KR20240017258A - Composition for preventing or treating metabolic disease comprising FSTL1 - Google Patents

Abstract

The present invention relates to a composition for preventing or treating metabolic diseases comprising the FSTL1 protein or the gene encoding the protein as an active ingredient.
The composition of the present invention can play a role in energy balance and blood sugar control in the hypothalamus, and can strengthen lipid metabolism by increasing the activity of genes related to lipid metabolism, and activate fatty acid oxidation-related or insulin signaling pathways in the hypothalamus. You can do it. According to this mechanism, the composition of the present invention can control blood sugar levels and cause loss of appetite and weight loss. As a result, it has the effect of preventing, treating or improving metabolic diseases, and can be used as a material for pharmaceuticals, food, feed compositions, etc. It can be utilized.

Description

Composition for preventing or treating metabolic disease comprising FSTL1 as an active ingredient {Composition for preventing or treating metabolic disease comprising FSTL1}

The present invention relates to a composition for preventing or treating metabolic diseases containing FSTL1 as an active ingredient.

Recently, there have been many changes in eating habits due to rapid economic growth and westernization of lifestyles. In particular, busy modern people are experiencing an increase in metabolic diseases due to high-fat diets such as fast food, consumption of foods with high sugar content, and low amounts of exercise. Metabolic disease is a general term for diseases caused by metabolic disorders in the body, and representative examples include obesity and diabetes.

Obesity is a chronic disease that increases the morbidity and mortality of various diseases due to excessive accumulation of fat tissue due to abnormal energy balance control function or hypernutrition. Obesity and related diseases are a common and very serious public health problem in the United States and around the world. According to the World Health Organization (WHO), more than 1 billion adults worldwide are overweight, and at least 3 million of them are overweight. It is clinically determined that he is obese.

In addition, diabetes is characterized by high blood sugar, which increases the concentration of glucose in the blood due to insufficient secretion of insulin or failure to function normally. Hyperglycemia causes various symptoms and signs and glucose is excreted in the urine. As a disease, it is known that fundamental treatment is difficult. In modern times, due to changes in eating habits and lack of exercise, there have been significant changes in the human body's inherent energy metabolism process, and as a result, the prevalence of chronic degenerative diseases such as diabetes is increasing. In Korea, the prevalence of diabetes is 5-10% and is continuously increasing. In the United States, diabetes has increased six-fold over the past 40 years, and the number of patients is expected to reach 26 million by 2050.

The etiology of type 2 diabetes (non-insulin dependent type), which accounts for more than 95% of diabetes, is known to be a complex disorder of insulin secretion disorder and insulin resistance. In other words, diabetes is a disease that shows symptoms of chronic hyperglycemia due to this complex disorder. As such, metabolic diseases are common among modern people, and there is a need to develop fundamental treatments. Effective and safe medicines that can be used in combination with diet and exercise from a long-term perspective to prevent or treat metabolic diseases are required. .

Recent studies have reported that FSTL1 plays diverse regulatory roles in biological functions including cell survival, proliferation, differentiation, migration, inflammation, metabolism, and immunity. Additionally, FSTL1 has been reported to be involved in the pathogenesis of various diseases, including heart failure, cancer, and immune diseases. FSTL1 is a glycoprotein secreted by the heart that not only functions as an anti-apoptotic protein, but is also known to play a role in maintaining myocardial cells and repairing heart damage. Additionally, it is known to play a role in inhibiting the proliferation, invasion, and survival of tumor cells derived from various tissues, including lung, breast, kidney, ovary, endometrium, and colon.

Against this background, the present inventors identified the role of FSTL1 in the regulation of energy balance and glucose homeostasis, confirmed that food intake and weight gain were suppressed when treated with FSTL1, and completed the present invention.

US Patent Publication No. 2009-0326053

The present invention aims to solve the above-described problems and other problems associated therewith.

An exemplary object of the present invention is to provide a pharmaceutical composition for preventing or treating metabolic diseases containing the FSTL1 protein or the gene encoding the protein as an active ingredient.

Another exemplary object of the present invention is to provide a quasi-drug composition for preventing, improving, or treating metabolic diseases containing the FSTL1 protein or the gene encoding the protein as an active ingredient.

Another exemplary object of the present invention is to provide a food composition for preventing or improving metabolic diseases containing the FSTL1 protein or the gene encoding the protein as an active ingredient.

Another exemplary object of the present invention is to provide a health functional food for preventing or improving metabolic diseases containing the FSTL1 protein or the gene encoding the protein as an active ingredient.

Another exemplary object of the present invention is to provide a feed composition for preventing or improving metabolic diseases containing the FSTL1 protein or the gene encoding the protein as an active ingredient.

The technical problem to be achieved according to the technical idea of the invention disclosed in this specification is not limited to the problem to solve the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Additionally, the scope of the present application cannot be considered limited by the specific description described below.

In one aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating metabolic diseases comprising the FSTL1 protein or the gene encoding the protein as an active ingredient.

In the present invention, 'FSTL1 (Follistatin-related protein 1)' is a protein similar to follistatin, a BMP-4 binding protein, and contains a FS module (follistatin-like sequence containing 10 conserved cysteine residues), a Cajal-type serine protease. It contains an inhibitor domain, two EF hand domains, etc. FSTL1 plays an important role in lung, central nervous system, and skeletal development and is known to play a cardioprotective role as well as upregulate pro-inflammatory mediators important in the pathology of arthritis.

In the present invention, 'gene encoding FSTL1 protein' refers to the FSTL1 gene, and may specifically be a polynucleotide encoding the FSTL1 protein or a vector containing the same, but is not limited thereto.

In the present invention, in the form of an activator that activates the function of the FSTL1 protein, various expression products including mRNA of the FSTL1 gene, or a means capable of expressing FSTL1 in addition to the vector, or an expression promoter that promotes the expression. It may be included as an active ingredient in the composition of the present invention.

The polynucleotide may be DNA or RNA, and the base sequence of the polynucleotide encoding FSTL1 can be easily confirmed by a person skilled in the art through known literature, and as an example, GENEBANK accession No. It includes the base sequence of NM_007085.5 (SEQ ID NO: 1), but is not limited thereto, considering mutations with biologically equivalent activity. In other words, the FSTL1 gene according to the present invention includes all or part of the FSTL1 sequence or the base sequence of SEQ ID NO: 1 known in the art, but includes variants with ‘substantial functional identity’. As an example, the FSTL1 gene may refer to a sequence showing more than 80% sequence homology and more than 90% sequence homology with the base sequence of SEQ ID NO: 1. As an example, the FSTL1 gene of the present invention has the base sequence 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 of SEQ ID NO: 1, within the range showing the biological activity of FSTL1. It may be a base sequence having sequence identity within 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

The type of the vector is not limited as long as it contains a gene encoding the FSTL1 protein. For example, it may be linear DNA, plasmid DNA, or a recombinant viral vector, but is not limited thereto.

In the present invention, 'metabolic disease' is also called metabolic syndrome and is a general term for diseases caused by metabolic disorders in the body, such as obesity, diabetes, hyperlipidemia, hypertension, hypercholesterolemia, It may be hyperinsulinemia, arteriosclerosis, dyslipidemia, hypertriglyceridemia, liver disease, fatty liver, stroke, myocardial infarction, cardiovascular disease, hyperglycemia, or insulin resistance disease, and preferably diabetes or obesity, but is limited thereto. It doesn't work.

The composition of the present invention plays a role in energy balance and blood sugar control in the hypothalamus, and the FSTL1 protein can regulate the expression of genes related to lipid metabolism or activate the insulin signaling pathway in the hypothalamus in the brain.

Specifically, it strengthens lipid metabolism by increasing the activity of lipid metabolism-related genes, regulates glucose levels through activation of fatty acid oxidation-related or insulin signaling pathways in the hypothalamus, and prevents metabolic diseases by causing loss of appetite and weight loss. It can have preventive, therapeutic or improving effects.

The lipid metabolism-related genes are related to the process of lipid synthesis and decomposition in cells, and refer to genes that contribute to the decomposition of lipids for energy production or the synthesis of lipids constituting cell membranes, specifically, lipolysis, Genes related to fatty acid utilization or lipogenesis include, for example, Pnpla2, Cpt1b, Cpt1c, Cd36, Fabp4, Acaca, and Fasn, but are not limited thereto.

As confirmed in one embodiment of the present invention, expression of genes related to lipid metabolism (lipolysis genes (Pnpla2, Cpt1b, Cpt1c), fatty acid utilization (Cd36, Fabp4), and lipogenesis genes (Acaca, Fasn)) before and after FSTL1 treatment. As a result of comparing the levels, the expression levels of Pnpla2, Cpt1b, Cpt1c, Fabp4, and Acaca genes were found to be significantly higher than the control group.

For example, the composition of the present invention may exhibit efficacy in preventing, improving, or treating metabolic diseases by increasing lipolysis by activating a signaling pathway related to fatty acid oxidation in the hypothalamus.

For example, the composition of the present invention may exhibit efficacy in preventing, improving, or treating metabolic diseases by activating AKT/mTOR signaling, an insulin signaling pathway in the hypothalamus, to regulate glucose homeostasis and improve insulin sensitivity.

For example, according to Example 2 of the present invention, after FSTL1 treatment, the expression level of the hypothalamic Agrp gene was significantly decreased, and in mice administered I3V FSTL1, anorexia and weight loss were induced compared to the control group.

For example, according to Example 3 of the present invention, the expression level of genes related to lipid metabolism was significantly increased in cells treated with FSTL1.

For example, according to Example 4 of the present invention, mice administered I3V FSTL1 showed food intake and weight loss effects, whereas mice knocking out ATGL, an enzyme that decomposes triglycerides, showed significant food intake and weight loss. No difference was observed, and when FSTL1 was administered as I3V, phosphorylation of ACC was reduced due to the interaction between b3-AR, and glucose levels were reduced due to activation of AKT/mTOR signaling.

In one aspect to achieve the above object, the pharmaceutical composition of the present invention may additionally include a brain-targeting peptide or a gene encoding the protein in addition to the FSTL1 protein or the gene encoding the protein.

The brain-targeting peptide refers to a peptide that can pass through the blood-brain barrier in order to exert blood sugar control, anorexia, and weight loss effects through the intracerebral action of FSTL1, i.e., expression in the hypothalamus. It may be a peptide, a hypothalamic targeting peptide, or a peptide derived from a receptor for FSTL1 in the brain, preferably a hypothalamic targeting peptide that causes FSTL1 to be expressed in the hypothalamus, or a peptide derived from a receptor for FSTL1 in the brain, but is not limited thereto.

The peptide derived from the FSTL1 receptor in the brain may be a peptide containing a sequence derived from a target or receptor of FSTL1 that can interact with FSTL1 in the brain and activate FSTL1-related signaling.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc.

Carriers for parenteral administration may also include water, suitable oils, saline solutions, aqueous glucose and glycols, and the like. Additionally, stabilizers and preservatives may be additionally included. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.

The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally, and parenteral administration methods include intravenous, intramuscular, intraarterial, intracentral, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, and intranasal. , may be administered enterally, topically, sublingually or intrarectally, and preferably centrally administered, but is not limited thereto.

The pharmaceutical composition of the present invention can be formulated into a preparation for oral administration or parenteral administration according to the administration route described above. When formulated, one or more buffers (e.g. saline or phosphate buffered saline (PBS)), antioxidants, bacteriostatic agents, chelating agents (e.g. EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g. For example, aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, solutions, gels, syrups, slurries, suspensions or capsules, etc. These solid preparations include the pharmaceutical composition of the present invention and at least one excipient, e.g. For example, starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol and maltitol. It can be prepared by mixing cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropylmethyl-cellulose, or gelatin. For example, tablets or dragees can be obtained by combining the active ingredient with solid excipients, grinding them, adding suitable auxiliaries, and processing them into a granule mixture.

In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water or liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, or preservatives may be included. there is. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may be additionally included. .

When administered parenterally, the pharmaceutical composition of the present invention can be formulated with a suitable parenteral carrier in the form of injections, transdermal administration, and nasal inhalation according to methods known in the art. The above injections must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. For injections, examples of suitable carriers may be, but are limited to, solvents or dispersions including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), mixtures thereof, and/or vegetable oils. That is not the case. More preferably, suitable carriers include Hanks' solution, Ringer's solution, PBS containing triethanolamine, or sterile water for injection, and isotonic solutions such as 10% ethanol, 40% propylene glycol, and 5% dextrose. . In order to protect the injection from microbial contamination, it may additionally contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Additionally, in most cases, the injection may additionally contain an isotonic agent such as sugar or sodium chloride.

In the case of transdermal administration, forms such as ointments, creams, lotions, gels, external solutions, paste preparations, linear preparations, and aerol preparations are included. Here, 'transdermal administration' means administering a pharmaceutical composition topically to the skin so that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin.

For inhalation administration, the compositions used according to the invention may be packaged in pressurized packs or using a suitable propellant, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered in the form of an aerosol spray from a nebulizer. For pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in Remington's Pharmaceutical Science, 15th Edition, 1975 Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a text generally known in all pharmaceutical chemistry.

In the pharmaceutical composition of the present invention, the amount of the active compound in the unit dose formulation may vary, and specifically, it may be adjusted to about 0.01 mg to about 1 g per time based on an average human weighing 70 kg. However, the administered dose may vary depending on the requirements of humans and mammals, the severity of the disease being treated, and the final composition of the compound used. It falls to those skilled in the art to determine the appropriate dosage for a particular situation.

The pharmaceutical composition of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, or methods using biological response modifiers.

In the present invention, 'active ingredient' refers to an ingredient that exhibits the desired activity alone or can exhibit activity in combination with a carrier that is not active on its own.

In the present invention, 'prevention' refers to any action that suppresses or delays the symptoms of a metabolic disease by administering the composition of the present invention, and 'improvement' in the present invention refers to the effect of alleviating the symptoms of a metabolic disease by applying the composition of the present invention. It means representing. In the present invention, 'treatment' refers to any action in which the symptoms of a metabolic disease are improved or beneficially changed by administration of the composition of the present invention.

The symptoms of the metabolic disease refer to all physical changes caused by the metabolic disease, and may include, for example, increased food intake, increased appetite, weight gain, decreased lipid metabolism, increased serum glucose level, decreased insulin sensitivity, etc. It is not limited.

In one aspect for achieving the above object, the present invention provides a quasi-drug composition for preventing, improving, or treating metabolic diseases containing the FSTL1 protein or the gene encoding the protein as an active ingredient.

The terms 'FSTL1 protein', 'gene encoding FSTL1 protein', 'metabolic disease', 'prevention', 'improvement', and 'treatment' are as described above.

In the present invention, "quasi-drugs" are articles used for the purpose of diagnosing, treating, alleviating, treating or preventing diseases in humans or animals, but are not instruments, machines, or devices, and do not have a pharmacological effect on the structure and function of humans or animals. It refers to products used for the purpose, excluding those that are not instruments, machines, or devices. One specific example may include preparations for internal use, but is not limited thereto, and the formulation method, dosage, usage method, and components of quasi-drugs etc. can be appropriately selected from conventional techniques known in the technical field.

In addition to the above ingredients, the quasi-drug composition of the present invention may further include pharmaceutically acceptable carriers, excipients, or diluents as needed. The pharmaceutically acceptable carrier, excipient, or diluent is not limited as long as it does not impair the effect of the present invention, and includes, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, etc. It can be included.

In one aspect for achieving the above object, the present invention provides a food composition for preventing or improving metabolic diseases comprising the FSTL1 protein or the gene encoding the protein as an active ingredient.

In another aspect, the present invention provides a food composition for suppressing appetite, suppressing weight gain, or reducing weight, comprising the FSTL1 protein or the gene encoding the protein as an active ingredient.

The terms ‘FSTL1 protein’, ‘gene encoding FSTL1 protein’, ‘metabolic disease’, ‘prevention’ and ‘improvement’ are as described above.

The food composition of the present invention includes all forms of functional food, nutritional supplement, health food, food additives, health functional food and feed, etc., for humans or livestock. Animals, including . Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

Food compositions of this type can be prepared in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruit and its processed foods (canned fruit, bottled food, jam, marmalade, etc.), fish, meat, and their processed foods (ham, sausage, corned beef, etc.) , bread and noodles (udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (butter, cheese, etc.), edible plant oils, margarine, vegetable protein, retort food, frozen food. , can be manufactured by adding the above active ingredients to various seasonings (soybean paste, soy sauce, sauce, etc.).

Additionally, nutritional supplements can be manufactured by adding the above-mentioned active ingredients to capsules, tablets, pills, etc. In addition, health functional foods are not limited to this, but for example, they can be manufactured in the form of tea, juice, and drinks and consumed by liquefying, granulating, encapsulating, and powdering so that they can be consumed as health drinks. Additionally, it can be prepared in the form of a composition by mixing it with one or more known active ingredients known to be effective in improving metabolic diseases.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, and preservatives. , may contain glycerin, alcohol, or carbonating agent.

In one aspect for achieving the above object, the present invention provides a feed composition for preventing or improving metabolic diseases comprising the FSTL1 protein or the gene encoding the protein as an active ingredient.

In another aspect, the present invention provides a feed composition for suppressing appetite, suppressing weight gain, or reducing body weight, comprising the FSTL1 protein or the gene encoding the protein as an active ingredient.

The terms ‘FSTL1 protein’, ‘gene encoding FSTL1 protein’, ‘metabolic disease’, ‘prevention’ and ‘improvement’ are as described above.

In the present invention, the feed composition may be formulated in the form of a normal feed and may include known feed ingredients. It can also be used as a feed additive, which is added to the feed used in the form of an additive. The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act, and includes mineral preparations such as sodium bicarbonate (sodium bicarbonate), bentonite, magnesium oxide, and complex minerals, and trace minerals such as zinc, copper, cobalt, and selenium. Preparations, vitamin preparations such as kerotene, vitamin E, vitamins A, D, E, nicotinic acid, and vitamin B complex, protective amino acid preparations such as methionine and lysic acid, protective fatty acid preparations such as fatty acid calcium salts, probiotics (lactic acid bacteria), yeast culture Water, live bacteria such as mold fermentation products, yeast, etc. may be additionally included.

The feed or feed additive of the present invention can be applied to a number of animal diets, including mammals, poultry, and fish.

The composition containing the FSTL1 protein of the present invention or the gene encoding the protein as an active ingredient can play a role in energy balance and blood sugar control in the hypothalamus. Specifically, it increases the activity of genes related to lipid metabolism and improves lipid metabolism. and may cause loss of appetite and weight loss.

In addition, FSTL1 regulates glucose levels through activation of hypothalamic insulin signaling, and as a result, has the effect of preventing, treating, or improving metabolic diseases, and can be used as a material for pharmaceuticals, food, and feed compositions.

Figure 1 shows the influence status of metabolically active tissues (mediobasal hypothalamus (A); skeletal muscle (B); brown adipose tissue (C); liver (D); mature adipocytes (E); stromal vascular fraction cells (F). This is a result of confirming the expression level of the FSTL1 gene that changes depending on the condition (Fed: mice fed randomly; Refed: mice fed for 2 hours after fasting for 24 hours; sHFD: mice fed a high-fat diet (HFD) for 2 days).
Figure 2 shows mid-basal hypothalamus of mice fed HFD (n = 34) and LFD (n = 6) for 12 weeks (A) and diet-induced obesity (DIO) and diet-resistant obesity (DR) groups (B). This is the result of comparing the expression level of the FSTL1 gene.
Figure 3 shows the results of immunofluorescence staining of FSTL1 confirming expression in NPY-expressing neurons in the hypothalamus of mice, confirming the colocalization and ratio of NPY and FSTL1 in the midbasal hypothalamus.
Figure 4 shows the results confirming that the expression of the Pomc gene and Agrp gene of FSTL1 is suppressed in N43/5 cells.
Figure 5 shows food intake and body weight gain in mice administered I3V with various forms of FSTL1 (recombinant FSTL1; FSTL1-3xHA, conditioned medium overexpressing N-terminally GST-tagged FSTL1 or C-terminally GST-tagged FSTL1). This is a result confirming that it has decreased.
Figure 6 shows the results of energy consumption (VO ₂ ) and respiratory quotient (RQ) confirming that FSTL1 does not affect energy consumption when administered to the central region of mice.
Figure 7 shows the results confirming that the expression levels of genes related to lipolysis, fatty acid utilization, lipogenesis, and pro-inflammation are increased by FSTL1 in N43/5 cells.
Figure 8 shows the results confirming that FSTL1 induces lipolysis through lipophagy, confirming that the level of lipid content is reduced when cells are transfected or treated with FSTL1.
Figure 9 confirms that central FSTL1-induced anorexia and weight loss are caused by changes in hypothalamic lipid metabolism mediated through ATGL in mice. 4 I3V administration of FSTL1-GST or control group in wild-type mice and ATGL KO mice. This is the result of comparing food intake (A) and weight gain (B) after time.
Figure 10 confirms that central FSTL1-induced anorexia and weight loss are mediated through b3-AR in the midbasal hypothalamus of mice. Pretreatment with a b3-AR antagonist increases the food intake (A) and body weight of mice. This result confirms that the action of FSTL1 on increase (B) is weakened.
Figure 11 shows the results confirming that serum glucose levels are reduced following central administration of FSTL1.
Figure 12 shows the results confirming that central FSTL1 activates AKT/mTOR signaling in the midbasal hypothalamus of mice. I3V administration of FSTL1-GST increases AKT activity at the Thr308 residue (A) and mTOR in the midbasal hypothalamus of mice. It was confirmed that activity (B) was significantly increased.
Figure 13 confirms the effect of central FSTL1 on improving peripheral glucose metabolism. It was confirmed that when FSTL1-GST was administered I3V, serum glucose levels were reduced and peripheral insulin sensitivity was improved.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Confirmation of FSTL1 expression level in metabolically active tissues

1.1. Animal experiments and immunostaining

(1) Animal testing

To determine the response to nutritional status of metabolic tissues (liver, skeletal muscle, adipose tissue, brown adipose tissue, hypothalamus) according to the level of FSTL1 mRNA expression, male C57BL/6 mice (n = 32, Orient Bio) were used. . The fed group (n = 8) was fed food (chow, 5L79, Orient Bio) ad libitum during the experiment, and the fasting group (n = 8) fasted for 24 hours. The re-feeding group (n = 8) was fed for 2 hours after a 24-hour fast. The sHFD group (n = 8) was fed a high-fat diet (HFD) for 2 days. Additionally, to confirm the response of FSTL1 mRNA expression to long-term HFD exposure, male C57BL/6 mice (n = 40) were used. The low-fat diet (LFD) group (n = 6) and the HFD group (n = 34) were fed LFD (D12450B, Research Diets) and HFD (D12451, Research Diets) ad libitum, respectively, for 12 weeks. To confirm the main effect of FSTL1 through ICV injection of FSTL1, male C57BL/6 mice (n = 18) were used and fed food ad libitum during the experiment.

Mice were raised indoors with constant temperature and humidity (22°C, 50% relative humidity) and housed individually in standard mouse cages with a 12-hour light/dark cycle and freely available food and water. All experimental procedures were performed in accordance with the guidelines for the ethical use of laboratory animals announced by the Korea University Animal Care Committee and the guidelines approved by the Korea University Animal Care System Committee.

(2) Intra-third cerebroventricular (I3V) surgery

To reduce pain and inflammation, carprofen (Rymadil, Pfizer) and antibiotics (Borgal) were injected subcutaneously 30 minutes before surgery. Mice were anesthetized with ketamine/xylazine and the surgical area was shaved. Placed in a stereotaxic frame on a heating pad to maintain body temperature, the head was secured with ear bars and the skull was exposed through a single incision. For a clean and dry skull, the pericranial tissue was removed and the cannula was fixed in a holder. When measuring dorsoventral (D-V) at bregma and lambda, it was confirmed that the gap between bregma and lambda was within 0.1 mm for the level of the skull. If D-V, mediolateral, and anteroposterior (A-P) at bregma are 0, -0.825 should be marked at the target point of A-P. A hole was drilled at the target point, 26 skull fragments were removed using forceps, two anchor screws were inserted obliquely from the center of the hole, and the cannula was inserted into the target point. The surface of the skull was always kept clean and dry. The surgical site was covered with dental cement and the cannula holder was removed. After completing the surgery, the mouse was placed under a lamp to maintain body temperature until it recovered from anesthesia.

(3) single or double I3V administration studies

To inject the protein into the third ventricle of the mouse, a 33-gauge injection cannula (Plastics One) was inserted through the cannula. This infusion cannula was connected to a microsyringe (8309020, CMA) driven by an infusion pump (Harvard Apparatus), and proteins were delivered to the mice in a volume of 2 μl over 1 min after a 1-h fast. To determine whether the anorexigenic effect of FSTL1 is mediated through 3-AR, we performed a dual I3V administration of SR 59230A followed by FSTL1-GST in mice. Additionally, SR 59230A was pre-administered into the third ventricle to block 3-AR 1 hour before I3V administration of FSTL1-GST to mice.

(4) Immunofluorescence

Mouse brains were obtained after perfusion using 4% paraformaldehyde (PFA)/0.1M NaKPB, and cultured overnight in fixative (4% PFA/0.1M NaKPB) at -4°C. The brain was cultured in 30% sucrose, then dehydrated and stored at -80°C. The brain was sectioned at 30 um thickness using a Microtome (HM440E, Microm). Incubate with 5% normal goat serum in 0.3% PBST (PBS containing 0.3% Triton-100) for 30 min, then in primary antibody against FSTL1 (1:200; MAB17381, R&D system) for 48 h at 4°C. Cultured overnight. Sections were then incubated with secondary antibodies (goat anti-mouse Alexa Fluor 488 (1:1000; A-11001, Thermo Fisher Scientific) and goat anti-mouse Alexa Fluor 555 (1:1000; A-21434, Thermo) for 2 h at room temperature. Fisher Scientific), and the sections were incubated with DAPI (1:1000; D9542, Sigma) for 5 minutes. Between all steps, sections were washed with PBS and mounted with mounting solution containing n-propyl gallate and glycerol. Fluorescence images were obtained using a confocal microscope (LSM700, Carl Zeiss).

1.2. Confirmation of FSTL1 expression level

(1) Expression level of FSTL1 according to various nutritional conditions

To evaluate the expression pattern of FSTL1 in metabolically active tissues under different nutritional conditions, mice fed ad libitum (Fed), fasted for 24 h (Fast), and fasted for 24 h and then re-fed for 2 h (RF). , liver, hypothalamus, skeletal muscle, white adipose tissue (mature adipocyte (MA), stromal vascular fraction (SVF)), and brown adipose tissue (BAT) in mice fed a HFD (sHFD) for 2 days. ), the expression of the FSTL1 gene was measured in metabolically active tissues (Figure 1A-F). The expression level of FSTL1 gene in the midbasal hypothalamus was significantly decreased in the RF group compared to the Fed group. The expression level of FSTL1 gene in stromal vascular fraction (SVF) cells of skeletal muscle, liver, BAT, and white adipose tissue was significantly lower in the Fast group compared to the Fed group. These results imply rapid responsiveness of FSTL1 gene expression in metabolically active tissues under various nutritional conditions.

Next, the expression level of the FSTL1 gene was measured in the midbasal hypothalamus between mice fed a high-fat diet (HFD) and a low-fat diet (LFD) for 12 weeks, or between diet-induced obesity (DIO) mice and diet-resistant (DR) mice. compared (Figure 2A, B). The expression level of the FSTL1 gene was elevated in the midbasal hypothalamus of the obese HFD group compared to the lean LFD group, but no significant differences were observed between the DIO and DR groups.

(2) Confirmation of the expression site of FSTL1

To confirm the direct action of FSTL1 on the regulation of energy balance in the brain, double immunofluorescence staining was performed in the midbasal hypothalamus of wild-type mice or NPY-hrGFP expressing mice to examine FSTL1 expression in NPY neurons and glial cells. As a result, it was confirmed that FSTL1 was intensely expressed in the hypothalamus around the third ventricle of mice and was co-expressed in some of the neurons expressing NPY (Figure 3A-D). FSTL1 was confirmed to be 60% colocalized with NPY (Figure 3E).

Example 2. Confirmation of the effect of FSTL1 on the energy balance regulation system

2.1. Cell line culture and confirmation of expression of related factors

(1) Mouse hypothalamic N43/5 cell line culture and processing

Embryonic mouse hypothalamic mHypoE N43/5 cell line (Cellutions Biosystems Inc., CLU127) was cultured in DMEM supplemented with 10% FBS (FBS-24A, Carpricorn) and 1% penicillin/streptomycin (PS-B, Carpricorn). HA, Carpricorn) was maintained at 37°C and 5% CO ₂ conditions. The POMC4 cell line was maintained in DMEM supplemented with 5% FBS and 1% penicillin/streptomycin at 37°C and 5% CO ₂ conditions. The HEK293T cell line was maintained in DMEM (DMEM-HPA) supplemented with 10% FBS (Sigma) and 1% penicillin/streptomycin (PS-B) at 37°C and 5% CO2. All cell lines were cultured under serum starvation during each experiment. PEI was used as a transfection reagent. On day 1, HEK293T cells were transfected with the FSTL1-3xHA plasmid and FSTL1-3xHAs with two EF hand domains removed (1-154 FSTL1-3xHA, 1-203 FSTL1-3xHA, △155-255 FSTL1-3xHA, △155-255 FSTL1-3xHA or FSTL1-3xHA). On the 2nd day, the existing medium was replaced with Opti-MEM (31985-070, Gibco), and the cells and medium were harvested on the 3rd day. In the caffeine treatment experiment, HEK293T cells were transfected with a plasmid containing FSTL1-3xHA, and the next day, the existing medium was removed and treated with caffeine (0, 5, 10mM) in Opti-MEM for 24 hours.

(2) Quantitative real-time PCR analysis

RNA was extracted from mouse tissues and harvested cells using TRIzol Reagent (Invitrogen, 15596018). To synthesize cDNA from the extracted RNA, the iScript cDNA synthesis kit (Biorad, 1708891) was used. Specifically, 1 ug of RNA was used along with 5x iScript reaction mix, iScript reverse transcriptase, and nuclease-free water. For quantitative real-time PCR (RT-qPCR), TaqMan primers, TaqMan Gene Expression Master Mix (Applied Biosystems, 4369016), and ABI 7500 were used. PCR conditions included two-step amplification (95°C for 10 seconds and annealing temperature of 60°C for 30 seconds). Ribosomal protein L32 was used as a housekeeping gene for normalization to the gene of interest. Gene expression in the hypothalamus of the control and FSTL1-GST treated groups was compared using approximate fold differences (Table 1).

gene name TaqMan Cat# Ribosomal protein L32 (Rpl32) Mm02528467_g1 Pomc (Pro-opiomelanocortin-alpha) Mm00435874_m1 Pnpla2 (Patatin-like phospholipase domain containing 2) Mm00503040_m1 Agrp(Agouti-related neuropeptide) Mm00475829_g1 Cpt1b (Carnitine palmitoyltransferase 1b) Mm00487191_g1 Carnitine palmitoyltransferase 1c (Cptlc) Mm00463960_m1 Cluster of differentiation 36 (Cd36) Mm01135198_m1 Fabp4 (Fatty acid binding protein 4) Mm00445878_m1 Acaca (Acetyl-coenzyme A carboxylase alpha) Mm01304257_m1 Fatty acid synthase (Fasn) Mm00662319_m1 IL6 (Interleukin 6) Mm00446190_m1 Tumor necrosis factor (TNF) Mm00443258_m1

(3) Western blot Western blot was performed to confirm protein expression in the cell line. Cells and media were harvested using 1x or 2x SDS sample buffer, and protein samples were loaded into the wells of an 8% acrylamide gel and gel run was performed (0.35A, 90 min). Then, the proteins in the gel were transferred to a PVDF (polyvinylidene difluoride) membrane by electrophoresis at 4°C for 120 minutes. Membranes were blocked with 5% skim milk or 5% BSA (A3059, Sigma) in TBST and incubated with primary antibody overnight at 4°C in TBST. The secondary antibody was washed with TBST and reacted with the membrane for 1 hour at room temperature. Membranes were washed with PBST, and proteins were detected using an ECL kit (EBP-1073, ELPIS biotec Inc.) for visualization.

Primary antibodies used: pACC(Ser79) (1:1000, #3661, Cell 24 Signaling Technology), ACC (1:1000, #3662, Cell Signaling Technology), pAKT(Thr308) (1:1000, #9275, Cell Signaling Technology), pAKT(Ser473)(1:1000, #9271, Cell Signaling Technology), AKT(1:1000, #9272, Cell Signaling Technology), pmTOR(Ser2448)(1:1000, #2971, Cell Signaling Technology) ), mTOR (1:1000, #2972, Cell Signaling Technology), HA-Tag (1:1000, sc-7392, Santa Cruz Biotechnology).

(4) Protein purification

C-terminally GST-tagged FSTL1 was obtained from transfected HEK293T cells. HEK293 cells at 80% confluency in a 150mm Petri dish were transfected by adding 40ug FSTL1-GST plasmid to Opti-MEM and 160ul PEI (1ug/ul). On the second day, 20 ml of new medium (Opti-MEM) was added to the transfected cells. The next day, cells and medium reacted overnight at 4°C with glutathione Sepharose (17513201, GE healthcare) were obtained. Glutathione Sepharose was washed with PBS and reacted with 10mM reduced glutathione to elute the protein from the beads. This protein was dialyzed with a 10 kDa molecular weight cutoff dialysis cassette (66380, Thermo Scientific), and the dialyzed protein was concentrated with Amicon ultra-0.5 (UFC501096, Millipore).

(5) Energy consumption

Energy expenditure was measured in FSTL1-GST ICV-injected mice using indirect calorimetry (Oxylet, Panlab). Each mouse was placed individually in a calorimetry chamber, and food and water were available ad libitum. VO ₂ and VCO ₂ values were measured continuously for 3 minutes at 30-minute intervals, energy expenditure was normalized to body weight and expressed as VO ₂ per mouse, and respiratory quotient (RQ) was calculated by dividing VCO ₂ by VO ₂ did.

2.2. Confirmation of the effect of FSTL1 on genes related to energy balance regulation

(1) Effect of FSTL1 on the expression of Agrp gene

To confirm the effect of FSTL1 on the energy balance regulation system, the expression levels of hypothalamic Agrp and Pomc genes and genes related to lipid metabolism and inflammation were compared in N43/5 cells after FSTL1-GST treatment. The expression level of Agrp gene was significantly lower in FSTL1-GST treated cells compared to control treated cells, but no significant differences in Pomc gene expression were observed between groups (Figure 4).

(2) Effects of central administration of FSTL1 on food intake and body weight gain

To investigate the key role of FSTL1 in energy balance regulation, food intake and body weight were compared at 24-hour intervals after I3V administration of various forms of recombinant FSTL1 protein into the third ventricle of mice. To test the effect of FSTL1 on energy balance in mice, four types of recombinant FSTL1 proteins were used: recombinant FSTL1 (R&D systems), secreted FSTL1-3xHA (cm-FSTL1), and N-terminally GST-tagged FSTL1. (GST-FSTL1, non-secretory form), and conditioned medium (CM) containing C-terminally GST-tagged FSTL1 (FSTL1-GST, secretory form). First, the effect of FSTL1 on energy balance was confirmed by treating mice with a commercially available recombinant FSTL1 protein. As a result, a significant decrease in food intake and body weight gain was confirmed in mice administered I3V with recombinant FSTL1 (10ug) compared to vehicle ( Figure 5A and B). Next, three types of recombinant FSTL1 proteins were produced to confirm the main effects of FSTL1 on energy balance. As a result, I3V administration of cm-FSTL1 significantly reduced food intake and body weight gain in mice compared to the cm-CON group (Figure 5C and D). Additionally, central GST-tagged FSTL1 significantly reduced food intake and body weight gain in mice compared with each GST-CON group ( Figure 5 E–H). In particular, it was confirmed that central administration of both the non-secretory form of FSTL1 (GST-FSTL1) and the secreted form of FSTL1 (cm-FSTL1 and FSTL1-GST) caused anorexia and weight loss in mice. This indicates that the anorexic action of FSTL1 is unrelated to the secretion process. Additionally, there was no significant difference in food intake and body weight gain between the untreated control group (GST) and the negative control group (NT), indicating that the process of producing the recombinant protein itself does not cause non-specific disease in mice. means (Figure 5G and H). Therefore, these results indicate that FSTL1 in the brain regulates energy balance.

(3) Effect of central FSTL1 on energy expenditure

To determine whether central FSTL1-induced weight loss was associated with changes in energy expenditure, energy expenditure and RQ were continuously monitored for 22 h in mice following I3V administration of FSTL1-GST or control. No significant differences in VO ₂ and RQ were observed between FSTL1-GST or control groups (Figures 6A and B). This means that central FSTL1-induced weight loss is mainly due to its anorexigenic effect in mice.

Example 3. Confirmation of the effect of FSTL1 on lipid metabolism and inflammatory activity

3.1. Lipid staining method - BODIPY staining

BODIPY staining was performed for neutral lipids, oils, and other non-polar lipids. Cells were cultured in 12-well cell culture dishes containing cover glasses. GFP-tagged FSTL1 was transfected into HEK293T cells or conditioned medium (CM) overexpressing FSTL1-3X-HA was treated with POMC4 cells. On the second day, cells were washed with 1ml of PBS and fixed by incubating in 4% PFA solution at room temperature for 30 minutes. After removing the 4% PFA solution, the cells were washed twice with 1 ml PBS and incubated with 2 uM BODIPY staining solution in PBS for 15 minutes at 37°C. Afterwards, the cells were washed twice in 1ml PBS and then cultured with DAPI solution. Using forceps, a cover glass was placed on a drop of mounting solution on a glass slide, and images were obtained using a confocal microscope (LSM700, Carl Zeiss).

3.2. Effect of FSTL1 on lipid metabolism-related gene expression

(1) Increased lipid metabolism and inflammatory activity by FSTL1

Hypothalamic lipid metabolism is known to be one of the regulators of energy balance in the brain. To investigate the mechanism of anorexia induced by central FSTL1, N43/5 cells treated with FSTL1-GST or GST (control) were examined. The expression levels of genes related to lipid metabolism (lipolysis genes (Pnpla2, Cpt1b, Cpt1c), fatty acid utilization (Cd36, Fabp4), and lipogenesis genes (Acaca, Fasn)) were compared. As a result, it was confirmed that the expression levels of Pnpla2, Cpt1b, Cpt1c, Fabp4, and Acaca genes were significantly higher in FSTL1-GST treated cells than in the control group (Figure 7). This means that FSTL1 contributes to increasing the activity of genes related to lipolysis, lipogenesis, and fatty acid utilization processes, and consequently enhances lipid metabolism.

(2) Promotion of lipolysis by FSTL1

To investigate the effect of FSTL1 on lipid metabolism, lipid content levels were compared by BODIPY staining in HEK209T cells transfected with GFP, Pnpla2-GFP, and FSTL1-GFP. As a result, the level of BODIPY staining was lower in cells transfected with FSTL1-GFP and Pnpla2-GFP than in cells transfected with GFP, and was similar between cells transfected with FSTL1-GFP and Pnpla2-GFP (Figure 8A).

Lipophagy is one of the main processes that removes intracellular lipids by decomposing lipid droplets through lysosomes. Hypothalamic autophagy rapidly increases intracellular fatty acid and AgRP expression to regulate energy balance and hypothalamic lipid Sensing is known to regulate energy intake through CPT1 and malonyl-CoA. Under this background, to determine whether the lipolytic action induced by FSTL1 is mediated through lipophagy, FSTL1-3xHA was administered together with two types of autophagy inhibitors such as chloroquine (CQ) and bafilomycin A1. Lipid content levels were compared in POMC4 cells by BODIPY staining. Conditioned medium (CM) from HEK293T cells overexpressing FSTL1-3xHA was used for FSTL1 protein, and similar to related studies using HEK293T cells overexpressing FSTL1-GFP, the level of BODIPY staining was significantly higher in POMC4 cells treated with CM compared to controls. It was lower. This indicates the lipolytic action of FSTL1, and when treated with CQ or bafilomycin A1, the decrease in lipid content of cells induced by FSTL1 was suppressed (Figure 8B). These results imply that the lipolytic action of FSTL1 is mediated through lipophagy.

Example 4. Confirmation of the effect of FSTL1 on anorexia and weight loss

4.1. Glucose and insulin tolerance tests

To investigate the effect of FSTL1 on glucose metabolism, oral glucose and intraperitoneal insulin tolerance tests were performed in mice administered I3V with FSTL1-GST or control. Glucose tolerance test (GTT) was performed by oral administration of glucose (1.5 g/kg, Sigma). Glucose levels were measured at time points of 0, 15, 30, 45, 60, and 120 min from a drop of blood from the mouse tail using a glucometer after cutting off 1–2 mm of the tip of the tail with sharp scissors (1110690879, Roche). . The insulin tolerance test (ITT) was performed by intraperitoneal administration of insulin (0.6 U/kg) and the procedure was the same as the glucose tolerance test (GTT). Blood glucose measurements were performed at 0, 15, 30, 45, and 60 minutes.

4.2. Confirmation of the mechanism of anorexia and weight loss caused by central FSTL1

(1) Confirmation of correlation with neutral fat hydrolyzing enzyme

To determine whether anorexia and weight loss induced by central FSTL1 are related to lipid metabolism in the central hypothalamus, which acts as a catalyst in the first step of hydrolyzing triglycerides in lipid droplets in adipocytes and non-adipocytes. ATGL KO mice, which are known to be key enzymes, were used. Comparison of food intake and body weight gain in wild-type mice or ATGL KO mice after I3V administration of FSTL1-GST or control showed that I3V administration of FSTL1-GST decreased food intake and body weight gain at 4 hours in wild-type mice compared to controls. significantly reduced. In contrast, no significant differences in food intake and body weight gain were observed between FSTL1-GST and control groups in ATGL KO mice (Figures 9A and B). These results imply that central FSTL1-induced anorexia and weight loss are associated with changes in hypothalamic lipid metabolism mediated through ATGL in mice.

(2) Confirmation of association with b3 adrenergic receptors

To determine whether central FSTL1-induced anorexia and weight loss are mediated through b3-AR, followed by increased lipolysis in the mesobasal hypothalamus, mice were treated with dual I3V administration of FSTL1-GST and SR 59230A or control group. Food intake and weight gain were compared. Consistently, co-administration of control and FSTL1-GST reduced food intake and body weight gain in mice compared to controls, and pretreatment with SR 59230A alleviated central FSTL1-induced anorexia and weight loss in mice (Figure 10A and B).

Next, to investigate whether the interaction between FSTL1 and b3-AR affects signaling pathways related to hypothalamic fatty acid oxidation in HEK293T cells overexpressing b3-AR, we measured the activity of ACC, i.e., the phosphorylation of ACC. Confirmed. As a result, compared to the control group, administration of FSTL1-GST decreased phosphorylation of ACC (pACC/ACC) in HEK293T cells overexpressing b3-AR, while in HEK293T cells that did not overexpress b3-AR, there was a decrease in phosphorylation between the control group and FSTL1-GST. No significant differences were observed (Figure 10C). Considering that increased hypothalamic ACC activity suppresses food intake in mice, these results imply that FSTL1 induces lipolysis through b3-AR in the regulation of energy homeostasis.

(3) Mechanism of blood sugar control through activation of AKT signal of FSTL1

To confirm the key role of FSTL1 in peripheral blood glucose control, serum glucose levels were measured after I3V administration of FSTL1-GST in mice. As a result, the FSTL1-GST administration group had reduced glucose levels compared to the control group (FIG. 11). In addition, it was confirmed that central FSTL1 activates AKT/mTOR signaling in the midbasal hypothalamus of mice compared to the control group (Figures 12A and B). These results imply that brain FSTL1 can regulate peripheral glucose homeostasis.

To confirm central FSTL1 regulation of peripheral glucose homeostasis, oral GTT was performed 2 h after I3V administration of FSTL1-GST in mice. Serum glucose levels were significantly higher 15 minutes after oral glucose challenge in the group administered I3V FSTL1-GST compared to the control group (Figure 13A). Next, intraperitoneal ITT was performed 1 hour after I3V administration of FSTL1-GST to mice. The percentage of initial glucose levels after insulin challenge was significantly lower in the group administered I3V FSTL1-GST than the control group (Figure 13B), indicating that insulin sensitivity was improved by FSTL1 in mice. As a result, central FSTL1 can improve peripheral glycemic control through activation of hypothalamic insulin signaling in mice.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Claims (15)

A pharmaceutical composition for preventing or treating metabolic diseases comprising the FSTL1 protein or the gene encoding the protein as an active ingredient.
According to paragraph 1,
The FSTL1 protein is a pharmaceutical composition that regulates the expression of genes related to lipid metabolism or activates the insulin signaling pathway in the hypothalamus in the brain.
According to paragraph 2,
A pharmaceutical composition wherein the FSTL1 protein increases or decreases the expression levels of Agrp, Pnpla2, Cpt1b, Cpt1c, Fabp4, and Acaca.
According to paragraph 1,
The above metabolic diseases include obesity, diabetes, hyperlipidemia, hypertension, hypercholesterolemia, hyperinsulinemia, arteriosclerosis, dyslipidemia, hypertriglyceridemia, liver disease, fatty liver, stroke, myocardial infarction, cardiovascular disease, hyperglycemia, and insulin resistance disease. At least one pharmaceutical composition selected from the group consisting of.
A quasi-drug composition for preventing, improving, or treating metabolic diseases, comprising the FSTL1 protein or the gene encoding the protein as an active ingredient.
According to clause 5,
The above metabolic diseases include obesity, diabetes, hyperlipidemia, hypertension, hypercholesterolemia, hyperinsulinemia, arteriosclerosis, dyslipidemia, hypertriglyceridemia, liver disease, fatty liver, stroke, myocardial infarction, cardiovascular disease, hyperglycemia, and insulin resistance disease. A quasi-drug composition, which is at least one selected from the group consisting of.
A food composition for preventing or improving metabolic diseases comprising the FSTL1 protein or the gene encoding the protein as an active ingredient.
In clause 7,
The above metabolic diseases include obesity, diabetes, hyperlipidemia, hypertension, hypercholesterolemia, hyperinsulinemia, arteriosclerosis, dyslipidemia, hypertriglyceridemia, liver disease, fatty liver, stroke, myocardial infarction, cardiovascular disease, hyperglycemia, and insulin resistance disease. A food composition, which is at least one selected from the group consisting of.
A food composition for suppressing appetite, suppressing weight gain, or reducing weight, comprising the FSTL1 protein or a gene encoding the protein as an active ingredient.
A health functional food for preventing or improving metabolic diseases containing the FSTL1 protein or the gene encoding the protein as an active ingredient.
According to clause 10,
The above metabolic diseases include obesity, diabetes, hyperlipidemia, hypertension, hypercholesterolemia, hyperinsulinemia, arteriosclerosis, dyslipidemia, hypertriglyceridemia, liver disease, fatty liver, stroke, myocardial infarction, cardiovascular disease, hyperglycemia, and insulin resistance disease. At least one health functional food selected from the group consisting of.
A health functional food for suppressing appetite, suppressing weight gain, or reducing weight, comprising the FSTL1 protein or the gene encoding the protein as an active ingredient.
A feed composition for preventing or improving metabolic diseases comprising the FSTL1 protein or the gene encoding the protein as an active ingredient.
According to clause 13,
The above metabolic diseases include obesity, diabetes, hyperlipidemia, hypertension, hypercholesterolemia, hyperinsulinemia, arteriosclerosis, dyslipidemia, hypertriglyceridemia, liver disease, fatty liver, stroke, myocardial infarction, cardiovascular disease, hyperglycemia, and insulin resistance disease. At least one feed composition selected from the group consisting of.
A feed composition for suppressing appetite, suppressing weight gain, or reducing body weight, comprising the FSTL1 protein or a gene encoding the protein as an active ingredient.