Use of inhbc as target in prevention and / or treatment of metabolic disease

Abstract

A use of INHBC as a target in the prevention and / or treatment of a metabolic disease. Provided is a use of a substance promoting the expression of an INHBC gene or improving the activity of an INHBC protein in the preparation of a product for preventing and / or treating a metabolic disease, wherein the metabolic disease comprises an energy metabolism dysfunction-induced disease, such as obesity, diabetes, fatty liver disease, or hyperlipidemia. Liver-specific overexpression or knock-in of INHBC can relieve liver steatosis and abnormal glucose metabolism in mice induced by a high-fat diet, and can also enhance the thermogenic activity of adipose tissues in the mice, thereby inhibiting obesity induced by the high-fat diet, showing significant application value in the prevention and / or treatment of metabolic diseases such as obesity, type 2 diabetes, and fatty liver diseases.

Description

Application of INHBC as a target in prevention and / or treatment of metabolic diseases TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of INHBC as a target in prevention and / or treatment of metabolic diseases. BACKGROUND

[0002] Obesity, type 2 diabetes mellitus (T2DM) and metabolic dysfunction-associated steatotic liver disease (MASLD) have become global chronic metabolic diseases. The conventional drugs for treating obesity and T2DM on the market all have certain side effects, and there is no safe and effective drug approved for treating MASLD. In addition, the specific causes, pathogenesis and effective cure methods of obesity, T2DM and MASLD are not clear. Therefore, finding the common mechanism of the onset of obesity, T2DM and MASLD and finding effective drug targets based on this are the key to solving this problem.

[0003] Related studies have shown that the inhibin subunit beta C (INHBC) gene is located on human chromosome 12, which may play a role in cancer suppression or promotion in some cancers, and the specific mechanism may be related to its function of regulating cell proliferation and differentiation. For example, in some tumors, high expression of the INHBC gene is related to the occurrence and development of tumors, and is regarded as a potential tumor marker and therapeutic target by researchers. The INHBC gene is expressed in multiple tissues, especially in the liver, but the role of the INHBC gene in obesity, T2DM and MASLD is not clear.

[0004] Therefore, the present application intends to take the INHBC gene as a therapeutic target for obesity, T2DM and MASLD, and expects to develop a new drug for preventing and / or treating metabolic diseases such as obesity, T2DM and MASLD. SUMMARY

[0005] The present application aims to at least solve one of the technical problems in the prior art. To this end, the present application provides use of a substance for promoting expression of an INHBC gene or increasing activity of a protein thereof in preparation of a product for preventing and / or treating metabolic diseases. The present application finds that overexpression or knock-in of INHBC in the liver can improve liver steatosis and abnormal glucose metabolism induced by a high-fat diet in mice, reduce the content of triglyceride in the liver and serum of mice, and at the same time reduce the content of lipid droplets in the liver. In addition, the present application finds that overexpression or knock-in of INHBC in the liver can enhance the thermogenic activity of adipose tissue in mice and thus inhibit obesity induced by a high-fat diet, which can provide a new treatment strategy for preventing and / or treating metabolic diseases such as obesity, type 2 diabetes and fatty liver disease.

[0006] The present application also provides a pharmaceutical composition for preventing and / or treating metabolic diseases.

[0007] In a first aspect of the present application, use of a substance for promoting expression of an INHBC gene or increasing activity of a protein thereof in preparation of a product for preventing and / or treating metabolic diseases is provided.

[0008] The present application discloses a model for overexpression, knockdown, knock-in or knockout of INHBC gene in the liver (such as an obese mouse model, a T2DM mouse or cell model, and a MASLD mouse or cell model, etc.), reveals the effect of INHBC as a target on the treatment of metabolic diseases, and specifically finds that:

[0009] (1) Overexpression or knock-in of INHBC in the liver can improve liver steatosis induced by a high-fat diet in mice, reduce the content of triglyceride in the liver and serum of mice, and at the same time reduce the content of lipid droplets in the liver; on the contrary, knockdown or knockout of INHBC in the liver can aggravate lipid accumulation in the liver of mice induced by a high-fat diet.

[0010] (2) INHBC can improve lipid metabolism disorder in the liver of mice induced by a high-fat diet through the AMPK-SREBP1c pathway. Compared with the control group, after overexpression or knock-in of INHBC in the liver, the expression level of mRNA of genes related to de novo synthesis of fatty acids in the liver decreases, in addition, the phosphorylated protein level of AMPK increases, and the expression levels of proteins FASN, SCD1 and ACC1 related to synthesis of fatty acids and proteins CIDEA and CIDEC related to formation of lipid droplets significantly decrease.

[0011] (3) Overexpression of INHBC can inhibit lipid synthesis in primary hepatocytes of mice induced by free fatty acids, and knockdown of INHBC can aggravate lipid deposition induced by free fatty acids. In addition, the protein expression level of INHBC in primary hepatocytes is significantly negatively correlated with the time of induction by free fatty acids.

[0012] (4) Liver-specific overexpression or knock-in of INHBC can up-regulate the phosphorylated protein level of LKB1, promote the translocation of LKB1 from the nucleus to the cytoplasm, activate the AMPK signaling pathway, inhibit the expression of SREBP1c, and thus improve the lipid deposition in the liver.

[0013] (5) After liver-specific overexpression of INHBC, the mRNA and protein expression levels of INHBC in the liver of mice are significantly increased, the fasting blood glucose level of mice is significantly reduced, the glucose tolerance of mice is significantly improved, and the mRNA and protein expression levels of gluconeogenesis-related genes in the liver are reduced. On the contrary, liver-specific knockdown of INHBC can lead to an increase in the fasting blood glucose level of mice, a significant impairment of glucose tolerance, and a significant up-regulation of the protein expression level of gluconeogenesis-related genes in the liver.

[0014] (6) Liver-specific overexpression of INHBC can significantly reduce the blood glucose level of db / db mice, improve the glucose tolerance and insulin sensitivity of the organism, and significantly reduce the mRNA and protein expression levels of gluconeogenesis-related genes.

[0015] (7) Liver-specific transgenic knock-in of INHBC can improve the glucose metabolism disorder of diabetic mice induced by high-fat diet and streptozotocin, the mRNA and protein expression levels of INHBC in the liver of mice are significantly increased, the fasting blood glucose level of mice is significantly reduced, the glucose tolerance and insulin sensitivity of the organism are significantly improved, and the mRNA and protein expression levels of gluconeogenesis-related genes in the liver are reduced. On the contrary, liver-specific transgenic knock-out of INHBC can exacerbate the glucose metabolism disorder of diabetic mice.

[0016] (8) Overexpression of INHBC can reduce the mRNA and protein expression levels of gluconeogenesis-related genes in mouse primary hepatocytes and reduce glucose production. On the contrary, knockdown of INHBC can up-regulate the mRNA and protein expression levels of gluconeogenesis-related genes in mouse primary hepatocytes and promote glucose production.

[0017] (9) Liver-specific overexpression or knock-in of INHBC can enhance the thermogenic activity of adipose tissue in mice, thereby inhibiting high-fat diet-induced obesity, significantly reducing the body weight of mice, reducing the accumulation of body fat, showing better cold tolerance, significantly increasing the mRNA and protein expression levels of genes related to thermogenesis, lipid hydrolysis, and fatty acid beta oxidation in adipose tissue, and relatively reducing the mRNA and protein expression levels of genes related to lipid synthesis, and increasing the whole-body energy consumption. On the contrary, liver-specific knockdown or knock-out of INHBC can lead to a decrease in the thermogenic level of adipose tissue in mice and exacerbate high-fat diet-induced obesity.

[0018] (10) The INHBC purified protein prepared in vitro can significantly increase the mitochondrial activity and heat production level of mouse primary adipocytes, and significantly increase the oxygen consumption rate of mouse primary adipocytes.

[0019] (11) INHBC can bind to DSG1β receptor on the surface of adipocytes, and when DSG1β is knocked down, the expression level of heat production related genes and proteins on the INHBC purified protein and the ability to promote energy consumption are obviously inhibited.

[0020] In some embodiments of the present application, the metabolic disease includes a disease caused by energy metabolism disorder.

[0021] In some embodiments of the present application, the metabolic disease includes at least one of obesity, diabetes, fatty liver disease, and hyperlipidemia.

[0022] In some embodiments of the present application, the substance for promoting INHBC gene expression or increasing its protein activity is selected from at least one of a nucleic acid drug, a polypeptide or protein drug, and a recombinant virus.

[0023] In some embodiments of the present application, the nucleic acid drug is selected from a nucleic acid drug containing an INHBC expression promoter or enhancer, a nucleic acid drug containing any truncated sequence or full-length sequence of INHBC mRNA containing at least the nucleotide sequence shown in SEQ ID NO: 1, and a nucleic acid drug encoding a polypeptide or protein drug containing any truncated sequence or full-length sequence of INHBC protein containing at least the amino acid sequence shown in SEQ ID NO: 5.

[0024] In some embodiments of the present application, the polypeptide or protein drug contains at least a polypeptide or protein drug containing any truncated sequence or full-length sequence of INHBC protein containing the amino acid sequence shown in SEQ ID NO: 5.

[0025] In some embodiments of the present application, the recombinant virus contains a nucleotide sequence expressing the INHBC gene.

[0026] In some embodiments of the present application, the functions of the product include at least one of A1) to A5):

[0027] A1) preventing and / or treating liver steatosis induced by high-fat diet;

[0028] A2) preventing and / or treating abnormal liver lipid metabolism induced by high-fat diet;

[0029] A3) preventing and / or treating diabetes induced by high-fat diet and streptozotocin or genetic mutation;

[0030] A4) inhibiting high-fat diet-induced obesity;

[0031] A5) inhibiting free fatty acid-induced lipid production in primary hepatocytes.

[0032] In some embodiments of the present application, the product includes a pharmaceutical product and a functional food.

[0033] In some embodiments of the present application, the pharmaceutical product is in a dosage form selected from the group consisting of at least one of a solid preparation, a liquid preparation, and a semi-solid preparation.

[0034] In some embodiments of the present application, the solid preparation includes a tablet, a granule, a powder, and a capsule.

[0035] In some embodiments of the present application, the liquid preparation includes an injection.

[0036] In some embodiments of the present application, the semi-solid preparation includes an ointment and a cream.

[0037] In some embodiments of the present application, the pharmaceutical product can further include a pharmaceutical excipient selected from at least one of a disintegrant, a diluent, a lubricant, a binder, a humectant, a flavoring agent, a suspending agent, a surfactant, and a preservative.

[0038] In some preferred embodiments of the present application, the pharmaceutical excipient is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient.

[0039] In some embodiments of the present application, the disintegrant is selected from at least one of corn starch, potato starch, cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, and alginic acid.

[0040] Preferably, the diluent is selected from at least one of lactose, sucrose, mannitol, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0041] Preferably, the lubricant is selected from at least one of microfine silica, magnesium stearate, calcium stearate, stearic acid, talc, and anhydrous silica.

[0042] Preferably, the binder is selected from at least one of acacia, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose, and polyvinyl pyrrolidone.

[0043] Preferably, the humectant is selected from sodium lauryl sulfate.

[0044] Preferably, the flavoring agent is selected from at least one of aspartame, stevioside, sucrose, maltitol, and citric acid.

[0045] Preferably, the suspending agent is selected from at least one of gum arabic, gelatin, methyl cellulose, sodium carboxymethyl cellulose, hydroxymethyl cellulose, aluminum stearate gel;

[0046] Preferably, the surfactant is selected from at least one of lecithin, sorbitan monooleate, glycerol monostearate.

[0047] Preferably, the preservative is selected from at least one of methyl or propyl paraben.

[0048] In some embodiments of the present application, the form of the functional food comprises at least one of a soup, a beverage, a candy, an oral liquid, a capsule, a tablet, a powder.

[0049] In a second aspect of the present application, a pharmaceutical composition for preventing and / or treating metabolic diseases is provided, comprising a substance promoting expression of an INHBC gene or increasing activity of a protein thereof, and a pharmaceutically acceptable carrier.

[0050] In some embodiments of the present application, the substance promoting expression of an INHBC gene or increasing activity of a protein thereof is selected from at least one of a nucleic acid drug, a polypeptide or protein drug, a recombinant virus.

[0051] In some embodiments of the present application, the nucleic acid drug is selected from a nucleic acid drug containing a promoter or enhancer of INHBC expression, a nucleic acid drug of any truncated sequence or full-length sequence of an INHBC mRNA comprising at least the nucleotide sequence shown in SEQ ID NO: 1, a polypeptide or protein drug of nucleic acid drug encoding any truncated sequence or full-length sequence of an INHBC protein comprising at least the amino acid sequence shown in SEQ ID NO: 5.

[0052] In some embodiments of the present application, the polypeptide or protein drug comprises at least a polypeptide or protein drug of any truncated sequence or full-length sequence of an INHBC protein comprising the amino acid sequence shown in SEQ ID NO: 5.

[0053] In some embodiments of the present application, the recombinant virus comprises a nucleotide sequence expressing an INHBC gene.

[0054] In some embodiments of the present application, the pharmaceutically acceptable carrier comprises at least one of water, saline, buffer, glycerol, ethanol, liposome, lipid, protein, protein-antibody conjugate, peptide substance, cellulose, nanogel.

[0055] The term "pharmaceutically acceptable" refers to those substances that are suitable for use in humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio.

[0056] The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of therapeutic agents including various excipients and diluents. The choice of carrier will be determined by the mode of administration, specifically according to the means known to those of ordinary skill in the art.

[0057] In a third aspect of the present application, a method for treating metabolic diseases is provided, comprising the steps of:

[0058] administering to the subject a substance that promotes expression of the INHBC gene or increases the activity of the protein thereof.

[0059] In some embodiments of the present application, the substance that promotes expression of the INHBC gene or increases the activity of the protein thereof is selected from at least one of a nucleic acid drug, a polypeptide or protein drug, and a recombinant virus.

[0060] In some embodiments of the present application, the nucleic acid drug is selected from a nucleic acid drug containing a promoter or enhancer of INHBC expression, a nucleic acid drug containing at least any truncated sequence or full-length sequence of an INHBC mRNA shown in SEQ ID NO: 1, and a nucleic acid drug encoding at least any truncated sequence or full-length sequence of an INHBC protein shown in SEQ ID NO: 5.

[0061] In some embodiments of the present application, the polypeptide or protein drug contains at least any truncated sequence or full-length sequence of an INHBC protein shown in SEQ ID NO: 5.

[0062] In some embodiments of the present application, the recombinant virus contains a nucleotide sequence expressing the INHBC gene.

[0063] In some embodiments of the present application, the metabolic disease includes at least one of obesity, diabetes, fatty liver disease, and hyperlipidemia.

[0064] Specifically, the method for treating metabolic diseases includes administering to the subject an effective dose of an INHBC overexpression vector or an INHBC protein.

[0065] In some embodiments of the present application, the INHBC overexpression vector contains a nucleotide sequence encoding the INHBC protein.

[0066] In some embodiments of the present application, the route of administration includes intravenous injection.

[0067] In some embodiments of the present application, the amino acid sequence of the INHBC protein is shown as SEQ ID NO: 5.

[0068] In some embodiments of the present application, the nucleotide sequence of the INHBC gene is shown as SEQ ID NO: 1.

[0069] In some embodiments of the present application, the overexpression vector comprises a lentivirus vector, an adenovirus vector, a gene knock-in vector.

[0070] In some embodiments of the present application, the adenovirus vector comprises an AAV8 vector.

[0071] In some embodiments of the present application, the gene knock-in vector comprises a CRISPR-Cas9 vector. Other features and advantages of the present application will be set forth in the following description. BRIEF DESCRIPTION OF DRAWINGS

[0072] The present application will be further described below in conjunction with the accompanying drawings and examples, in which:

[0073] Figure 1 is a volcano plot of gene expression in the liver of subjects with BMI < 24 kg / m 2 and BMI ≥ 24 kg / m 2 ; B is a heat map of the expression of differential genes in the liver of normal diet mice and high-fat diet mice fed for 27 weeks; C is the detection result of INHBC mRNA expression level in different organs of wild-type mice; D is the detection result of INHBC mRNA expression level in the liver of mice under different high-fat diet feeding conditions (fed for 11 weeks, 27 weeks, and 40 weeks, respectively); E is the detection result of INHBC protein expression level in the liver of mice under different high-fat diet feeding conditions (fed for 11 weeks, 27 weeks, and 40 weeks, respectively); F is the detection result of INHBC mRNA expression level in the liver of db / db and ob / ob mice; G is the detection result of INHBC protein expression level in the liver of db / db and ob / ob mice.

[0074] Figure 2 is the related detection result of the experiment of the effect of liver-specific overexpression of INHBC on the fatty degeneration of the liver of mice induced by a high-fat diet, wherein A is the detection result of the INHBC mRNA expression level in the liver of mice; B is the detection result of the INHBC protein expression level in the liver of mice; C is the phenotype diagram of the liver of mice; D is the statistical result of the liver weight and body weight ratio of mice; E is the detection result of the triglyceride content in the liver of mice; F is the detection result of the triglyceride content in the serum of mice; G is the detection result of the alanine aminotransferase content in the serum of mice; H is the detection result of the aspartate aminotransferase content in the serum of mice; I is the result of H&E staining and oil red O staining of the liver of mice and its statistical diagram; and J is the shooting result of the Micro CT of the liver of mice and the result of the CT value ratio of the liver and the spleen.

[0075] Figure 3 is the related detection result of the experiment of the effect of liver-specific transgenic knock-in INHBC on the fatty degeneration of the liver of mice induced by a high-fat diet, wherein A is the detection result of the INHBC mRNA expression level in the liver of mice; B is the detection result of the INHBC protein expression level in the liver of mice; C is the phenotype diagram of the liver of mice; D is the statistical result of the liver weight and body weight ratio of mice; E is the detection result of the triglyceride content in the liver of mice; F is the detection result of the alanine aminotransferase content in the serum of mice; G is the detection result of the aspartate aminotransferase content in the serum of mice; H is the result of H&E staining and oil red O staining of the liver of mice and its statistical diagram; and I is the shooting result of the Micro CT of the liver of mice and the result of the CT value ratio of the liver and the spleen.

[0076] Figure 4 is the related detection result of the experiment of the effect of liver-specific knockdown of INHBC on the lipid accumulation of the liver of mice induced by a high-fat diet, wherein A is the detection result of the INHBC mRNA expression level in the liver of mice; B is the detection result of the INHBC protein expression level in the liver of mice; C is the phenotype diagram of the liver of mice; D is the statistical result of the liver weight and body weight ratio of mice; E is the detection result of the triglyceride content in the liver of mice; F is the detection result of the triglyceride content in the serum of mice; G is the detection result of the alanine aminotransferase content in the serum of mice; H is the detection result of the aspartate aminotransferase content in the serum of mice; I is the result of H&E staining and oil red O staining of the liver of mice and its statistical diagram; and J is the shooting result of the Micro CT of the liver of mice and the result of the CT value ratio of the liver and the spleen.

[0077] Figure 5 is the related detection results of the experiment of the effect of liver-specific transgenic knockout INHBC on high-fat diet-induced mouse liver lipid accumulation, wherein A is the detection results of INHBC mRNA expression level in mouse liver; B is the detection results of INHBC protein expression level in mouse liver; C is the phenotype diagram of mouse liver; D is the statistical results of the ratio of mouse liver weight to body weight; E is the detection results of triglyceride content in mouse liver; F is the detection results of triglyceride content in mouse serum; G is the detection results of alanine aminotransferase content in mouse serum; H is the detection results of aspartate aminotransferase content in mouse serum; I is the results of H&E staining and oil red O staining of mouse liver and its statistical diagram; J is the shooting results of mouse liver Micro CT and the results of liver to spleen CT value ratio.

[0078] Figure 6 is the related detection results of the experiment of the effect of liver-specific overexpression or knock-in INHBC on high-fat diet-induced mouse liver lipid metabolism-related pathway gene mRNA and protein expression level, wherein A is the results of liver-specific overexpression of INHBC liver transcriptome sequencing GO enrichment analysis; B is the detection results of lipid de novo synthesis-related gene mRNA expression level after liver-specific overexpression of INHBC; C is the detection results of lipid de novo synthesis-related gene mRNA expression level after liver-specific transgenic knock-in of INHBC; D is the results of liver-specific overexpression of INHBC liver transcriptome sequencing KEGG enrichment analysis; E is the detection results of AMPK phosphorylated protein level after liver-specific overexpression of INHBC; F is the detection results of AMPK phosphorylated protein level after liver-specific transgenic knock-in of INHBC; G is the detection results of fatty acid synthesis-related protein and lipid droplet formation-related protein expression level after liver-specific overexpression of INHBC; H is the detection results of fatty acid synthesis-related protein and lipid droplet formation-related protein expression level after liver-specific transgenic knock-in of INHBC.

[0079] Figure 7 is the related detection results of the experiment of the effect of overexpression of INHBC on free fatty acid-induced mouse primary hepatocyte lipid generation, wherein A is the detection results of relative expression amount of INHBC mRNA in primary hepatocytes after overexpression of INHBC; B is the detection results of triglyceride content in primary hepatocytes after overexpression of INHBC; C is the detection results of mRNA expression level of fatty acid synthesis-related genes in primary hepatocytes after overexpression of INHBC; D is the results of oil red O staining of primary hepatocytes after overexpression of INHBC and its statistical diagram; E is the detection results of lipid metabolism-related protein expression level in primary hepatocytes after overexpression of INHBC; F is the detection results of INHBC mRNA expression level after free fatty acid treatment of primary hepatocytes for different time; G is the detection results of INHBC protein expression level after free fatty acid treatment of primary hepatocytes for different time.

[0080] Figure 8 is related detection results of the experiment of the effect of knocking down INHBC on the lipid generation of free fatty acid-induced mouse primary hepatocytes, wherein A is the detection result of the relative expression amount of INHBC mRNA in primary hepatocytes after knocking down INHBC; B is the detection result of the triglyceride content in primary hepatocytes after knocking down INHBC; C is the detection result of the mRNA expression level of fatty acid synthesis-related genes in primary hepatocytes after knocking down INHBC; D is the result of oil red O staining of primary hepatocytes after knocking down INHBC and its statistical chart; E is the detection result of the expression level of lipid metabolism-related proteins in primary hepatocytes after knocking down INHBC.

[0081] Figure 9 is part of the related detection results of the experiment of the effect of INHBC on the LKB1-AMPK signal axis, wherein A is the detection result of the triglyceride content in primary hepatocytes after overexpressing INHBC and adding AMPK inhibitor Compound C; B is the detection result of the mRNA expression level of de novo lipid synthesis-related genes in primary hepatocytes after overexpressing INHBC and adding AMPK inhibitor Compound C; C is the detection result of the expression level of AMPK and lipid metabolism-related proteins in primary hepatocytes after overexpressing INHBC and adding AMPK inhibitor Compound C; D is the detection result of the triglyceride content in primary hepatocytes after overexpressing INHBC and adding LKB1 inhibitor Pim1; E is the detection result of the mRNA expression level of de novo lipid synthesis-related genes in primary hepatocytes after overexpressing INHBC and adding LKB1 inhibitor Pim1; F is the detection result of the expression level of AMPK and lipid metabolism-related proteins in primary hepatocytes after overexpressing INHBC and adding LKB1 inhibitor Pim1; G is the detection result of the expression level of LKB1-related proteins in the liver after liver-specific overexpression of INHBC; H is the detection result of the expression level of LKB1-related proteins in the liver after liver-specific transgenic knock-in of INHBC.

[0082] Figure 10 is another part of the related detection results of the experiment of the effect of INHBC on the LKB1-AMPK signal axis, wherein A is the detection result of the expression level of phosphorylated proteins of AMPK and LKB1 in primary hepatocytes after overexpressing INHBC; B is the detection result of the expression level of phosphorylated proteins of AMPK and LKB1 in primary hepatocytes after knocking down INHBC; C is the detection result of the expression level of LKB1 and AMPK-related proteins in the nucleus and cytoplasm of primary hepatocytes after overexpressing INHCB; D is the detection result of the expression level of LKB1 and AMPK-related proteins in the nucleus and cytoplasm of primary hepatocytes after knocking down INHCB.

[0083] Figure 11 is the related detection results of the experiment of the effect of liver-specific overexpression of INHBC on blood glucose level and glucose tolerance of mice, wherein A is the detection results of INHBC mRNA expression level in the liver of mice; B is the detection results of INHBC protein expression level in the liver of mice; C is the detection results of random blood glucose and blood glucose level after fasting for 6 and 16 hours of mice; D is the detection and statistical results of glucagon tolerance test; E is the detection and statistical results of pyruvate tolerance test; F-H are the detection results of gluconeogenesis-related gene mRNA expression level in the liver of mice; I is the detection results of gluconeogenesis-related protein expression level in the liver of mice.

[0084] Figure 12 is the related detection results of the experiment of the effect of liver-specific knockdown of INHBC on blood glucose level and glucose tolerance of mice, wherein A is the detection results of INHBC mRNA expression level in the liver of mice; B is the detection results of INHBC protein expression level in the liver of mice; C is the detection results of random blood glucose and blood glucose level after fasting for 6 and 16 hours of mice; D is the detection and statistical results of glucagon tolerance test; E is the detection and statistical results of pyruvate tolerance test; F is the detection results of gluconeogenesis-related protein expression level in the liver of mice.

[0085] Figure 13 is the related detection results of the experiment of the effect of liver-specific overexpression of INHBC on blood glucose level and glucose tolerance of db / db mice, wherein A is the detection results of INHBC mRNA expression level in the liver of mice; B is the detection results of INHBC protein expression level in the liver of mice; C is the detection results of random blood glucose level of mice per week; D is the detection results of random blood glucose and blood glucose level after fasting for 6 and 16 hours of mice; E is the detection and statistical results of glucagon tolerance test; F is the detection and statistical results of pyruvate tolerance test; G is the detection and statistical results of insulin tolerance test; H is the detection and statistical results of glucose tolerance test; I is the detection results of gluconeogenesis-related gene mRNA expression level in the liver of mice; J is the detection results of gluconeogenesis-related protein expression level in the liver of mice.

[0086] Figure 14 is the related detection results of the experiment of the effect of liver-specific transgenic knockout of INHBC on blood glucose level and glucose tolerance of high-fat diet combined with streptozotocin-induced diabetic mice, wherein A is the detection result of INHBC mRNA expression level in the liver of mice; B is the detection result of INHBC protein expression level in the liver of mice; C is the detection result of random blood glucose and blood glucose level after fasting for 6 and 16 hours of mice; D is the detection and statistical result of glucagon tolerance test; E is the detection and statistical result of pyruvate tolerance test; F is the detection and statistical result of insulin tolerance test; G is the detection and statistical result of glucose tolerance test; H is the detection result of gluconeogenesis-related gene mRNA expression level in the liver of mice; I is the detection result of gluconeogenesis-related protein expression level in the liver of mice.

[0087] Figure 15 is the related detection results of the experiment of the effect of liver-specific transgenic knockout of INHBC on blood glucose level and glucose tolerance of high-fat diet combined with streptozotocin-induced diabetic mice, wherein A is the detection result of INHBC mRNA expression level in the liver; B is the detection result of INHBC protein expression level in the liver; C is the detection result of random blood glucose and blood glucose level after fasting for 6 and 16 hours of mice; D is the detection and statistical result of glucagon tolerance test; E is the detection and statistical result of pyruvate tolerance test; F is the detection and statistical result of glucose tolerance test; G is the detection result of gluconeogenesis-related gene mRNA expression level in the liver of mice; H is the detection result of gluconeogenesis-related protein expression level in the liver of mice.

[0088] Figure 16 is the related detection results of the experiment of the effect of overexpression of INHBC on glucose production of mouse primary hepatocytes, wherein A is the detection result of INHBC mRNA expression level in mouse primary hepatocytes; B is the detection result of INHBC protein expression level in mouse primary hepatocytes; C is the detection result of gluconeogenesis-related gene mRNA expression level in mouse primary hepatocytes; D is the detection result of gluconeogenesis-related protein expression level in mouse primary hepatocytes; E is the detection result of glucose production amount in mouse primary hepatocytes; F is the detection result of glucose production amount in mouse primary hepatocytes under glucagon stimulation; G and H are the detection results of gluconeogenesis-related gene mRNA expression level in mouse primary hepatocytes under glucagon stimulation.

[0089] Figure 17 is related detection results of the experiment of the effect of INHBC knockdown on glucose production in mouse primary hepatocytes, wherein A is the detection result of INHBC mRNA expression level in mouse primary hepatocytes; B is the detection result of INHBC protein expression level in mouse primary hepatocytes; C is the detection result of gluconeogenesis related gene mRNA expression level in mouse primary hepatocytes; D is the detection result of gluconeogenesis related protein expression level in mouse primary hepatocytes; E is the detection result of glucose production amount in mouse primary hepatocytes; F is the detection result of glucose production amount in mouse primary hepatocytes under glucagon stimulation; G and H are the detection results of gluconeogenesis related gene mRNA expression level in mouse primary hepatocytes under glucagon stimulation.

[0090] Figure 18 is related detection results of the first part of the experiment of the effect of liver-specific overexpression of INHBC on high-fat diet induced mouse obesity, wherein A is the detection result of INHBC mRNA expression level in mouse liver; B is the detection result of INHBC protein expression level in mouse liver; C is the body map of mouse; D is the monitoring curve of mouse body weight change; E is the statistical result of mouse daily food intake; F is the detection result of mouse body composition; G is the detection result of rectal temperature of mouse under low temperature induction for 6 hours; H-I are the detection and statistical results of oxygen consumption rate of mouse under different temperatures; J-K are the detection and statistical results of energy consumption of mouse under different temperatures.

[0091] Figure 19 is related detection results of the second part of the experiment of the effect of liver-specific overexpression of INHBC on high-fat diet induced mouse obesity, wherein A is the detection result of weight of each organ or different adipose tissue of mouse; B is the result of H&E staining of mouse inguinal white adipose tissue and brown adipose tissue; C is the detection result of mRNA expression level of thermogenesis, lipid hydrolysis, fatty acid beta oxidation and lipid synthesis related genes in mouse inguinal white adipose tissue; D is the detection result of mRNA expression level of thermogenesis, lipid hydrolysis, fatty acid beta oxidation and lipid synthesis related genes in mouse brown adipose tissue.

[0092] Figure 20 is related detection results of the third part of the experiment of the effect of liver-specific overexpression of INHBC on high-fat diet induced mouse obesity. A is the detection result of thermogenesis, lipid hydrolysis and mitochondrial respiratory chain protein complex related protein expression level in mouse inguinal white adipose tissue; B is the detection result of thermogenesis, lipid hydrolysis and mitochondrial respiratory chain protein complex related protein expression level in mouse brown adipose tissue.

[0093] Figure 21 is the results of the first part of the experiment of the effect of liver-specific transgenic knock-in INHBC on high-fat diet-induced mouse obesity, wherein A is the detection result of INHBC mRNA expression level in mouse liver; B is the detection result of INHBC protein expression level in mouse liver; C is the body shape of the mouse; D is the monitoring curve of the change of mouse body weight; E is the statistical result of the daily food intake of the mouse; F is the detection result of the body composition of the mouse; G is the detection result of the rectal temperature of the mouse under low temperature induction for 6 hours; H-I are the detection and statistical results of the oxygen consumption rate of the mouse at different temperatures; J-K are the detection and statistical results of the energy consumption of the mouse at different temperatures.

[0094] Figure 22 is the results of the second part of the experiment of the effect of liver-specific transgenic knock-in INHBC on high-fat diet-induced mouse obesity, wherein A is the detection result of the weight of each organ or different fat tissue of the mouse; B is the H&E staining result of the inguinal white adipose tissue and brown adipose tissue of the mouse; C is the detection result of the mRNA expression level of the heat production, lipid hydrolysis, fatty acid beta oxidation and lipid synthesis related genes in the inguinal white adipose tissue of the mouse; D is the detection result of the mRNA expression level of the heat production, lipid hydrolysis, fatty acid beta oxidation and lipid synthesis related genes in the brown adipose tissue of the mouse.

[0095] Figure 23 is the results of the third part of the experiment of the effect of liver-specific transgenic knock-in INHBC on high-fat diet-induced mouse obesity, A is the detection result of the expression level of heat production, lipid hydrolysis and mitochondrial respiratory chain protein complex related proteins in the inguinal white adipose tissue of the mouse; B is the detection result of the expression level of heat production, lipid hydrolysis and mitochondrial respiratory chain protein complex related proteins in the brown adipose tissue of the mouse.

[0096] Figure 24 is the results of the first part of the experiment of the effect of liver-specific knockdown of INHBC on high-fat diet-induced mouse obesity, wherein A is the detection result of INHBC mRNA expression level in mouse liver; B is the detection result of INHBC protein expression level in mouse liver; C is the body shape of the mouse; D is the monitoring curve of the change of mouse body weight; E is the statistical result of the daily food intake of the mouse; F is the detection result of the body composition of the mouse; G is the detection result of the rectal temperature of the mouse under low temperature induction for 6 hours; H-I are the detection and statistical results of the oxygen consumption rate of the mouse at different temperatures; J-K are the detection and statistical results of the energy consumption of the mouse at different temperatures.

[0097] Figure 25 is the results of the second part of the experiment of the effect of liver-specific knockdown of INHBC on high-fat diet-induced obesity in mice, in which A is the detection results of the weight of each organ or different fat tissues of mice; B is the results of H&E staining of the inguinal white adipose tissue and brown adipose tissue of mice; C is the detection results of the mRNA expression levels of thermogenesis, lipid hydrolysis, fatty acid beta oxidation and lipid synthesis-related genes in the inguinal white adipose tissue of mice; D is the detection results of the mRNA expression levels of thermogenesis, lipid hydrolysis, fatty acid beta oxidation and lipid synthesis-related genes in the brown adipose tissue of mice.

[0098] Figure 26 is the results of the third part of the experiment of the effect of liver-specific knockdown of INHBC on high-fat diet-induced obesity in mice, A is the detection results of the expression levels of thermogenesis, lipid hydrolysis and mitochondrial respiratory chain protein complex-related proteins in the inguinal white adipose tissue of mice; B is the detection results of the expression levels of thermogenesis, lipid hydrolysis and mitochondrial respiratory chain protein complex-related proteins in the brown adipose tissue of mice.

[0099] Figure 27 is the results of the first part of the experiment of the effect of liver-specific knockdown of INHBC on high-fat diet-induced obesity in mice, in which A is the detection results of the mRNA expression level of INHBC in the liver of mice; B is the detection results of the protein level of INHBC in the liver of mice; C is the body shape of mice; D is the monitoring curve of the body weight change of mice; E is the statistical results of the daily food intake of mice; F is the detection results of the body composition of mice; G is the detection results of the rectal temperature of mice under low temperature induction for 6 hours; H-I are the detection and statistical results of the oxygen consumption rate of mice at different temperatures; J-K are the detection and statistical results of the energy consumption of mice at different temperatures.

[0100] Figure 28 is the results of the second part of the experiment of the effect of liver-specific knockdown of INHBC on high-fat diet-induced obesity in mice, in which A is the detection results of the weight of each organ or different fat tissues of mice; B is the results of H&E staining of the inguinal white adipose tissue and brown adipose tissue of mice; C is the detection results of the mRNA expression levels of thermogenesis, lipid hydrolysis, fatty acid beta oxidation and lipid synthesis-related genes in the inguinal white adipose tissue of mice; D is the detection results of the mRNA expression levels of thermogenesis, lipid hydrolysis, fatty acid beta oxidation and lipid synthesis-related genes in the brown adipose tissue of mice.

[0101] Figure 29 is the results of the third part of the experiment of the effect of liver-specific knockdown of INHBC on high-fat diet-induced obesity in mice, A is the detection results of the expression levels of thermogenesis, lipid hydrolysis and mitochondrial respiratory chain protein complex-related proteins in the inguinal white adipose tissue of mice; B is the detection results of the expression levels of thermogenesis, lipid hydrolysis and mitochondrial respiratory chain protein complex-related proteins in the brown adipose tissue of mice.

[0102] Figure 30 is part of the related detection results of the experiment of the effect of INHBC purified protein on the thermogenic level of mouse primary adipocytes, wherein A is the detection result of the mitochondrial fluorescence activity of mouse primary beige adipocytes after treatment with INHBC purified protein (40 ng / mL) and / or CL316243 (40 nM); B is the detection result of the mitochondrial fluorescence activity of mouse primary brown adipocytes after treatment with INHBC purified protein (40 ng / mL) and / or CL316243 (40 nM); C is the detection result of the mRNA expression level of mouse primary beige adipocytes related to thermogenesis, lipid hydrolysis and fatty acid β oxidation; D is the detection result of the mRNA expression level of mouse primary brown adipocytes related to thermogenesis, lipid hydrolysis and fatty acid β oxidation.

[0103] Figure 31 is another part of the related detection results of the experiment of the effect of INHBC purified protein on the thermogenic level of mouse primary adipocytes, A is the detection result of the expression level of mouse primary beige adipocyte mitochondrial respiratory chain protein complex, UCP1 and other thermogenic related proteins; B is the detection result of the expression level of mouse primary brown adipocyte mitochondrial respiratory chain protein complex, UCP1 and other thermogenic related proteins; C is the detection result of the oxygen consumption rate of mouse primary beige adipocytes; D is the detection result of the oxygen consumption rate of mouse primary brown adipocytes.

[0104] Figure 32 is part of the related detection results of the experiment of determining the receptor of INHBC on the surface of mouse primary adipocytes, wherein A is two experimental schemes for finding the surface receptor of adipocytes; B is the detection result of protein quantification before magnetic bead pull-down; C is the silver staining detection result of protein after magnetic bead pull-down; D is the mass spectrometry detection result of protein after magnetic bead pull-down; E is the signal pathway mechanism diagram of INHBC synergistically enhancing mitochondrial activity and promoting energy consumption of adipocytes through DSG1 β-p38 MAPK-ATF2 and DSG1 β-cAMP-PKA-CREB axes on the surface of adipocytes; F is the co-immunoprecipitation detection result of INHBC and DSG1 β after adding BS3 crosslinking agent; G is the co-immunoprecipitation detection result of INHBC and DSG1 β without adding BS3 crosslinking agent.

[0105] FIG. 33 is another part of the relevant detection results of the experiment for determining the receptor of INHBC on the surface of mouse primary adipocytes, wherein A is the detection results of the mRNA expression levels of DSG1 β, thermogenesis, lipid hydrolysis and fatty acid β oxidation related genes after knocking down DSG1 β in mouse primary beige adipocytes; B is the detection results of the mRNA expression levels of DSG1 β, thermogenesis, lipid hydrolysis and fatty acid β oxidation related genes after knocking down DSG1 β in mouse brown adipocytes; C is the detection results of the protein expression levels of DSG1 β, thermogenesis, lipid hydrolysis and fatty acid β oxidation related genes after knocking down DSG1 β in mouse primary beige adipocytes; D is the detection results of the protein expression levels of DSG1 β, thermogenesis, lipid hydrolysis and fatty acid β oxidation related genes after knocking down DSG1 β in mouse primary brown adipocytes. DETAILED DESCRIPTION

[0106] The concept and technical effects of the present application will be described below in conjunction with the embodiments so as to fully understand the objects, features and effects of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative effort fall within the scope of protection of the present application.

[0107] The word "preferably" and the like in the present application means the embodiments of the present application which can provide certain beneficial effects in certain cases. However, other embodiments can also be preferred in the same or other cases. In addition, the description of one or more preferred embodiments does not mean that other embodiments cannot be used, nor is it intended to exclude other embodiments from the scope of the present application.

[0108] In the description of the present application, the reference term "and / or" includes all and any combinations of one or more of the related listed items.

[0109] In the detailed description of the present application, unless otherwise specified, the humidity of all mouse breeding is 50-60% 12 / 12 hour light / dark cycle, and the temperature is 23-24℃; the feed fed during the conventional breeding stage contains 10kcal% fat, 70kcal% carbohydrate and 20kcal% protein; the feed (D12492) fed during the high-fat diet stage contains 60kcal% fat, 20kcal% carbohydrate and 20kcal% protein. In addition, unless otherwise specified, all experiments use male mice.

[0110] In the detailed description of the present application, unless specifically indicated, the experiments and methods described in the embodiments are basically carried out according to the conventional methods well known in the art and described in various references.

[0111] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0112] Inventive concept:

[0113] Previous research in this invention identified INHBC as a potential molecular target influencing metabolic diseases. This invention targets individuals with a BMI <24 kg / m². 2 and BMI ≥ 24 kg / m 2 Gene expression level analysis was performed on the livers of the subjects, revealing 9 upregulated and 4 downregulated genes (critical value: P < 0.05, |Log2(fold change)| > 0.3, as shown in Figure 1A). Further investigation revealed that INHBC was highly expressed in the livers of normal mice (as shown in Figure 1C). Specifically, a high-fat diet was found to decrease INHBC expression in the livers of mice (as shown in Figure 1B), with INHBC mRNA expression levels significantly downregulated in the livers of mice fed a high-fat diet for 11, 27, and 40 weeks, as well as in db / db and ob / ob mice (as shown in Figure 1D and F). INHBC protein expression levels were also significantly reduced in the livers of mice fed a high-fat diet for 11, 27, and 40 weeks, as well as in db / db and ob / ob mice (as shown in Figure 1E and G).

[0114] Based on this, the present invention aims to further reveal the impact of INHBC as a target on the treatment of metabolic diseases by constructing relevant models of liver-specific overexpression, knockdown, knock-in or knockout of the INHBC gene (such as obese mouse model, T2DM mouse or cell model, and MASLD mouse or cell model, etc.).

[0115] The following detailed description is provided through specific embodiments.

[0116] Example 1: Effect of liver-specific overexpression of INHBC on high-fat diet-induced hepatic steatosis in mice

[0117] This embodiment investigated the effect of liver-specific overexpression of INHBC on high-fat diet-induced hepatic steatosis in mice, specifically including the following:

[0118] 1. Experimental Methods

[0119] (1) Acquisition of overexpression viral vector: recombinant adeno-associated virus pAAV-TBG-INHBC-3xFLAG-P2A-GdGreen-WPRE (hereinafter referred to as AAV8-INHBC) carrying INHBC as an overexpression plasmid vector is constructed, wherein AAV is an adeno-associated virus vector, TBG is a promoter element, GdGreen is green fluorescence (non-fusion), and 3xFLAG is a tag sequence.

[0120] The nucleotide sequence of INHBC is shown in SEQ ID NO: 1 (5'-3'):

[0121] The control group vector is GL3009 pAAV-TBG-GdGreen-WPRE vector, hereinafter referred to as AAV8-GFP.

[0122] (2) Construction of liver-specific overexpression INHBC mouse model: 5-week-old C57BL / 6J mice were taken, and 1x10 11 GC / mL of AAV8-INHBC virus to construct a liver-specific overexpression INHBC mouse model as an experimental group, and 1x10 11 GC / mL of AAV8-GFP virus as a control group.

[0123] (3) High-fat diet-induced mouse liver steatosis: after injection of the virus for three weeks, the mice were continued to be fed with a high-fat diet for 13 weeks.

[0124] (4) Detection index: the mRNA and protein expression levels of the gene in the mouse liver, liver steatosis, triglyceride content, and liver function indicators were detected, the generation of lipid droplets in the liver was detected by H&E and oil red O staining methods, and the mouse steatosis was detected by high-resolution small animal Micro CT.

[0125] 2. Experimental results

[0126] The detection results are shown in Figure 2. Compared with the control group, the expression of INHBC mRNA in the liver of the mice with liver-specific overexpression of INHBC was significantly increased by about 22 times (as shown in A of Figure 2), and the expression level of INHBC protein in the liver was also significantly increased (as shown in B of Figure 2). After liver-specific overexpression of INHBC, the fatty liver phenotype of the mice was improved (as shown in C of Figure 2), the ratio of liver weight to body weight was decreased (as shown in D of Figure 2), and the contents of triglyceride in the liver and serum of the mice were significantly decreased compared with the control group (as shown in E and F of Figure 2). The detection results of liver function indicators showed that the levels of alanine aminotransferase and aspartate aminotransferase in the serum were significantly reduced after liver-specific overexpression of INHBC (as shown in G and H of Figure 2), and the lipid droplets in the liver of the mice were significantly reduced after liver-specific overexpression of INHBC (as shown in I of Figure 2). The detection results of high-resolution small animal Micro CT showed that the CT value ratio of the liver to the spleen was increased after overexpression of INHBC, indicating the improvement of fatty liver (as shown in J of Figure 2).

[0127] The above results show that liver-specific overexpression of INHBC can improve the fatty liver induced by high-fat diet in mice.

[0128] Example 2: Effect of liver-specific transgenic knock-in INHBC on fatty liver induced by high-fat diet in mice

[0129] This example detects the effect of liver-specific transgenic knock-in INHBC on fatty liver induced by high-fat diet in mice, which specifically includes the following contents.

[0130] 1. Experimental method

[0131] (1) Construction of transgenic knock-in mice: Cas9 mRNA and gRNA are generated by in vitro transcription, and a donor vector is fused. The plasmid structure comprises a 5' homologous arm (3.3 kb), a knock-in, and a 3' homologous arm (3.3 kb); a mixture of Cas9 mRNA, gRNA, and donor vector is microinjected into fertilized eggs, and then three F0 mice identified by PCR and sequencing are generated; the F0 positive mice are crossed with wild-type C57BL / 6J mice to generate F1 mice, and then homozygous mice INHBC WT are obtained. The mice are crossed with Alb-Cre mice, and finally liver-specific transgenic knock-in INHBC mice are obtained, which are represented by INHBC LKI .

[0132] (2) Fatty liver induced by high-fat diet in mice: 5-week-old transgenic mice are fed with high-fat diet for 13 weeks.

[0133] (3) Detection index: refer to the description in Example 1.

[0134] 2. Experimental results

[0135] The detection results are shown in Figure 3. Compared with the control group, the expression of INHBC mRNA in the liver of the liver-specific transgenic knock-in INHBC mice was significantly increased (as shown in Figure 3A), and the expression level of INHBC protein in the liver was also significantly increased (as shown in Figure 3B). After liver-specific transgenic knock-in INHBC, the fatty liver phenotype of the mice was significantly improved (as shown in Figure 3C), the ratio of liver weight to body weight was significantly decreased (as shown in Figure 3D), and the content of triglyceride in the liver of the mice was significantly decreased (as shown in Figure 3E) compared with the control group. The detection results of liver function indicators showed that the liver-specific transgenic knock-in INHBC significantly reduced the levels of alanine aminotransferase and aspartate aminotransferase in the serum (as shown in Figures 3F and 3G), and the lipid droplets in the liver of the mice were significantly reduced (as shown in Figure 3H) after liver-specific transgenic knock-in INHBC. The detection results of high-resolution small animal Micro CT showed that the CT value ratio of liver to spleen was increased after liver-specific transgenic knock-in INHBC, indicating the improvement of fatty degeneration (as shown in Figure 3I).

[0136] The above results show that liver-specific transgenic knock-in INHBC can improve the lipid deposition in the liver of mice induced by high-fat diet.

[0137] Example 3: Effect of liver-specific knockdown of INHBC on lipid accumulation in the liver of mice induced by high-fat diet

[0138] This example detects the effect of liver-specific knockdown of INHBC on lipid accumulation in the liver of mice induced by high-fat diet, which specifically includes the following contents.

[0139] 1. Experimental method

[0140] (1) Construction of INHBC knockdown related virus: a recombinant adeno-associated virus AAV8-shINHBC carrying shINHBC was constructed as a knockdown plasmid vector, wherein the shINHBC interference sequence has three sequences, which are shINHBC-1, shINHBC-2 and shINHBC-3, and the nucleotide sequences are shown in SEQ ID NO: 2-SEQ ID NO: 4 (5'-3') respectively:

[0141] shINHBC-1: GCCACCCAGACCATGAATATA (SEQ ID NO: 2);

[0142] shINHBC-2: CCTCTGTCTTTGCTCTACTAT (SEQ ID NO: 3);

[0143] shINHBC-3: CCTCACCTTGACAAGTCAGTA (SEQ ID NO: 4).

[0144] (2) Establishment of a liver-specific knockdown INHBC mouse model: Five-week-old C57BL / 6J mice were injected with 5×10⁻⁶ mmol / L via the tail vein. 11 A liver-specific knockdown mouse model of INHBC was constructed using GC / mL AAV8-shINHBC virus as the experimental group, and mice were injected with 5×10⁶ GC / mL AAV8-shINHBC virus. 11 A GC / mL AAV8-shScramble virus (without shINHBC interference sequence) was used as a control group.

[0145] (3) High-fat diet-induced steatosis in mouse liver: See Example 1 for details.

[0146] (4) Detection indicators: Please refer to Example 1 for details.

[0147] 2. Experimental Results

[0148] The results are shown in Figure 4. qPCR and Western blot results showed that liver-specific knockdown of INHBC significantly decreased the expression levels of INHBC mRNA and protein in the liver (Figure 4, A and B), exacerbated hepatic steatosis in mice (Figure 4, C), and increased the liver weight to body weight ratio (Figure 4, D). Liver-specific knockdown of INHBC significantly increased triglyceride levels in both the liver and serum (Figure 4, E and F), and significantly impaired liver function in mice (Figure 4, G and H). Furthermore, H&E staining, Oil Red O staining, and high-resolution microCT scans of small animals all showed that liver-specific knockdown of INHBC aggravated high-fat diet-induced lipid accumulation in the liver of mice (Figure 4, I and J).

[0149] The above results indicate that liver-specific knockdown of INHBC exacerbates lipid accumulation in the liver of mice induced by a high-fat diet.

[0150] Example 4: Effect of liver-specific transgenic knockout INHBC on high-fat diet-induced hepatic steatosis in mice

[0151] This embodiment investigated the effect of liver-specific transgene knockout INHBC on high-fat diet-induced hepatic steatosis in mice, specifically including the following:

[0152] 1. Experimental Methods

[0153] (1) Construction of liver-specific transgenic knockout INHBC mouse model: The INHBC gene was modified by fertilized egg homologous recombination. Specifically, Cas9 mRNA and gRNA were obtained by in vitro transcription, and then a homologous recombination vector (donor vector) was constructed by In-Fusion cloning, which contains a 3.0 kb 5' homologous arm, a 1.5 kb flox region, and a 3.0 kb 3' homologous arm. Finally, Cas9 mRNA, gRNA and donor vector were microinjected into fertilized eggs of C57BL / 6J mice to obtain positive mice. Then the homozygous mouse INHBC f / f was obtained, which was then crossed with Alb-Cre mice to obtain liver-specific transgenic knockout INHBC mice. LKO

[0154] (2) High-fat diet-induced liver steatosis in mice: See Example 2 for details.

[0155] (3) Detection index: See Example 1 for details.

[0156] 2. Experimental results

[0157] The detection results are shown in Figure 5. qPCR and Western blot results showed that after liver-specific transgenic knockout of INHBC, the expression levels of INHBC mRNA and protein in the liver were significantly decreased (as shown in Figures 5A and 5B), and the degree of liver steatosis in mice was aggravated (as shown in Figure 5C), and the ratio of liver weight to body weight increased (as shown in Figure 5D). After liver-specific transgenic knockout of INHBC, the content of triglyceride in the liver and serum was significantly increased (as shown in Figures 5E and 5F), and the liver function of mice was further impaired (as shown in Figures 5G and 5H). In addition, the results of H&E staining, oil red O staining and high-resolution small animal Micro CT detection all showed that liver-specific transgenic knockout of INHBC further aggravated the lipid accumulation in the liver of high-fat diet-induced mice (as shown in Figures 5I and 5J).

[0158] The above results show that liver-specific transgenic knockout of INHBC aggravates lipid accumulation in the liver of high-fat diet-induced mice.

[0159] Example 5: Effect of INHBC on lipid metabolism signaling pathway in high-fat diet-induced mice

[0160] To study the mechanism of INHBC in improving liver steatosis in mice, this example detected the effect of liver-specific overexpression or knock-in of INHBC on the lipid metabolism signaling pathway in the liver of high-fat diet mice, which included the following contents.​

[0161] 1. Experimental method

[0162] This example relates to the construction of a liver-specific overexpression and knock-in INHBC mouse model: refer to Examples 1 and 2 for details. After injection of the relevant viral vectors for three weeks or 5-week-old transgenic mice for 13 weeks, the mice were euthanized, and the liver RNA was extracted for transcriptome sequencing, and the expression levels of mRNA and protein of lipid metabolism-related genes in the mouse liver were detected.

[0163] 2. Experimental results

[0164] The detection results are shown in Figure 6. GO enrichment analysis found that fatty acid metabolism and lipid metabolism pathways were significantly enriched (as shown in Figure 6A). The results of bioinformatics analysis were verified by qPCR experiments, and it was found that compared with the control group, the expression levels of mRNA of lipid de novo synthesis-related genes were significantly down-regulated after liver-specific overexpression or knock-in of INHBC (as shown in Figures 6B and 6C). KEGG enrichment analysis further found that INHBC may regulate the AMPK signaling pathway (as shown in Figure 6D). Western Blot results showed that after liver-specific overexpression or knock-in of INHBC, the phosphorylated protein level of AMPK was up-regulated (as shown in Figures 6E and 6F), and the expression levels of lipid de novo synthesis-related proteins FASN, SCD1 and ACC1, and lipid droplet formation proteins CIDEA and CIDEC were significantly down-regulated (as shown in Figures 6G and 6H).

[0165] The above results show that INHBC may improve lipid metabolism disorder in the liver of mice induced by high-fat diet through the AMPK-SREBP1c pathway.

[0166] Example 6: Effect of overexpression of INHBC on free fatty acid-induced lipid deposition in primary hepatocytes

[0167] This example detects the effect of overexpression of INHBC on lipid synthesis in free fatty acid-induced mouse primary hepatocytes, which includes the following contents.

[0168] 1. Experimental method

[0169] (1) Extraction of mouse primary hepatocytes:

[0170] S1, spread mouse tail collagen: dilute 100x mouse tail collagen to 1x with PBS, plate in 6-well plates (0.5 mL per well) or 24-well plates (150 μL per well), and preheat PBS, medium and complete medium in a 37°C water bath. Before plating the primary hepatocytes, wash the 6-well or 24-well plates twice with PBS and reserve for use.

[0171] S2, clean the peristaltic pump and remove air: clean twice in the order of ultrapure water and 75% alcohol, and remove air in the tube. Buffer (prepared from D-glucose 1.8 g / L, magnesium chloride 0.0468 g / L, potassium chloride 0.34 g / L, sodium chloride 7.0 g / L, disodium hydrogen phosphate 0.1 g / L, sodium dihydrogen phosphate 0.18 g / L, and sodium bicarbonate 1.26 g / L) is loaded into a 50 mL centrifuge tube and preheated in a 37°C water bath.

[0172] S3, perfusion and digestion: inject anesthetic into the abdominal cavity of the mouse, and perform perfusion (about 100 mL buffer per mouse) after anesthesia. Dissolve 30 mg collagenase II in 50 mL buffer, and divide into two centrifuge tubes, each adding 1.125 mL of 100 mM calcium chloride, and supplementing with buffer to 30 mL. When about 5 mL of buffer remains, add the collagenase solution to digest the liver cells, and after digestion, turn off the peristaltic pump, separate the liver in a biological safety cabinet, filter with a 100 μm cell strainer, and centrifuge at 750 rpm for 4 minutes, and discard the supernatant.

[0173] S4, live cell screening: prepare a Percoll solution (30 mL in total) in a ratio of PBS:Percoll=2:1, and add to the centrifuge tube containing the liver cells to separate live cells and dead cells. After centrifugation at 750 rpm for 4 minutes, resuspend with 30 mL of medium, and centrifuge again at 750 rpm for 4 minutes, and discard the supernatant.

[0174] S5, cell counting: resuspend with 10 mL of complete medium, and perform cell counting, and plate according to the required cell density.

[0175] (2) Cell viral transfection:

[0176] Thaw the virus liquid on ice, and gently pipette to mix the thawed virus particles, and then take an appropriate amount of Ad-GFP (control group) or Ad-INHBC (experimental group) virus liquid into an appropriate amount of medium (about 100 μL / cm 2 ), and gently mix. The initial MOI for transduction of cells is 5, the original culture dish is washed to remove dead cells, the original medium is aspirated, the medium containing the virus is added, the virus liquid is gently shaken to cover each cell, and the culture plate is placed in a 37°C, 5% carbon dioxide incubator for 6 hours. Change the medium, aspirate the medium containing the virus, add complete medium, and place in a 37°C, 5% carbon dioxide incubator for 48 hours. Observe the fluorescence effect under a fluorescence microscope to check whether the transfection is successful.

[0177] (3) Construction of an MASLD in vitro model:

[0178] For example, using a six-well plate, plate the mouse liver primary cells at 1.2 x 10 6The cells were placed in a six-well plate and incubated overnight in a 37°C carbon dioxide incubator. Then the cells were washed twice with PBS, and Ad-GFP or Ad-INHBC virus (the nucleotide sequence of INHBC is shown in SEQ ID NO: 1) diluted in culture medium was added, and after 6 hours of infection, the complete culture medium was replaced. After 48 hours of virus infection, free fatty acids (prepared from oleic acid and palmitic acid at a ratio of 2: 1) at a working concentration of 1 mM were added to each well, and the cells were treated for 6 hours, 12 hours or 24 hours to induce the MASLD model. Finally, the expression levels of the relevant genes in the cells were detected.

[0179] 2. Experimental results

[0180] The results are shown in Figure 7. The qPCR results showed that the expression level of INHBC mRNA in the mouse primary hepatocytes was significantly increased after overexpression of INHBC (as shown in Figure 7A), and the content of triglyceride in the primary hepatocytes and the expression level of the mRNA of the fatty acid synthesis-related gene were significantly decreased (as shown in Figures 7B and 7C), the oil red O staining results showed that the lipid droplets in the cells were significantly reduced after overexpression of INHBC (as shown in Figure 7D), the phosphorylated protein level of AMPK in the mouse primary hepatocytes was up-regulated, and the expression level of SREBP1c protein was down-regulated (as shown in Figure 7E). In addition, the mouse primary hepatocytes were treated with free fatty acids for 6 hours, 12 hours and 24 hours to construct the MASLD in vitro model, and the qPCR results showed that the INHBC mRNA level was significantly decreased after 12 hours and 24 hours of treatment (as shown in Figure 7F), and the Western Blot results showed that the INHBC protein level also showed a downward trend (as shown in Figure 7G).

[0181] The above results show that overexpression of INHBC can inhibit the lipid synthesis of mouse primary hepatocytes induced by free fatty acids.

[0182] Example 7: Effect of knockdown of INHBC on free fatty acid-induced lipid deposition in primary hepatocytes

[0183] This example detects the effect of knockdown of INHBC on the lipid synthesis of mouse primary hepatocytes induced by free fatty acids, which specifically includes the following contents.

[0184] 1. Experimental method

[0185] (1) The extraction of mouse primary hepatocytes, virus infection and the method for constructing the MASLD in vitro are specifically referred to in Example 6, and the nucleotide sequences are shown in SEQ ID NO: 2-SEQ ID NO: 4.

[0186] 2. Experimental results

[0187] The detection results are shown in FIG. 8. The qPCR detection results show that the expression level of INHBC mRNA in the mouse primary hepatocytes is reduced by about 50% after knocking down INHBC (as shown in A of FIG. 8), the content of triglyceride in the primary hepatocytes and the expression levels of the mRNA of the fatty acid synthesis related genes are significantly up-regulated (as shown in B and C of FIG. 8), and the oil red O staining results show that knocking down INHBC promotes the lipid accumulation induced by free fatty acids (as shown in D of FIG. 8). Similarly, after knocking down INHBC, the phosphorylation level of the AMPK protein in the mouse primary hepatocytes is inhibited, and the expression of the SREBP1c protein is promoted (as shown in E of FIG. 8).

[0188] The above results show that knocking down INHBC promotes the lipid generation of the mouse primary hepatocytes induced by free fatty acids.

[0189] Example 8: Effect of INHBC on LKB1-AMPK signal axis

[0190] In this example, the downstream signal pathway of INHBC is detected to verify whether the regulation of INHBC on lipid metabolism depends on the LKB1-AMPK signal pathway, which specifically includes the following contents.

[0191] 1. Experimental method

[0192] (1) Extracting primary hepatocytes from INHBC WT The primary hepatocytes are extracted from the mouse, and the purpose of overexpressing INHBC is achieved by Cre adenovirus, and free fatty acids and the AMPK inhibitor Compound C (10 μM) are given for treatment for 24 hours.

[0193] (2) Extraction of mouse primary hepatocytes, infection of viruses, and construction of MASLD in vitro: For details, refer to the description in Example 6.

[0194] 2. Experimental results

[0195] The detection results are shown in FIG. 9 and FIG. 10. After overexpressing INHBC, the content of triglyceride in the primary hepatocytes and the expression levels of the mRNA and protein of the lipid metabolism related genes are significantly reduced, but after adding Compound C, the improvement effect of INHBC is reversed (as shown in A-C of FIG. 9), which indicates that INHBC regulates lipid synthesis through the AMPK signal pathway. In order to confirm whether the upstream kinase of AMPK is LKB1, the primary hepatocytes are extracted from the INHBC WTPrimary hepatocytes were extracted from mice and treated with Cre adenovirus, free fatty acids and LKB1 inhibitor Pim1 (5 mM) for 24 hours. The results showed that LKB1 inhibitor could reverse the improvement of INHBC on lipid deposition (as shown in D and F of FIG. 9). The phosphorylation level of LKB1 protein in the liver of mice with liver-specific overexpression or knock-in of INHBC was then detected, and the results showed that INHBC up-regulated the phosphorylation level of LKB1 protein (as shown in G and H of FIG. 9).

[0196] In addition, consistent results were also obtained in primary hepatocytes in vitro. After overexpression of INHBC, the phosphorylation levels of LKB1 and AMPK proteins were up-regulated; after knockdown of INHBC, the phosphorylation levels of LKB1 and AMPK proteins were down-regulated (as shown in A and B of FIG. 10). The cell localization of LKB1 was then detected, and the results showed that after overexpression of INHBC, LKB1 exited the nucleus into the cytoplasm, and the phosphorylation level increased; while after knockdown of INHBC, the opposite results were obtained (as shown in C and D of FIG. 10).

[0197] The above results showed that INHBC activated LKB1, up-regulated the phosphorylation level of LKB1 protein, and then activated the AMPK signaling pathway to improve liver steatosis induced by high-fat diet.

[0198] Example 9: Effect of liver-specific overexpression of INHBC on improving blood glucose level and glucose tolerance of mice

[0199] This example explores the effect of liver-specific overexpression of INHBC on improving blood glucose level and glucose tolerance of mice, which specifically includes the following contents.

[0200] 1. Experimental method

[0201] (1) The overexpression virus vector and liver-specific overexpression of INHBC mouse model involved in this example were constructed, which was specifically referred to in Example 1.

[0202] (2) All mice were given normal diet and water, and the relevant indicators were detected after 3 weeks.

[0203] (3) Pyruvate tolerance test: the mice were fasted overnight for 16 hours, and then given sodium pyruvate by intraperitoneal injection. The dose of sodium pyruvate in non-diabetic model mice was 2 g / kg; in HFD-STZ model mice, it was 1 g / kg; in db / db mice, it was 0.5 g / kg. At 0, 15, 30, 45, 60, 90 and 120 minutes after injection, the blood glucose level was measured by tail blood sampling.

[0204] (4) Glucagon tolerance test: Mice were fasted for 6 hours, then given glucagon by intraperitoneal injection. The dose of glucagon in non-diabetic and HFD-STZ model mice was 10 μg / kg; in db / db mice, it was 5 μg / kg. At 0, 15, 30, 45, 60, 90 and 120 minutes after injection, blood glucose levels were measured by tail blood sampling.

[0205] 2. Experimental results

[0206] The results are shown in Figure 11. After liver-specific overexpression of INHBC, qPCR and Western Blot results showed that the expression levels of INHBC mRNA and protein in mouse liver were significantly increased (as shown in A and B of Figure 11), the fasting blood glucose levels of mice for 6 or 16 hours were significantly reduced (as shown in C of Figure 11), and the glucagon tolerance and pyruvate tolerance tests showed that the blood glucose levels of mice were significantly reduced and the area under the curve was significantly reduced (as shown in D and E of Figure 11). In addition, the mRNA and protein expression levels of liver gluconeogenesis-related genes were significantly down-regulated (as shown in F-I of Figure 11).

[0207] The above results show that liver-specific overexpression of INHBC can reduce blood glucose levels and improve glucose tolerance in mice.

[0208] Example 10: Effect of liver-specific knockdown of INHBC on blood glucose levels and glucose tolerance in mice

[0209] This example investigates the effect of liver-specific knockdown of INHBC on blood glucose levels and glucose tolerance in mice, which includes the following contents.

[0210] 1. Experimental methods

[0211] (1) The knockdown viral vector and liver-specific INHBC knockdown mouse model involved in this example are constructed as described in Example 3.

[0212] (2) All mice were given normal diet and water, and the relevant indicators were detected after 3 weeks.

[0213] (3) Detection method of pyruvate tolerance test and glucagon tolerance test: refer to Example 9.

[0214] 2. Experimental results

[0215] The detection results are shown in Figure 12. After liver-specific knockdown of INHBC, qPCR and Western Blot results showed that the expression levels of INHBC mRNA and protein in the liver of mice were significantly reduced (as shown in Figures 12A and 12B), the fasting blood glucose levels of mice were significantly increased (as shown in Figure 12C), and the glucagon tolerance and pyruvate tolerance tests showed that the blood glucose levels of mice were significantly increased and the area under the curve was significantly increased (as shown in Figures 12D and 12E). The expression levels of liver gluconeogenesis-related proteins were significantly increased (as shown in Figure 12F).

[0216] The above results show that liver-specific knockdown of INHBC can cause glucose metabolism disorder in mice.

[0217] Example 11: Effect of liver-specific overexpression of INHBC on improving blood glucose level and glucose tolerance of db / db diabetic mice

[0218] This example investigates the effect of liver-specific overexpression of INHBC on improving blood glucose level and glucose tolerance of db / db diabetic mice, which specifically includes the following contents.

[0219] 1. Experimental method

[0220] (1) db / db diabetic mouse model: 4-week-old male BKS-db / db (genotype (Lepr) KO / KO) and BKS-db / m (Lepr) KO / wt) mice were purchased from Jiangsu Jiqie Pharmaceutical Biotechnology Co., Ltd. and adapted to the environment for one week. The blood glucose level was detected, and the mice meeting the standard were included in the experiment.

[0221] (2) Construction of liver-specific overexpression of INHBC db / db mouse model: 5-week-old BKS-db mice were taken, and 1×10 11 GC / mL of AAV8-INHBC virus was used to construct a liver-specific overexpression of INHBC db / db mouse model as the experimental group, and 1×10 11 GC / mL of AAV8-GFP virus was used as the control group. All mice were given normal diet and water.

[0222] (3) Pyruvate tolerance and glucagon tolerance test: refer to the description in Example 9.

[0223] (4) Insulin tolerance test: the mice were fasted for 6 hours, and then insulin was given by intraperitoneal injection. The dose of insulin in non-diabetic model mice was 1 U / kg; the dose in HFD-STZ model mice was 2 U / kg; and the dose in db / db mice was 2 U / kg. At 0, 15, 30, 45, 60, 90 and 120 minutes after injection, the blood glucose level was measured by tail blood collection.

[0224] (5) Glucose tolerance test: Mice were fasted overnight for 16 hours, then given sodium pyruvate by intraperitoneal injection. The dose of glucose in non-diabetic model mice was 2 g / kg; in HFD-STZ model mice, it was 1 g / kg; in db / db mice, it was 0.5 g / kg. Blood glucose levels were measured by tail blood sampling at 0, 15, 30, 45, 60, 90 and 120 minutes after injection.

[0225] 2. Experimental results

[0226] The results are shown in Figure 13. After liver-specific overexpression of INHBC, qPCR and Western Blot results showed that the mRNA and protein expression levels of INHBC in the liver of db / db mice were significantly up-regulated (as shown in Figures 13 A and B), regular monitoring of the blood glucose levels of mice found that the random blood glucose levels of db / db mice with liver-specific overexpression of INHBC were significantly lower than those of control mice every week (as shown in Figure 13 C), the fasting blood glucose levels of mice for 6 or 16 hours were significantly reduced (as shown in Figure 13 D), and the glucagon tolerance, pyruvate tolerance, insulin tolerance and glucose tolerance tests all showed that the blood glucose levels of mice were significantly reduced and the area under the curve was significantly reduced (as shown in Figures 13 E-H). The mRNA and protein expression levels of liver gluconeogenesis-related genes were significantly down-regulated (as shown in Figures 13 I and J).

[0227] The above results show that liver-specific overexpression of INHBC can reduce the blood glucose levels and improve the glucose tolerance of db / db mice.

[0228] Example 12: Effect of liver-specific transgenic knock-in of INHBC on blood glucose levels and glucose tolerance of high-fat diet combined with streptozotocin-induced diabetic mice

[0229] This example detects the effect of liver-specific transgenic knock-in of INHBC on the blood glucose levels and glucose tolerance of high-fat diet combined with streptozotocin-induced diabetic mice, which specifically includes the following contents.

[0230] 1. Experimental method

[0231] (1) Construction of liver-specific transgenic knock-in INHBC mouse model: refer to Example 2 for details.

[0232] (2) Feeding of model mice: Five-week-old male C57BL / 6J mice were selected and fed a high-fat diet for 12 weeks to induce insulin resistance and obesity models. After fasting for 4 hours, streptozotocin was injected intraperitoneally with freshly prepared 0.1M citrate buffer (pH 4.5) at a final concentration of 50 mg / kg body weight once a day for 5 consecutive days. On days 3-7 after the last injection, fasting blood glucose was measured after fasting for 6 hours. Only mice with a blood glucose concentration ≥11.1 mM were included in the experiment.

[0233] (3) Pyruvate tolerance, glucagon tolerance, insulin tolerance and glucose tolerance tests: see Examples 9 and 11 for details.

[0234] 2. Experimental Results

[0235] The results are shown in Figure 14. qPCR and Western blot results indicated that after liver-specific transgene knock-in of INHBC, the expression levels of INHBC mRNA and protein in mouse liver were significantly increased (as shown in A and B of Figure 14). Compared with the control group, fasting blood glucose levels at 6 or 16 hours were significantly reduced in mice (as shown in C of Figure 14). Glucagon tolerance, pyruvate tolerance, insulin tolerance, and glucose tolerance tests all showed significantly reduced blood glucose levels and significantly decreased areas under the curve (as shown in DG of Figure 14). The expression levels of liver gluconeogenesis-related genes mRNA and protein were significantly downregulated (as shown in H and I of Figure 14).

[0236] The above results indicate that liver-specific transgene knock-in of INHBC can improve glucose metabolism disorder induced by a high-fat diet combined with streptozotocin in diabetic mice, reduce blood glucose levels, and improve glucose tolerance.

[0237] Example 13: Effects of liver-specific transgenic knockout INHBC on blood glucose levels and glucose tolerance in high-fat diet combined with streptozotocin-induced diabetic mice

[0238] This embodiment investigated the effect of liver-specific transgene knockout INHBC on glucose metabolism in diabetic mice induced by a high-fat diet combined with streptozotocin, specifically including the following:

[0239] 1. Experimental Methods

[0240] (1) Constructing a liver-specific transgene knockout INHBC mouse model: Refer to Example 4 for details.

[0241] (2) Feeding of model mice: Please refer to Example 12 for details.

[0242] (3) Detection methods for pyruvate tolerance, glucagon tolerance and glucose tolerance tests: Please refer to Examples 9 and 11 for details.

[0243] 2. Experimental results

[0244] The results are shown in Figure 15. The qPCR and Western Blot results showed that the expression levels of INHBC mRNA and protein in the liver of mice were significantly reduced after liver-specific transgenic knockout of INHBC (as shown in A and B of Figure 15). The blood glucose levels of mice were significantly increased after fasting for 6 or 16 hours (as shown in C of Figure 15), and the results of glucagon tolerance, pyruvate tolerance and glucose tolerance tests showed that the blood glucose levels of mice were significantly increased and the area under the curve was significantly increased (as shown in D-F of Figure 15), and the mRNA and protein expression levels of liver gluconeogenesis-related genes were significantly up-regulated (as shown in G and H of Figure 15).

[0245] The above results show that liver-specific transgenic knockout of INHBC can promote gluconeogenesis in diabetic mice induced by high-fat diet combined with streptozotocin, significantly increase the blood glucose level of the body, and impair the glucose tolerance of the body.

[0246] Example 14: Effect of overexpression of INHBC on glucose production in mouse primary hepatocytes

[0247] This example detects the effect of overexpression of INHBC on glucose production in mouse primary hepatocytes, which specifically includes the following contents.

[0248] 1. Experimental method

[0249] (1) Extraction of mouse primary hepatocytes and method of viral transfection of cells: refer to the description in Example 6 for details.

[0250] (2) Glucose content determination: after washing twice with PBS, the cells were incubated in glucose production buffer (consisting of glucose-free medium (pH 7.4) without phenol red, supplemented with 20 mM sodium lactate, 2 mM sodium pyruvate and 1 mM glycerol) containing 100 nM glucagon for 4 hours. The culture medium was collected, and the glucose concentration was detected using a colorimetric glucose assay kit.

[0251] 2. Experimental results

[0252] The detection results are shown in Figure 16. The qPCR results show that the expression level of INHBC mRNA in the mouse primary hepatocytes is increased by nearly 20,000 times after overexpression of INHBC (as shown in Figure 16A), the expression level of INHBC protein is also significantly increased (as shown in Figure 16B), the expression levels of gluconeogenesis-related gene mRNA and protein are significantly decreased (as shown in Figures 16C and 16D), and the production of glucose is significantly reduced (as shown in Figure 16E). In addition, the production of glucose is significantly reduced under the stimulation of glucagon (as shown in Figure 16F), and it is found that the expression levels of gluconeogenesis-related gene mRNA are all significantly decreased (as shown in Figures 16G and 16H).

[0253] The above results show that overexpression of INHBC can reduce the production of glucose in mouse primary hepatocytes.

[0254] Example 15: Effect of knockdown of INHBC on the production of glucose in mouse primary hepatocytes

[0255] This example detects the effect of knockdown of INHBC on the production of glucose in mouse primary hepatocytes, which specifically includes the following contents.

[0256] 1. Experimental method

[0257] (1) Extraction of mouse primary hepatocytes and cell viral transfection method: refer to the description in Example 6.

[0258] (2) Determination of glucose content: refer to the description in Example 14.

[0259] 2. Experimental results

[0260] The detection results are shown in Figure 17. The qPCR results show that the expression level of INHBC mRNA in the mouse primary hepatocytes is decreased by about 70% after knockdown of INHBC (as shown in Figure 17A), the expression level of INHBC protein is significantly decreased (as shown in Figure 17B), the expression levels of gluconeogenesis-related gene mRNA and protein are significantly increased (as shown in Figures 17C and 17D), the production of glucose is significantly increased (as shown in Figure 17E), the production of glucose is significantly increased under the stimulation of glucagon (as shown in Figure 17F), and it is found that the expression levels of gluconeogenesis-related gene mRNA are all significantly increased (as shown in Figures 17G and 17H).

[0261] The above results show that knockdown of INHBC can increase the production of glucose in mouse primary hepatocytes.

[0262] Example 16: Effect of liver-specific overexpression of INHBC on high-fat diet-induced obesity in mice

[0263] This example detects the effect of liver-specific overexpression of INHBC on high-fat diet-induced obesity in mice, which specifically includes the following.

[0264] 1. Experimental method

[0265] (1) This example relates to the construction of a virus vector overexpressing and a liver-specific INHBC overexpressing mouse model: please refer to Example 1 for details.

[0266] (2) After injection of the relevant virus vector for three weeks, the mice were fed with a high-fat diet for 10 weeks, during which the body weight, body composition and other physical indicators of the mice were monitored, and then the mice were euthanized, and liver and adipose tissue samples were extracted, and the expression levels of lipid metabolism-related genes and proteins in the adipose tissue of the mice were detected.

[0267] (3) Comprehensive energy metabolism monitoring system for experimental animals: Comprehensive Laboratory Animal Monitoring System (CLAMS) was used. Specifically, the mice were individually housed in metabolic chambers under a 12 / 12 hour light / dark cycle. Mice had free access to food and water. The system can detect oxygen consumption, carbon dioxide output, energy consumption, total locomotor activity and food intake in each individual cage.

[0268] 2. Experimental results

[0269] The detection results are shown in Figures 18, 19 and 20, and the qPCR and Western Blot results show that after liver-specific overexpression of INHBC, the mRNA and protein levels of INHBC in the liver of the mice are significantly up-regulated (as shown in A and B of Figure 18); the body weight of the liver-specific INHBC overexpressing mice is significantly reduced (as shown in C and D of Figure 18), but the daily food intake is not different (as shown in E of Figure 18); the body composition analysis results show that the liver-specific INHBC overexpressing mice have reduced fat accumulation in the body, but the lean body mass does not have significant difference (as shown in F of Figure 18); the results of monitoring the rectal temperature of the mice at different time points in a 4℃ environment show that the liver-specific INHBC overexpressing mice exhibit better cold tolerance, with higher rectal temperature than the control group (as shown in G of Figure 18). The results of the laboratory animal monitoring system show that the liver-specific INHBC overexpressing mice exhibit high oxygen consumption rate and energy consumption (as shown in H-K of Figure 18).

[0270] In addition, the liver-specific overexpression of INHBC mice showed less fat accumulation, and the weights of epididymal fat and subcutaneous fat tissues were significantly reduced (as shown in A of FIG. 19), according to the pathological result analysis, the cell size of the inguinal white adipose tissue of the liver-specific overexpression of INHBC mice was significantly reduced (as shown in B of FIG. 19), the qPCR results showed that the mRNA expression levels of heat production, lipid hydrolysis and fatty acid β oxidation related genes in the inguinal white adipose and brown adipose tissues of the liver-specific overexpression of INHBC mice were significantly increased, while the mRNA expression levels of lipid synthesis related genes were relatively reduced (as shown in C and D of FIG. 19), and the Western Blot results showed that the protein expression levels of mitochondrial respiratory chain complex, p-HSL, DIO2 and UCP1 in the inguinal white adipose and brown adipose tissues of the liver-specific overexpression of INHBC mice were significantly increased (as shown in A and B of FIG. 20).

[0271] The above results show that the liver-specific overexpression of INHBC can inhibit high-fat diet-induced obesity by enhancing the thermogenic activity of mouse adipose tissue.

[0272] Example 17: Effect of liver-specific transgenic knock-in INHBC on high-fat diet-induced obesity in mice

[0273] This example detects the effect of liver-specific transgenic knock-in INHBC on high-fat diet-induced obesity in mice, which specifically includes the following contents.

[0274] 1. Experimental method

[0275] (1) Constructing a liver-specific transgenic knock-in INHBC mouse model: for details, refer to the description in Example 2.

[0276] (2) Take 8-week-old mice, feed them with high-fat diet for 10 weeks, monitor the body weight, body composition and other body indicators of the mice during the period, then euthanize the mice, extract liver and fat sample tissues, and detect the expression levels of lipid metabolism related genes and proteins in the fat tissues of the mice.

[0277] 2. Experimental results

[0278] The results are shown in Figures 21, 22 and 23. The qPCR and Western Blot results showed that the expression levels of INHBC mRNA and protein in the liver of the mice were significantly increased after the liver-specific transgenic knock-in of INHBC (as shown in A-B of Figure 21). Compared with the control group, the body weight of the mice with liver-specific transgenic knock-in of INHBC was significantly decreased, and the body composition analysis showed that the fat weight was significantly reduced (as shown in C-F of Figure 21). The rectal temperature of the mice was monitored at different time points under a 4°C environment, and the results showed that the mice with liver-specific transgenic knock-in of INHBC exhibited stronger cold tolerance, and the rectal temperature was significantly higher than that of the control group (as shown in G of Figure 21). The results of the experimental animal energy monitoring system further showed that the mice with liver-specific transgenic knock-in of INHBC had higher oxygen consumption rate and energy consumption level (as shown in H-K of Figure 21).

[0279] In addition, the mice exhibited less fat accumulation, and the weights of epididymal fat and subcutaneous fat were significantly reduced (as shown in A of Figure 22). According to the analysis of the pathological results, the cell size of the inguinal white adipose tissue of the mice with liver-specific transgenic knock-in of INHBC was significantly reduced (as shown in B of Figure 22). The qPCR detection results showed that the mRNA expression levels of the genes related to thermogenesis, lipid hydrolysis and fatty acid β-oxidation in the inguinal white adipose tissue and brown adipose tissue of the mice with liver-specific transgenic knock-in of INHBC were significantly up-regulated, while the mRNA expression levels of the genes related to lipid synthesis were decreased (as shown in C and D of Figure 22). The Western Blot detection results showed that the expression levels of the proteins related to energy metabolism, such as mitochondrial respiratory chain complex, p-HSL, DIO2 and UCP1, in the inguinal white adipose tissue and brown adipose tissue of the mice were significantly increased (as shown in A and B of Figure 23).

[0280] The above results showed that the liver-specific transgenic knock-in of INHBC could enhance the thermogenic function of adipose tissue and increase energy consumption, thereby alleviating the obesity induced by high-fat diet.

[0281] Example 18: Effect of liver-specific knockdown of INHBC on high-fat diet-induced obesity in mice

[0282] This example detected the effect of liver-specific knockdown of INHBC on high-fat diet-induced obesity in mice, which specifically included the following contents.

[0283] 1. Experimental method

[0284] (1) The knockdown virus vector and the liver-specific knock-in INHBC mouse model involved in this example were constructed, which were specifically described in Example 3.

[0285] (2) Experimental animal comprehensive energy metabolism monitoring system setting: refer to Example 16 for details.

[0286] 2、Experimental results

[0287] The detection results are shown in Figures 24, 25 and 26. The qPCR and Western Blot results show that after liver-specific knockdown of INHBC, the mRNA and protein expression levels of INHBC in the liver of mice are significantly down-regulated (as shown in A and B of Figure 24); the body weight of the liver-specific INHBC knockdown mice is significantly increased (as shown in C and D of Figure 24), but there is no difference in daily food intake (as shown in E of Figure 24), and the body composition analysis shows that the fat accumulation in the liver-specific INHBC knockdown mice is significantly increased, but there is no significant difference in lean body mass (as shown in F of Figure 24), and the results of monitoring the rectal temperature of mice placed in a 4℃ environment at different time points show that the cold tolerance of liver-specific INHBC knockdown mice is significantly decreased, and the rectal temperature is lower than that of the control group (as shown in G of Figure 24). The results of the experimental animal energy monitoring system show that the liver-specific INHBC knockdown mice exhibit low oxygen consumption rate and energy consumption (as shown in H-K of Figure 24).

[0288] In addition, the liver-specific INHBC knockdown mice exhibit more fat accumulation, and the weights of epididymal fat and subcutaneous fat are significantly increased (as shown in A of Figure 25), and according to the analysis of pathological results, the cell size of the inguinal white adipose tissue and brown adipose tissue of the liver-specific INHBC knockdown mice is significantly increased (as shown in B of Figure 25), and the qPCR results show that the mRNA expression levels of the heat production, lipid hydrolysis and fatty acid beta oxidation related genes in the inguinal white adipose tissue and brown adipose tissue of the liver-specific INHBC knockdown mice are significantly decreased, while the mRNA levels of the lipid synthesis related genes are relatively increased (as shown in C and D of Figure 25). The Western Blot results show that the protein expression levels of the mitochondrial respiratory chain complex, p-HSL, DIO2 and UCP1 in the inguinal white adipose tissue and brown adipose tissue of mice are significantly decreased (as shown in A and B of Figure 26).

[0289] The above results show that liver-specific knockdown of INHBC can lead to down-regulation of the heat production level of adipose tissue in mice, thereby aggravating high-fat diet induced obesity.

[0290] Example 19: Effect of liver-specific transgenic knock-out of INHBC on high-fat diet induced obesity in mice

[0291] This example detects the effect of liver-specific transgenic knock-out of INHBC on high-fat diet induced obesity in mice, which specifically includes the following contents.

[0292] 1、Experimental method

[0293] (1) Construction of liver-specific transgenic knockout INHBC mouse model: refer to Example 4 for details.

[0294] (2) Experimental animal comprehensive energy metabolism monitoring system setup: refer to Example 16 for details.

[0295] 2. Experimental results

[0296] The results are shown in Figures 27, 28 and 29. The qPCR and Western Blot results showed that after liver-specific transgenic knockout of INHBC, the mRNA and protein of INHBC in the liver of mice were almost not expressed (as shown in A-B of Figure 27), the body weight of liver-specific transgenic knockout INHBC mice was significantly increased, but there was no difference in daily food intake (as shown in C-E of Figure 27), and the body composition analysis showed that the fat accumulation in the liver-specific transgenic knockout INHBC mice was significantly increased, while the lean body mass had no significant difference (as shown in F of Figure 27). The results of monitoring the rectal temperature of mice placed in a 4°C environment at different time points showed that the cold tolerance of liver-specific transgenic knockout INHBC mice was significantly impaired, and the rectal temperature was lower than that of the control group (as shown in G of Figure 27). The results of the experimental animal energy monitoring system showed that the liver-specific transgenic knockout INHBC mice showed low oxygen consumption rate and energy consumption (as shown in H-K of Figure 27).

[0297] In addition, the liver-specific transgenic knockout INHBC mice showed more fat accumulation, and the weight of epididymal fat and subcutaneous fat was significantly increased (as shown in A of Figure 28), according to the analysis of pathological results, the cell size of inguinal white adipose tissue and brown adipose tissue of liver-specific transgenic knockout INHBC mice was significantly increased (as shown in B of Figure 28), the qPCR results showed that the mRNA expression levels of heat production, lipid hydrolysis and fatty acid beta oxidation related genes in the inguinal white adipose tissue and brown adipose tissue of liver-specific transgenic knockout INHBC mice were down-regulated, while the mRNA levels of lipid synthesis related genes were relatively increased (as shown in C and D of Figure 28), and the Western Blot results showed that the protein expression levels of mitochondrial respiratory chain complex, p-HSL, DIO2 and UCP1 in the inguinal white adipose tissue and brown adipose tissue of liver-specific transgenic knockout INHBC mice were significantly decreased (as shown in A and B of Figure 29).

[0298] The above results showed that liver-specific transgenic knockout of INHBC could lead to down-regulation of heat production in adipose tissue of mice, thereby aggravating high-fat diet-induced obesity.

[0299] Example 20: Effect of INHBC purified protein on thermogenic level of mouse primary adipocytes

[0300] This example detects the effect of INHBC purified protein on the thermogenic level of mouse primary adipocytes, which specifically includes the following contents.

[0301] 1. Experimental method

[0302] (1) Extraction of mouse primary adipocytes:

[0303] Select 7-day-old young mice, cut the back skin, and remove the white and brown adipose tissue subcutaneously, respectively. Add collagenase I for digestion, centrifuge after digestion, and culture in DMEM / F12 medium containing 10% (v / v) FBS. After reaching 100% confluence, induce cell differentiation by replacing the culture medium with induction medium (containing 10% FBS, 0.5mM IBMX, 125μM indomethacin, 1μM dexamethasone, 1nM T3 and 20nM insulin). After two days of induction, culture the cells in differentiation medium (containing 10% FBS, 1nM T3 and 20nM insulin). Change the cells to fresh differentiation medium every 2 days until complete differentiation.

[0304] (2) Recombinant protein acquisition:

[0305] S1, Obtain INHBC gene sequence: Obtain the nucleotide sequence of the INHBC gene from public databases (such as NCBI).

[0306] S2, primer design and PCR amplification: 1) primer design: design specific primers for the INHBC gene, ensuring that the primers contain appropriate restriction enzyme sites for subsequent cloning, for example, NdeI and Xhol are commonly used restriction enzyme sites; 2) PCR amplification: use appropriate template DNA (such as cDNA library) and designed primers to amplify the INHBC gene by PCR.

[0307] S3, cloning into expression vector: 1) selection of expression vector: including pET series (for E. coli expression) and pCDNA series (for mammalian cell expression); 2) restriction enzyme digestion and ligation: restriction enzyme digestion of PCR-amplified INHBC gene and expression vector, insertion of INHBC gene into expression vector using DNA ligase; 3) transformation and screening: transform the recombinant plasmid into E. coli (such as DH5α), obtain positive clones by antibiotic screening, extract the plasmid and perform sequencing verification to ensure that the INHBC gene is correctly inserted and has no mutations.

[0308] S4, recombinant protein expression: 1) selection of expression system: E. coli has the advantages of high efficiency and low cost, yeast is suitable for eukaryotic expression, mammalian cells such as HEK293 or CHO cells are suitable for protein expression that requires complex post-translational modification; 2) transformation or transfection: transform the recombinant plasmid into the appropriate E. coli expression host (such as BL21(DE3)), transfect the recombinant plasmid into HEK293 or CHO cells; 3) expression induction: induce protein induction in E. coli by adding IPTG, and start expression in mammalian cells by adding specific inducers (such as tetracycline).

[0309] S5, protein purification: 1) cell lysis: collect cells expressing INHBC and lyse to release intracellular proteins; 2) preliminary purification: remove cell debris using centrifugation or filtration to obtain crude protein extract; 3) affinity purification: add His tag to the N or C terminus of the recombinant protein, use Ni-NTA affinity column for purification, load the crude extract onto the Ni-NTA column, bind the His-tagged INHBC protein, remove non-specifically bound proteins by washing with low-concentration imidazole, and elute the target protein with high-concentration imidazole; 4) further purification: further purify by ion exchange chromatography and gel filtration chromatography to improve protein purity and activity.

[0310] S6, verification of recombinant protein: amino acid sequencing analysis of the purified protein shows that the results are consistent with expectations, and the amino acid sequence information of the recombinant protein INHBC is as follows:

[0311] MTSSLLLAFLLLAPTTVATPRAGGQCPACGGPTLELESQRELLLDLAKRSILDKLHLTQRPTLNRPVSRAALRTALQHLHGVPQGALLEDNREQECEIISFAETGLSTINQTRLDFHFSSDRTAGDREVQQASLMFFVQLPSNTTWTLKVRVLVLGPHNTNLTLATQYLLEVDASGWHQLPLGPEAQAACSQGHLTLELVLEGQVAQSSVILGGAAHRPFVAARVRVGGKHQIHRRGIDCQGGSRMCCRQEFFVDFREIGWHDWIIQPEGYAMNFCIGQCPLHIAGMPGIAASFHTAVLNLLKANTAAGTTGGGSCCVPTAQRPLSLLYYDRDSNIVKTDIPDMVVEACGCS*(SEQ ID NO:5). Wherein "*" is the stop codon.

[0312] (3) Recombinant INHBC protein treatment:

[0313] After the primary adipocytes were matured, the purified recombinant protein was added into the maintenance medium (final concentration of 40 ng / mL), and the blank group was added with BSA (final concentration of 40 ng / mL). After 24 hours of treatment, the protein and RNA were collected for subsequent experimental operation.

[0314] (4) Mitochondrial fluorescence staining:

[0315] After the primary adipocytes were matured, the purified recombinant protein was added into the maintenance medium (final concentration of 40 ng / mL), and the blank group was added with BSA (final concentration of 40 ng / mL). After 24 hours of treatment, the protein and RNA were collected for subsequent experimental operation.

[0316] (5) Seahorse experiment:

[0317] The Agilent Seahorse XF was used to detect the energy metabolism of the primary adipocytes after the addition of the INHBC purified protein for stimulation. The primary adipocytes 1x10 4 cells were seeded in a 96-well plate specially used for Seahorse at a density of 1x10

[0318] 2. Experimental results

[0319] The detection results are shown in FIGS. 30 and 31. After the treatment of the INHBC purified protein (40 ng / mL) and / or CL316243 (40 nM), the mitochondrial fluorescence activity of the primary beige adipocytes and the primary brown adipocytes was enhanced (as shown in A and B of FIG. 30). The qPCR results showed that the mRNA expression levels of the related genes such as thermogenesis, lipid hydrolysis and fatty acid beta oxidation in the primary beige adipocytes and the primary brown adipocytes were significantly up-regulated (as shown in C and D of FIG. 30). The Western Blot results also showed that the protein expression levels of the mitochondrial respiratory chain complex, PGC1a, DIO2 and UCP1 were significantly up-regulated (as shown in A and B of FIG. 31). The Seahorse metabolic analysis results showed that the oxygen consumption rate and the maximum respiratory capacity of the primary beige adipocytes and the primary brown adipocytes were significantly enhanced after the treatment of the INHBC purified protein, which indicated that the mitochondrial respiratory function was significantly activated (as shown in C and D of FIG. 31).

[0320] The above results show that INHBC purified protein can increase the thermogenic level of mouse primary adipocytes.

[0321] Example 21: DSG1β is a specific receptor of INHBC on the surface of mouse primary adipocytes

[0322] This example verifies the experimental results that DSG1β is a specific receptor of INHBC on the surface of mouse primary adipocytes, and specifically includes the following.

[0323] 1. Experimental method

[0324] (1) To identify the potential membrane receptor of INHBC in primary beige adipocytes, two complementary experimental schemes were designed, including cell environment-based binding experiments (Scheme 1) and in vitro binding experiments based on extracted membrane proteins (Scheme 2).

[0325] Scheme 1: After primary beige adipocytes are differentiated and matured, BSA (control group; 100 ng / mL) or INHBC-Flag recombinant protein (experimental group; 100 ng / mL) is added, and BS3 crosslinking agent is added at the same time and incubated at 4°C for 30 minutes to promote the binding of INHBC-Flag to cell membrane receptors. After incubation, the cells are washed with PBS to remove unbound proteins, and cell membrane protein components are extracted. The obtained membrane protein solution is incubated with pre-equilibrated Anti-Flag magnetic beads to capture receptor proteins bound to INHBC. After multiple washes of the magnetic beads, the bound proteins are eluted for mass spectrometry analysis to identify potential binding proteins.

[0326] Scheme 2: After primary beige adipocytes are differentiated and matured, total membrane proteins are extracted and incubated with BSA (control group; 100 ng / mL) or INHBC-Flag recombinant protein (experimental group; 100 ng / mL) in an in vitro system, while BS3 crosslinking agent is added and incubated at 4°C for 30 minutes. After the incubation reaction is completed, pre-equilibrated Anti-Flag magnetic beads are added for enrichment, non-specific binding proteins are removed by washing, and then the target proteins are eluted. The eluted products are also used for mass spectrometry analysis to identify possible INHBC membrane receptors.

[0327] The eluted products of each group were reduced and subjected to SDS-PAGE separation, and the overall protein band was detected by Coomassie blue staining to confirm the abundance and specificity of the immunoprecipitated proteins, and to guide the segmentation and subsequent digestion of the gel for mass spectrometry samples. The differential bands visible by Coomassie blue and the whole eluted protein solution were digested with Trypsin or Lys-C enzyme to generate peptides, which were then desalted and purified by C18 reverse phase chromatography column, and then analyzed by nanoLC coupled with high-resolution mass spectrometry (such as Thermo TMOrbitrap Fusion TM Lumos TM or Q Exactive TM Plus) were used for detection. The mass spectrometer was operated in positive ion mode with a resolution setting of 60,000-120,000, and the target peptide signals were captured in data-dependent acquisition (DDA) or parallel reaction monitoring (PRM) mode. The obtained raw data were analyzed by database searching using Proteome Discoverer, MaxQuant or Mascot software, aligned with the Uniprot mouse protein database, and the high-confidence protein identification results were screened with a threshold of 1% FDR (false discovery rate).

[0328] By comparing the experimental group and the control group combined with the protein spectrum results, combined with enrichment analysis (GO, KEGG) and literature screening, the candidate proteins of the membrane receptors that INHBC may combine were determined, providing a basis for subsequent functional verification.

[0329] (2) siRNA transfection

[0330] The primary beige adipocytes and primary brown adipocytes inoculated in the 6-well plate were differentiated and matured. On the transfection day, the complete culture medium without antibiotics was replaced. The NC siRNA (50 nM; sequence: UUCUCCGAACGUGUCACGUTT, SEQ ID NO: 6) and the siRNA of DSG1β (50 nM; sequence: AGCAGCAGGGCAGCUAUCCUG, SEQ ID NO: 7) were respectively diluted with the transfection reagent (Lipo3000; 5 μL / well) in the serum-free Opti-MEM medium, and after being mixed gently, the transfection complex was incubated at room temperature for 5-20 minutes. After the transfection was completed, the cells were collected, and the mRNA and protein expression levels of the target genes were detected by qPCR and / or Western Blot to evaluate the transfection efficiency.

[0331] 2. Experimental results

[0332] To identify the potential membrane receptors of INHBC in primary beige adipocytes, two experimental protocols were adopted for membrane protein capture and mass spectrometry analysis (as shown in FIG. 32A). There was no difference between the two groups of proteins before magnetic bead sorting, indicating that the protein amount was the same before sorting (as shown in FIG. 32B). After immunoprecipitation by Anti-Flag magnetic beads and separation by SDS-PAGE, silver staining detection showed that more protein bands were detected in INHBC-Flag treated samples than in BSA treated samples, indicating that INHBC can interact with specific proteins on the surface of adipocyte membranes (as shown in FIG. 32C). Mass spectrometry analysis showed that seven potential INHBC binding proteins were identified by both experimental protocols, namely Bmal2, Pof1b, Got1, INHBC, and three new candidate protein receptors Pkp1, Jup, and DSG1β (as shown in FIG. 32D). Based on existing literature reports and functional prediction analysis, we speculate that INHBC may interact with membrane receptors such as DSG1β, Jup, and Pkp1, activate downstream signaling pathways, including p38 MAPK-ATF2 and cAMP-PKA-CREB axis, thereby promoting the transcription of key thermogenic genes such as PGC1α, PRDM16, and UCP1 mRNA, enhancing mitochondrial activity in adipocytes, and promoting heat consumption (as shown in FIG. 32E). Immunoprecipitation experiment results showed that after the addition of INHBC-Flag and BS3 cross-linking, specific binding signals of DSG1β were detected in cell membrane proteins, while no obvious bands were observed in the control group, suggesting that DSG1β may be a receptor protein of INHBC (as shown in FIG. 32F). Further repeated experiments in an in vitro membrane protein binding system without the addition of BS3 cross-linking agent also observed specific binding signals, further supporting the direct binding relationship between the two (as shown in FIG. 32G).

[0333] In addition, after specific knockdown of DSG1β using siRNA, the transcriptional activation effect of INHBC purified protein on primary beige adipocytes and primary brown adipocytes was significantly weakened (as shown in FIG. 33A and B). Western Blot results showed that in control cells, INHBC purified protein treatment can up-regulate the expression levels of mitochondrial respiratory chain complexes (CI-CV), PGC1α, p-HSL, DIO2, and UCP1 proteins, while in the DSG1β knockdown group, the expression levels of these proteins were significantly reduced or could not be effectively activated by INHBC purified protein (as shown in FIG. 33C and D).

[0334] The above results show that INHBC can bind to the DSG1 beta receptor on the surface of adipocytes, and when DSG1 beta is knocked down, the ability of INHBC purified protein to up-regulate the expression level of heat production related gene mRNA and protein and promote energy consumption is obviously inhibited.

[0335] In summary, by constructing a liver-specific overexpression, knockdown, knock-in or knockout INHBC gene related model (such as an obese mouse model, a T2DM mouse or cell model, and a MASLD mouse or cell model, etc.), the present application reveals the influence of INHBC as a target on the treatment of metabolic diseases. Specifically, the present application finds that:

[0336] (1) Liver-specific overexpression or knock-in of INHBC can improve high-fat diet-induced liver steatosis in mice, reduce the content of triglyceride in the liver and serum of mice, and at the same time reduce the content of lipid droplets in the liver. On the contrary, liver-specific knockdown or knockout of INHBC will aggravate high-fat diet-induced liver lipid accumulation in mice.

[0337] (2) INHBC can improve high-fat diet-induced liver lipid metabolism disorder in mice through the AMPK-SREBP1c pathway. Compared with the control group, after liver-specific overexpression or knock-in of INHBC, the expression level of fatty acid de novo synthesis related gene mRNA in the liver decreases, in addition, the phosphorylated protein level of AMPK increases, and the expression levels of fatty acid synthesis related proteins FASN, SCD1 and ACC1 and lipid droplet formation proteins CIDEA and CIDEC significantly decrease.

[0338] (3) Overexpression of INHBC can inhibit the lipogenesis of free fatty acid-induced mouse primary hepatocytes, and knockdown of INHBC can aggravate the lipid deposition induced by free fatty acids. Secondly, the protein expression level of INHBC in primary hepatocytes is significantly negatively correlated with the time of free fatty acid induction.

[0339] (4) Liver-specific overexpression or knock-in of INHBC can up-regulate the phosphorylated protein level of LKB1, promote the transfer of LKB1 from the nucleus to the cytoplasm, activate the AMPK signaling pathway, inhibit the expression of SREBP1c, and thus improve liver lipid deposition.

[0340] (5) After liver-specific overexpression of INHBC, the mRNA and protein expression levels of INHBC in the liver of mice are significantly increased, the fasting blood glucose level of mice is significantly reduced, the glucose tolerance of mice is obviously improved, and the mRNA and protein expression levels of liver gluconeogenesis related genes are reduced. On the contrary, liver-specific knockdown of INHBC can cause the fasting blood glucose level of mice to increase, the glucose tolerance of mice to be significantly impaired, and the expression level of liver gluconeogenesis related proteins to be significantly up-regulated.

[0341] (6) Liver-specific overexpression of INHBC can significantly reduce the blood glucose level of db / db mice, improve the glucose tolerance and insulin sensitivity of the body, and significantly reduce the mRNA and protein expression levels of gluconeogenesis-related genes in the liver.

[0342] (7) Liver-specific transgenic knock-in of INHBC can improve the glucose metabolism disorder of diabetic mice induced by high-fat diet and streptozotocin, the mRNA and protein expression levels of INHBC in the liver of the mice are significantly increased, the fasting blood glucose level of the mice is significantly reduced, the glucose tolerance and insulin sensitivity of the body are significantly improved, and the mRNA and protein expression levels of gluconeogenesis-related genes in the liver are reduced; on the contrary, liver-specific transgenic knockout of INHBC aggravates the glucose metabolism disorder of diabetic mice.

[0343] (8) Overexpression of INHBC can reduce the mRNA and protein expression levels of gluconeogenesis-related genes in mouse primary hepatocytes and reduce glucose production; on the contrary, knockdown of INHBC can up-regulate the mRNA and protein expression levels of gluconeogenesis-related genes in mouse primary hepatocytes and promote glucose production.

[0344] (9) Liver-specific overexpression or knock-in of INHBC can enhance the thermogenic activity of mouse adipose tissue to inhibit high-fat diet-induced obesity, the body weight of the mice is significantly reduced, the accumulation of body fat is reduced, better cold tolerance is exhibited, the mRNA and protein expression levels of adipose tissue thermogenesis, lipid hydrolysis and fatty acid beta oxidation-related genes are significantly increased, while the mRNA and protein expression levels of lipid synthesis-related genes are relatively reduced, and the whole body energy consumption is increased. On the contrary, liver-specific knockdown or knockout of INHBC can lead to down-regulation of the thermogenic level of mouse adipose tissue and aggravation of high-fat diet-induced obesity.

[0345] (10) Treatment of mouse primary adipocytes with INHBC purified protein prepared in vitro can significantly increase the mitochondrial activity and thermogenic level of the mouse primary adipocytes, and significantly increase the oxygen consumption rate of the mouse primary adipocytes.

[0346] (11) INHBC can bind to the DSG1β receptor on the surface of adipocytes, and when DSG1β is knocked down, the ability of INHBC purified protein to up-regulate the expression levels of thermogenic-related genes and proteins and promote energy consumption is significantly inhibited. The above embodiments of the present application are described in detail, but the present application is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a substance that promotes expression of an inhibin beta-C chain gene (INHBC) or increases protein activity thereof in the preparation of a product for preventing and / or treating a metabolic disease.

2. Use according to claim 1, characterized in that: The metabolic disease includes a disease caused by energy metabolism disorder; Preferably, the metabolic disease includes at least one of obesity, diabetes, fatty liver disease, hyperlipidemia.

3. Use according to claim 1, characterized in that: The substance that promotes expression of the INHBC gene or increases protein activity thereof is selected from at least one of a nucleic acid drug, a polypeptide or protein drug, and a recombinant virus; Preferably, the nucleic acid drug is selected from a nucleic acid drug containing a promoter or enhancer of INHBC expression, a nucleic acid drug of any truncated sequence or full-length sequence of an INHBC mRNA comprising at least the nucleotide sequence shown in SEQ ID NO: 1, and a nucleic acid drug of a polypeptide or protein drug encoding any truncated sequence or full-length sequence of an INHBC protein comprising at least the amino acid sequence shown in SEQ ID NO: 5; Preferably, the polypeptide or protein drug is at least a polypeptide or protein drug of any truncated sequence or full-length sequence of an INHBC protein comprising at least the amino acid sequence shown in SEQ ID NO: 5; Preferably, the recombinant virus comprises a nucleotide sequence expressing an INHBC gene.

4. Use according to claim 1, characterized in that: The functions of the product include at least one of A1) to A5): A1) preventing and / or treating liver steatosis induced by a high-fat diet; A2) preventing and / or treating abnormal liver lipid metabolism induced by a high-fat diet; A3) preventing and / or treating diabetes induced by a high-fat diet and streptozotocin or genetic mutation; A4) inhibiting obesity induced by a high-fat diet; A5) inhibiting lipogenesis of primary hepatocytes induced by free fatty acids.

5. Use according to any one of claims 1 to 4, characterized in that: The product includes a pharmaceutical product and a functional food product.

6. Use according to claim 5, characterized in that: The raw materials for preparing the pharmaceutical product can further include at least one of a pharmaceutical excipient selected from a disintegrant, a diluent, a lubricant, a binder, a humectant, a flavoring agent, a suspending agent, a surfactant, and a preservative; Preferably, the dosage form of the pharmaceutical product is at least one of a solid preparation, a liquid preparation, and a semi-solid preparation.

7. Use according to claim 5, characterized in that: The form of the functional food product includes at least one of a decoction, a beverage, a candy, an oral liquid, a capsule, a tablet, and a powder.

8. A pharmaceutical composition for preventing and / or treating metabolic diseases, characterized by: The substance that promotes expression of the INHBC gene or increases protein activity thereof, and a pharmaceutically acceptable carrier; The pharmaceutically acceptable carrier includes at least one of water, saline, a buffer, glycerol, ethanol, a liposome, a lipid, a protein, a protein-antibody conjugate, a peptide substance, cellulose, and a nanogel.

9. The pharmaceutical composition of claim 8, wherein: The substance that promotes expression of the INHBC gene or increases protein activity thereof is selected from at least one of a nucleic acid drug, a polypeptide or protein drug, and a recombinant virus; Preferably, the nucleic acid drug is selected from the group consisting of a nucleic acid drug containing a promoter or enhancer of INHBC expression, a nucleic acid drug of any truncated sequence or full-length sequence of INHBC mRNA comprising at least the nucleotide sequence shown in SEQ ID NO: 1, a nucleic acid drug of a polypeptide or protein drug encoding any truncated sequence or full-length sequence of INHBC protein comprising at least the amino acid sequence shown in SEQ ID NO: 5; Preferably, the polypeptide or protein drug is a polypeptide or protein drug of any truncated sequence or full-length sequence of INHBC protein comprising at least the amino acid sequence shown in SEQ ID NO: 5; Preferably, the recombinant virus comprises a nucleotide sequence expressing an INHBC gene.

10. A method for treating metabolic diseases, comprising the steps of: administering to the subject a substance that promotes the expression of INHBC gene or increases the protein activity thereof.

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