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WO2020189970A1 - Composition pharmaceutique pour prévenir ou traiter une stéatose hépatique non alcoolique, comprenant tcf7l2 en tant que composant efficace - Google Patents

Composition pharmaceutique pour prévenir ou traiter une stéatose hépatique non alcoolique, comprenant tcf7l2 en tant que composant efficace Download PDF

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WO2020189970A1
WO2020189970A1 PCT/KR2020/003514 KR2020003514W WO2020189970A1 WO 2020189970 A1 WO2020189970 A1 WO 2020189970A1 KR 2020003514 W KR2020003514 W KR 2020003514W WO 2020189970 A1 WO2020189970 A1 WO 2020189970A1
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tcf7l2
fatty liver
liver disease
alcoholic fatty
protein
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Korean (ko)
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오경진
배광희
김원곤
이다솜
이상철
이은우
한백수
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

Definitions

  • the present invention relates to a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease induced by increasing fat biosynthesis (De novo lipogenesis) containing TCF7L2 protein or gene as an active ingredient.
  • Fatty liver refers to a state in which fat is accumulated in hepatocytes, and the ratio of fat to normal liver is about 5%, and a state in which more fat is accumulated is called fatty liver.
  • the fatty liver deteriorates and the fat mass in the hepatocyte becomes large, the important components of the cells including the nucleus are pushed to one side and the function of the hepatocyte decreases, and the expanded hepatocytes due to the accumulated fat in the cells press the microvessels and lymph glands between the liver cells Thus, the circulation of blood and lymph fluid in the liver is impaired. In this case, the liver cells cannot properly receive oxygen and nutrients, and liver function is degraded.
  • Fatty liver can be divided into alcoholic fatty liver and non-alcoholic fatty liver.
  • Alcoholic fatty liver is caused by alcohol. The more alcohol you drink, the more likely it occurs. If you continue to drink alcohol, your liver's ability to metabolize alcohol decreases, making fatty liver more severe. It also occurs well when nutrition is poor. Some of the alcoholic fatty liver can lead to alcoholic hepatitis and cirrhosis, leading to death.
  • Non-alcoholic fatty liver is a case of increased fat accumulation in the liver (more than 5% of hepatocytes) regardless of alcohol or viral infection, and is caused by a problem of increased fat synthesis and excretion in the liver.
  • About 59% of patients with non-alcoholic fatty liver caused by an imbalance in the input and output of free fatty acids are caused by increased absorption of free fatty acids from the outside, and about 26% of patients are in the liver. It is known to be caused by an increase in the input of free fatty acids due to an increase in fat biosynthesis.
  • TCF7L2 Transcription factor 7-like 2 is a transcription factor that controls other genes and is known to regulate the Wnt signaling pathway, a key mechanism that regulates cell development-growth. Mutations in either pair of TCF7L2 genes increase the likelihood of developing type 2 diabetes by 40%.
  • non-alcoholic fatty liver disease a composition for the prevention or treatment of non-alcoholic hepatitis containing silkworms having silk silk protein is disclosed in Korean Patent No. 1856448, and GDF15 protein in Korean Patent No. 1727506
  • a pharmaceutical composition for preventing or treating fatty liver containing a polynucleotide encoding it as an active ingredient is disclosed, but non-alcoholic fatty liver disease induced by increased fat biosynthesis containing the TCF7L2 protein or gene of the present invention as an active ingredient
  • the present invention was derived from the above demands, in an animal model in which the expression of TCF7L2 was specifically suppressed in liver tissue, by carrying out diet conditions to reproduce two main factors that can induce non-alcoholic fatty liver, fatty acid in liver
  • the gene expression level in liver tissues was confirmed under conditions of inducing fatty liver due to re-feeding that induces fat biosynthesis in the liver or a high-fat diet injected with glycolysis and over-expression of TCF7L2 under similar conditions of re-feeding and carbohydrate diet.
  • the present invention is to prevent or prevent non-alcoholic fatty liver disease induced by increasing fat biosynthesis (De novo lipogenesis) containing a vector containing a TCF7L2 protein or a polynucleotide encoding the TCF7L2 protein as an active ingredient. It provides a pharmaceutical composition for treatment.
  • the present invention provides a method for inhibiting fat biosynthesis in liver by administering a vector containing a TCF7L2 protein or a polynucleotide encoding the TCF7L2 protein to an individual.
  • the present invention provides a composition for diagnosing non-alcoholic fatty liver disease induced by an increase in fat biosynthesis (De novo lipogenesis), including an agent for measuring the expression level of TCF7L2 protein or its mRNA.
  • the present invention provides a kit for diagnosing non-alcoholic fatty liver disease induced by increasing fat biosynthesis (De novo lipogenesis) comprising the composition.
  • the present invention comprises the steps of: (a) measuring the expression level of TCF7L2 protein or its mRNA from a biological sample derived from a patient suspected of developing non-alcoholic fatty liver disease induced by increased fat biosynthesis (De novo lipogenesis); And (b) comparing the measured TCF7L2 protein or its mRNA expression level with the level measured from a sample of a normal person, and when it decreases, it is determined that non-alcoholic fatty liver disease induced by an increase in de novo lipogenesis has occurred. It provides a method of diagnosing non-alcoholic fatty liver disease induced by an increase in fat biosynthesis (De novo lipogenesis), including the step of, and a method of providing information for diagnosis.
  • the present invention comprises the steps of: (1) treating a candidate substance in a sample taken from a patient suffering from non-alcoholic fatty liver disease induced by an increase in fat biosynthesis (De novo lipogenesis);
  • step (3) comparing the expression level measured in step (2) with the mRNA or protein expression level of TCF7L2 in a sample not treated with the candidate substance; Induced by increased de novo lipogenesis, including A method for screening a therapeutic agent for non-alcoholic fatty liver disease is provided.
  • the present invention relates to a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease induced by increased fat biosynthesis containing TCF7L2 protein or gene as an active ingredient.
  • the present invention confirms that the TCF7L2 deficient animal model specifically in liver tissue induces fatty liver through a high carbohydrate diet, and when TCF7L2 is overexpressed under similar conditions of a high carbohydrate diet, the expression of increased fat biosynthesis-related genes is reduced. I did. Therefore, TCF7L2 of the present invention has the effect of specifically and strongly regulating fat biosynthesis, which is the main cause of non-alcoholic fatty liver induced by a high carbohydrate diet.
  • the TCF7L2 protein or gene of the present invention is fat It can be used as a therapeutic agent for non-alcoholic fatty liver disease induced by increased biosynthesis.
  • HFD is a high fat diet group
  • Refed is a group that re-fed for 24 hours after fasting.
  • a high fat diet is a condition that provides free fatty acids
  • a refeed is a carbohydrate diet that induces fat biosynthesis.
  • the screening results were expressed as the result of dividing the value measured in the liver-specific TCF7L2 deficient mouse (TCF7L2 LKO) by the value measured in the mouse normally expressing TCF7L2 (TCF7L2 WT).
  • Figure 3 is a liver tissue of a liver-specific TCF7L2 deficient mouse (TCF7L2 LKO), free diet, fasting, re-feeding for 6 hours after fasting, and re-feeding for 24 hours after fasting fat biosynthesis (corresponding process + fat synthesis process) related genes (GLUT2, GK, LPK, Acly, ACC, FAS and DGAT1) is the result of confirming the expression level.
  • TCF7L2 WT is a mouse that normally expresses TCF7L2.
  • FIG. 4 is a result of confirming the effect of promoting fat biosynthesis (corresponding process + fat synthesis process) in liver tissue of liver-specific TCF7L2 deficient mice (TCF7L2 LKO) re-fed with a high carbohydrate diet.
  • TCF7L2 WT is a mouse that normally expresses TCF7L2.
  • Ad-TCF7L2 is an adenovirus vector expressing TCF7L2
  • Ad-GFP is an adenovirus vector that does not express TCF7L2.
  • TCF7L2 WT is a mouse that normally expresses TCF7L2
  • TCF7L2 LKO is a liver-specific TCF7L2 deficient mouse.
  • TCF7L2 WT-GFP is a control group in which an adenovirus that does not express TCF7L2 is injected into mice that normally express TCF7L2, and TCF7L2 LKO-GFP is an adenovirus that does not express TCF7L2 in mice lacking the expression of TCF7L2 specifically in liver tissue.
  • TCF7L2 LKO-TCF7L2 is a group in which the expression of TCF7L2 was restored by injecting an adenovirus expressing TCF7L2 into a mouse whose TCF7L2 expression was specifically deficient in liver tissue.
  • (A) is the result of confirming the TG content in the liver by free diet, fasting, and re-feeding according to the regulation of TCF7L2 expression
  • (B) is the TG content in the liver by fasting, re-feeding, and high-carbohydrate re-feeding according to the regulation of TCF7L2 expression
  • (C) is the result of confirming the TG content as a result of fat biosynthesis according to the regulation of TCF7L2 expression in the primary liver cell line
  • (D) is the TG in the liver by fasting and high carbohydrate refeeding according to the regulation of TCF7L2 expression.
  • TCF7L2 WT is a mouse that normally expresses TCF7L2, and TCF7L2 LKO is a liver-specific TCF7L2 deficient mouse.
  • TCF7L2 WT-GFP is a control group in which an adenovirus that does not express TCF7L2 is injected into mice that normally express TCF7L2, and TCF7L2 LKO-GFP is an adenovirus that does not express TCF7L2 in mice lacking the expression of TCF7L2 specifically in liver tissue.
  • TCF7L2 LKO-TCF7L2 is a group in which the expression of TCF7L2 was restored by injecting an adenovirus expressing TCF7L2 into a mouse whose TCF7L2 expression was specifically deficient in liver tissue.
  • Ad-GFP is an adenovirus-treated group that does not express TCF7L2
  • Ad-TCF7L2 is an adenovirus-treated group that expresses TCF7L2.
  • TCF7L2 LKO liver defect of TCF7L2
  • Ad-TCF7L2 overexpression of TCF7L2
  • B overexpression of TCF7L2
  • TCF7L2 WT is a mouse that normally expresses TCF7L2
  • TCF7L2 LKO is a liver-specific TCF7L2 deficient mouse.
  • Ad-GFP is a group injected with adenovirus not expressing TCF7L2
  • Ad-TCF7L2 is a group in which TCF7L2 expression is restored by injection of adenovirus expressing TCF7L2.
  • Figure 11 is a result of confirming the fatty liver induction effect according to the TCF7L2 deletion in the liver of mice that have been on a high carbohydrate diet (HCD) and a high fat diet (HFD) for 22 weeks by fat staining (A) and the result of confirming the TG content in the liver (B). .
  • GFP is a control group
  • GFP-labeled adenovirus (Ad-GFP) is injected into the mouse tail vein
  • TCF7L2 is an experimental group
  • GFP-labeled adenovirus (Ad-TCF7L2) expressing TCF7L2 is injected into the mouse tail vein. It was injected.
  • NBD general diet
  • HCD high carbohydrate diet
  • HFD high fat diet
  • TCF7L2 LKO liver-specific TCF7L2 deficient mouse
  • neutrophils macrophages.
  • the present invention relates to a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease induced by an increase in fat biosynthesis (De novo lipogenesis) containing a TCF7L2 protein or a vector containing a polynucleotide encoding the TCF7L2 protein as an active ingredient. .
  • the TCF7L2 protein is composed of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.
  • the scope of the TCF7L2 protein according to the present invention includes a protein having the amino acid sequence of SEQ ID NO: 1 and functional equivalents of the protein.
  • the term "functional equivalent” is at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% of the amino acid sequence of SEQ ID NO: 1 as a result of addition, substitution or deletion of amino acids. It refers to a protein having the above sequence homology and exhibiting substantially the same physiological activity as a protein having the amino acid sequence of SEQ ID NO: 1.
  • substantially homogeneous physiological activity means the prophylactic or therapeutic activity of non-alcoholic fatty liver disease induced by increased fat biosynthesis.
  • the vector including the polynucleotide encoding the TCF7L2 protein may be a recombinant viral vector, a plasmid vector, a cosmid vector, or a bacteriophage vector, and is preferably a recombinant viral vector, but is not limited thereto.
  • the recombinant virus may be any one selected from the group consisting of adenovirus, adeno-associated virus, retrovirus, herpes simplex virus, and lentivirus, preferably adenovirus, but is limited thereto. It is not.
  • the TCF7L2 protein or the polynucleotide encoding the TCF7L2 protein has an effect of inhibiting fat biosynthesis in the liver.
  • the non-alcoholic fatty liver disease is preferably non-alcoholic simple fatty liver, non-alcoholic steatohepatitis, or non-alcoholic liver cirrhosis, but is not limited thereto.
  • the increase in fat biosynthesis may be due to a high carbohydrate diet or type 2 diabetes.
  • the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the TCF7L2 protein or the polynucleotide encoding the TCF7L2 protein.
  • oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols may be formulated and used in the form of external preparations, suppositories, and sterile injectable solutions according to a conventional method, but is not limited thereto. .
  • Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the extract, such as starch, calcium carbonate, and sucrose. ) Or lactose (Lactose), gelatin, etc. are mixed to prepare.
  • lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. have.
  • Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.
  • the non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.
  • As a base for suppositories Witepsol, macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
  • a suitable dosage of the pharmaceutical composition according to the present invention may be prescribed in various ways depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity. Can be.
  • the concentration of the active ingredient contained in the composition of the present invention can be determined in consideration of the purpose of treatment, the condition of the patient, the required period, etc., and is not limited to a specific range of concentration.
  • the present invention relates to a method of inhibiting fat biosynthesis in liver by administering to an individual a vector containing a TCF7L2 protein or a polynucleotide encoding the TCF7L2 protein.
  • the biosynthesis of fat in the liver may be induced by a high carbohydrate diet.
  • the individual may be an animal other than humans, but is not limited thereto.
  • the present invention relates to a composition for diagnosing non-alcoholic fatty liver disease induced by an increase in fat biosynthesis (De novo lipogenesis), including an agent for measuring the expression level of TCF7L2 protein or its mRNA.
  • diagnosis of the present invention means to confirm the presence or characteristics of a pathological condition.
  • diagnosis can be interpreted as confirming the onset of non-alcoholic fatty liver disease induced by an increase in fat biosynthesis.
  • the agent measuring the expression level of the TCF7L2 protein may be an antibody that specifically binds to the TCF7L2 protein, and the agent measuring the expression level of the TCF7L2 mRNA May be at least one selected from probes, primers, antisense oligonucleotides, and aptamers that specifically bind to TCF7L2 mRNA, but is not limited thereto.
  • the present invention relates to a kit for diagnosing non-alcoholic fatty liver disease induced by increasing fat biosynthesis (De novo lipogenesis) comprising the composition.
  • the kit may be an RT-PCR kit, a DNA chip kit, or a protein chip kit, but is not limited thereto.
  • the RT-PCR kit in addition to each primer pair specific for the TCF7L2 gene, includes a test tube or other suitable container, reaction buffer (varies in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase. Such enzymes, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like may be included.
  • the DNA chip kit may include a substrate to which cDNA corresponding to the TCF7L2 gene or fragment thereof is attached as a probe, and reagents, agents, enzymes, etc. for preparing a fluorescently labeled probe.
  • the protein chip kit kit is not particularly limited thereto, but may include a substrate, a suitable buffer solution, a secondary antibody labeled with a color developing enzyme or a fluorescent substance, a color developing substrate, and the like for immunological detection
  • the present invention comprises the steps of: (a) measuring the expression level of TCF7L2 protein or its mRNA from a biological sample derived from a patient suspected of developing non-alcoholic fatty liver disease induced by increased fat biosynthesis (De novo lipogenesis); And
  • the expression level of the TCF7L2 protein is Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, and rocket.
  • Rocket immunoelectrophoresis immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography, FACS analysis It may be measured by any one method selected from the group consisting of (fluorescence activated cell sorter analysis) and protein chip technology assay, but is not limited thereto.
  • the TCF7L2 mRNA expression level was determined by reverse transcriptase polymerase reaction (RT-PCR), competitive reverse transcriptase polymerase reaction (competitive RT-PCR), real time quantitative RT-PCR, and RNase protection assay (RNase). protection method), Northern blotting, or DNA chip technology assay, but is not limited thereto.
  • RT-PCR reverse transcriptase polymerase reaction
  • competitive RT-PCR competitive reverse transcriptase polymerase reaction
  • RNase RNase protection assay
  • Northern blotting or DNA chip technology assay, but is not limited thereto.
  • the present invention comprises the steps of: (a) measuring the expression level of TCF7L2 protein or its mRNA from a biological sample derived from a patient suspected of developing non-alcoholic fatty liver disease induced by increased fat biosynthesis (De novo lipogenesis); And
  • the sample is liver It is preferably any one selected from tissue, lymph fluid, saliva, serum, plasma, and blood, but is not limited thereto.
  • the present invention comprises the steps of: (1) treating a candidate substance in a sample taken from a patient suffering from non-alcoholic fatty liver disease induced by an increase in fat biosynthesis (De novo lipogenesis);
  • step (3) comparing the expression level measured in step (2) with the mRNA or protein expression level of TCF7L2 in a sample not treated with the candidate substance; Induced by increased de novo lipogenesis, including It relates to a method for screening a therapeutic agent for non-alcoholic fatty liver disease.
  • the sample is preferably any one selected from liver tissue, lymph fluid, saliva, serum, plasma, and blood, but is not limited thereto,
  • the candidate material is not particularly limited as long as it can be used as a therapeutic agent for non-alcoholic fatty liver disease induced by an increase in fat biosynthesis (De novo lipogenesis).
  • the candidate substance can be determined as a substance for the treatment of non-alcoholic fatty liver disease induced by increased fat biosynthesis.
  • the expression of TCF7L2 was decreased in the group (HCD) chronically high carbohydrate diet (HCD) for 22 weeks compared to the general diet group (NCD), and the expression of the liposynthetic enzymes FAS and ACC was increased. . Through this, it was confirmed that when fatty liver is induced by high carbohydrate, the expression of TCF7L2 is reduced.
  • TCF7L2 LKO C57BL/6 mice with a liver-specific TCF7L2 expression deficient were prepared.
  • TCF7L2 WT C57BL/6 mice
  • Example 3 Screening for expression of genes related to lipid metabolism and glucose metabolism in liver tissue of liver-specific TCF7L2 deficient mice
  • the amount of gene expression change was indicated by the change in the expression amount of TCF7L2 LKO compared to TCF7L2 WT.As a result, as disclosed in FIG.2, a high-fat diet (60% fat, D1249) and a group that was fasted for 24 hours and then re-fed for 24 hours (carbohydrate diet Condition, Teklad gloval 18% protein, 2018S), the expression of genes related to lipid metabolism and glucose metabolism was increased, and in particular, expression of genes related to lipid metabolism and glucose metabolism was significantly increased under the carbohydrate diet condition, which was re-fed after fasting for 24 hours. .
  • Example 4 Expression of adipose biosynthesis related gene in liver tissue of liver-specific TCF7L2 deficient mice following refeeding
  • Example 3 changes in the expression of genes related to fat biosynthesis (de novo lipogenesis) were confirmed under the carbohydrate diet condition where the expression of genes related to lipid metabolism and sugar metabolism was the greatest. Since fat biosynthesis in liver tissue consists of glycolysis and fat synthesis, changes in fat biosynthesis in liver tissue were confirmed through changes in the expression of genes related to glycolysis and fat synthesis.
  • the experimental group was a free diet group (Ad, ad libitum), fasting group (F, fasting), fasting for 24 hours, and refeeding for 6 hours (6(R), 6 refeeding) and 24 using mice of 10 weeks of age. After fasting for hours, they were classified into groups that were re-feeding for 24 hours (24(R), 24 refeeding).
  • the expression of adipose biosynthesis-related genes was increased in TCF7L2 LKO mice compared to TCF7L2 WT, and in particular, a fat biosynthesis-related gene under the condition (24(R)) re-feeding for 24 hours, which is a condition for promoting fat biosynthesis.
  • Phosphorus GLUT2 Glucose transporter 2
  • GK Glycerol kinase
  • LPK Liver type pyruvate kinase
  • Acly ATP citrate lyase
  • ACC Alcohol citrate lyase
  • FAS Fatty acid synthase
  • GPAT1 Glycerol-3-
  • the expression of phosphate acyltransferase 1 was significantly increased in TCF7L2 LKO mice compared to TCF7L2 WT.
  • Example 5 Expression of adipose biosynthesis-related gene in liver tissues of liver-specific TCF7L2 deficient mice following high carbohydrate refeeding
  • Example 4 since the expression of genes related to fat biosynthesis was increased according to refeeding, which is a carbohydrate diet, the experiment was conducted through high carbohydrate refeeding to confirm whether the increase in expression of fat biosynthesis related genes by the high carbohydrate diet was further promoted.
  • the experimental group consisted of a fasting group (F, fasting), fasting for 24 hours, and refeeding for 24 hours (refeeding, Teklad gloval 18% protein, 2018S) using 10-week-old mice, and 24 hours after fasting for 24 hours.
  • the carbohydrate diet (70% carbohydrate, TD. 98090) was classified into the refeeding group (HCR, High-carbohydrate refeeding).
  • Example 6 Changes in blood glucose and insulin concentration of liver-specific TCF7L2 deficient mice according to refeeding
  • the experimental group was a free diet group (Ad, ad libitum), fasting group (F, fasting), fasting for 24 hours, and refeeding for 6 hours (6(R), 6 refeeding) and 24 using mice of 10 weeks of age. After fasting for hours, they were classified into groups that were re-feeding for 24 hours (24(R), 24 refeeding).
  • Example 7 Changes in adipose biosynthesis related protein according to liver-specific TCF7L2 deletion and repair
  • Ad-TCF7L2 GFP-labeled adenovirus
  • liver tissue was excised 5 days after injection, and changes in fat biosynthesis related proteins were confirmed.
  • Ad-GFP adenovirus labeled with only GFP without TCF7L2 gene was injected and used in the same manner.
  • TCF7L2 protein which was deleted in the TCF7L2 LKO mouse, as disclosed in FIG. 6, recovered normally 5 days after the injection of Ad-TCF7L2. It was confirmed that the expression of ACC and FAS decreased after the expression of TCF7L2 was restored.
  • HCR high-carbohydrate refeeding
  • Example 9 Induction and control of fatty liver according to TCF7L2 expression regulation
  • TG triglycerides
  • hepatocytes were isolated from liver tissue, and after 4 hours, Ad-GFP and Ad-TCF7L2 were infected, and after 24 hours, 1 ⁇ Ci [ 14 C] glucose and 100 nM insulin were treated in a serum-free culture medium. After reacting for 48 hours, the TG content in the cells was checked. Glucose and insulin are the main substances that induce fat biosynthesis by carbohydrates.
  • the TG content in the cell which was increased by the deletion of TCF7L2, decreased when the expression of TCF7L2 was restored, showing a content similar to that of TCF7L2 WT-GFP.
  • TCF7L2 regulates the degree of fat accumulation by regulating fat biosynthesis in the liver.
  • Example 10 Changes in fat biosynthesis according to TCF7L2 overexpression in primary hepatocyte cell line
  • the overexpression of TCF7L2 in the primary hepatocyte line is a control that does not overexpress TCF7L2 by treatment with glucose and insulin, which are major substances in the fat biosynthesis process by carbohydrates (Ad-GFP).
  • Ad-GFP the overexpression of TCF7L2 in the primary hepatocyte line
  • TCF7L2 plays a role in reducing fat biosynthesis and TG content in the liver under conditions of inducing fat biosynthesis by carbohydrates.
  • TCF7L2 LKO mice fed a high-fat diet for 12 weeks showed little change in liver TG content compared to TCF7L2 WT mice.
  • TCF7L2 specifically controls the TG content in the liver by a high carbohydrate diet that regulates fat biosynthesis.
  • Example 12 Effect of inducing fatty liver according to TCF7L2 defect in liver under conditions of inducing fatty liver according to diet
  • liver The effect of inducing fatty liver and TG content in liver according to TCF7L2 deficiency was confirmed in the liver of mice that were fed high carbohydrate diet (HCD) and high fat diet (HFD) for 22 weeks.
  • HCD carbohydrate diet
  • HFD high fat diet
  • liver tissue-specific TCF7L2 deficient mice fed with high carbohydrate diet for 22 weeks significantly promoted fat accumulation in the liver compared to TCF7L2 WT mice, but mice fed high fat diet for 22 weeks were subjected to the above. Similar to Example 11, the difference in fat accumulation due to the deletion of TCF7L2 could not be confirmed.
  • TCF7L2 specifically regulates fat accumulation in the liver by a high carbohydrate diet.
  • Example 13 In an animal model in which fatty liver was induced by a high carbohydrate diet (HCD), the change in the expression level of the body weight, triglyceride (TG) content in the liver, and de novo lipogenesis-related genes according to TCF7L2 injection was confirmed
  • TCF7L2 expression was induced in the experimental group, and the weight of the experimental group in which TCF7L2 expression was induced compared to the control group that did not induce TCF7L2 expression did not increase, but the triglyceride content in the liver was remarkably.
  • the expression level of fat biosynthesis-related genes SREBP1c, ChREBP ⁇ , ChREBP ⁇ , GK, LPK, ACLY, ACC, FAS, DGAT2 and GPAT was statistically significantly reduced (Fig. 12).
  • Example 14 Confirmation of changes in expression of steatohepatitis-related genes in liver tissue according to diet using liver-specific TCF7L2 deficient mice
  • TNF ⁇ , neutrophils, macrophages, and monocytes following TCF7L2 deficiency in livers of normal diet group (NCD), high carbohydrate diet group (HCD) and high fat diet group (HFD) mice for 22 weeks. monocytes), inflammation-related CD11, KC (keratinocyte chemokine), F4/80, MIP1 ⁇ and MCP1 gene expression levels were confirmed.
  • the liver tissue-specific TCF7L2 deficient mice fed with high carbohydrate diet for 22 weeks had a statistically significant increase in the expression of steatohepatitis-related factors compared to the TCF7L2 WT mice.
  • the expression level of steatohepatitis factor due to the deletion of TCF7L2 in mice that had been fed for 22 weeks, and it was confirmed that the expression of steatohepatitis-related factors was significantly increased in mice that were on a high fat diet.

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Abstract

La présente invention concerne une composition pharmaceutique comprenant une protéine ou un gène TCF7L2 en tant que composant efficace pour prévenir ou traiter une stéatose hépatique non alcoolique induite par une augmentation de la lipogenèse de novo. La composition de la présente invention, comprenant la protéine ou le gène TCF7L2 en tant que composant efficace, peut être avantageusement utilisée en tant qu'agent thérapeutique pour une stéatose hépatique non alcoolique induite par un régime riche en glucides, ou par une lipogenèse de novo induite par un régime riche en glucides.
PCT/KR2020/003514 2019-03-15 2020-03-13 Composition pharmaceutique pour prévenir ou traiter une stéatose hépatique non alcoolique, comprenant tcf7l2 en tant que composant efficace Ceased WO2020189970A1 (fr)

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KR1020190029866A KR102020031B1 (ko) 2019-03-15 2019-03-15 Tcf7l2를 유효성분으로 함유하는 비알코올성 지방간의 예방 또는 치료용 약학 조성물

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KR102020031B1 (ko) * 2019-03-15 2019-09-09 한국생명공학연구원 Tcf7l2를 유효성분으로 함유하는 비알코올성 지방간의 예방 또는 치료용 약학 조성물
KR102341336B1 (ko) 2019-11-20 2021-12-21 숙명여자대학교산학협력단 만성간질환의 예후 예측용 바이오마커 조성물
KR102288299B1 (ko) 2019-11-20 2021-08-11 숙명여자대학교산학협력단 만성간질환의 진행 단계 판별용 바이오마커 조성물
WO2023008673A1 (fr) 2021-07-30 2023-02-02 숙명여자대학교산학협력단 Marqueur complexe basé sur l'apprentissage automatique permettant de déterminer la stéatohépatite non alcoolique et son utilisation

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