, and UMP refers to disodium uridylic acid. [Figure 14] Figure 14 shows the effect of cytidylic acid on increasing the myotubular diameter of C2C12 cells in Example 10. In the figure, CMP means disodium cytidylic acid, error bars mean standard error, and * means p<0.05. [Figure 15] Figure 15 shows the effect of uridylic acid on increasing the myotubular diameter of C2C12 cells in Example 10. In the figure, UMP: disodium uridylate, error bars: standard error, *: p<0.05. [Figure 16] Figure 16 shows the effect of cytidine on increasing the myotubular diameter of C2C12 cells in Example 10. In the figure, error bars represent the standard error, and * indicates p<0.05. [Figure 17] Figure 17 shows the effect of uridine on increasing the myotubular diameter of C2C12 cells in Example 10. In the figure, error bars represent the standard error, and * indicates p<0.05. [Modes for carrying out the invention]

[0020] The present invention relates to a PGC-1α expression promoter, muscle-enhancing agent, or mitochondrial activator containing a pyrimidine nucleotide or its precursor as an active ingredient. Hereinafter, when simply referred to as "agent," it is a general term for the PGC-1α expression promoter, muscle-enhancing agent, and mitochondrial activator. Furthermore, in the agents of the present invention, the PGC-1α expression promoting effect, muscle-enhancing effect, and mitochondrial activating effect may occur simultaneously and in correlation with each other.

[0021] In the present invention, PGC-1α expression enhancement means that when the PGC-1α expression enhancer of the present invention is administered, the amount of mRNA measured increases statistically significantly compared to the control.

[0022] 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 can contribute to the alleviation, prevention, and protection of these diseases or symptoms.

[0023] In the present invention, muscle enhancement means that when the muscle-enhancing agent of the present invention is administered, at least one of the following effects (1) to (3) occurs, causing muscle fibers to thicken and muscles to be strengthened. (1) The expression of genes involved in myotubular cell differentiation, such as myogenin, is promoted, thereby promoting the differentiation of myotubular cells. (2) The expression of genes related to proteins that make up muscle, such as Myh7, is promoted. (3) A significant increase in the diameter of myotubes.

[0024] The mitochondrial activation effect in the present invention refers to an increase in the number of mitochondrial DNA copies. Add In its evaluation, this can be measured using known methods such as real-time PCR, which can then be used as an indicator of the degree of activation.

[0025] In this specification, pyrimidine nucleotides mean cytidylic acid and uridylic acid.

[0026] Cytidylic acid (cytidine monophosphate, 5'-phosphate, CMP) is a compound represented by CAS registry number 63-37-6. In this specification, the term "cytidylic acid" includes salts of cytidylic acid.

[0027] In this specification, when the mass of cytidylic acid is stated, it shall be the mass converted to disodium cytidylic acid salt (CMP,2Na). When the concentration (%) of cytidylic acid is stated, unless otherwise specified, it shall be the mass / volume percentage concentration (w / v%), and the mass of cytidylic acid shall be the mass converted to 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 amount of cytidylic acid shall be used as the basis, and the mass shall be converted to CMP,2Na.

[0028] Uridylic acid (uridine monophosphate, UMP) is a compound represented by CAS registry number 58-97-9. In this specification, the term "uridylic acid" includes salts of uridylic acid.

[0029] In this specification, when the mass of uridylic acid is stated, it shall be the mass converted to disodium uridylate (UMP,2Na). When the concentration (%) of uridylic acid is stated, unless otherwise specified, it shall be the mass-volume percentage concentration (w / v%), and the mass of uridylic acid shall be the UMP,2Na converted mass. 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 amount of uridylic acid shall be used as the basis, and the mass shall be converted to UMP,2Na.

[0030] In this specification, a pyrimidine nucleotide precursor means a compound that can be metabolized to pyrimidine nucleotides, i.e., cytidylic acid and / or uridylic acid. Whether a compound is included as a pyrimidine nucleotide precursor is determined by whether or not it is known to be converted to a pyrimidine nucleotide. Specifically, cytidine diphosphate, cytidine triphosphate, uridine diphosphate, and uridine triphosphate (Isao Matsuoka, "Ectonucleotidase in the Nervous System", Clinical Chemistry 33:11-18, 2004), which are known to be broken down to cytidylic acid and / or uridylic acid by the action of ectonucleotidases, etc., and cytidine, cytosine, uridine, and uracil (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.), which are known to be phosphorylated to cytidylic acid and / or uridylic acid by the action of kinases, are exemplified as pyrimidine nucleotide precursors in this specification.

[0031] Examples of pyrimidine nucleotides or their precursors in the present invention include, as mentioned above, cytidine, cytosine, cytidylic acid, cytidine diphosphate, cytidine triphosphate, uridine, uracil, uridylic acid, uridylic acid, and uridylic triphosphate. Among these, cytidylic acid, uridylic acid, cytidine, and uridine are preferred.

[0032] As mentioned above, the concept of cytidylic acid in the present invention includes salts. Examples of salts of cytidylic 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. Preferably, it is an alkali metal salt such as sodium salt. Specifically, examples of such alkali metal salts include monosodium cytidylate and disodium cytidylate, and disodium cytidylate is preferred from the standpoint of ease of handling.

[0033] As mentioned above, the concept of uridylic acid in the present invention includes salts. Examples of uridylic 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. Preferably, it is an alkali metal salt such as sodium salt. Specifically, examples of such alkali metal salts include monosodium uridylate and disodium uridylate, and disodium uridylate is preferred from the standpoint of ease of handling.

[0034] The aforementioned active ingredients may be used alone or in combination of two or more.

[0035] There are no particular restrictions on the origin of the active ingredients, but those derived from natural products such as yeast, bacteria, fish and shellfish, animals, and plants are preferred.

[0036] The agent of the present invention can be put into practical use as a composition of food and beverages, supplements, powdered milk, enteral nutrition formulas, health foods (including foods for specified health uses and foods with functional claims), additives for animal feed, pharmaceuticals for humans or non-human animals, etc.

[0037] When the agent of the present invention is used as food or beverage, health food or powdered milk, etc., by appropriately adding the active ingredient to known food or beverages, it is possible to create food or beverages that have PGC-1α expression promoting effects, muscle strengthening effects, or mitochondrial activation effects. Examples of target food and beverages include milk and dairy products, seasonings, beverages, confectionery, bread, noodles, oils and fats, processed meat products, processed seafood products, processed agricultural products, frozen foods, instant foods, etc.

[0038] Furthermore, the active ingredient can be mixed into food and beverage ingredients to produce new foods and beverages that have PGC-1α expression promoting effects, muscle strengthening effects, or mitochondrial activation effects. The target foods and beverages can be in various forms, such as tablets, granules, capsules, powders, solutions, syrups, milky substances, or pastes. In the production of these foods and beverages, in addition to the active ingredient of the present invention, various excipients and flavorings that can be used as food can be added as appropriate.

[0039] The aforementioned food and beverages may be provided and sold as food and beverages with health claims such as PGC-1α expression promoting effect, muscle strengthening effect, or mitochondrial activation effect. The act of "labeling" includes all acts to inform consumers of the aforementioned uses, and any expression that can evoke or infer the aforementioned uses, regardless of the purpose of the labeling, the content of the labeling, or the object or medium on which it is displayed, falls under the act of "labeling" of this technology.

[0040] The aforementioned "display" is preferably made in a manner that allows consumers to directly recognize the above-mentioned use. Specifically, this includes acts such as transferring, delivering, displaying for transfer or delivery, or importing food and beverage products or product packaging on which the above-mentioned use is described; displaying or distributing advertisements, price lists, or transaction documents related to products that describe the above-mentioned use; or providing information containing such information by electromagnetic means (such as the Internet).

[0041] The content of the display should preferably be a display approved by the government or other administrative body (for example, a display approved under various systems established by the government and made in accordance with such approval). Furthermore, it is preferable to attach such display content to packaging, containers, catalogs, brochures, point-of-sale (POP) materials and other documents used at sales sites.

[0042] Furthermore, when the agent of the present invention is put into practical use as a pharmaceutical, supplement, enteral nutritional supplement, etc., the active ingredient can be formulated alone or in combination with formulation aids, etc. The method of administration of the formulation may be orally or parenterally, but oral or enteral administration is preferred.

[0043] The form of the aforementioned preparation can be, if administered orally, tablets, granules, capsules, powders, solutions, syrups, emulsions, etc., or if administered parenterally, injections, sprays, ointments, patches, etc.

[0044] In the aforementioned formulation, in addition to the active ingredient of the present invention, any formulation aids such as excipients, binders, disintegrants, lubricants, flavoring and odor-correcting agents, solubilizers, suspending agents, and coating agents may be used in appropriate combinations according to their respective delivery forms.

[0045] 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, or symptom relief, etc.), the age of the target person, the method of administration or intake, the dosage form, etc.

[0046] The dosage or intake of the agent of the present invention may vary depending on the age, weight, severity of symptoms, and method of administration or intake of the subject, but it can be appropriately selected from a range of approximately 1 mg to 800 g per day. [Examples]

[0047] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention should not be interpreted as being limited by these examples.

[0048] (Example 1) Effect of promoting the expression of the 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), seeded in a 24-well plate, and cultured in an incubator at 37°C and 5% CO2 until the cell density reached 70-90%. The growth medium was removed and the cells were replaced with differentiation induction medium containing 1 mM disodium cytidylate or 5 mM disodium cytidylate (Dulbecco's modified Eagle's Medium containing 2% adult bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin), or differentiation induction medium without disodium cytidylate. After culturing for 6 days with the medium changed every two days, the medium was removed and total RNA was extracted from the cells using the RNeasy Mini kit (QIAGEN). In addition, unless otherwise specified, the following examples were also conducted with n=6 samples.

[0049] Using this total RNA as a template, a reverse transcription reaction mixture was prepared using the ReverTra Ace(R) qPCR RT Kit (Toyobo). The mRNA expression levels of PGC-1α and β-actin (as an internal standard) were measured using the reverse transcription reaction mixture, GoTaq(R) qPCR Master Mix (Promega), and a real-time PCR instrument, the Thermal Cycler Dice Real Time System (Takara Bio). Analysis was performed by relative quantification, and the mRNA expression level was corrected using β-actin mRNA expression as an endogenous control. Dunnett's multiple comparison test was performed for statistical analysis. The threshold for statistical significance was set at 5%. The results are shown in Figure 1.

[0050] As shown in Figure 1, PGC-1α mRNA expression levels were significantly and in a concentration-dependent manner by cytidylic acid. These results indicate that cytidylic acid has a very excellent effect in promoting PGC-1α expression.

[0051] (Example 2) Effect of promoting PGC-1α gene expression (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), seeded in a 24-well plate, and cultured in an incubator at 37°C and 5% CO2 until the cell density reached 70-90%. The growth medium was removed, and the culture medium was replaced with differentiation induction 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 differentiation induction medium without the test substance. After culturing the cells for 4-6 days, changing the culture medium every two days, the medium was removed and total RNA was extracted from the cells using either the RNeasy Mini kit (QIAGEN) or NucleoSpin RNA (Takara Bio).

[0052] Using this total RNA as a template, a reverse transcription reaction solution was prepared using the ReverTra Ace(R) qPCR RT Kit (Toyobo). The mRNA expression levels of PGC-1α and β-actin (as an internal standard) were measured using the reverse transcription reaction solution, GoTaq(R) qPCR Master Mix (Promega), and a real-time PCR instrument, the Thermal Cycler Dice Real Time System (Takara Bio). Analysis was performed by relative quantification, and the mRNA expression level was corrected using β-actin mRNA expression as an endogenous control. For statistical analysis, a t-test or Dunnett's multiple comparison test was performed using a differentiation induction medium treatment group without the test substance as a control. The threshold for statistical significance was set at 5%. The results are shown in Figure 2.

[0053] As shown in Figure 2, PGC-1α mRNA expression levels were significantly and in a concentration-dependent manner by cytidylic acid and uridylic acid treatment. These results indicate that cytidylic acid and uridylic acid have excellent PGC-1α expression-promoting effects.

[0054] (Example 3) Effect of promoting Myogenin gene expression (I) The expression level of Myogenin mRNA, a marker gene for myotubular cell differentiation, was evaluated using the method described below. Mouse myoblast cell line C2C12 cells were suspended in growth medium and seeded in a 24-well plate. They were cultured in a 37°C, 5% CO2 incubator until the cell density reached 70-90%. The growth medium was removed and replaced with 0.1 mM disodium cytidylate, 1 mM disodium cytidylate, or a cytidylic acid-free differentiation induction medium. The cells were cultured for 6 days, changing the medium every two days. Total RNA extraction and reverse transcription were performed in the same manner as in Example 1. Myogenin and β-actin mRNA expression levels (as an internal standard) were measured by real-time PCR analysis. Statistical analysis was performed in the same manner as in Example 1. The results are shown in Figure 3.

[0055] As shown in Figure 3, myogenin mRNA expression levels were significantly and in a concentration-dependent manner by cytidylic acid. These results indicate that cytidylic acid has a very excellent effect in promoting myotubular differentiation.

[0056] (Example 4) Effect of promoting Myogenin gene expression (II) The expression level of Myogenin mRNA, a marker gene for myotubular cell differentiation, was evaluated using the method described below. Cell culture, test substance treatment, total RNA extraction, and reverse transcription were carried out 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. These results are shown in Figures 4 and 5.

[0057] As shown in Figure 4, myogenin mRNA expression levels were significantly increased by cytidylic acid and uridylic acid treatment. As shown in Figure 5, myogenin mRNA expression levels were significantly increased by cytidine treatment. From these results, it was found that cytidylic acid, uridylic acid, and cytidine have excellent effects in promoting myotubular differentiation.

[0058] (Example 5) Effect of promoting Myh7 gene expression (I) The expression level of Myh7 mRNA encoding slow-twitch muscle myosin protein was evaluated using the method described below. Mouse myoblast cell line C2C12 cells were suspended in growth medium, seeded in a 24-well plate, and cultured in a 37°C, 5% CO2 incubator until the cell density reached 70-90%. The growth medium was removed and replaced with differentiation induction medium containing 5 mM disodium cytidylate or differentiation induction medium without cytidylate. After culturing for 4 days, changing the medium every two days, the medium was removed and total RNA extraction and reverse transcription were performed in the same manner as in Example 1. The mRNA expression levels of Myh7 and β-actin as an internal standard were measured by real-time PCR analysis. Welch's t-test was performed for statistical analysis. The threshold for statistical significance was set at 5%. The results are shown in Figure 6.

[0059] As a result, as shown in Figure 6, the mRNA expression level of Myh7 was significantly enhanced by cytidylic acid. These results indicate that cytidylic acid has a very excellent effect in promoting the expression of slow-twitch muscle myosin proteins.

[0060] (Example 6) Effect of promoting Myh7 gene expression (II) The expression level of Myh7 mRNA encoding slow-twitch muscle myosin protein was evaluated using the method described below. Cell culture, test substance treatment, total RNA extraction, and reverse transcription were carried out 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. These results are shown in Figures 7 and 8.

[0061] As shown in Figure 7, Myh7 mRNA expression levels were significantly increased by cytidylic acid and uridylic acid treatment. As shown in Figure 8, Myh7 mRNA expression levels were significantly increased by cytidine treatment. From these results, it was found that cytidylic acid, uridylic acid, and cytidine have excellent effects in promoting the expression of slow-twitch muscle myosin proteins.

[0062] (Example 7) Effect of increasing mitochondrial DNA copy number (I) Cell culture and test substance addition were carried out in the same manner as in Example 1. After culturing for 6 days, changing the differentiation induction medium containing the test substance every two days, the medium was removed and total DNA was extracted from the cells using the DNeasy Blood & Tissue kit (QIAGEN). Using the extracted DNA, GoTaq(R) qPCR Master Mix (Promega), and the Thermal Cycler Dice Real Time System (Takara Bio), COX2 (Cytochrome c oxidase subunit 2), a gene encoded in mitochondrial DNA, and an internal standard were used. mouseWe measured PPIA (Cyclophilin A), a gene encoded by DNA. The amount of COX2 relative to the amount of PPIA was evaluated as mitochondrial DNA copy number. Statistical analysis was performed using the same method as in Example 1. This example was investigated with n=3. The results are shown in Figure 9.

[0063] As shown in Figure 9, mitochondrial DNA copy number was significantly and in a concentration-dependent manner by cytidylic acid. These results indicate that cytidylic acid has a very excellent mitochondrial activating effect.

[0064] (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 culture medium was removed and total DNA was extracted from the cells using the DNeasy Blood & Tissue kit (QIAGEN). Using the extracted DNA, GoTaq(R) qPCR Master Mix (Promega), and the Thermal Cycler Dice Real Time System (Takara Bio), COX2 (Cytochrome c oxidase subunit 2), a gene encoded in mitochondrial DNA, and an internal standard were used. mouse We measured PPIA (Cyclophilin A), a gene encoded by DNA. The amount of COX2 relative to the amount of PPIA was evaluated as mitochondrial DNA copy number. Statistical analysis was performed using the same method as in Example 1. This example was investigated with n=3. The results are shown in Figure 10.

[0065] As shown in Figure 10, mitochondrial DNA copy number was significantly and in a concentration-dependent manner increased by cytidylic acid and uridylic acid treatment. These results indicate that cytidylic acid and uridylic acid have excellent mitochondrial activation effects.

[0066] (Example 9) Effect on increasing myotubular cell diameter (I) Cell culture and test substance addition were carried out in the same manner as in Example 1. After culturing for 6 days, changing the differentiation induction medium containing the test substance every two days, photographs were taken at 100x magnification at five locations near the center of each well. Using the image analysis software ImageJ, the diameters of 10 myotubes were measured for each photograph, in descending order of diameter, and the average of 50 measurements was taken as the myotube diameter of 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 the average myotube diameter measurement are shown in Table 1 and Figure 12.

[0067] [Table 1]

[0068] As shown in Figures 11 and 12, myotubular diameter was significantly increased by cytidylic acid in a concentration-dependent manner. These results indicate that cytidylic acid has a very effective effect in increasing myotubular diameter.

[0069] (Example 10) Effect of increasing myotubular 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, changing the differentiation induction medium containing the test substance every two days, photographs were taken at 100x magnification at five locations near the center of the wells. Representative photographs of each group are shown in Figure 13. Furthermore, using the image analysis software ImageJ, the diameters of 10 myotubes were measured for each photograph, in descending order of diameter, and the average of these 50 measurements was taken as the myotube diameter for each well. Statistical analysis was also performed using the same method as in Example 2. The results are shown in Figures 14-17.

[0070] As shown in Figure 13, the diameter of myotubes was significantly increased by treatment with cytidylic acid, uridylic acid, cytidine, and uridine. As shown in Figure 14, cytidylic acid treatment significantly increased myotube diameter in a concentration-dependent manner. As shown in Figure 15, uridylic acid treatment significantly increased myotube diameter. As shown in Figure 16, cytidine treatment significantly increased myotube diameter in a concentration-dependent manner. As shown in Figure 17, uridine treatment significantly increased myotube diameter in a concentration-dependent manner. From these results, it was found that cytidylic acid, uridylic acid, cytidine, and uridine have excellent effects in increasing myotube diameter.

Claims

1. A PGC-1α expression promoter containing cytidylic acid or uridylic acid as an active ingredient, for preventing or treating diseases or symptoms caused by low expression of PGC-1α.

2. A mitochondrial activator containing cytidylic acid or uridylic acid as an active ingredient.

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