KR20210051381A - Composition for treating metabolic diseases comprising thrap3 as active ingredient - Google Patents

Abstract

The present invention relates to a composition for treating metabolic diseases containing Thrap3 as an active component, wherein Thrap3 binds to PPAR-α in the liver, and the composition for treating metabolic diseases containing Thrap3 as an active component can provide an effect of improving glucose and lipid metabolism in the liver by specifically binding to PPAR-α and regulating transcriptional activity thereof. In addition, the composition for treating metabolic diseases containing Thrap3 as an active component can be usefully used to treat metabolic diseases such as obesity, diabetes, hypertension, cardiovascular diseases, and hyperlipidemia.

Description

A composition for treating metabolic diseases containing Thrap3 as an active ingredient {COMPOSITION FOR TREATING METABOLIC DISEASES COMPRISING THRAP3 AS ACTIVE INGREDIENT}

The present invention relates to a composition for treating metabolic diseases comprising Thrap3 as an active ingredient, and more specifically, to a composition for treating metabolic diseases, comprising Thrap3 as an active ingredient, and wherein Thrap3 binds to PPAR-α in the liver. About.

Peroxisome is one of the organelles in the cells that cause this metabolic abnormality, and it has long been considered to play an insufficient role in cell function. However, through many recent studies, it is abundantly present in the liver and kidney and regulates cell proliferation and differentiation. , It plays an important role in the regulation of inflammatory mediators, and has been reported to have a wide effect on the metabolism of oxygen, glucose, lipids, and hormones. Through lipid metabolism and glucose metabolism, peroxisomes are known to play an important role in aging and tumor formation through not only insulin sensitivity, but also cell membranes and adipocyte formation, and through the influence of oxidative stress.

Peroxisome proliferator refers to a compound capable of increasing the number of peroxisomes. These include fats and fatty acids, fibrate, and prostaglandin, which are treatments for hyperlipidemia. The nuclear transcription factor receptor, which has such a peroxisome growth factor as a ligand, is collectively referred to as a peroxisom proliferator activated receptor (hereinafter referred to as "PPAR").

Over the past decade, it has been reported that nuclear hormone receptors called peroxisome growth factor activating receptors will be good targets as a pharmacological approach for various diseases. Peroxisome growth factor activating receptors are largely divided into PPAR-α, PPAR-δ/β, and PPAR-γ. PPAR-α is mainly expressed in the liver and is involved in oxidation of fatty acids or neutralization of toxic substances, and is associated with inflammatory reaction In addition, it is known that PPAR-α inhibits the production of inflammatory mediators and erythema by ultraviolet rays. PPAR-γ is known to be involved in the differentiation and fat accumulation of several cells, including adipocytes. Although the physiological function of PPAR-δ/β has not been well elucidated, it has been reported that it is evenly distributed in the living body and is involved in embryo implantation. The unique roles of each of these PPARs appear to be manifested by binding to different ligands.

PPAR-α is mainly induced by eicosanoids, carbaprostacyclin, fibrates, and nonsteroidal anti-inflammatory drugs (NSAIDs). In addition, PPAR-γ specifically binds to thiazolidinediones (TZDs), a therapeutic agent for diabetes, and is a metabolite of prostaglandin, 15-deoxy -△ ^12.14 -prostaglandin J2 (PGJ2), unsaturated fatty acids ( polyunsaturated fatty acid) and the NSAID. In particular, it has been reported that PPAR-α regulates lipid metabolism, inflammation and arteriosclerosis. Recently, many studies on PPARα as a kind of target protein for improving and treating inflammation, arteriosclerosis, obesity, and cancer have been conducted.

Metabolic disease (metabolic disease or metabolic syndrome) is rapidly spreading, showing characteristics such as obesity, type 2 diabetes, hypertension, cardiovascular disease, and hyperlipidemia. These metabolic disorders are deeply associated with abnormal lipid homeostasis due to excessive lipid intake and lack of exercise. Recent studies have reported that when PPAR-α activity is artificially activated in adipocytes and muscle cells, the expression of genes involved in fatty acid oxidation and energy consumption is increased.

Obesity refers to a disease caused by the loss of energy balance in the human body due to genetic factors, environmental factors, mental factors, or lifestyle habits such as diet and lack of exercise. The number of obese patients continues to increase, and the World Health Organization recently reported that more than 1 billion children are overweight among adults around the world, and at least 300 million of them are obese. Obesity, in particular, has a higher prevalence and mortality than other diseases, and has been found to be associated with complications of various adult diseases. For example, obese patients have a mortality rate of about 4 times higher in diabetic disease than those in normal weight, 2 times in case of mild hepatic disease, 1.6 times in cerebrovascular disease, and 1.8 times in case of coronary artery disease. It is reported to be high. Currently, diet, exercise therapy, and drug therapy including diuretics, laxatives, and the like are being tried in a variety of ways, but the use of it is limited temporarily and limitedly to the occurrence of side effects such as nutritional imbalance, immunity reduction, and depression.

In particular, it has been reported that lipid accumulation is decreased as a result of treatment with a ligand or agonist for PPAR-α in experimental obese mice (Wang Y.X. et al., Cell, 113: 159, 2003). In addition, Wy14,643, the most well-known PPARα ligand, reduces triglyceride (TG) and blood cholesterol levels in human metabolism, while high density lipoprotein (HDL) in the intestine and liver. It is known to actively regulate cholesterol remodeling and cholesterol outflow (Caroline Duval et al.. Biochemicaet Biophtsica Acta, 1771: 961-971, 2007). In addition, it is known that the ligand of PPAR-α stimulates the ABCA1 pathway, promotes cholesterol removal from blood vessels, and reduces the formation of dysfunction (Chinetti et al. Nat Med 7: 53-58, 2001).

In addition, fibrates, which are specific ligands for PPAR-α, are known to be involved in glucose homeostasis by lowering fasting insulin and glucose concentrations in hyperinsulinic patients with metabolic syndrome. In addition, Apo AI and A-II related to PPAR-α lipid metabolism are major proteins of HDL, and show the effect of preventing vascular disease by moving excess cholesterol in tissues to the liver. PPAR-α is Apo AI and A-II. It is known to decrease the incidence of arteriosclerosis by increasing the HDL concentration by increasing the expression of the gene (Stael B. et al., Circulation 98: 2088-2093, 1998).

Through these research reports, it can be seen that PPAR-α is widely involved in lipolysis and is an important target protein for metabolic diseases caused by obesity. Furthermore, the ligand of PPAR-α may have a high potential as a therapeutic agent for the metabolic disease. However, studies on factors that regulate the transcriptional activity of PPAR-α have not yet been identified in various ways.

On the other hand, insulin is a peptide secreted from the beta cells of the pancreas and is a substance that plays a very important role in regulating blood sugar in the body. Diabetes is a metabolic disease in which the concentration of glucose in the blood increases due to insufficient secretion of insulin or the normal functioning. Type 1 diabetes is a case where the pancreas cannot secrete insulin and blood sugar rises, and type 2 is when insulin is not secreted properly or the secreted insulin does not work properly in the body and the blood sugar in the body is not controlled and rises. It is called diabetes. In the case of type 1 diabetes, the administration of insulin is indispensable, whereas in the case of type 2 diabetes, an oral hypoglycemic agent based on a chemical substance is used for treatment, and some patients are treated with insulin. .

Because there is no abnormality in the secretion of insulin in type 2 diabetes, it is also called non-insulindependent diabetes mellitus. Type 2 diabetes is often combined with obesity due to intake of high-calorie and high-protein diets and lack of exercise, and can also be caused by genetic factors, infections, certain drugs, and pancreatic surgery. Currently, the best treatment for type 2 diabetes is to lose weight through diet changes and exercise lifestyle corrections. In addition, in some cases, drugs such as insulin sensitivity improving agents are administered. However, until now, no suitable therapeutic agent or treatment method that can directly treat type 2 diabetes has been developed.

Therefore, the present inventors are a novel inhibitor that modulates the transcriptional activity of PPAR-α, and a thyroid hormone receptor-associated protein (hereinafter referred to as "Thrap3") specifically binds to PPAR-α to activate transcription. The present invention was completed on the basis of affecting sugar and lipid metabolism in the liver by controlling it.

Wang Y.X. et al., Cell, 113: 159, 2003 Caroline Duval et al.. Biochemicaet Biophtsica Acta, 1771: 961-971, 2007 Chinetti et al. Nat Med 7: 53-58, 2001 Stael B. et al., Circulation 98: 2088-2093, 1998

In order to solve the above-described problem, the present invention comprises Thrap3 as an active ingredient, and the Thrap3 is characterized in that it binds with PPAR-α in the liver, and an object of the present invention is to provide a composition for treating metabolic diseases.

In addition, the second object of the present invention is to provide Thrap3 as a novel inhibitor that modulates the transcriptional activity of PPAR-α in the liver.

In order to achieve the above object, the composition for treating metabolic diseases according to the present invention includes Thrap3 as an active ingredient, and the Thrap3 may bind to PPAR-α in the liver.

Here, the site binding to PPAR-α may be a c-terminal of Thrap3 domains.

Here, the Thrap3 may be an inhibitor that regulates the transcriptional activity of PPAR-α in the liver.

Here, Thrap3 may induce oxidation of fatty acids by binding to PPAR-α in the liver.

Here, Thrap3 can regulate sugar metabolism by binding to PPAR-α in the liver.

Here, the Thrap3 can regulate lipid metabolism by binding to PPAR-α in the liver.

Here, the metabolic diseases may include obesity, diabetes, hypertension, cardiovascular disease, and hyperlipidemia.

Here, the diabetes is preferably type 2 diabetes.

Meanwhile, the present invention can provide Thrap3 as a novel inhibitor that regulates the transcriptional activity of PPAR-α in the liver.

Here, the c-terminal of the Thrap3 domain may bind to PPAR-α in the liver.

The composition for treating metabolic diseases comprising Thrap3 as an active ingredient of the present invention according to the above can provide an effect of improving sugar and lipid metabolism in the liver by specifically binding to PPAR-α to regulate transcriptional activity. have.

In addition, the composition for treating metabolic diseases comprising Thrap3 of the present invention as an active ingredient may be usefully used to treat metabolic diseases such as obesity, diabetes, hypertension, cardiovascular disease and hyperlipidemia.

1 shows a mouse in which Thrap3 expression is knocked out specifically only in the liver using the albumin-cre recombinase system in a mouse model according to an embodiment of the present invention.
FIG. 2 shows the results of phenotypic analysis according to a general diet of a liver-specific Thrap3 knockout mouse (hereinafter referred to as “Example 1”) according to an embodiment of the present invention.
3 is a diagram showing the results of phenotypic analysis according to a high fat diet of mice whose expression of Thrap3 is knocked out specifically only in the liver according to an embodiment of the present invention.
FIG. 4 shows the results of RNA sequencing of mice in which Thrap3 expression is knocked out specifically only in the liver according to an embodiment of the present invention.
5 is a qPCR result of the expression pattern of metabolic genes involved in fat synthesis in mice in which Thrap3 expression is knocked out specifically only in the liver and in hepatocytes in which Thrap3 is knocked down according to an embodiment of the present invention. Is shown.
6 is a qPCR result of the expression patterns of major genes involved in fatty acid oxidation in mice in which Thrap3 expression is specifically knocked out only in the liver and in hepatocytes in which Thrap3 is knocked down according to an embodiment of the present invention. Is shown.
7 is a metabolic gene involved in glycolysis in relation to energy generation in a mouse in which Thrap3 expression is specifically knocked out only in the liver and in hepatocytes in which Thrap3 is knocked down according to an embodiment of the present invention. It shows the qPCR results on the expression pattern of the
8 is Acetyl-CoA produced by fatty acid oxidation in relation to energy production in mice in which Thrap3 expression is specifically knocked out only in the liver and in hepatocytes in which Thrap3 is knocked down according to an embodiment of the present invention. It shows the result of confirming whether the production amount of and Acetyl-CoA affects the ketone production process.
9 shows the expression patterns of major genes targeting PPAR-α in mice in which Thrap3 expression is specifically knocked out only in the liver and in hepatocytes in which Thrap3 is knocked down according to an embodiment of the present invention. It shows the qPCR results.
FIG. 10 shows immunoprecipitation and Western blot results confirming binding to Thrap3 when PPAR-α is overexpressed in hepatocytes knocked down Thrap3 according to an embodiment of the present invention.
FIG. 11 shows the results of a luciferase assay and western blot to confirm whether Thrap3 regulates the transcriptional activity of PPAR-α in hepatocytes knocked down Thrap3 according to an embodiment of the present invention. .
12 shows qPCR results on the expression patterns of major genes involved in fatty acid oxidation when treated with Wy14643 as a specific receptor agonist for PPAR-α in hepatocytes knocked down Thrap3 according to an embodiment of the present invention. will be.
13 is a photograph showing a result of inducing a fatty liver model through oleic acid in hepatocytes knocked down Thrap3 according to an embodiment of the present invention.
14 shows qPCR results of expression patterns of metabolic genes involved in fat synthesis in a fatty liver model induced through oleic acid in hepatocytes knocked down Thrap3 according to an embodiment of the present invention.

The terms used in the present application are only used to describe specific examples. So, for example, a singular expression includes a plural expression unless the context clearly has to be singular. In addition, terms such as "include" or "include" used in the present application are used to clearly refer to the existence of features, steps, functions, components or combinations thereof described in the specification, but other features It should be noted that it is not used to preliminarily exclude the presence of elements, steps, functions, components, or combinations thereof.

In the present specification, "knockout" is a genetic engineering generally used in a mouse experimental model, and refers to a method of completely eliminating the expression of a target gene.

In the present specification, "knockdown" refers to a method of reducing the expression (mRNA level) of a target gene by using siRNA or shRNA.

Meanwhile, unless otherwise defined, all terms used in the present specification should be viewed as having the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention belongs. Therefore, unless clearly defined in the specification, a specific term should not be interpreted as an excessively ideal or formal meaning.

As a result of researching to solve the above-described problems, the inventors have come up with the following invention.

The present specification discloses a composition for treating metabolic diseases, comprising Thrap3 as an active ingredient, wherein Thrap3 binds to PPAR-α in the liver. Hereinafter, with respect to the composition for treating metabolic diseases containing Thrap3 as an active ingredient of the present invention, it will be described in detail by each technical feature.

In the present invention, the composition for treatment of metabolic diseases comprising Thrap3 as an active ingredient may specifically bind to the PPAR-α in the liver by the c-terminal of the Thrap3 domain. In this regard, specific details will be described in detail in the following <Examples and Evaluation>.

In addition, in the present invention, the Thrap3 may be utilized as an inhibitor that regulates the transcriptional activity of PPAR-α in the liver, but is not limited thereto. Specifically, Thrap3 induces oxidation of fatty acids by binding with PPAR-α in the liver, and can regulate sugar metabolism and lipid metabolism. In this regard, specific details will be described in detail in the following <Examples and Evaluation>.

In addition, in the present invention, the metabolic disease may include obesity, diabetes, high blood pressure, cardiovascular disease, and hyperlipidemia, but is not limited thereto. Specifically, the metabolic disease is deeply associated with abnormalities in lipid homeostasis due to excessive lipid intake and lack of exercise. For example, when the activity of PPAR-α is artificially activated in adipocytes and muscle cells, the expression of genes involved in fatty acid oxidation and energy consumption may be increased.

In addition, in the present invention, the diabetes may be type 2 diabetes, but is not limited thereto. The diabetes is a disease in which the ability to control blood glucose levels is impaired because the patient partially loses the ability to respond appropriately to the action of insulin. Specifically, the type 2 diabetes refers to a case where insulin is not secreted properly or the secreted insulin does not function properly in the body, so that blood sugar in the body cannot be controlled and rises.

Meanwhile, the present specification further discloses Thrap3 as a novel inhibitor that regulates the transcriptional activity of PPAR-α in the liver.

In addition, in the present invention, the c-terminal of Thrap3 domains can specifically bind to PPAR-α in the liver. In this regard, specific details will be described in detail in the following <Examples and Evaluation>.

Hereinafter, what is claimed by the present specification will be described in more detail with reference to the accompanying drawings and embodiments. However, the drawings or examples presented in the present specification may be modified in various ways by a person skilled in the art to have various forms, and the description of the present specification is not limited to a specific disclosure form. It should be viewed as including all equivalents or substitutes included in the spirit and scope of the present invention. In addition, the accompanying drawings are provided to help those skilled in the art understand the present invention more accurately, and may be exaggerated or reduced than in actuality.

{Examples and evaluation}

Example 1. Preparation of mice in which Thrap3 expression was knocked out specifically only in the liver (in vivo test)

First, the purchased Thrap3 floxed mouse sperm is obtained through an IVF (In Vitro Fertilization) experiment. Thereafter, through mating between Thrap3 (floxed/wild-type) mice, Thrap3 (floxed/floxed) mice (home genotype) are selected through genotyping, and then mated with Albumin-cre mice (hereinafter, floxed = F, wild-type = +, Albumin-cre = cre). A Thrap3(F/F) mouse and a Trap3(cre/+) mouse were crossed, and Thrap3(F/+) mouse and Thrap3(F/+); (cre/+) Gain a mouse. Thereafter, Thrap3 (F/F) mice and Thrap3 (F/+); (cre/+) mice were crossed, and Thrap3(F/+) mice, Thrap3(F/F) mice, Thrap3(F/+); (cre/+) mouse and Thrap3 (F/F); (cre/+) Gain a mouse. Among the mice from the above generation, a sufficient number of generational species of children is secured by crossing the mice with the cre gene with Thrap3(F/+) mice or Thrap3(F/F) mice. Then, Thrap3 (F/F); (cre/+) mouse and Thrap3(F/F); (cre/+) mice were crossed and finally Thrap3(F/F) mice and Thrap3(F/F); (cre/cre) You can secure a mouse. Here, Thrap3(F/F); The (cre/cre) mouse was selected as a mouse (hereinafter referred to as'Example 1') whose expression of Thrap3 was knocked out specifically only in the liver corresponding to an embodiment of the present invention.

1 shows a mouse in which Thrap3 expression is knocked out specifically only in the liver using the albumin-cre recombinase system in a mouse model according to an embodiment of the present invention.

Referring to FIG. 1, in order to confirm the effect of Thrap3 as a novel inhibitor that regulates the transcriptional activity of PPAR-α in the liver of the present invention, it was confirmed that the mouse was specifically knocked out of Thrap3 expression. I can.

Comparative Example 1. Preparation of control mice

Thrap3 (F/F) mice and Thrap3 (F/F) finally secured by performing the same as in Example 1; Among the (cre/cre) mice, Thrap3(F/F) mice were selected as mice corresponding to the control of the present invention (hereinafter, referred to as'Comparative Example 1').

Preparation Example 1. Preparation of hepatocytes knocked down Thrap3

After removing the liver tissue through dissection from the mouse in which Thrap3 expression was knocked out specifically only in the liver of Example 1, a protein sample and an RNA sample were obtained through homogenization to proceed with qPCR. I did. In the case of knockdown hepatocytes, HepG2 hepatocytes were used. Cell culture was performed in FBS DMEM, and siRNA having a complementary sequence to knock down Thrap3 was produced, and transfection was performed on HepG2 cells with RNAi max reagent. At this time, the siRNA having a complementary sequence that knocks down Thrap3 is an siRNA corresponding to SEQ ID NO: 1. Thereafter, PCR was performed with cDNA through RNA sampling, primers of genes related to sugar and lipid metabolism including fatty acid oxidation were prepared, and qPCR was performed to confirm the difference in gene expression level.

Preparation Example 2. Preparation of hepatocytes knocked down Thrap3

Hepatocytes knocked down Thrap3 in the same manner as in Preparation Example 1, except that the siRNA having a complementary sequence that knocks down Thrap3 is the siRNA corresponding to SEQ ID NO: 2 Was prepared.

On the other hand, the sequence of the siRNA having a complementary sequence (sequence) knocking down Thrap3 of SEQ ID NO: 1 and SEQ ID NO: 2 are shown in Table 1 below.

SEQ ID NO: 1 (KD#1) CGAUCUGUCUCUCGUUCAA SEQ ID NO: 2 (KD#2) UGGCAGAACUACCGGCAAG

Comparative Preparation Example 1. Preparation of control hepatocytes

Control hepatocytes in the same manner as in Preparation Example 1, except that the liver tissue of the control mouse of Comparative Example 1 was removed, and the siRNA used siRNA that binds to another sequence that does not knock down Thrap3. Was prepared.

Evaluation 1. Confirmation of weight change, GTT and ITT results according to diet in mice whose expression of Thrap3 is knocked out specifically in the liver and control mice

In the present invention, measurement of weight change according to a normal chow diet and a high fat diet for a mouse in which Thrap3 expression is knocked out and a control mouse (hereinafter referred to as "Comparative Example 1"), GTT (glucose tolerance test, glucose tolerance test) and ITT (insulin tolerance test, insulin resistance test) were performed.

2 is a diagram illustrating a result of phenotypic analysis according to a general diet of a liver-specific Thrap3 knockout mouse (hereinafter referred to as “Example 1”) according to an embodiment of the present invention.

Table 2 below shows the weight change amount of Example 1 and Comparative Example 1 according to a general diet, measured every week.

1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks Normal
Diet Example 1 24.7 g 25.4 g 26.7 g 27.8 g 28.8 g 29.1 g 29.6 g Comparative Example 1 24.7 g 25.7 g 26.7 g 27.4 g 28.1 g 28.5 g 28.8 g

2A and 2, there is little difference in weight change between Example 1 and Comparative Example 1 as a control, and referring to FIGS. 2B and 2C, the Comparative Example 1 as a control. It can be seen that there is little difference in glucose change over time compared to 1.

Meanwhile, FIG. 3 shows the results of phenotypic analysis according to a high-fat diet of mice whose expression of Thrap3 is knocked out specifically only in the liver according to an embodiment of the present invention.

Table 3 below shows the weight change of Example 1 and Comparative Example 1 according to a high fat diet, measured every week.

1 week 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks High fat
Diet Example 1 28.5 g 31.3 g 33.1 g 36.2 g 39.1 g 41.4 g 41.5 g Comparative Example 1 28.1 g 29.7 g 31.0 g 33.7 g 36.0 g 38.0 g 38.5 g

Referring to Figures 3a and 3, in the case of Example 1 in the present invention, it can be seen that the weight increase rate is lower than that of Comparative Example 1 as a control. Referring to Figures 3B and 3C, the Example 1 It can be seen that the rate of glucose reduction over time is higher than that of Comparative Example 1, which is a control group.

Therefore, it can be inferred that Example 1 is due to the effect of expression of Thrap3 through a remarkable reduction in body weight and remarkable improvement in insulin sensitivity when a high-fat diet is performed.

Evaluation 2. Confirmation of RNA sequencing results of mice whose expression of Thrap3 is knocked out specifically in the liver and control mice

FIG. 4 shows the results of RNA sequencing of mice in which Thrap3 expression is knocked out specifically only in the liver according to an embodiment of the present invention.

4A and 4B, it can be seen that in the case of Example 1 in the present invention, lipid metabolism and sugar metabolism were significantly increased in the metabolic signaling pathway compared to Comparative Example 1 as a control.

Therefore, in the present invention, as a result of RNA sequencing of mice whose expression of Thrap3 is knocked out specifically only in the liver, it can be inferred that lipid metabolism and sugar metabolism are significantly increased due to the influence of the expression of Thrap3.

Evaluation 3. Confirmation of expression of genes involved in fat synthesis and fatty acid oxidation

In the present invention, qPCR was performed to investigate the expression patterns of metabolic genes and major genes involved in fat synthesis and fatty acid oxidation in mice in which Thrap3 expression is knocked out and in hepatocytes in which Thrap3 is knocked down.

5 is a qPCR result of the expression pattern of metabolic genes involved in fat synthesis in mice in which Thrap3 expression is knocked out specifically only in the liver and in hepatocytes in which Thrap3 is knocked down according to an embodiment of the present invention. Is shown.

Referring to FIG. 5A, it can be seen that the expression pattern of metabolic genes involved in fat synthesis of Example 1 was significantly reduced. Referring to FIG. 5B, metabolism involved in fat synthesis in hepatocytes knocked down Thrap3. It can be seen that the expression pattern of the genes was significantly reduced.

6 is a qPCR result of the expression patterns of major genes involved in fatty acid oxidation in mice in which Thrap3 expression is specifically knocked out only in the liver and in hepatocytes in which Thrap3 is knocked down according to an embodiment of the present invention. Is shown.

Referring to Figure 6a, it can be seen that the expression pattern of major genes involved in fatty acid oxidation in mice whose expression of Thrap3 is knocked out specifically only in the liver is significantly increased, and referring to Figure 6b, Thrap3 is knocked out. It can be seen that the expression patterns of major genes involved in fatty acid oxidation in (knockdown) hepatocytes were remarkably increased.

Therefore, in the present invention, it can be confirmed that the fatty acid oxidation process occurs actively in the liver due to the expression of Thrap3, and as a result, lipid metabolism is remarkably improved through processes such as reduction of fat synthesis and activation of ATP production. have. Furthermore, by controlling the expression of Thrap3, it can be effectively used in the treatment of metabolic diseases including obesity, diabetes, hypertension, cardiovascular disease, and hyperlipidemia.

Evaluation 4. Confirmation of expression of genes involved in glycolysis

In the present invention, qPCR was performed to investigate the expression patterns of metabolic genes involved in glycolysis in mice in which Thrap3 expression is knocked out and in hepatocytes in which Thrap3 is knocked down.

7 is a metabolic gene involved in glycolysis in relation to energy generation in a mouse in which Thrap3 expression is specifically knocked out only in the liver and in hepatocytes in which Thrap3 is knocked down according to an embodiment of the present invention. It shows the qPCR results for the expression pattern of the.

Referring to Figure 7a, it can be seen that the expression pattern of metabolic genes involved in the glycolysis of mice whose expression of Thrap3 is knocked out specifically only in the liver is significantly increased, and referring to Figure 7b, Thrap3 is knocked down. It can be seen that the expression patterns of metabolic genes involved in glycolysis in the (knockdown) hepatocytes were significantly increased.

Therefore, in the present invention, due to the influence of Thrap3 expression, glycolysis occurs actively in the liver, and as a result, it can be confirmed that sugar metabolism is remarkably improved. Furthermore, by controlling the expression of Thrap3, it can be effectively used in the treatment of metabolic diseases including obesity, diabetes, hypertension, cardiovascular disease, and hyperlipidemia.

Evaluation 5. Confirmation of the amount of Acetyl-CoA produced by fatty acid oxidation

In the present invention, through the change in the glycolysis process during metabolism, it is additionally important to participate in the mitochondrial TCA cycle, which is the next pathway in the glycolysis process, and the amount of Acetyl-CoA produced by the fatty acid oxidation process was confirmed.

8 is Acetyl-CoA produced by fatty acid oxidation in relation to energy production in mice in which Thrap3 expression is specifically knocked out only in the liver and in hepatocytes in which Thrap3 is knocked down according to an embodiment of the present invention. It shows the result of confirming whether the production amount of and Acetyl-CoA affects the ketone production process.

Referring to FIG. 8A, it can be seen that Acetyl-CoA was somewhat increased in hepatocytes knocked down Thrap3. This means that the fatty acid oxidation process actively occurred in the liver due to the expression of Thrap3.

On the other hand, the Acetyl-CoA moves in the form of a ketone body through the process of producing ketones so as to affect the metabolism of the surrounding tissues as well as the generation of energy in the mitochondria, thereby affecting energy metabolism. Whether or not the generated Acetyl-CoA exhibits the above tendency was confirmed through a mouse in which Thrap3 expression was knocked out specifically in the liver and through hepatocytes in which Thrap3 was knocked down. Referring to, it can be seen that there is no significant difference from the control group.

Therefore, it can be inferred that the effect of the generated Acetyl-CoA on the production of ketones is small, and instead, energy generation is remarkably increased through the TCA circuit and the electron transport system in the mitochondria, and metabolic improvement is greatly improved.

Evaluation 6. Identification of Thrap3 as an inhibitor regulating the transcriptional activity of PPAR-α

9 shows the expression patterns of major genes targeting PPAR-α in mice in which Thrap3 expression is specifically knocked out only in the liver and in hepatocytes in which Thrap3 is knocked down according to an embodiment of the present invention. It shows the qPCR results.

Referring to FIG. 9, the expression patterns of genes targeted for PPAR-α are significantly increased in both mice whose expression of Thrap3 is knocked out specifically only in the liver and in hepatocytes that have knocked down Thrap3. I can confirm.

From the above results, it is assumed that Thrap3 will play a role in regulating the activity of PPAR-α, and to clarify this, the interaction between Thrap3 and PPAR-α was confirmed.

FIG. 10 shows immunoprecipitation and Western blot results confirming binding to Thrap3 when PPAR-α is overexpressed in hepatocytes knocked down Thrap3 according to an embodiment of the present invention.

Referring to FIG. 10A, when PPAR-α is overexpressed in hepatocytes knocked down Thrap3, it can be seen that overexpressed PPAR-α binds to Thrap3. More specifically, after transfection into cells with Flag-tagged Vehicle and PPAR-α, respectively, binding of PPAR-α and Thrap3 present in the cell as endogenous can be confirmed through IP (immunoprecipitation).

In addition, referring to FIGS. 10B and 10C, it can be seen that the overexpressed PPAR-α specifically binds to the c-terminal of Thrap3 domains. More specifically, referring to FIG. 10B, based on the results of FIG. 10A, flag-tagged PPAR-α, HA-tagged Vechicle, and domains of Thrap3 are mutated, respectively, in the form of DNA plasmids. As a result of confirming which domain of Thrap3 by co-transfection, PPAR-α binds to Thrap3 through IP (immunoprecipitation), it can be confirmed that PPAR-α binds to the C-Terminal of Thrap3.

In addition, referring to FIG. 10C, as a result of more clearly showing the results of FIG. 10B, it can be additionally confirmed that PPAR-α binds to the C-terminal following sequence 640.

FIG. 11 shows the results of a luciferase assay and western blot to confirm whether Thrap3 regulates the transcriptional activity of PPAR-α in hepatocytes knocked down Thrap3 according to an embodiment of the present invention. .

Referring to Figure 11a, the activity (activity) of the PPAR-α is increased in the hepatocytes knocked down the Thrap3 through the luciferase assay (luciferase assay), the specific receptor agonist of the PPAR-α (agonist) When treated with Wy14643 known as, it can be seen that the activity of PPAR-α is remarkably increased.

In addition, referring to FIG. 11B, when PPAR-α is overexpressed in hepatocytes knocked down Thrap3 and then treated with Wy14643, the binding of PPAR-α and Thrap3 decreases, whereas PPAR-α In the case of SRC-1, which is known as a co-activator in the transcriptional activity of, it can be seen that the binding significantly increased.

In addition, referring to FIG. 11C, after transfection of Thrap3 by dose based on the results of FIGS. 11A and 11B, the amount of Thrap3 was divided into the group not treated with Wy-14643 and the group treated with the treatment. As this increases, it can be seen that the level of SRC-1 by Wy-14643 decreases. FIG. 12 is a specific receptor agonist of PPAR-α in hepatocytes knocked down Thrap3 according to an embodiment of the present invention. When treated with Wy14643, the qPCR results of the expression patterns of major genes involved in fatty acid oxidation are shown.

Here, "Scr NT" refers to a sample that was transfected with Scr (control siRNA) and not treated with Wy-14643, and "Scr WY" refers to a sample that was transfected with Scr (control siRNA) and treated with Wy-14643. it means. In addition, "#1 NT" refers to a sample that was transfected with Thrap3 siRNA #1 (SEQ ID NO: 1), and is not treated with Wy-14643, and "#1 Wy" is transfected with Thrap3 siRNA #1 (SEQ ID NO: 1). And means a sample treated with Wy-14643. On the other hand, "#2 NT" refers to a sample that was transfected with Thrap3 siRNA #2 (SEQ ID NO: 2), and is not treated with Wy-14643, and "#2 Wy" is transfected with Thrap3 siRNA #2 (SEQ ID NO: 2). Referring to FIG. 12, when the Thrap3 was knocked down with Wy14643 in hepatocytes, the expression patterns of genes involved in fatty acid oxidation were remarkably increased. I can confirm.

Therefore, the Thrap3 interacts with PPAR-α in the liver to regulate the transcriptional activity of PPAR-α, and accordingly, the fatty acid oxidation process occurs actively in the liver, resulting in a reduction in fat synthesis and energy generation (ATP production). It can be seen that lipid metabolism is remarkably improved through processes such as activation. Furthermore, by regulating the activity of PPAR-α in the liver by the expression of Thrap3, it can be effectively used in the treatment of metabolic diseases including obesity, diabetes, hypertension, cardiovascular disease and hyperlipidemia.

Evaluation 7. Confirmation of the expression of genes involved in lipid synthesis by inducing a fatty liver model

In the present invention, to further confirm the effect of Thrap3 on fat metabolism in hepatocytes knocked down Thrap3 in the present invention, qPCR was performed to determine the result by inducing a fatty liver model in the hepatocytes through oleic acid. I did.

13 is a photograph showing a result of inducing a fatty liver model through oleic acid in hepatocytes knocked down Thrap3 according to an embodiment of the present invention.

Referring to FIGS. 13A and 13B, it can be seen that the expression pattern of Thrap3 is remarkably increased when the fatty liver model is induced through oleic acid in hepatocytes in which Thrap3 is knocked down.

14 shows qPCR results of expression patterns of metabolic genes involved in fat synthesis in a fatty liver model induced through oleic acid in hepatocytes knocked down Thrap3 according to an embodiment of the present invention.

Here, "Scr NT" refers to a sample that was transfected with Scr (control siRNA) and not treated with oleic acid, and "Scr OA" refers to a sample that was transfected with Scr (control siRNA) and treated with oleic acid. . In addition, "siThrap3 NT" refers to a sample transfected with Thrap3 siRNA and not treated with Oleic acid, and "siThrap3 OA" refers to a sample transfected with Thrap3 siRNA and treated with Oleic acid.

Referring to FIG. 14, it can be seen that when a fatty liver model is induced through oleic acid in hepatocytes knocked down Thrap3, the expression pattern of genes involved in lipid synthesis is remarkably changed.

Therefore, the Thrap3 interacts with PPAR-α in the liver to regulate the transcriptional activity of PPAR-α, and accordingly, the fatty acid oxidation process occurs actively in the liver, resulting in a reduction in fat synthesis and energy generation (ATP production). It can be seen that lipid metabolism is remarkably improved through processes such as activation. Furthermore, by regulating the activity of PPAR-α in the liver by the expression of Thrap3, it can be effectively used in the treatment of metabolic diseases including obesity, diabetes, hypertension, cardiovascular disease and hyperlipidemia.

The composition for treating metabolic diseases comprising Thrap3 as an active ingredient of the present invention according to the above can provide an effect of improving sugar and lipid metabolism in the liver by specifically binding to PPAR-α to regulate transcriptional activity. have.

In addition, the composition for treating metabolic diseases comprising Thrap3 of the present invention as an active ingredient may be usefully used to treat metabolic diseases such as obesity, diabetes, hypertension, cardiovascular disease and hyperlipidemia.

The above description is merely illustrative of the technical idea of the present invention, and those of ordinary skill in the art to which the present invention pertains will be able to make various modifications and variations without departing from the essential characteristics of the present invention.

Accordingly, the embodiments disclosed in the present invention are not intended to limit the technical idea of the present invention, but to explain the technical idea, and the scope of the technical idea of the present invention is not limited by these embodiments. The scope of protection of the present invention should be construed by the following claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.

<110> UNIST(ULSAN NATIONAL INSTITUTE OF SCIENCE AND TECHNOLOGY) <120> COMPOSITION FOR TREATING METABOLIC DISEASES COMPRISING THRAP3 AS ACTIVE INGREDIENT <130> KL-P-02238 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> RNA <213> Homo sapiens <400> 1 cgaucugucu cucguucaa 19 <210> 2 <211> 19 <212> RNA <213> Homo sapiens <400> 2 uggcagaacu accggcaag 19

Claims (10)

Containing Thrap3 as an active ingredient, wherein Thrap3 is characterized in that it binds with PPAR-α in the liver, a composition for treating metabolic diseases.
The method of claim 1,
The site binding to the PPAR-α is characterized in that the c-terminal of the domain of Thrap3, metabolic disease treatment composition.
The method of claim 1,
The Thrap3 is a composition for treating metabolic diseases, characterized in that it is an inhibitor that modulates the transcriptional activity of PPAR-α in the liver.
The method of claim 1,
The Thrap3 is a composition for treating metabolic diseases, characterized in that it induces oxidation of fatty acids by binding with PPAR-α in the liver.
The method of claim 1,
The Thrap3 is a composition for treating metabolic diseases, characterized in that it regulates sugar metabolism by binding to PPAR-α in the liver.
The method of claim 1,
The Thrap3 is a composition for treating metabolic diseases, characterized in that it regulates lipid metabolism by binding with PPAR-α in the liver.
The method of claim 1,
The metabolic disease is characterized in that including obesity, diabetes, hypertension, cardiovascular disease and hyperlipidemia, metabolic disease treatment composition.
The method of claim 7,
The diabetes is characterized in that the type 2 diabetes, metabolic disease treatment composition.
Thrap3 as a novel inhibitor regulating the transcriptional activity of PPAR-α in the liver.
The method of claim 9,
Thrap3, characterized in that the c-terminal of the Thrap3 domain binds to PPAR-α in the liver.

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