Use of alpha-ketobutyric acid in promoting skeletal muscle fiber type transition from fast to slow

Abstract

The application discloses application of alpha-ketobutyric acid in promoting transformation of skeletal muscle fiber type from fast muscle to slow muscle, and belongs to the technical field of animal nutrition. In the application, pig skeletal muscle satellite cells are treated by alpha-ketobutyric acid, mRNA expression levels of slow muscle genes MyHC I and MyHC IIa are significantly up-regulated, and expression level of fast muscle gene MyHC IIb is significantly down-regulated. Alpha-ketobutyric acid treatment significantly increases expression of slow muscle protein and reduces expression of fast muscle protein. In the myotube of the alpha-ketobutyric acid treatment group, MyHC-slow positive staining area is obviously higher than that of the control group, and MyHC-fast positive staining area is obviously lower than that of the control group. The above results show that the muscle fiber can be transformed from fast muscle to slow muscle after alpha-ketobutyric acid treatment. Therefore, the application provides a brand-new technical means and thought for regulating muscle physiology and improving meat quality.

Description

Technical Field

[0001] This invention relates to the field of animal nutrition technology, specifically to the application of α-ketobutyric acid in promoting the transformation of animal skeletal muscle fiber type from fast-twitch to slow-twitch muscle. Background Technology

[0002] Skeletal muscle is a vital organ for locomotion in animals and a source of meat. Its function and quality are determined by the type of muscle fibers. Based on the myosin heavy chain subtypes they express, muscle fibers are mainly classified into four types: MyHCⅠ (slow oxidative), MyHCⅡa (rapid oxidative-glycolytic), MyHCⅡx (rapid glycolytic), and MyHCⅡb (rapid glycolytic). Slow-twitch muscle fibers (MYHCⅠ) contract slowly but have high endurance, are rich in mitochondria, and rely on aerobic metabolism; fast-twitch fibers (Ⅱa, Ⅱx, Ⅱb) contract quickly and have great strength but are easily fatigued, primarily relying on anaerobic glycolysis for energy. The ratio of muscle fiber types directly affects an animal's athletic performance, energy metabolism efficiency, and the flavor, juiciness, and nutritional value of meat. In animal husbandry, increasing the proportion of slow-twitch and oxidative fast-twitch fibers (MyHCⅠ and MyHCⅡa) in livestock through nutritional regulation and other means is an effective strategy for improving meat quality and producing high-end meat products.

[0003] α-Ketobutyric acid (α-KB), also known as 2-oxobutyric acid, is a naturally occurring endogenous keto acid produced during the metabolism of amino acids such as threonine and methionine. Currently, its known functions mainly focus on its role as a metabolic intermediate in the tricarboxylic acid cycle (TCA cycle) and the generation of succinyl-CoA. No research has yet reported on the specific regulatory role of α-ketobutyric acid in skeletal muscle fiber type conversion; its potential function and value as a feed additive for improving meat quality remain unexplored.

[0004] Currently, there are very limited methods to effectively and safely regulate the conversion of muscle fiber types to slow-twitch muscle fibers. While long-term low-intensity endurance training can induce slow-twitch conversion, it is difficult to implement in large-scale animal husbandry. Although some drugs or hormones can affect muscle growth, their application is strictly limited due to concerns about drug residues, safety issues, and ethical controversies. Therefore, the livestock industry urgently needs to develop a new type of natural, safe, and effective nutritional regulator that can promote the conversion of fast-twitch muscle fibers to slow-twitch fibers. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide an application of α-ketobutyric acid in promoting the transformation of animal skeletal muscle fiber types from fast-twitch to slow-twitch, thereby providing a novel, natural, safe, and effective nutritional regulator that can promote the transformation of fast-twitch to slow-twitch muscle fibers. This nutritional regulation method increases the proportion of slow-twitch and oxidized fast-twitch muscle fibers in livestock, thereby improving pork quality.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: providing an application of α-ketobutyric acid in promoting the transformation of animal skeletal muscle fiber type from fast muscle to slow muscle.

[0007] Furthermore, the animal is a pig.

[0008] This invention provides a method for promoting the conversion of porcine skeletal muscle fiber type from fast-twitch to slow-twitch, comprising the following steps: (1) Cell culture and differentiation: porcine skeletal muscle satellite cells were cultured in growth medium until they fused, and then the porcine skeletal muscle satellite cells were transferred to differentiation medium for further culture; (2) When porcine skeletal muscle satellite cells begin to differentiate, add α-ketobutyric acid solution to the culture medium and continue culturing for 48-96 hours.

[0009] Furthermore, the concentration of the α-ketobutyric acid solution in step (2) is 1-30 μmol / L.

[0010] This invention provides a nutritional regulator that promotes the conversion of fast-twitch muscle fibers in pigs to slow-twitch muscle fibers, the nutritional regulator including α-ketobutyric acid.

[0011] Furthermore, the dosage forms of nutritional regulators include any one of tablets, capsules, oral liquids, granules, and sustained-release formulations.

[0012] The present invention also provides a method for increasing the proportion of slow-twitch muscle fibers in pig skeletal muscle fibers by adding the above-mentioned nutritional regulator to the basal diet of pigs.

[0013] This invention offers the following beneficial effects: It utilizes α-ketobutyric acid (QBPA) to treat porcine skeletal muscle satellite cells. RT-PCR results showed that, compared to the control group, the mRNA expression levels of slow-twitch muscle genes MyHCⅠ and MyHCⅡa were significantly upregulated in the 20 μmol / L QBPA treatment group, while the expression level of the fast-twitch muscle gene MyHCⅡb was significantly downregulated to 50% of the control group. Western Blot results indicated that QBPA treatment significantly increased the expression of slow-twitch muscle proteins and decreased the expression of fast-twitch muscle proteins, consistent with changes in gene expression. Immunofluorescence results showed that the area of ​​MyHC-slow positive staining in the muscle tubes of the QBPA treatment group was significantly higher than that in the control group, while the area of ​​MyHC-fast positive staining was significantly lower, indicating that QBPA treatment can promote the conversion of muscle fibers from fast to slow-twitch muscle fibers, thereby increasing the proportion of slow-twitch muscle fibers in skeletal muscle fibers. Therefore, this invention reveals for the first time the biological function of the natural metabolite QBPA in promoting the conversion of fast to slow-twitch muscle fibers, providing a novel technical means and approach for regulating muscle physiology and improving meat quality, filling a gap in existing technologies. Attached Figure Description

[0014] Figure 1 Image showing the isolation, culture, and identification of porcine skeletal muscle satellite cells; Figure 2 Figure showing the effect of different concentrations of α-ketobutyrate on the viability of porcine skeletal muscle satellite cells; Figure 3 Figure showing the effect of α-ketobutyrate on the expression of myofiber type-related genes in porcine skeletal muscle satellite cells as detected by real-time quantitative PCR. Figure 4 Figure showing the effect of α-ketobutyrate on the expression of myofiber type-related proteins in porcine skeletal muscle satellite cells detected by Western blotting. Figure 5 The graph shows the change in the area of ​​MyHC-slow positive staining in myotubes by immunofluorescence staining. Figure 6 The graph shows the change in the area of ​​MyHC-fast positive staining in myotubes observed by immunofluorescence staining. Detailed Implementation

[0015] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0016] Example 1: In vitro culture of porcine skeletal muscle satellite cells Longissimus dorsi muscle samples were collected from healthy piglets (Duroc × Landrace × Large White) and placed in DMEM / F12 serum-free culture medium. The tissue was cut into pieces approximately 1 mm thick in a laminar flow hood. 3 Cell fragments were digested with type II collagenase. The digested cell suspension was filtered through 200-mesh and 400-mesh cell sieves, and the filtrate was collected and centrifuged to obtain cell pellet. The cells were resuspended and seeded into cell culture dishes and cultured in a 37°C, 5% CO2 incubator. Satellite cells could be further purified by differential adhesion. Cells were seeded into 96-well plates, fixed with 4% paraformaldehyde at room temperature for 30 min, permeabilized for 10 min, blocked with BSA for 2 h, and washed with PBS; incubated with porcine skeletal muscle satellite cell marker protein Pax7 antibody, washed with PBS; incubated with allogeneic secondary antibody for 2 h, washed with PBS; incubated with DAPI for 10 min, washed with PBS, mounted, photographed under a microscope, and detected with porcine skeletal muscle satellite cell marker protein Pax7 to confirm successful cell culture. Figure 1 ).

[0017] Example 2: Verification of the effect of α-ketobutyric acid on the fiber type conversion of porcine skeletal muscle satellite cells The MTT assay was used to screen the effects of different concentrations (0, 1, 5, 10, 20, and 30 μmol / L) of α-ketobutyrate on the viability of porcine skeletal muscle satellite cells. The results showed that treatment with α-ketobutyrate at concentrations not exceeding 30 μmol / L for 48 h or 96 h had no significant inhibitory effect on cell viability. Figure 2 (A and 2B).

[0018] Porcine skeletal muscle satellite cells were cultured to confluence in culture medium, then replaced with differentiation medium to induce myotube formation. At the start of differentiation, the experimental group received 20 μmol / L α-ketobutyrate in the culture medium, while the control group received an equal amount of PBS. After 96 hours of differentiation, total RNA was extracted from the cells and reverse transcribed into cDNA. Real-time quantitative PCR was performed using the SYBR Green assay to detect changes in the expression of myofiber type marker genes. Total protein was extracted from the cells, and the expression changes of fast-twitch and slow-twitch muscle proteins were detected by Western blotting. The results showed that compared with the control group, the mRNA expression levels of slow-twitch muscle genes MyHCⅠ and MyHCⅡa were significantly upregulated in the 20 μmol / L α-ketobutyrate treatment group (P<0.05), while the expression level of fast-twitch muscle gene MyHCⅡb was significantly downregulated to 50% of that in the control group (P<0.05). Figure 3 Western blot analysis revealed that α-ketobutyrate treatment significantly increased the expression of slow-twitch muscle protein and decreased the expression of fast-twitch muscle protein (P<0.05), consistent with changes in gene expression. Figure 4 (A and 4B).

[0019] Immunofluorescence results: In the myotubes of the α-ketobutyrate treatment group, the area of ​​MyHC-slow positive staining was significantly higher than that of the control group. Figure 5 The area of ​​MyHC-fast positive staining was significantly lower than that of the control group ( Figure 6 This indicates that α-ketobutyric acid treatment can promote the transformation of muscle fibers from fast-twitch to slow-twitch muscle fibers.

[0020] Existing research indicates that slow-twitch muscle fibers have higher myoglobin content, stronger oxidative metabolism, and richer mitochondrial density, which can significantly improve meat color redness, increase muscle water retention, and reduce shear force, thereby improving tenderness (see: ①Joo ST, Kim GD, Hwang YH, Ryu YC. Control of fresh meat quality through manipulation of muscle fiber characteristics. Meat Sci. 2013;95(4):828-836. doi:10.1016 / j.meatsci.2013.04.044.;②Li Y, Feng Y, Chen X, et al.Dietary short-term supplementation of grape seed proanthocyanidin extract improves pork quality and promotes skeletal muscle fiber type conversion infinishing pigs. Meat Sci. 2024;210:109436. doi:10.1016 / j.meatsci.2024.109436). Furthermore, slow-twitch muscle fibers are generally positively correlated with intramuscular fat deposition, jointly enhancing the flavor and juiciness of meat (see: Zhang L, Li F, Guo Q, et al. Balanced branched-chain aminoacids modulate meat quality by adjusting muscle fiber type conversion and intramuscular fat deposition in finishing pigs. J Sci Food Agric. 2022;102(9):3796-3807. doi:10.1002 / jsfa.11728.). Therefore, the method of treating muscle fibers with α-ketobutyric acid to promote the conversion of fast-twitch muscle fibers to slow-twitch muscle fibers, as described in this invention, provides a new strategy for improving pork quality.

[0021] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of α-ketobutyric acid in promoting the conversion of animal skeletal muscle fiber type from fast-twitch to slow-twitch.

2. The application according to claim 1, characterized in that, The animal in question is a pig.

3. A method for promoting the transformation of porcine skeletal muscle fiber type from fast-twitch to slow-twitch muscle, characterized in that, Includes the following steps: (1) Cell culture and differentiation: porcine skeletal muscle satellite cells were cultured in growth medium until they fused, and then the porcine skeletal muscle satellite cells were transferred to differentiation medium for further culture; (2) When porcine skeletal muscle satellite cells begin to differentiate, add α-ketobutyric acid solution to the culture medium and continue culturing for 48-96 hours.

4. The method according to claim 3, characterized in that, The concentration of the α-ketobutyric acid solution in step (2) is 1-30 μmol / L.

5. A method for increasing the proportion of slow-twitch muscle fibers in porcine skeletal muscle fibers, characterized in that, The proportion of slow-twitch muscle fibers in pig skeletal muscle fibers can be increased by adding α-ketobutyric acid to the basal diet of pigs.

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