WO2020189970A1 - Pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising tcf7l2 as effective component - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising a TCF7L2 protein or gene as an effective component for preventing or treating non-alcoholic fatty liver disease induced by an increase in de novo lipogenesis. The composition of the present invention, comprising the TCF7L2 protein or gene as an effective component, may be beneficially used as a therapeutic agent for non-alcoholic fatty liver disease induced by a high carbohydrate diet, or by de novo lipogenesis induced by a high carbohydrate diet.

Description

Pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease containing TCF7L2 as an active ingredient

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. When 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.

The cause and severity of fatty liver disease have been identified, and symptoms should be improved through diet control and exercise, but fatty liver patients are often unable to practice this. Therefore, there is a need for the development of effective fatty liver treatment drugs.

On the other hand, 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%.

On the other hand, as a prior art related to 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 Or 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 There is no disclosure regarding a pharmaceutical composition for the prevention or treatment of

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. Changes in expression were confirmed, and expression of genes related to fat biosynthesis due to deletion and repair of TCF7L2 under high carbohydrate refeeding conditions were confirmed.If TCF7L2 gene is defective, chronic high carbohydrate diet conditions specifically promote fatty liver production. In an animal model in which the TCF7L2 gene was not deleted, fatty liver was induced through a high-carbohydrate diet, and GFP-labeled adenovirus (Ad-TCF7L2) expressing TCF7L2 was injected into the tail vein of the mouse to induce the expression of TCF7L2. It was confirmed that the expression of the gene related to fat biosynthesis was decreased, and the weight was not changed, but the triglyceride content in the liver tissue was decreased, thereby completing the present invention.

In order to solve the above problems, 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.

In addition, 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.

In addition, 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.

In addition, the present invention provides a kit for diagnosing non-alcoholic fatty liver disease induced by increasing fat biosynthesis (De novo lipogenesis) comprising the composition.

In addition, 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.

In addition, 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);

(2) measuring the mRNA or protein expression level of TCF7L2 in the sample treated with the candidate substance of step (1); And

(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.

On the other hand, in an animal model in which the TCF7L2 gene was not deleted, fatty liver was induced through a high carbohydrate diet, and GFP-labeled adenovirus (Ad-TCF7L2) expressing TCF7L2 was injected into the mouse tail vein to induce the expression of TCF7L2. It was confirmed that the expression of the gene related to fat biosynthesis was decreased, and the weight was not changed, but the triglyceride content in the liver tissue was decreased.

The excessive intake of carbohydrates of Asians, including Korea, is closely related to the induction of non-alcoholic fatty liver disease, and a high carbohydrate diet can control the induction of fatty liver disease due to increased fat biosynthesis in the liver. Therefore, 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.

1 is a result of confirming the protein expression levels of TCF7L2 and liposynthetic enzymes (FAS and ACC) in the liver of an animal model that was fed a high carbohydrate diet for 22 weeks.

2 is a result of screening the expression of genes related to lipid metabolism and glucose metabolism in liver tissues of liver-specific TCF7L2 deficient mice. HFD is a high fat diet group, and Refed is a group that re-fed for 24 hours after fasting. A high fat diet is a condition that provides free fatty acids, and 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.

5 is a result of confirming the content of insulin and glucose in the blood in the normal diet re-feeding (A) and high carbohydrate diet re-feeding conditions (B) according to the TCF7L2 defect in the liver.

6 is a result of confirming the expression of liposynthetic enzymes (SREBP1, ChREBP, ACC, FAS) proteins according to defects and repairs in the liver of TCF7L2 by high carbohydrate refeeding (HCR). Ad-TCF7L2 is an adenovirus vector expressing TCF7L2, and Ad-GFP is an adenovirus vector that does not express TCF7L2. TCF7L2 WT is a mouse that normally expresses TCF7L2, and TCF7L2 LKO is a liver-specific TCF7L2 deficient mouse.

7 is a result of confirming the expression of genes related to fat biosynthesis (GK, LPK, ACC, FAS, SCD1 and DGAT2) according to TCF7L2 liver defect and repair under high carbohydrate refeeding conditions. 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.

8 is a result of confirming the effect of inducing and controlling fatty liver according to the regulation of TCF7L2 expression. (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, and (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, and (D) is the TG in the liver by fasting and high carbohydrate refeeding according to the regulation of TCF7L2 expression. This is the result of checking the content. 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.

9 is a situation in which glucose and insulin, which are major substances inducing fat biosynthesis by carbohydrates, are treated in primary liver cell lines, TG content change according to fat biosynthesis by TCF7L2 overexpression (A) and fat biosynthesis related genes by TCF7L2 overexpression This is the result of confirming expression (B). Ad-GFP is an adenovirus-treated group that does not express TCF7L2, and Ad-TCF7L2 is an adenovirus-treated group that expresses TCF7L2.

10 is a result of confirming the TG content in the liver due to a liver defect of TCF7L2 (TCF7L2 LKO) (A) or an overexpression of TCF7L2 (Ad-TCF7L2) (B) under a high fat dietary condition that provides free fatty acid from the outside. TCF7L2 WT is a mouse that normally expresses TCF7L2, and TCF7L2 LKO is a liver-specific TCF7L2 deficient mouse. Ad-GFP is a group injected with adenovirus not expressing TCF7L2, and 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). .

12 is a general mouse model in which fatty liver was induced by a high carbohydrate diet (HCD), according to TCF7L2 injection, body weight (A), liver triglyceride (TG) content (B), and changes in the expression levels of genes related to fat biosynthesis ( This is the result of checking C). GFP is a control group, and GFP-labeled adenovirus (Ad-GFP) is injected into the mouse tail vein, TCF7L2 is an experimental group, and GFP-labeled adenovirus (Ad-TCF7L2) expressing TCF7L2 is injected into the mouse tail vein. It was injected.

13 is an inflammatory cytokine TNFα according to a general diet (NCD), a high carbohydrate diet (HCD) and a high fat diet (HFD) in the liver tissue of a liver-specific TCF7L2 deficient mouse (TCF7L2 LKO), neutrophils, macrophages. This is the result of confirming the expression levels of CD11, KC (keratinocyte chemokine), F4/80, MIP1α and MCP1 genes related to inflammation in (macrophage) and monocytes.

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. The term "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. In addition, 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. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. 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. In addition to simple excipients, 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. As 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.

In addition, 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.

In addition, 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.

The term "diagnosis" of the present invention means to confirm the presence or characteristics of a pathological condition. In the present invention, the diagnosis can be interpreted as confirming the onset of non-alcoholic fatty liver disease induced by an increase in fat biosynthesis.

In the composition for diagnosing non-alcoholic fatty liver disease induced by increased fat biosynthesis of the present invention, 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.

In addition, 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 of the antibody.

In addition, 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 determining that non-alcoholic fatty liver disease induced by an increase in fat biosynthesis (De novo lipogenesis) when it decreases It relates to a method for diagnosing non-alcoholic fatty liver disease, comprising the steps of.

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.

In addition, 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 determining that non-alcoholic fatty liver disease induced by an increase in fat biosynthesis (De novo lipogenesis) when it decreases It relates to a method of providing information for diagnosing non-alcoholic fatty liver disease induced by increased de novo lipogenesis, comprising the steps of.

In the method for diagnosing non-alcoholic fatty liver disease induced by the increase in fat biosynthesis (De novo lipogenesis) or the method for providing information for diagnosing non-alcoholic fatty liver disease induced by increasing de novo lipogenesis, the sample is liver It is preferably any one selected from tissue, lymph fluid, saliva, serum, plasma, and blood, but is not limited thereto.

In addition, 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);

(2) measuring the mRNA or protein expression level of TCF7L2 in the sample treated with the candidate substance of step (1); And

(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.

In the screening method for the treatment of non-alcoholic fatty liver disease induced by the increase in fat biosynthesis (De novo lipogenesis), 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).

In the screening method for a therapeutic agent for non-alcoholic fatty liver disease induced by the increase in fat biosynthesis, when the expression level measured in step (2) is higher than the mRNA or protein expression level of TCF7L2 in a sample not treated with the candidate substance, the The candidate substance can be determined as a substance for the treatment of non-alcoholic fatty liver disease induced by increased fat biosynthesis.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for describing the present invention in more detail, and it is apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Example 1. Changes in expression of TCF7L2 in liver tissue of high carbohydrate diet mice

8-week-old C557BL/6 male mice were fed a high-carbohydrate diet (70% (w/w) carbohydrate, TD.98090) or a regular diet (Teklad gloval 18% protein, 2018S) for 22 weeks, and then TCF7L2 in liver tissue Expression and protein expression levels of FAS and ACC, which are key liposynthetic enzymes, were confirmed using Western blot.

As a result, as disclosed in FIG. 1, 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.

Example 2. Establishment of liver-specific TCF7L2 deficient mice

Using the Cre-LoxP system, C57BL/6 mice (hereinafter referred to as TCF7L2 LKO) with a liver-specific TCF7L2 expression deficient were prepared.

After the albumin promoter gene, which is specifically activated in the liver, a cre mouse with a cre gene and a loxP mouse with a loxP gene before and after the TCF7L2 gene were crossed, and a portion of the loxP gene was cleaved by the cre protein specifically expressed in liver tissue. The liver-specific TCF7L2 deficient mice were established.

As a control group, cre mice and loxP mice were crossed, but C57BL/6 mice (hereinafter referred to as TCF7L2 WT) that do not express the cre gene and express TCF7L2 normally were used.

Example 3. Screening for expression of genes related to lipid metabolism and glucose metabolism in liver tissue of liver-specific TCF7L2 deficient mice

Using the liver-specific TCF7L2 deficient mice established in Example 2 above, RNA sequencing analysis of the expression of genes related to lipid metabolism and sugar metabolism (RNA) to confirm changes in lipid metabolism and sugar metabolism in liver tissues according to diet. -seq) was used to screen.

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

In 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).

As a result, as shown in FIG. 3, 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 (Acetyl-CoA carboxylase), FAS (Fatty acid synthase) and 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

In 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).

As a result, as disclosed in FIG. 4, the expression of adipose biosynthesis-related genes was increased in TCF7L2 LKO mice compared to TCF7L2 WT, and in particular, the increase in genes was remarkable in the high carbohydrate refeeding group.

Through this, it was confirmed that fat biosynthesis in the liver was significantly increased in TCF7L2 LKO mice by a high carbohydrate diet compared to TCF7L2 WT.

Example 6. Changes in blood glucose and insulin concentration of liver-specific TCF7L2 deficient mice according to refeeding

Changes in blood glucose and insulin concentrations of liver-specific TCF7L2 deficient mice according to refeeding were measured.

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).

As a result, as disclosed in FIG. 5A, blood glucose and insulin concentrations in the free diet, fasting, and 6-hour refeeding group showed little change in TCF7L2 LKO mice compared to TCF7L2 WT, but the TCF7L2 WT showed little change in the group re-fed for 24 hours. In comparison, blood glucose and insulin concentrations were increased in TCF7L2 LKO mice.

In addition, in order to measure the change in blood glucose and insulin concentration of liver-specific TCF7L2 deficient mice due to high carbohydrate refeeding, the experimental group was fasted (F, fasting), fasted for 24 hours, and then re-fed for 24 hours (refeeding, Teklad gloval 18% protein, 2018S) and a high-carbohydrate diet (70% carbohydrate, TD. 98090) for 24 hours after fasting for 24 hours were classified into the group (HCR, High-carbohydrate refeeding), and the experiment was conducted. In the experiment in which the carbohydrate diet was re-fed, it was confirmed that when the high carbohydrate was re-fed as disclosed in FIG. 5B, the change in blood glucose concentration was slight, but the blood insulin concentration rapidly increased.In order to maintain a constant level of blood glucose concentration, blood insulin It could be seen that the concentration was increased.

Example 7. Changes in adipose biosynthesis related protein according to liver-specific TCF7L2 deletion and repair

Under high carbohydrate diet conditions, TCF7L2 LKO mouse tail vein with GFP-labeled adenovirus (Ad-TCF7L2) expressing TCF7L2 to confirm that the expression of adipose biosynthetic gene increased by liver-specific TCF7L2 deletion is indicated by TCF7L2. After tail vein injection, liver tissue was excised 5 days after injection, and changes in fat biosynthesis related proteins were confirmed. As a control, adenovirus (Ad-GFP) labeled with only GFP without TCF7L2 gene was injected and used in the same manner.

As a result, it was confirmed that the 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.

Through this, it was confirmed that fat biosynthesis in the liver caused by a high carbohydrate diet is regulated by TCF7L2.

Example 8. Changes in genes related to fat biosynthesis following liver-specific TCF7L2 deletion and repair

Under high-carbohydrate refeeding (HCR) conditions, changes in the expression of adipose biosynthesis-related genes were confirmed due to the deletion and repair of liver-specific TCF7L2 expression.

As a result, as shown in Fig.7, under high-carbohydrate refeeding conditions, the expression of fat biosynthesis-related genes GK, LPK, ACC, FAS, SCD1 and DGAT2, which was increased by the deletion of TCF7L2 (TCF7L2 LKO-GFP), was expressed in TCF7L2. After recovering (TCF7L2 LKO-TCF7L2), it was confirmed that it was reduced to a similar degree to that of TCF7L2 WT (TCF7L2 WT-GFP).

Example 9. Induction and control of fatty liver according to TCF7L2 expression regulation

The content of triglycerides (TG) in the liver according to the deletion and expression recovery of TCF7L2 was confirmed.

As a result, it was confirmed that the TG content in the liver increased due to the deletion of TCF7L2 under refeeding and high carbohydrate refeeding conditions as disclosed in FIGS. 8A and 8B, and the TG content in the liver was further increased under the high carbohydrate refeeding condition.

In addition, primary hepatocytes were isolated from liver tissue, and after 4 hours, Ad-GFP and Ad-TCF7L2 were infected, and after 24 hours, 1 μCi [ 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.

As a result, as disclosed in FIG. 8C, 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.

In addition, when the expression of TCF7L2 was restored through tail vein injection of adenovirus to liver-specific TCF7L2 deficient mice under high carbohydrate diet conditions, the increased TG content in the liver decreased to a similar degree as TCF7L2 WT as disclosed in FIG. 8D. I could confirm that.

Through this, it was confirmed that 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

In a situation where the primary hepatocyte cell line isolated from normal mice was treated with glucose and insulin, which are major substances inducing fat biosynthesis by carbohydrates, fat biosynthesis and related gene expression by TCF7L2 overexpression were confirmed.

As a result, as disclosed in FIG. 9, the overexpression of TCF7L2 in the primary hepatocyte line (Ad-TCF7L2) 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). In contrast, it was confirmed that fat biosynthesis was significantly reduced, and the expression of fat biosynthesis-related genes (GK, LPK, FAS and ACC) was also significantly reduced.

Through this, it was confirmed that TCF7L2 plays a role in reducing fat biosynthesis and TG content in the liver under conditions of inducing fat biosynthesis by carbohydrates.

Example 11. Regulation of fat biosynthesis in liver of TCF7L2 according to a high fat diet

In high fat dietary conditions that provide free fatty acids from the outside, changes in TG content in the liver due to liver defects of TCF7L2 (TCF7L2 LKO) or TCF7L2 overexpression (Ad-TCF7L2) were confirmed.

As a result, as disclosed in FIG. 10A, TCF7L2 LKO mice fed a high-fat diet for 12 weeks showed little change in liver TG content compared to TCF7L2 WT mice.

In addition, after tail vein injection of Ad-TCF7L2 or Ad-GFP into wild-type C57BL/6 mice fed a high fat diet for 12 weeks, the results of analyzing the TG content in liver tissue 5 days later were also found in the results of liver tissue-specific TCF7L2 deficient mice. Similarly, there was little change in the TG content in the liver.

Through this, it was confirmed that 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

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.

As a result, as disclosed in FIG. 11, 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.

Through this, it was confirmed that 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

8-week-old C557BL/6 male mice were fed a high-carbohydrate diet (70% (w/w) carbohydrate, TD.98090), and the experimental group was treated with GFP-labeled adenovirus (Ad-CF 7 L2) expressing TCF7L2 at 21 weeks. Tail vein injection was performed, and adenovirus (Ad-GFP) labeled with only GFP without TCF7L2 gene was injected into the control group by the same method, and then a high carbohydrate diet was added for 1 week, and then liver tissue was After extraction, the content of triglycerides and genes related to fat biosynthesis were confirmed. Each body weight was measured and compared before sacrifice of the animal model.

As a result, as disclosed in FIG. 12, 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. In the experimental group in which TCF7L2 expression was induced, the expression level of fat biosynthesis-related genes (SREBP1c, ChREBPα, ChREBPβ, 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

TNFα, 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, MIP1α and MCP1 gene expression levels were confirmed.

As a result, as shown in FIG. 13, 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. However, there was little difference in 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.

Claims (17)

A pharmaceutical composition for the prevention or treatment of 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. According to claim 1, wherein the TCF7L2 protein is a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease induced by increased fat biosynthesis, characterized in that consisting of the amino acid sequence of SEQ ID NO: 1. The method of claim 1, wherein the vector containing the polynucleotide encoding the TCF7L2 protein is a recombinant viral vector, a plasmid vector, a cosmid vector, or a bacteriophage vector.The non-alcoholic fatty liver disease induced by increased fat biosynthesis Pharmaceutical composition for prophylaxis or treatment. The method of claim 3, wherein the recombinant virus is any one selected from the group consisting of adenovirus, adeno-associated virus, retrovirus, herpes simplex virus, and lentivirus. Pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease induced by. The pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease according to claim 1, wherein the TCF7L2 protein or the polynucleotide encoding the TCF7L2 protein inhibits fat biosynthesis in the liver. The method of claim 1, wherein the non-alcoholic fatty liver disease is non-alcoholic simple fatty liver, non-alcoholic steatohepatitis, or non-alcoholic liver cirrhosis for the prevention or treatment of non-alcoholic fatty liver disease induced by increased fat biosynthesis. Pharmaceutical composition. The pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease according to claim 1, wherein the increased fat biosynthesis is caused by a high carbohydrate diet or type 2 diabetes. A method of inhibiting fat biosynthesis in liver by administering to an individual a vector comprising a TCF7L2 protein or a polynucleotide encoding the TCF7L2 protein. A composition for diagnosing non-alcoholic fatty liver disease, which is induced by an increase in fat biosynthesis, comprising an agent measuring the level of TCF7L2 protein or its mRNA expression. The method of claim 9, wherein the agent for measuring the expression level of TCF7L2 protein or its mRNA is at least one selected from antibodies, probes, primers, antisense oligonucleotides, and aptamers that specifically bind to TCF7L2 protein or mRNA. A composition for diagnosing non-alcoholic fatty liver disease induced by increased fat biosynthesis. A kit for diagnosing non-alcoholic fatty liver disease induced by an increase in fat biosynthesis comprising the composition of claim 9. The kit for diagnosing non-alcoholic fatty liver disease according to claim 11, wherein the kit is an RT-PCR kit, a DNA chip kit, or a protein chip kit. (a) measuring the expression level of TCF7L2 protein or its mRNA from a biological sample derived from a patient suspected of developing a non-alcoholic fatty liver disease induced by increased fat biosynthesis; And (b) comparing the measured TCF7L2 protein or mRNA expression level thereof with the level measured from a sample of a normal person, and determining that non-alcoholic fatty liver disease induced by an increase in fat biosynthesis when decreased, Method for diagnosing non-alcoholic fatty liver disease induced by increased fat biosynthesis. (a) measuring the expression level of TCF7L2 protein or its mRNA from a biological sample derived from a patient suspected of developing a non-alcoholic fatty liver disease induced by increased fat biosynthesis; And (b) comparing the measured TCF7L2 protein or mRNA expression level thereof with the level measured from a sample of a normal person, and determining that non-alcoholic fatty liver disease induced by an increase in fat biosynthesis when decreased, A method of providing information for diagnosing non-alcoholic fatty liver disease induced by increased fat biosynthesis. (1) treating a candidate substance in a sample taken from a patient suffering from non-alcoholic fatty liver disease induced by increased fat biosynthesis; (2) measuring the mRNA or protein expression level of TCF7L2 in the sample treated with the candidate substance of step (1); And (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; non-alcoholic fatty liver disease therapeutic agent comprising an increase in fat biosynthesis Screening method. 16. The method of claim 15, wherein the sample is any one selected from liver tissue, lymph fluid, saliva, serum, plasma, and blood. The method of claim 15, wherein when the expression level measured in step (2) is higher than the mRNA or protein expression level of TCF7L2 in the sample not treated with the candidate substance, the candidate substance is non-alcoholic induced by increased fat biosynthesis. A method of screening for a therapeutic agent for non-alcoholic fatty liver disease, characterized in that it is determined as a substance for treating fatty liver disease.

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