CD73 gene and application of protein encoded by CD73 gene in regulation and control of fat metabolism

Abstract

The invention discloses a CD73 gene and an application of a protein encoded by the CD73 gene in regulation and control of fat metabolism. A nucleotide sequence of the CD73 gene is shown as SEQ ID NO: 1. The expression quantity of the CD73 gene and the encoded protein thereof is in negative correlation with the fat content of the tissue. The CD73 gene and the encoding protein thereof influence the content of triglyceride and the number of lipid droplets and maintain the steady state of fat metabolism by regulating and controlling fat metabolism protein and fat metabolism signal channels. By increasing the expression quantity of the CD73 gene and the encoded protein thereof, fat degradation can be promoted, lipid droplets and triglyceride can be reduced, and fat deposition can be reduced. The CD73 gene and the expression quantity of the encoded protein thereof can be applied to regulation and control of pork fat deposition, improvement of carcass quality and improvement of production efficiency, and can also be used as a molecular target to develop related targeting technologies such as seed selection, identification and treatment. CD73 has a wide application prospect and a potential value in the aspect of fat metabolism regulation and control.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to an application of a CD73 gene and its encoded protein in regulating fat metabolism. Background Art

[0002] Lipid metabolism in organisms is a crucial physiological process that significantly influences the flavor, nutritional value, and quality of meat products. Therefore, during poultry and livestock production, it is necessary to rationally regulate lipid metabolism to improve meat quality and ensure that the final product meets market demand and has a high market value. Fat deposition is the result of a coordinated distribution of nutrients within the animal body, and moderate fat reserves are essential for maintaining normal life activities. However, excessive fat deposition is a key indicator of an imbalance in energy homeostasis, resulting in wasted feed resources and reduced meat quality. Therefore, regulating fat deposition is crucial for improving carcass quality and increasing the efficiency of pig production.

[0003] The CD73 protein encoded by the CD73 gene, also known as extracellular-5'-nucleotidase, is the main enzyme that catalyzes adenosine monophosphate (AMP) to produce extracellular adenosine outside the cell. At present, a large number of studies are focusing on exploring CD73 as a metabolic immune checkpoint. Patent CN116617259A uses CD73 as a sensitive immune marker for adipose-derived mesenchymal stem cells and develops technology for the use of CD73 in the preparation of products that enhance adipose-derived mesenchymal stem cell angiogenesis. There is a certain correlation between the CD73 gene and its encoded protein and lipid metabolism, such as blood lipid content. However, the specific mechanism of the CD73 gene and its encoded protein on fat metabolism is still unclear, which hinders the development and direct application of related technologies for the CD73 gene and its encoded protein in fat metabolism. Summary of the Invention

[0004] To address the above issues, the present invention provides an application of the CD73 gene and its encoded protein in regulating fat metabolism. Increasing the expression of the CD73 gene and its encoded protein in adipocytes can enhance the intensity of fat metabolism signals, increase the expression of proteins involved in fat degradation, reduce the number of lipid droplets and triglyceride content, and reduce fat deposition. Conversely, it promotes fat deposition. The present invention utilizes the mechanism of the CD73 gene and its encoded protein in regulating fat metabolism, providing a theoretical basis for the development of related technologies.

[0005] A CD73 gene and its encoded protein are used to regulate fat metabolism. The nucleotide sequence of the CD73 gene is shown in SEQ ID NO: 1.

[0006] Preferably, the expression of the CD73 gene is negatively correlated with fat deposition. When the expression of the CD73 gene and its encoded protein is inhibited or downregulated, the signal intensity of fat metabolism is weakened, and the expression of fat degradation-related proteins is also reduced accordingly. This leads to the obstruction of the lipid droplet decomposition process, increased triglyceride accumulation, and thus promoted fat deposition in cells.

[0007] Preferably, inhibiting the expression of the CD73 gene in adipocytes comprises the following steps:

[0008] Cell culture: Primary adipocytes were cultured in complete medium until contact inhibition occurred.

[0009] Inducing differentiation: adding differentiation induction medium to primary adipocytes;

[0010] Inhibition of CD73 gene: On the first day after differentiation induction, inhibitors were added to primary adipocytes;

[0011] Preferably, the complete culture medium is DMEM / F12 culture medium containing 10% fetal bovine serum, 0.05% penicillin and 0.05% streptomycin.

[0012] Preferably, the differentiation induction medium is a DMEM / F12 medium containing 5% fetal bovine serum, 0.05% penicillin, 0.05% streptomycin, 50 nM dexamethasone, 50 mM octanoic acid, 50 μM oleic acid and 50 nM insulin.

[0013] In complete culture medium and induced differentiation medium, fetal bovine serum provides primary cells with growth factors, hormones, and other substances that promote cell growth. Penicillin and streptomycin act as dual antibiotics to prevent bacterial contamination during cell culture and exert antibacterial effects. In induced differentiation medium, dexamethasone, a commonly used differentiation inducer, can encourage cells to differentiate in specific directions. Caprylic acid acts as a signaling molecule in primary cells to regulate fat metabolism. Oleic acid can affect adipocyte differentiation and lipid accumulation. Insulin can regulate cell metabolism and growth, promoting adipocyte differentiation.

[0014] Preferably, the interval between inducing differentiation and culturing cells is no less than 24 hours. When cells exhibit contact inhibition, it indicates that the cells are in a normal and stable growth state in complete culture medium. After one day, the cell proliferation rate slows down. Inducing differentiation during this period ensures that the cells differentiate in a relatively stable state, which is conducive to obtaining more reliable and consistent differentiation results.

[0015] Preferably, the temperature for inducing differentiation is 35-40°C, the number of times of inducing differentiation is not less than 4 times, the interval between each time is not less than 48 hours, and the induction differentiation medium is replaced each time differentiation is induced. The temperature for inducing differentiation is close to the body temperature of most mammals, and the universality of the experimental results is stronger. During the process of inducing differentiation, cells will consume a large amount of nutrients, produce metabolic waste and harmful substances, and the secreted metabolites may change the conditions of the culture medium. Therefore, it is necessary to replace the culture medium multiple times to ensure that the cells can obtain sufficient nutritional supplements during the differentiation process, remove these metabolic wastes, stabilize the culture medium conditions, and maintain the normal physiological functions and metabolic activities of the cells. At the same time, multiple inductions are also beneficial to gradually guide the primary cells to differentiate into the target direction, i.e., adipocytes, gradually increase the proportion of differentiated cells, improve the differentiation efficiency, and ensure the integrity and accuracy of the differentiation process.

[0016] Preferably, the inhibitor is LY-3475070, and the inhibitor concentration is 10 μM.

[0017] Preferably, the application of the CD73 gene and its encoded protein in regulating fat metabolism includes any one of the following applications:

[0018] Application of CD73 gene and its encoded protein in regulating fat deposition;

[0019] Application of CD73 gene and its encoded protein as differential target genes for fat metabolism regulation.

[0020] Preferably, the application of the CD73 gene and its encoded protein in regulating fat deposition includes any one of the following applications:

[0021] Application of CD73 gene and its encoded protein in regulating triglyceride content;

[0022] Application of CD73 gene and its encoded protein in regulating lipid droplet number;

[0023] Application of CD73 gene and its encoded protein in regulating fat metabolism proteins;

[0024] Application of CD73 gene and its encoded protein in regulating fat metabolism signaling pathway;

[0025] Application of CD73 gene and its encoded protein in maintaining fat metabolism homeostasis.

[0026] Preferably, the fat metabolism proteins include adipose triglyceride lipase, carnitine palmitoyltransferase 1A, and perilipin. Adipose triglyceride lipase (ATGL) is a key enzyme for fat decomposition. Reduced expression of ATGL will lead to a large accumulation of triglycerides in adipocytes and other tissues, promoting fat deposition. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme in the fatty acid oxidation process, catalyzing the transport of fatty acids to the mitochondrial matrix and further decomposition by β-oxidation. Perilipin (PERLIN) has a dual regulatory effect on the metabolism of triglycerides in adipose tissue. When the number of fat droplets is too low, PERLIN can prevent lipase from approaching lipolysis. When the number of fat droplets is too high, PERLIN can promote lipase secretion, stimulate fat decomposition, and maintain the stability of fat metabolism in the body.

[0027] Preferably, the use of the CD73 gene and its encoded protein as a differential target gene for fat metabolism regulation includes any one of the following applications:

[0028] The application of the CD73 gene and its encoded protein in identifying the fat deposition phenotype; by detecting the expression level of the CD73 gene and its encoded protein, it can be determined whether an individual is at risk of abnormal fat deposition, which helps to detect fat deposition-related diseases at an early stage.

[0029] The application of CD73 gene and its encoded protein in the development of fat deposition breeding marker technology; by detecting the expression level of CD73 gene and its encoded protein, animal breeds with excellent fat deposition traits can be screened, which helps to improve the quality and yield of meat.

[0030] The application of the CD73 gene and its encoded protein in the development of targeted detection technologies for fat metabolism; using CD73 as a target, targeted detection technologies for fat metabolism can be developed. These technologies can monitor the dynamic changes of fat metabolism in real time, providing important evidence for clinical diagnosis and treatment.

[0031] The application of CD73 gene and its encoded protein in the development of targeted treatment technology for obesity; by promoting CD73 gene expression, increasing the abundance of lipolytic proteins, improving fat metabolic efficiency, and reducing fat deposition, it helps to alleviate and treat obesity and other related diseases.

[0032] Compared with the prior art, the beneficial effect of the present invention lies in that the core of the present invention is that the expression of the CD73 gene and its encoded protein is negatively correlated with the fat content. When the expression of the CD73 gene and its encoded protein is increased, the abundance of fat decomposition-related proteins in adipocytes, including adipose triglyceride lipase and carnitine palmitoyltransferase 1A, will increase, promoting fat degradation, reducing the number of fat droplets and triglyceride content, and reducing the degree of fat deposition. By utilizing this negative correlation, the fat deposition of pork can be changed by regulating the expression of CD73, thereby improving the quality of pork carcasses and improving production efficiency. CD73 can also be used as a molecular target with broad application prospects and potential value in the regulation of fat deposition and fat metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0035] In the attached figure:

[0036] Figure 1 Figure 2 shows the triglyceride test results of fat serum in Lantang pigs and Landrace pigs, where * indicates a significant difference between the two groups (P<0.05).

[0037] Figure 2 These are the expression analysis results of CD73 protein in adipose tissue of Lantang pigs and Landrace pigs, where β-actin is actin.

[0038] Figure 3 These are the expression analysis results of lipid metabolism proteins in Lantang pigs and Landrace pigs, among which β-actin is actin, FASN is fatty acid synthase, CD63 is a transmembrane protein, CPT1A is carnitine palmitoyltransferase 1A, and ATGL is adipose triglyceride lipase.

[0039] Figure 4 The results of Nile red staining of adipocyte lipid droplets after adding CD73 inhibitors were observed. CON is the control group with added water, and LY-3475070 is the treatment group with added CD73 inhibitors.

[0040] Figure 5Figure 3: Changes in triglyceride content in adipocytes after adding CD73 inhibitors. CON is the control group with water addition, and LY-3475070 is the treatment group with CD73 inhibitors addition. * indicates a significant difference between the two groups (P<0.05).

[0041] Figure 6 The expression analysis results of CD73 protein in adipocytes after adding CD73 inhibitors, where CON is the control group with added water and LY-3475070 is the treatment group with added CD73 inhibitors.

[0042] Figure 7 These are the expression analysis results of fat metabolism proteins in adipocytes after adding CD73 inhibitors. CON is the control group with water addition, LY-3475070 is the treatment group with CD73 inhibitor addition, β-actin is actin, PERLIN is perilipin, CPT1A is carnitine palmitoyltransferase 1A, and ATGL is adipose triglyceride lipase. DETAILED DESCRIPTION

[0043] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Any modifications or substitutions to the methods, steps, or conditions of the present invention, without departing from the spirit and substance of the present invention, fall within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, and instruments used in the examples of the present invention are commercially available; unless otherwise specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art.

[0044] Example 1 Comparison of fat metabolism differences between Lantang pigs and Landrace pigs

[0045] Blood samples were collected from 3-day-old Lantang pigs and Landrace pigs, respectively. The blood samples were placed in a centrifuge at a speed of 3000 rpm / min and centrifuged for 15 minutes. The supernatant was collected and the triglyceride content was determined by a one-step endpoint colorimetric method.

[0046] like Figure 1 As shown, the results showed that the triglyceride content in the blood of Lantang pigs was greater than 0.4mM, which was significantly higher than that of Landrace pigs, indicating that Lantang pigs were of high fat deposition type and Landrace pigs were of low fat deposition type.

[0047] Subcutaneous adipose tissue samples were collected from 3-day-old Lantang pigs (high fat deposition) and Landrace pigs (low fat deposition), and protein expression was analyzed by Western blot. 15 μg of protein was loaded into each well. The expression levels of CD73 protein and other fat metabolism-related proteins in Lantang pigs and Landrace pigs were detected.

[0048] like Figure 2The results showed that the content of CD73 protein was lower in tissue samples of Lantang pigs with high fat deposition, while the content of CD73 protein was higher in tissue samples of Landrace pigs with low fat deposition, indicating that the content of CD73 protein was negatively correlated with the degree of fat deposition.

[0049] like Figure 3 As shown, the results showed that there was no significant difference in the content of proteins related to fat synthesis, including fatty acid synthase (FASN) and transmembrane protein (CD63), in the tissues of two different pig breeds with different fat deposition. In the tissue samples of Lantang pigs with high fat deposition, the content of proteins related to fat degradation, including carnitine palmitoyltransferase 1A (CPT1A) and adipose triglyceride lipase (ATGL) was significantly lower than that in the tissue samples of Landrace pigs with low fat deposition. This shows that low levels of CD73 protein will reduce the activity of fat-degrading enzymes, slow down the rate of fat decomposition, and promote fat deposition. High levels of CD73 protein will increase the expression of fat-degrading enzymes, increase the rate of fat decomposition, and slow down fat deposition.

[0050] Example 2 CD73 gene regulates pig fat deposition

[0051] Cell culture: Primary adipocytes were cultured in DMEM / F12 complete medium containing 10% fetal bovine serum, 0.05% penicillin, and 0.05% streptomycin until contact inhibition occurred.

[0052] Differentiation Induction: After contact inhibition occurs, add differentiation induction medium to the primary adipocytes 24 hours later and transfer the cells to 37°C for culture. The differentiation induction medium consists of DMEM / F12 medium supplemented with 5% fetal bovine serum, 0.05% penicillin, 0.05% streptomycin, 50 nM dexamethasone, 50 mM octanoic acid, 50 μM oleic acid, and 50 nM insulin. Day 0 is the day of addition of differentiation induction medium.

[0053] Inhibition of CD73 gene: On the first day of differentiation induction, the inhibitor LY-3475070 was added to the primary adipocytes at a concentration of 10 μM.

[0054] Multiple induction: Replace the differentiation medium on days 2, 4, 6, and 8 of differentiation induction.

[0055] After the induction of differentiation, the number of lipid droplets in adipocytes was observed by Nile red staining, the triglyceride content was determined by one-step endpoint colorimetry, and the changes in the levels of fat metabolism-related proteins were analyzed by Western blot.

[0056] like Figure 4As shown, after adding the inhibitor LY-3475070, the tissue coloration part was significantly increased compared with the control group (CON) added with clear water. The results showed that inhibiting the expression of CD73 protein would increase the number of lipid droplets and promote fat deposition.

[0057] like Figure 5 As shown, the results showed that after adding the inhibitor LY-3475070, the concentration of triglycerides was significantly increased after CD73 was inhibited, compared with the control group (CON) added with water, thereby promoting fat deposition.

[0058] like Figure 6 As shown, the results showed that after the addition of the inhibitor LY-3475070, the protein content of CD73 was significantly reduced compared with the control group (CON) added with clean water.

[0059] like Figure 7 The results showed that after adding the inhibitor LY-3475070, compared with the control group (CON) with the addition of water, the levels of proteins related to lipolysis, including carnitine palmitoyltransferase 1A (CPT1A) and adipose triglyceride lipase (ATGL), were significantly reduced after CD73 inhibition, indicating that lipolysis was inhibited and fat deposition was favored. The increase in the content of perilipin (PERLIN) indicates that the number of lipid droplets is in an unbalanced state, indicating that CD73 protein can regulate fat metabolism.

[0060] Based on the above experimental results, the expression of the CD73 gene and its encoded protein is negatively correlated with fat deposition. When the expression of the CD73 gene and its encoded protein is increased, the abundance of fat-decomposition-related proteins in adipocytes, including adipose triglyceride lipase and carnitine palmitoyltransferase 1A, increases, promoting fat degradation, reducing the number of lipid droplets and triglyceride content, and reducing the degree of fat deposition. Utilizing this negative correlation, CD73 can effectively regulate the fat content of pork and improve pork quality. CD73 also has broad application prospects and potential value as a molecular target in the regulation of fat deposition and fat metabolism.

[0061] It is understandable that the above embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent scope of the present invention. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, the above technical features can be freely combined, and several deformations and improvements can be made, all of which fall within the scope of protection of the present invention. Therefore, all equivalent changes and modifications made to the scope of the claims of the present invention should fall within the scope of coverage of the claims of the present invention.

Claims

1. An application of the CD73 gene and its encoded protein in regulating fat metabolism, characterized in that: The nucleotide sequence of the CD73 gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The expression level of the CD73 gene is negatively correlated with fat deposition.

3. The use according to claim 2, characterized in that Inhibiting the expression of the CD73 gene in adipocytes comprises the following steps: Cell culture: Primary adipocytes were cultured in complete medium until contact inhibition occurred. Inducing differentiation: adding differentiation induction medium to primary adipocytes; Inhibition of the CD73 gene: After inducing differentiation, the inhibitor is added to the primary adipocytes. The use according to claim 3, characterized in that the complete culture medium is DMEM / F12 culture medium containing 10% fetal bovine serum, 0.05% penicillin and 0.05% streptomycin.

4. The use according to claim 3, characterized in that The differentiation induction medium is a DMEM / F12 medium containing 5% fetal bovine serum, 0.05% penicillin, 0.05% streptomycin, 50 nM dexamethasone, 50 mM octanoic acid, 50 μM oleic acid and 50 nM insulin.

5. The use according to claim 3, characterized in that The interval between the differentiation induction and the cell culture is no less than 24 hours.

6. The use according to claim 3, characterized in that The temperature for inducing differentiation is 35-40° C., the number of times of inducing differentiation is no less than 4 times, each time interval is no less than 48 hours, and the inducing differentiation medium is replaced each time differentiation is induced.

7. The use according to claim 3, characterized in that The inhibitor is LY-3475070.

8. The use according to claim 1, characterized in that Includes any of the following applications: Application of CD73 gene and its encoded protein in regulating fat deposition; Application of CD73 gene and its encoded protein as differential target genes for fat metabolism regulation.

9. The use according to claim 8, characterized in that The application of the CD73 gene and its encoded protein in regulating fat deposition includes any one of the following applications: Application of CD73 gene and its encoded protein in regulating triglyceride content; Application of CD73 gene and its encoded protein in regulating lipid droplet number; Application of CD73 gene and its encoded protein in regulating fat metabolism proteins; Application of CD73 gene and its encoded protein in regulating fat metabolism signaling pathway; Application of CD73 gene and its encoded protein in maintaining fat metabolism homeostasis.

10. The use according to claim 9, characterized in that The fat metabolism proteins include adipose triglyceride lipase, carnitine palmitoyltransferase 1A, and perilipin.

11. The use according to claim 8, characterized in that The application of the CD73 gene and its encoded protein as differential target genes for fat metabolism regulation includes any one of the following applications: Application of CD73 gene and its encoded protein in identifying fat deposition phenotype; Application of CD73 gene and its encoded protein in developing fat deposition breeding marker technology; Application of CD73 gene and its encoded protein in the development of targeted detection technology for fat metabolism; Application of CD73 gene and its encoded protein in the development of targeted treatment technology for obesity.

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