KR101727506B1 - Pharmaceutical composition for the prevention or treatment of fat liver comprising GDF15 protein or polynucleotide encoding GDF15 as an effective ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating fatty liver and containing a growth differentiation factor 15 (GDF15) protein or a polynucleotide encoding the same as an active ingredient. When the GDF15 protein or the polynucleotide encoding the same is administered to an ob/ob mouse model, which is an obesity animal model, an effect in suppressing an increase in body weight is shown. Also, lipogenesis in liver tissue and fat tissue is decreased, and the expression of genes in charge of lipolysis and genes related to the improvement of mitochondrial functions and the expression of genes related to beta-oxidation are increased. Additionally, when different concentrations of recombinant GDF15 proteins are treated in vitro on muscle cells and hepatocytes, it has been finally confirmed that mitochondrial functions are improved by increasing mitochondrial oxygen consumption amounts and lipolysis. Therefore, the GDF15 protein or the polynucleotide encoding the same can increase mitochondrial functions, and thus can be effectively used as a pharmaceutical composition for preventing or treating fatty liver.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating fatty liver comprising GDF15 protein or polynucleotide encoding the GDF15 protein as an active ingredient,

The present invention relates to a therapeutic pharmaceutical composition containing, as an active ingredient, a growth factor 15 (GDF 15) protein or a polynucleotide encoding the same for the prevention or treatment of fat liver.

The liver is a large organs located in the right and upper abdominal cavity of the body and weighs about 1.5 Kg in adults. The normal liver is grossly reddish and the surface is smooth. The shape and size of the liver are changed when the liver disease develops. As a typical example, when a lot of alcohol is consumed and grease accumulates in the liver, the size of the liver becomes larger and it becomes yellowish by the grease. Conversely, when cirrhosis is severe, the size of the liver is reduced and the surface changes to rugose.

The liver is a very important organ with various functions, and its functions are summarized as follows. First, the liver functions properly to treat various nutrients in our body, that is, metabolic function. Foods that are absorbed from the bowel are appropriately changed in the liver for use in various tissues of our body, and nutrients are used in various tissues, and the remaining waste is transported and processed again to the liver. Second, the liver functions to store some nutrients needed for our body. The third is to create bile acid, a substance necessary for nutrient absorption in the intestines, and to discharge it to the intestines through the bile duct. If these bile acids are not produced, nutrients will not be absorbed and nutritional deficiencies will occur. Fourth, our bodies function to make substances (albumin, blood clotting proteins, cholesterol, etc.) that are absolutely necessary for proper functioning. Finally, drinking (alcohol), medications, and we are acting to decrypt the number of toxins in the body caused (Eugene Brainward, et al. Harrison 's principles of internal medicine. 15 th edition. 2001).

Fat liver refers to a state of abnormally accumulating fat within hepatocytes, and medically refers to a pathological state in which the neutral lipid content exceeds 5% of the total liver weight. Alcoholic fatty liver disease (ALD), which is caused by persistent and excessive drinking, and nonalcoholic fatty liver, which is similar to alcoholic fatty liver, have.

Alcoholic fatty liver is common in Korea, and alcohol consumption promotes fat synthesis in the liver and normal energy metabolism is not done. It can develop into hepatitis or cirrhosis according to the degree of alcohol consumption. In addition, non-alcoholic fatty liver disease (NAFLD) can be caused by causes of obesity, diabetes, hyperlipidemia, drug, etc. regardless of drinking, and simple fatty liver refers to a wide range of diseases including non-alcoholic steatohepatitis (NASH), advanced fibrosis, and cirrhosis, which exhibit steatosis and hepatocellular inflammation .

Currently, there are two types of treatments that are used in patients with alcoholic or nonalcoholic fatty liver disease. Firstly, there are two types of treatments for obesity (orlistat), insulin resistance treatment (metformin, pioglitazone, (ursodeoxycholic acid and taurine) as a drug for the recovery of damaged hepatocytes and liver function independently of the risk factors of fatty liver, and secondly, for the treatment of hyperlipidemia and rosiglitazone, clofibrate, gemfibrozil, bezafibrate, atorvastatin and simvastatin. , Antioxidants (vitamine E), and nutritional supporters (lectin, betaine, N-acetylcystein).

However, the above-mentioned existing therapeutic agents are drugs that are used as symptoms remedy, not an essential therapeutic agent in terms of efficacy, and thus can not be regarded as a target effect. In addition, the medicines approved by the Korea Food and Drug Administration due to the efficacy of treatment for fatty liver are currently one item in Korea, and the market size of these drugs has been decreasing continuously since 2008. This is a fundamental problem . ≪ / RTI > Therefore, there are few pharmaceutical preparations that can treat pharmacologically fatty liver so far, so it is urgent to develop appropriate therapeutic agents that have no side effects and are safe even when taken for a long time.

On the other hand, mitochondrial dysfunction and dysfunction may manifest as symptoms of various diseases in various organs or tissues in the human body, rather than a single symptom or disease, so that the possibility of a specific treatment for diseases and diseases associated with mitochondrial dysfunction However, until now, there is no precise target or proper treatment technology, and future research and development is urgent. In particular, there is strong evidence that increased oxidative stress due to increased reactive oxygen species (ROS) in cells is a molecular biologic underlying mechanism in degenerative neurological diseases, as well as fatty liver and metabolic diseases, Treatment methods using metabolic disease drugs or antioxidants used in preclinical stages are still controversial.

GDF15 (Growth differentiation factor 15) is a protein belonging to the transfection growth factor β (TGF- β) superfamily. Other names include macrophage inhibitory cytokine 1, placental TGF-β prostate-derived factor (PDF), nonsteroidal anti-inflammatory drug-activator gene. GDF15 protein is expressed low in the brain, large intestine, and bone marrow, but it is known to be expressed in the placenta. It is a protein known to be highly expressed in gastric cancer, breast cancer, colon cancer, prostate cancer and the like.

Thus, the expression pattern of GDF15 protein is very diverse, but there is little known precise mechanism of action. Recent studies have shown that they are not expressed or expressed very weakly in the resting state, but they are strongly expressed in hypoxia, inflammation, UV exposure, and various carcinogenesis, which are the stimulation or stress exposure conditions of various cells This is well known. It is also known that it is involved in the regulation of biomass energy homeostasis.

Therefore, the present inventors conducted an in vitro experiment to confirm the possibility of GDF15 protein as a therapeutic agent for treating fatty liver through improvement of mitochondrial function, and confirmed that GDF15 protein inhibits fatty liver and improves metabolic disease Thus completing the present invention.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating fatty liver, which contains GDF15 protein or a polynucleotide encoding the same as an active ingredient.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for prevention or treatment of fatty liver comprising GDF15 (Growth differentiation factor 15) protein as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of a liver comprising a polynucleotide encoding a GDF15 protein or a cell containing the vector as an active ingredient.

In addition, the present invention provides a health functional food for improving liver fat containing GDF15 protein as an active ingredient.

In the present invention, when the GDF15 (growth factor 15) protein or a polynucleotide encoding the GDF15 protein was administered to an ob / ob mouse model, the body weight gain was suppressed and lipogenesis ) Was decreased. It was confirmed that gene expression and beta-oxidation-related gene expression associated with improvement of genes involved in lipolysis and mitochondrial function were increased. In addition, in vitro (in vitro) was by treatment in muscle cells and liver cells a recombinant GDF15 protein at different concentrations mitochondrial increase the degradation (lipolysis) of the oxygen consumption and fat because improve mitochondrial function, GDF15 protein or polynucleotide encoding it from improves mitochondrial function Thereby being useful as a pharmaceutical composition for prevention or treatment of fatty liver.

1 is vitro (in vitro) treatment of recombinant GDF15 protein at different concentrations in the muscle cells of C2C12 and stem cells of hepatocyte in the mitochondrial respiration rate (OCR, O ₂ consumption rate) to be analyzed using the Seahorse XF analyzer basal respiration, proton leak, maximal respiratory capacity Fig.
A: muscle cells (C2C12); And
B: Hepatocyte.
FIG. 2 is a graph showing the amount of glycerol secreted into the medium by analysis of lipolysis by treatment with recombinant GDF15 protein in 3T3L1 adipocytes in vitro. FIG.
SB-431542: TGF-beta inhibitor;
PD98059: Ere1 / 2 inhibitor; And
SB203580: p38 inhibitor.
FIG. 3 is a graph showing changes in food intake and weight change after treatment with recombinant GDF15 protein (500 ug / kg / day) in an ob / ob mouse model of an obese animal model:
A: Diagram showing changes in food intake after 3 weeks of treatment with GDF15 protein:
B: Body weight change at 5-day intervals with GDF15 protein treated for 3 weeks (** p <0.001).
FIG. 4 is a graph showing oxygen consumption, carbon dioxide generation, and energy expenditure measured at 12-hour intervals using an indirect calorimetry apparatus after 3 weeks of treatment of recombinant GDF15 protein in ob / ob mice;
A: oxygen consumption;
B: Carbon dioxide generation amount; And
C: Energy consumption.
FIG. 5 shows results of beta-oxidation, lipogenesis, lipolysis, and qRT-PCR on mitochondrial-related proteins by separating RNA from liver and adipose tissue of FIG. 4 Fig.
A: liver tissue; And
B: Adipose tissue (epididymal white adipose tissue, eWAT).
FIG. 6 is a graph showing the treatment of ob / ob mice with recombinant GDF15 protein and vehicle, separating liver and adipose tissue, and performing H / E (Hematoxylin and Eosin) staining. In particular, oil-red-o staining was performed to confirm lipid accumulation in liver tissue.
Figure 7 is a diagram showing the treatment of recombinant GDF15 protein and vehicle in ob / ob mice and using inflammatory response markers to confirm the inflammatory response in liver tissue:
A: Whether phosphorylation of NF-kB p65, p38 and JNK1 / 2; And
B: Expression and secretion of TNF-α gene.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for prevention or treatment of fatty liver comprising GDF15 (Growth differentiation factor 15) protein as an active ingredient.

The GDF15 protein preferably includes the amino acid sequence of SEQ ID NO: 2, more preferably SEQ ID NO: 1.

SEQ ID NO: order
GDF15 (SEQ ID NO: 2)
MPGQELRTVN GSQMLLVLLV LSWLPHGGAL SLAEASRASF PGPSELHSED SRFRELRKRY EDLLTRLRAN QSWEDSNTDL VPAPAVRILT PEVRLGSGGH LHLRISRAAL PEGLPEASRL HRALFRLSPT ASRSWDVTRP LRRQLSLARP QAPALHLRLS PPPSQSDQLL AESSSARPQL ELHLRPQAAR GRRRARARNG DHCPLGPGRC CRLHTVRASL EDLGWADWVL SPREVQVTMC IGACPSQFRA ANMHAQIKTS LHRLKPDTVP APCCVPASYN PMVLIQKTDT GVSLQTYDDL LAKDCHCI


rGDF15 (SEQ ID NO: 1)

RRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI

In the present invention, the fatty liver is preferably non-alcoholic fatty liver disease (NAFLD), but is not limited thereto. In addition, it is preferable that the fatty liver is a disease due to malfunction or malfunction of mitochondria.

Non-alcoholic fatty liver disease can be caused by causes such as obesity, diabetes, hyperlipidemia, and drugs regardless of alcohol, and steatosis and hepatocellular inflammation, which do not accompany inflammation, Non-alcoholic steatohepatitis (NASH), advanced fibrosis, and cirrhosis. In addition, recent studies have shown that mitochondrial dysfunction due to various stress mechanisms is one of the major mechanisms of metabolic diseases including fatty liver.

In the experimental example of the present invention, in order to examine the effect of the GDF15 protein of the present invention on the fatty liver, vitro) from the result of this experiment was treated GDF15 protein, it was confirmed that the oxygen respiration by mitochondria enhancement is increased (it was confirmed that, see FIG. 1), inducing an exploded (lypolysis) of the fat by the mitochondrial function improvement (Fig. 2). In addition, when GDF15 protein was administered to an obesity animal model mouse (ob / ob), it was confirmed that the body weight was decreased (see FIG. 3), and the oxygen consumption and energy consumption of the mouse were also significantly increased Reference). In addition, when the GDF15 protein was administered, it was confirmed that the thermogenic genes related to oxidation, lipolysis and energy consumption in the adipose tissues of mice were highly expressed, thereby improving the function of mitochondria (see FIG. 5) (See FIG. 5), and the size of lipid droplets in the liver was significantly reduced by the GDF15 protein in the liver (See FIG. 6). In addition, we examined the phosphorylation of inflammatory response markers by GDF15 protein. As a result, we confirmed that the phosphorylation of NF-kB p65, p38 and JNK1 / 2 was significantly reduced in adipose tissue and the expression of TNF- And the amount of secretion was also decreased (see Fig. 7).

Accordingly, a pharmaceutical composition containing the GDF15 protein of the present invention or a polynucleotide encoding the same as an active ingredient can be usefully used for prevention or treatment of fatty liver.

The present invention also provides a pharmaceutical composition for preventing or treating fatty liver, comprising a vector comprising a polynucleotide encoding the GDF15 protein or a cell containing the vector as an active ingredient.

The vector may be a linear DNA, a plasmid vector, a vector comprising a viral expression vector or a recombinant retrovirus vector, a recombinant adenovirus vector, an adeno-associated virus , An AAV) vector, a recombinant herpes simplex virus vector or a recombinant lentivirus vector, but is not limited thereto.

The cells are preferably selected from the group consisting of hematopoietic stem cells, dendritic cells, autologous tumor cells and established tumor cells, It is not limited.

In addition, the fatty liver is preferably a non-alcoholic fatty liver, and the fatty liver is preferably a disease due to mitochondrial dysfunction and insufficiency.

When the pharmaceutical composition is formulated, it is prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like which are usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules, troches and the like, which may contain one or more excipients in the compound of formula (I) of the present invention, Starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included. have.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 81, cacao butter, taurine, glycerol, gelatin and the like.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the compound according to the present invention may vary depending on the age, sex, and body weight of the patient. In general, 0.1 mg to 100 mg, preferably 0.5 mg to 10 mg per kg of body weight is administered daily or every other day Or one to three times a day. However, the dosage may be varied depending on the route of administration, the severity of the disease, sex, weight, age, and the like, and therefore the dose is not limited to the scope of the present invention by any means.

In the case of a vector comprising a polynucleotide encoding the GDF15 protein of the present invention, the vector preferably contains 0.05 to 500 mg, more preferably 0.1 to 300 mg, and the encoding of the GDF15-derived peptide includes a polynucleotide , It is preferable that the recombinant virus contains 10 ³ to 10 ¹² IU (10 to 10 ¹⁰ PFU), more preferably 10 ⁵ to 10 ¹⁰ IU, but is not limited thereto.

In addition, in the case of a cell containing a polynucleotide encoding the GDF15 protein of the present invention, it is preferable that the cell contains 10 ³ to 10 ^8, more preferably 10 ⁴ to 10 ⁷ , but is not limited thereto .

The effective dose of a vector containing a polynucleotide encoding the GDF15 protein of the present invention or a composition containing cells as an active ingredient is 0.05 to 12.5 mg / kg in the case of a vector per 1 kg of body weight, 10 ⁷ to 10 ¹¹ viral particles (10 ⁵ to 10 ⁹ IU) / kg, in the case of cells, 10 ³ to 10 ⁶ cells / kg, preferably 0.1 to 10 mg / kg in the case of vector, 10 ⁸ to 10 ¹⁰ particles, the case of (10 ⁶ to 10 ⁸ IU) / ㎏, cells, and 10 ² to 10 ⁵ cells / ㎏, it may be administered twice or three times a day. Such composition is not necessarily limited to this, but may vary depending on the condition of the patient and the degree of neurological disease.

In addition, the compositions of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

Also, the present invention provides a health functional food for improving liver fat, which contains GDF15 (Growth differentiation factor 15) protein as an active ingredient, and the GDF15 protein preferably contains the amino acid sequence shown in SEQ ID NO: 2, It is more preferable to include SEQ ID NO: 1.

As used herein, the term "health functional food" is produced by using raw materials or ingredients (functional raw materials) having functions useful for nutrients or human body that are likely to be deficient in daily eating, and is intended to maintain the normal function of the human body, But is not limited to, and is not meant to exclude health food in the usual sense.

The composition of the present invention can be added directly to food or used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). In general, the amount of the compound in the health functional food may be 0.01 to 90 parts by weight based on the total weight of the food. However, when consumed for a long period of time for the purpose of health and hygiene or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention is not particularly limited to other components other than those containing the above-mentioned composition as an essential ingredient at the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As a flavor other than the above, a natural flavoring agent {tau martin, stevia extract (for example, rebaudioside A, glycyrrhizin etc.)} and synthetic flavorings (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

These components may be used independently or in combination. The ratio of such additives is not so important, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited by the following Examples and Experimental Examples.

< Example  1> Recombination GDF15  protein( rGDF15 )

To prepare the recombinant GDF15 protein, the amino acid sequence of human GDF15 was cloned into pGEX plasmid (promega) and the GST-GDF15 protein was introduced into E. coli using IPTG. The GST-GDF15 protein was purified by GST column Pure recombinant GDF15 protein was prepared by treating thrombin and used in the experiment.

In addition, recombinant GDF15 protein was purchased (R / D system, # 8146-GD / CF) according to need and used in the experiment.

< Experimental Example  1> Recombination GDF15  Confirmation of increase of oxygen consumption of mitochondria by protein

I (in the inventors in vitro to determine whether the enhancement of mitochondrial function by recombinant GDF15 protein vitro experiments were performed to determine the effect of mitochondrial cellular respiration rate on recombinant GDF15 protein.

Specifically, the recombinant GDF15 protein (rGDF15) prepared in Example 1 was treated with C2C12 muscle cell line and hepatocyte primary hepatocyte (0, 100, 300 ng / ml) The oxygen consumption rate (OCR) in mitochondria was measured using a seahorse XF analyzer while treating cell respiratory inhibitors such as oligomycin, oligo, carbonic cyanide, m-chlorophenyl hydrazone (CCCP) and rotenone Respectively.

As a result, as shown in Fig. 1, it was confirmed that the oxygen consumption (cell respiration rate) according to the improvement of mitochondrial function was increased in both the muscle cell line and the liver tissue cell depending on the recombinant GDF15 protein concentration (Fig. 1).

< Experimental Example  2> Recombination GDF15  Degradation of adipocytes by mitochondria by protein lipolysis ) Increase confirmation

In addition to increasing oxygen respiration rates in mitochondria, beta-oxidation of mitochondria is a typical function of mitochondria producing energy and is associated with lipolysis of adipocytes. In mitochondria, since fat is used as a raw material and glycerol, which is its metabolite, is produced, the function of mitochondria can be confirmed indirectly by measuring the concentration of glycerol. Therefore, the present inventors conducted the following experiment to confirm the extent of lipolysis of adipocytes by the recombinant GDF15 protein (rGDF15).

More specifically, in vitro (in vitro) and then to differentiate the undifferentiated 3T3L1 fat cells made with fat cells from the amount of glycerol that is secreted into the <Example 1> A recombinant GDF15 protein to 100ng / ml in the manufacture process and the culture medium was measured. In addition, SB-431542, PD98059, and SB203580, which are various protein inhibitors involved in lipid metabolism, were treated with 5 μM, 25 μM, and 10 μM of GDF15 protein, respectively, to confirm the resolution of adipocytes.

As a result, as shown in FIG. 2, when the recombinant GDF15 protein was treated, the lipid in the adipocyte was decomposed into glycerol and secreted into the culture medium and accumulated (FIG. 2). In addition, by confirming that the production of glycerol is restrained by the simultaneous treatment of various protein inhibitors (SB-431542, TGF-beta inhibitor / PD98059, Ere1 / 2 inhibitor / SB203580, p38 inhibitor) It was confirmed that recombinant GDF15 protein induces degradation of adipocytes, which is also related to improvement of mitochondrial function.

< Experimental Example  3> Recombination in obese animal models GDF15  Identification of dietary and weight changes by protein

The ob / ob mouse model, an obesity animal model, is an animal model showing an obesity phenotype due to a deficiency of the leptin gene. It is also well known that ob / ob mice have abnormally suppressed or abnormal mitochondrial function.

In order to examine the effect of GDF15 protein on mitochondrial function and inhibition of obesity, the recombinant GDF15 protein (rGDF15, 500 ug / kg / day) prepared in Example 1 was administered to ob / Were administered by IP method for 3 weeks. Changes in food intake and body weight were observed. The vehicle and recombinant GDF15 protein were administered to male ob / ob mice at 6 weeks of age (n = 5 per group), and the changes in food intake and body weight by recombinant GDF15 protein were checked after 2 weeks.

As a result, as shown in Fig. 3, it was confirmed that the recombinant GDF15 protein did not change the amount of the mouse food intake (Fig. 3A). However, when the recombinant GDF15 protein was treated, a significant decrease in the body weight of the mice was observed compared with the vehicle-treated group (FIG. 3B). It was confirmed that the body weight gain tended to decrease until about 10 days when the recombinant GDF15 protein was administered, but it was confirmed that the increase rate of the body weight was statistically decreased from the 2 weeks after the treatment of the recombinant GDF15 protein, , The body weight was significantly reduced by GDF15 administration.

< Experimental Example  4> Recombination in obese animal models GDF15  Confirmation of increased oxygen consumption and energy consumption by protein

The present inventors administered the vehicle and the recombinant GDF15 protein (rGDF15) of the present invention to the ob / ob mouse model in order to directly confirm the phenotype of mouse metabolism in the above Experimental Example 3, and then, using indirect calorimetry equipment Oxygen consumption (VO ₂ ), carbon dioxide generation (VCO ₂ ) and energy expenditure (EE) were measured directly.

Experiments were carried out for 24 to 72 hours with autoclaved food and water using a physical cage system (Oxylet, Panlab, Cornella, Spain). The standard feeds and high fat diets fed to mice were placed in individual metabolic chambers with no restriction on access to water and food, and the oxygen consumption (VO ₂ ), carbon dioxide production (VCO ₂ ) Energy expenditure (EE) and movement behavior were analyzed according to the indirect calorimetry method using physical cage system. The obtained data were analyzed using METABOLISM software version 2.2, and the average value with and without light was calculated, and the P value was calculated using t-test.

As a result, as shown in FIG. 4, it was confirmed that the oxygen consumption of the mouse group treated with the recombinant GDF15 protein was significantly increased (FIG. 4A) and the amount of carbon dioxide generation was significantly increased (FIG. 4B) . In addition, it was confirmed that the energy consumption (EE) was also increased (FIG. 4C). Also, it was confirmed that the same results were obtained even when night and day were separately analyzed.

< Experimental Example  5> Recombination in obese animal models GDF15  By protein Liver tissue  Identification of Metabolic Gene Expression Changes in Adipose Tissue

The present inventors observed the expression of metabolic genes in liver tissue and adipose tissue in order to understand more specifically the metabolic phenotypes in Experimental Example 4 above.

Specifically, the beta-oxidation-related genes (Ppara, Pdp) in the vehicle and the epididymal white adipose tissue (eWAT) of the mouse administered with the recombinant GDF15 protein prepared in Example 1 above for 3 weeks, Expression of genes related to lipogenesis (Fasn, Pparγ), lipolysis (Hsl, Atgl) and mitochondrial metabolism related genes (Pgc1α, Cpt1α, Cox 1 and Ndufa9) -PCR experiment.

As a result, as shown in FIG. 5, the expression of Ppara, AcadL and AcadM, which are beta-oxidation related genes, was significantly increased in the case of liver tissues and the expression of Hsl and Atgl genes related to lipolysis Expression was also increased, and expression of Pgc1α, Cpt1α, Cox 1 and Ndufa9, which are related to mitochondrial metabolism, was also increased. On the other hand, there was no increase in the expression of Fasn and Ppar gamma, genes related to lipogenesis (FIG. 5A).

In the case of adipose tissue, expression of beta-oxidation related genes Ppara, AcadL and AcadM is increased by the recombinant GDF15 protein and expression of Hsl and Atgl genes related to lipolysis are also increased, The expression of Pgc1?, Cptl?, Cox 1 and Ndufa9, which are genes related to the mitochondrial metabolism, was also increased (Fig. 5B).

As a result of confirming the expression of specific genes in liver tissue and adipose tissue as described above, high expression of thermogenic genes related to oxidation, lipolysis and energy consumption by recombinant GDF15 protein was confirmed, The same results are obtained as the phenotypes are improved.

< Experimental Example  6> Recombination in obese animal models GDF15  Fatty liver and fat droplets by protein lipid droplet ) Reduction confirmation

In the above Experimental Example 3, the liver tissues and adipose tissues were extracted from each of the mouse groups to which the recombinant GDF15 protein was administered for 3 weeks, and H / E staining was performed for histological examination.

Mice that were fasted for 16 hours were treated with CO ₂ After treatment, the cells were fixed with 4% paraformaldehyde at room temperature for 1 hour. Paraffin blocks were prepared and histological examination of liver and adipose tissues was performed using standard H / E staining.

In addition, oil-red-O staining was performed using frozen interstitial tissue to confirm the amount of lipid more accurately. Prepare a 0.5% (w / v) oil red O solution, dilute it with sterilized distilled water at a ratio of 3: 2, and let it sit at room temperature for 1 hour before using it as a staining reagent for fat cells. Immediately before, it was passed through a 0.2-μm filter. The frozen sections were washed twice with 1 × PBS, and the cells were fixed with 3.7% (w / v) formalin (in PBS) at 4 ° C for 1 hour. After fixation, 300 의 of Oil Red O Working solution was treated for 1 hour, washed twice with distilled water and confirmed by microscope.

As a result, as shown in FIG. 6, a phenotype of fatty liver disease was observed in the vehicle-treated mouse group, but the lipid droplet in the liver tissue of the mouse group treated with the recombinant GDF15 protein was significantly decreased (Fig. 6).

This is a direct result of the suppression or improvement of fatty liver symptoms by the GDF15 protein. In addition, histological changes in the adipose tissue (eWAT) treated with recombinant GDF15 protein and H / E staining showed that lipid droplet size was similar to that in liver tissue, Group was significantly reduced by 50% or more (Fig. 6).

< Experimental Example  7> Recombination in obese animal models GDF15  Reduction of inflammatory response of adipose tissue by protein

In order to investigate the phosphorylation of the inflammation-related gene using the mouse liver tissue of the above <Experimental Example 3>, the present inventors treated the vehicle and the recombinant GDF15 protein, and lysed the liver tissue, The phosphorylation of kB p65, p38 and JNK1 / 2 was confirmed by western blot experiments.

As a result, as shown in Fig. 7, phosphorylation of the inflammatory markers NF-kB p65, p38 and JNK1 / 2 was markedly reduced in the group treated with the recombinant GDF15 protein (Fig. 7A). In addition, the amount of TNF-α secretion and secretion, which are well-known as inflammation-related cytokines, was confirmed in liver tissues and it was confirmed that the amount of TNF-α gene expression and secretion were significantly reduced by GDF15 protein (FIG. 7B)

<110> Chungnam National University Industry-University Collaboration <120> Pharmaceutical composition for the prevention or treatment of fat          liver comprising GDF15 protein or polynucleotide encoding GDF15          as an effective ingredient <130> 2016P-04-042 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> rGDF15 <400> 1 Arg Arg Ala Arg Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly   1 5 10 15 Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly              20 25 30 Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys          35 40 45 Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln      50 55 60 Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro  65 70 75 80 Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr                  85 90 95 Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp             100 105 110 Cys His Cys Ile         115 <210> 2 <211> 308 <212> PRT <213> Artificial Sequence <220> <223> GDF15 <400> 2 Met Pro Gly Gln Glu Leu Arg Thr Val Asn Gly Ser Gln Met Leu Leu   1 5 10 15 Val Leu Leu Val Leu Ser Trp Leu Pro His Gly Gly Ala Leu Ser Leu              20 25 30 Ala Glu Ala Ser Arg Ala Ser Phe Pro Gly Pro Ser Glu Leu His Ser          35 40 45 Glu Asp Ser Arg Phe Arg Glu Leu Arg Lys Arg Tyr Glu Asp Leu Leu      50 55 60 Thr Arg Leu Arg Ala Asn Gln Ser Trp Glu Asp Ser Asn Thr Asp Leu  65 70 75 80 Val Pro Ala Pro Ala Val Arg Ile Leu Thr Pro Glu Val Arg Leu Gly                  85 90 95 Ser Gly Gly His Leu His Leu Arg Ile Ser Arg Ala Ala Leu Pro Glu             100 105 110 Gly Leu Pro Glu Ala Ser Arg Leu His Arg Ala Leu Phe Arg Leu Ser         115 120 125 Pro Thr Ala Ser Arg Ser Trp Asp Val Thr Arg Pro Leu Arg Arg Gln     130 135 140 Leu Ser Leu Ala Arg Pro Gln Ala Pro Ala Leu His Leu Arg Leu Ser 145 150 155 160 Pro Pro Ser Ser Gln Ser Asp Gln Leu Leu Ala Glu Ser Ser Ser Ala                 165 170 175 Arg Pro Gln Leu Glu Leu His Leu Arg Pro Gln Ala Ala Arg Gly Arg             180 185 190 Arg Arg Ala Arg Ala Arg Asn Gly Asp His Cys Pro Leu Gly Pro Gly         195 200 205 Arg Cys Cys Arg Leu His Thr Val Arg Ala Ser Leu Glu Asp Leu Gly     210 215 220 Trp Ala Asp Trp Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys 225 230 235 240 Ile Gly Ala Cys Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln                 245 250 255 Ile Lys Thr Ser Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro             260 265 270 Cys Cys Val Pro Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr         275 280 285 Asp Thr Gly Val Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp     290 295 300 Cys His Cys Ile 305

Claims (12)

A pharmaceutical composition for prevention or treatment of fatty liver comprising, as an active ingredient, a C-terminal domain of a GDF15 (Growth differentiation factor 15) protein having an amino acid sequence represented by SEQ ID NO: 1.
delete The pharmaceutical composition for prevention or treatment of fatty liver according to claim 1, wherein the fatty liver is a non-alcoholic fatty liver.
The pharmaceutical composition for prevention or treatment of fatty liver according to claim 1, wherein the fatty liver is a disease due to mitochondrial dysfunction and dysfunction.
delete delete delete delete delete A health functional food for improving liver fat, comprising the C-terminal domain of GDF15 (Growth differentiation factor 15) protein having the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.
11. The health functional food for fatty liver improvement according to claim 10, wherein the fatty liver is a non-alcoholic fatty liver.
[10] The health functional food for fatty liver improvement as set forth in claim 10, wherein the fatty liver is a disease due to mitochondrial dysfunction and dysfunction.

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