Application of glutamic acid and alpha-KG in promoting differentiation and fusion of skeletal muscle myoblasts
By using glutamate and α-KG to promote the differentiation and fusion of skeletal muscle myoblasts, the problem of insufficient myoblast differentiation and fusion in the existing technology is solved, and significant proliferation and development of animal muscles are achieved, which has broad application prospects.
Patent Information
- Application Number
- CN202510831688.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-20
- Publication Date
- 2025-09-23
AI Technical Summary
Existing technologies fail to effectively promote the differentiation and fusion of skeletal muscle myoblasts, affecting the meat production performance of meat animals.
Glutamate and α-ketoglutarate (α-KG) are used as promoters to promote myoblast differentiation and fusion by contacting with myoblasts, increase the number of myotubes, and enhance the expression level of myogenic differentiation factors.
It significantly improves the differentiation and fusion rate of myoblasts, increases the number of myotubes, promotes animal muscle development, and is used in muscle variety improvement and livestock breeding in the livestock industry.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of glutamic acid and α-KG in promoting the differentiation and fusion of skeletal muscle myoblasts. Background Art
[0002] Skeletal muscle, a vital component of the animal body, accounts for approximately 40% of the total body weight. Its growth and development is a key factor in determining the meat production performance of meat animals. Therefore, in-depth research on the regulatory mechanisms of skeletal muscle growth and development is not only an important foundation for improving the meat production performance of mutton sheep, but also a core scientific issue for achieving improved breed breeding and industrial upgrading.
[0003] Gene transcription and cellular metabolism are two fundamental, mutually regulated biological processes. Metabolic selection plays a key role in cell proliferation and differentiation, with metabolites supporting energy needs and regulating various cellular signaling processes. Studies have shown that upregulation or altered expression of sugar metabolism genes during glycolysis and gluconeogenesis is the primary driving force behind enhanced aerobic glycolysis in tumor cells. The mutual regulation between cellular metabolism and gene expression through a feedback mechanism constitutes a unique characteristic of tumor cells. Cellular metabolic products also participate in the differentiation of skeletal muscle satellite cells. Under differentiation conditions, ATP promotes satellite cell differentiation and inhibits proliferation by upregulating myogenin MyoG and the cell cycle regulator p21. Glutamate is a key amino acid in animals and a reactant in alanine metabolism. α-KG is catalyzed by alanine aminotransferase to produce α-KG. α-KG is an intermediate in the tricarboxylic acid (TCA) cycle and a cofactor for multiple dioxygenases, such as TET enzymes and histone demethylases. It participates in DNA and histone demethylation, thereby influencing gene expression. Summary of the Invention
[0004] The present invention aims to provide a method for promoting the differentiation and fusion of skeletal muscle myoblasts by using glutamate and α-KG (α-ketoglutarate). The technical problems to be solved are not limited to the technical subject matter described above, and those skilled in the art will clearly understand other technical subjects not mentioned herein through the following description.
[0005] To achieve the above objectives, the present invention first provides the use of glutamic acid or a physiologically acceptable salt thereof, and / or α-ketoglutaric acid or a physiologically acceptable salt thereof in any of the following:
[0006] A1) Application in promoting myoblast differentiation;
[0007] A2) Application in promoting myoblast fusion;
[0008] A3) Application in increasing the number of myotubes of myoblasts;
[0009] A4) Application in increasing the expression level of myogenic differentiation factors;
[0010] A5) Use in the preparation of a product for promoting myoblast differentiation and / or fusion;
[0011] A6) Use in promoting animal muscle development or preparing a product for promoting animal muscle development.
[0012] The present invention also provides the use of the composition in any of the following:
[0013] B1) Application in promoting myoblast differentiation;
[0014] B2) Application in promoting myoblast fusion;
[0015] B3) Application in increasing the number of myotubes of myoblasts;
[0016] B4) Application in increasing the expression level of myogenic differentiation factors;
[0017] B5) Use in the preparation of a product for promoting myoblast differentiation and / or fusion;
[0018] B6) Use in promoting animal muscle development or in preparing products for promoting animal muscle development;
[0019] The composition comprises glutamic acid or a physiologically acceptable salt thereof, and / or α-ketoglutaric acid or a physiologically acceptable salt thereof.
[0020] In the above applications, the composition may further comprise a physiologically acceptable carrier.
[0021] The physiologically acceptable carrier can be selected from excipients, preservatives, protective agents, cosolvents, diluents (such as water, physiological saline, PBS (phosphate buffered saline), ethanol, polyethylene glycol, propylene glycol, PEG-400, dimethyl sulfoxide, etc.), wetting agents, disintegrants (such as dry starch, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, etc.), lubricants (such as sorbitan trioleate, soybean lecithin, lecithin, oleic acid, magnesium stearate, sodium lauryl sulfate, etc.), fillers (such as starch, dextrin, etc.), adhesives (such as gelatin, pectin, gum arabic, hydroxypropyl cellulose (CP), PVP, CMC-Na, etc.), penetration enhancers (such as Brij-78). , pH regulator, stabilizer (such as sodium sulfite, citric acid, tartaric acid, EDTA, etc.), surfactant (such as Tween, Span, eucalyptus oil, polysorbate-80, sodium lauryl sulfate, soybean lecithin, sodium cholate, sodium deoxycholate, etc.), absorption accelerator (such as chitosan), thickener (such as sodium hyaluronate, sodium carboxymethyl cellulose, polyvinyl alcohol, etc.), antioxidant (such as sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, vitamin C, etc.), plasticizer (such as glycerol, sorbitol, phthalate, etc.), propellant (such as hydrofluoroalkane, dimethyl ether, etc.), aerosolizing agent, suspending agent, dispersant, colorant (such as TiO2, pigment, etc.) and flavoring agent. Those skilled in the art know that a carrier usually has multiple functions. For example, starch can be used as a disintegrant and as a binder. Suitable carriers can be selected according to actual needs.
[0022] In the above application, the myogenic differentiation factor can be selected from MyoG.
[0023] In the above applications, the product can be selected from reagents, preparations, foods, feeds, feed additives, myoblast fusion promoters, muscle growth promoters and muscle enhancement supplements.
[0024] In the above applications, the myoblasts may include skeletal muscle myoblasts.
[0025] The present invention also provides a method for promoting myoblast fusion, comprising contacting the myoblasts with glutamate or a physiologically acceptable salt thereof and / or α-ketoglutarate or a physiologically acceptable salt thereof, or the composition described herein.
[0026] In the above method, the contacting step may comprise contacting the myoblasts with a biologically effective amount of glutamate or a physiologically acceptable salt thereof and / or α-ketoglutarate or a physiologically acceptable salt thereof, or a biologically effective amount of the composition described herein.
[0027] In the above method, the contacting may include oral administration and injection.
[0028] In the above method, the myoblasts may include skeletal muscle myoblasts.
[0029] The present invention also provides a product for promoting muscle development or skeletal muscle development in animals, wherein the product contains glutamic acid or a physiologically acceptable salt thereof and / or α-ketoglutaric acid or a physiologically acceptable salt thereof, or the composition described herein.
[0030] Furthermore, the product may be a reagent, a preparation, a food, a feed, a feed additive, a myoblast fusion promoter, a muscle growth promoter, a muscle enhancement supplement, etc.
[0031] Furthermore, the animals include but are not limited to: deer, sheep, chicken, duck, fish, human, rat, mouse, guinea pig, hamster, nude mouse, rabbit, pig, dog, monkey, horse, cow, etc.
[0032] Herein, a biologically effective amount may refer to an amount of an exogenous substance (eg, glutamate and / or α-ketoglutarate) sufficient to produce a desired biological effect (eg, promoting differentiation of animal myoblasts).
[0033] Herein, physiologically acceptable carriers are intended to encompass various types of additives or ingredients that can be included in the compositions of the present invention that do not pose adverse safety or toxicity issues to an organism.
[0034] As used herein, a physiologically acceptable salt refers to an addition salt present in conjunction with the acidic or basic portion of a compound that is non-toxic to an organism. Physiologically acceptable salts may include sodium salts, potassium salts, magnesium salts, calcium salts, ammonium salts, non-toxic amine salts, and the like.
[0035] As used herein, "comprising" is not intended to be limiting, but rather inclusive and means that there may be other elements other than the listed elements, and may be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of". The terms "including" and "comprising" are used interchangeably herein.
[0036] After extensive and in-depth research, the inventors of this application have discovered for the first time two metabolites that promote skeletal muscle myoblast fusion: glutamate and α-ketoglutarate. Using mouse C2C12 cells, the present invention demonstrated that the transition from proliferation to differentiation is a glutamate-consuming process. Without the addition of glutamate, glutamate content significantly decreased, indicating that myoblast fusion requires glutamate. The present invention further demonstrated the effect of exogenous glutamate addition on cell fusion using mouse C2C12 cells and sheep skeletal muscle myoblasts. Adding 2mM and 4mM glutamate to the culture medium significantly promoted the fusion of mouse C2C12 cells and sheep myoblasts. The present invention also demonstrated the effect of exogenous α-KG on cell fusion using mouse C2C12 cells and sheep skeletal muscle myoblasts. Adding 2mM α-KG to the culture medium significantly promoted the fusion of mouse C2C12 cells and sheep myoblasts.
[0037] Beneficial effects of the present invention: The present invention uses mouse C2C12 cells and sheep skeletal muscle myoblasts to explore the functions of glutamate and α-KG in myoblast fusion. These two substances are highly correlated with the fusion of skeletal muscle myoblasts and have the effect of promoting myoblast differentiation and / or fusion. They can be prepared into feed, feed additives, myoblast fusion promoters and other products for promoting animal muscle development and breeding high-yield and high-quality meat livestock products. Experiments have shown that adding appropriate amounts of glutamate and α-KG during the muscle cell differentiation process can significantly increase the fusion rate of mouse C2C12 cells and sheep skeletal muscle myoblasts, increase the number of myotubes formed, and promote the differentiation level of the two cells. The present invention has broad application prospects in the fields of muscle variety improvement, livestock breeding, muscle disease treatment, muscle regeneration and repair in the livestock industry. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 The figure shows the changes in glutamate content during the differentiation of mouse C2C12 cells in Example 1.
[0039] Figure 2 The myotube formation of mouse C2C12 cells after adding different concentrations of glutamate in Example 2 is shown.
[0040] Figure 3 The expression of the myogenic differentiation factor MyoG in mouse C2C12 cells after adding different concentrations of glutamate in Example 2 is shown.
[0041] Figure 4 This is the myotube formation of sheep myoblasts after adding 4 mM glutamate in Example 2.
[0042] Figure 5 This is the expression of the myogenic differentiation factor MyoG in sheep myoblasts after adding 4 mM glutamate in Example 2.
[0043] Figure 6 This is the myotube formation of mouse C2C12 cells after adding 2 mM α-KG in Example 3.
[0044] Figure 7 The expression of the myogenic differentiation factor MyoG in mouse C2C12 cells after adding 2 mM α-KG in Example 3.
[0045] Figure 8 This is the myotube formation of sheep myoblasts after adding 2 mM α-KG in Example 3.
[0046] Figure 9 The expression of the myogenic differentiation factor MyoG in sheep myoblasts after adding 2 mM α-KG in Example 3. DETAILED DESCRIPTION
[0047] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.
[0048] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.
[0049] The mouse C2C12 cells in the following examples were purchased from the cell bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
[0050] The sheep skeletal muscle myoblasts in the following examples were isolated from Hu sheep about 1 day after birth.
[0051] In the following examples, FBS is fetal bovine serum; PS is penicillin-streptomycin solution; high-glucose DMEM: cell culture medium containing pyruvate but not L-glutamine (purchased from Thermo Fisher Scientific, brand: Gbico, catalog number C11960500BT). In the following examples, the MyoG antibody was purchased from DSHB, USA, the Lamin B1 antibody was purchased from Beyotime, China, the α-Tubulin antibody was purchased from Cell Signaling Technology, USA, and glutamate and α-KG were purchased from Sigma-Aldrich, USA.
[0052] Example 1: Glutamate consumption during myoblast fusion
[0053] To investigate the role of glutamate in myoblast fusion, two groups of mouse C2C12 cells were prepared without the addition of glutamate. One group of mouse cells was cultured in proliferation medium (DMEM without L-glutamine containing 10% FBS and 1% PS). When the cell density reached about 90%, the cells were replaced with differentiation medium (DMEM without L-glutamine containing 1 μM dexamethasone, 1 μg / mL linoleic acid, and 1 μM insulin). Differentiation was induced for 48 hours, and the cells were digested and collected. The other group of cells was cultured in proliferation medium (DMEM without L-glutamine containing 10% FBS and 1% PS). When the cell density reached about 90%, the cells were digested and collected. The glutamate content in the two groups of cells was detected using the kit (Catalog Number AKAM002M) from Beijing Box Biotechnology Co., Ltd. The results are shown in Figure 2. Figure 1 The results showed that the glutamate content in cells decreased significantly as differentiation was induced, indicating that glutamate is consumed during cell fusion and plays an important role in muscle fusion.
[0054] Example 2: Exogenous addition of glutamate promotes fusion of mouse C2C12 cells and sheep myoblasts
[0055] In order to verify the effect of glutamate on the fusion of mouse C2C12 cells, three groups of mouse C2C12 cells were cultured in proliferation medium (DMEM without L-glutamine containing 10% FBS and 1% PS) without adding glutamate. When the cell density reached about 90%, the control group (NC) was replaced with induction differentiation medium (DMEM without L-glutamine containing 1μM dexamethasone, 1μg / mL linoleic acid, and 1μM insulin). The other two groups of C2C12 cells were replaced with induction differentiation medium containing 2mM and 4mM glutamate, respectively. The three groups of cells were induced to differentiate for 48h, and the formation of myotubes was detected. The cells were digested and collected, and the expression of myogenic differentiation factor MyoG was detected by Western Blot technology. The results are shown in Figure 2. Figure 2 As shown in Figure 2, the number of myotubes increased significantly after adding 2mM and 4mM glutamate. Figure 3 As shown in Figure 3, the expression of the myogenic differentiation factor MyoG was also significantly increased.
[0056] In order to verify the effect of glutamate on the fusion of sheep myoblasts, two groups of sheep skeletal muscle myoblasts were cultured in proliferation medium (DMEM without L-glutamine containing 10% FBS and 1% PS) without adding glutamate. When the cell density reached about 90%, the control group (NC) was replaced with induction differentiation medium (DMEM without L-glutamine containing 1μM dexamethasone, 1μg / mL linoleic acid, and 1μM insulin), and the other group of cells was replaced with induction differentiation medium containing 4mM glutamate. The two groups of cells were induced to differentiate for 48h, and the formation of myotubes was detected. The cells were digested and collected, and the expression of myogenic differentiation factor MyoG was detected by Western Blot technology. The results are shown in Figure 2. Figure 4 As shown in Figure 2, the number of myotubes increased significantly after adding 4 mM glutamate. Figure 5 As shown in Figure 3, the expression of the myogenic differentiation factor MyoG was also significantly increased.
[0057] In this example, the method for detecting myotube formation is as follows: replacing the differentiation induction medium, differentiating for 48 hours, and observing myotube formation under a 10x microscope. Myotubes are multinuclear myocytes formed by the fusion of mononuclear myocytes. They are much longer and have a significantly larger cell volume than mononuclear cells. Therefore, randomly selected fields of view can be photographed under a microscope, and then the myotube formation of the experimental and control groups can be compared.
[0058] The method for Western Blot detection of MyoG expression was as follows: (1) The protein concentration was detected according to the instructions of the BCA protein concentration assay kit (Beijing Adlai Biological), and 6 concentration gradients were set: 5 mg / mL, 2.5 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0 mg / mL.
[0059] (2) Add 6× SDS-PAGE loading buffer (Biyuntian Biotechnology Co., Ltd.) to the protein in proportion and boil in 100°C water bath for 5 min.
[0060] (3) Prepare the SDS-PAGE gel according to Table 1. Add 20 μg of nuclear protein to the prepared gel wells. Adjust the voltage to 80 V. After the marker is running, switch to 120 V and continue running the gel for about 1 hour.
[0061] (4) Gently transfer the protein gel onto a PVDF membrane, remove any bubbles, and place it in transfer buffer for electroporation at 320 mA for 1 h.
[0062] (5) Remove the PVDF membrane, wash it once with TBST, and then place it in TBST containing 5% skim milk powder and shake it at room temperature for 1.5 hours to block it.
[0063] (6) Dilute the primary antibody proportionally with TBST, transfer the membrane to the diluted primary antibody, and incubate overnight at 4°C. Aspirate the primary antibody and wash the membrane three times with TBST, each wash at high speed for 5-10 minutes.
[0064] (7) Dilute the secondary antibody in TBST in proportion, transfer the membrane to the diluted secondary antibody, incubate at room temperature for 1 hour, and wash three times with TBST, each wash at high speed for 5 minutes.
[0065] (8) Add solution A and solution B from the ECL chemiluminescence kit (Suzhou Xinsaimei Biotechnology Co., Ltd.) in a volume ratio of 1:1, shake off the liquid on the PVDF membrane, and place it in a protein imager for imaging.
[0066] Table 1 SDS-PAGE gel composition (5 mL system
[0067]
[0068] Example 3: Exogenous addition of α-KG promotes fusion of mouse C2C12 cells and sheep myoblasts
[0069] In order to verify the effect of α-KG (α-ketoglutarate) on the fusion of mouse C2C12 cells, two groups of mouse C2C12 cells were cultured in proliferation medium (DMEM containing 10% FBS, 1% PS) without adding α-KG. When the cell density reached about 90%, the control group (NC) replaced the induction differentiation medium (DMEM without L-glutamine containing 1μM dexamethasone, 1μg / mL linoleic acid, and 1μM insulin), and the other group of C2C12 cells replaced the induction differentiation medium containing 2mM α-KG. The two groups of cells were induced to differentiate for 48h, and the formation of myotubes was detected. The cells were digested and collected, and the expression of myogenic differentiation factor MyoG was detected by Western Blot technology. The results are shown in Figure 2. Figure 6 As shown in Figure 2, the number of myotubes increased significantly after adding 2 mM α-KG. Figure 7 As shown in Figure 3, the expression of the myogenic differentiation factor MyoG was also significantly increased.
[0070] In order to verify the effect of α-KG on the fusion of sheep myoblasts, two groups of sheep skeletal muscle myoblasts were cultured in proliferation medium (DMEM containing 10% FBS and 1% PS) without adding α-KG. When the cell density reached about 90%, the control group (NC) was replaced with induction differentiation medium (DMEM without L-glutamine containing 1μM dexamethasone, 1μg / mL linoleic acid, and 1μM insulin), and the other group of cells was replaced with induction differentiation medium containing 2mM α-KG. The two groups of cells were induced to differentiate for 48h, and the formation of myotubes was detected. The cells were digested and collected, and the expression of myogenic differentiation factor MyoG was detected by Western Blot technology. The results are shown in Figure 2. Figure 8 As shown in Figure 2, the number of myotubes increased significantly after adding 2 mM α-KG. Figure 9 As shown in Figure 3, the expression of the myogenic differentiation factor MyoG was also significantly increased.
[0071] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.
Claims
1. Use of glutamic acid or a physiologically acceptable salt thereof, and / or α-ketoglutaric acid or a physiologically acceptable salt thereof in any of the following: A1) Application in promoting myoblast differentiation; A2) Application in promoting myoblast fusion; A3) Application in increasing the number of myotubes of myoblasts; A4) Application in increasing the expression level of myogenic differentiation factors; A5) Use in the preparation of a product for promoting myoblast differentiation and / or fusion; A6) Use in promoting animal muscle development or preparing a product for promoting animal muscle development.
2. Use of the composition in any of the following: B1) Application in promoting myoblast differentiation; B2) Application in promoting myoblast fusion; B3) Application in increasing the number of myotubes of myoblasts; B4) Application in increasing the expression level of myogenic differentiation factors; B5) Use in the preparation of a product for promoting myoblast differentiation and / or fusion; B6) Use in promoting animal muscle development or in preparing products for promoting animal muscle development; The composition comprises glutamic acid or a physiologically acceptable salt thereof, and / or α-ketoglutaric acid or a physiologically acceptable salt thereof.
3. The use according to claim 2, characterized in that The composition also includes a physiologically acceptable carrier.
4. The use according to any one of claims 1 to 3, characterized in that The myogenic differentiation factor is selected from MyoG.
5. The use according to any one of claims 1 to 4, characterized in that: The product is selected from the group consisting of reagents, preparations, foods, feeds, feed additives, myoblast fusion promoters, muscle growth promoters, and muscle enhancement supplements.
6. The use according to any one of claims 1 to 5, characterized in that: The myoblasts include skeletal muscle myoblasts.
7. A method for promoting myoblast fusion, characterized in that: The method comprises contacting myoblasts with glutamate or a physiologically acceptable salt thereof and / or α-ketoglutarate or a physiologically acceptable salt thereof, or the composition of claim 2 or 3.
8. The method according to claim 7, characterized in that The contacting step comprises contacting the myoblasts with a biologically effective amount of glutamic acid or a physiologically acceptable salt thereof and / or α-ketoglutaric acid or a physiologically acceptable salt thereof, or a biologically effective amount of the composition according to claim 2 or 3.
9. The method according to claim 7 or 8, characterized in that Such exposure includes oral administration and injection.
10. A product for promoting muscle development or skeletal muscle development in animals, characterized in that: The product contains glutamic acid or a physiologically acceptable salt thereof and / or α-ketoglutaric acid or a physiologically acceptable salt thereof, or the composition according to claim 2 or 3.