KR101215759B1 - Uses of Compositions Comprising Orosomucoid - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of metabolic diseases comprising an ORM (Orosomucoid) gene or protein. The composition of the present invention includes ORM which is a natural substance present in abundance in vivo, and has a selective regulating activity on various targets, so that it can be effectively used for preventing or treating symptoms caused by metabolic diseases, and is easy to manufacture and has no side effects. It is characterized by a low probability of occurrence.

Description

Uses of Compositions Comprising Orosomucoid

The present invention relates to a composition for preventing or treating metabolic diseases, and more particularly, to a composition comprising ORM which is a molecule in vivo and having selective regulatory activity against various in vivo targets.

As obesity of modern people increases due to changes in living environment, the onset of metabolic diseases including diabetes, hypertension, lipid metabolism abnormality, insulin resistance, etc. is increasing rapidly. These diseases increase the risk of mutual development and are common diseases associated with multiple metabolic changes such as aging, stress, and immune dysfunction.

According to 2005 National Health and Nutrition Survey results, 32% of Korean adults over 20 years old were obese (35.2% of adult men and 28.3% of women). The incidence of childhood obesity in Korea has also increased rapidly.In 2005, 11.3% of elementary school students, 10.7% of middle school students, and 16% of high school students were classified as obese (BMI ≥ 25 kg / m ² ), and overweight (BMI ≥ 23 kg / m ^2). Or 17% of obese adolescents had metabolic syndrome.

This increase in overweight and obesity leads to an increase in the prevalence of chronic diseases such as hypertension (30.2% for men, 25.6% for women), diabetes (9.0% for men, 7.2% for women) and hypercholesterolemia The prevalence of hemophilia (7.5% for men and 8.8% for women) is much higher than for other countries.

The socio-economic loss due to obesity is estimated at 1.1.7 trillion won per year. As a result, the government aims to reduce the adult obesity rate to less than 20% and the youth obesity rate to less than 15%. As a strategy to achieve the goal, I decided to find an accurate definition of obesity and how to measure it.

Obesity can be best treated with diet, exercise, and behavior modification therapy. However, obesity treatment or diet products are being used because these methods are time consuming and laborious. However, orlistat, which is currently used as a treatment for obesity, has side effects such as fatty stool, intestinal gas generation, and bloating. Sibutramine has side effects such as headache, dry mouth, anorexia, insomnia, and constipation. Also, orlistat inhibits the absorption of vitamin E and vitamin D, while phentermine and sibutramine have side effects that increase heart rate, palpitations or dizziness.

As the side effects of synthetic medicines have overcome the limitations of various chronic diseases, the development of biopharmaceuticals that regulate metabolic-related biomolecules for the development of treatments for these metabolic diseases is underway. Little is known about endogenous mediators and mechanisms that restore energy homeostasis.

The present inventors have tried to find in vivo molecules effective for the prevention and treatment of metabolic diseases. As a result, the inventors have found that the composition comprising ORM inhibits the inflammatory activity of adipocyte-derived ORM by adipocytes and monocytes / macrophages, reduces macrophage infiltration into adipocytes, and promotes the action of insulin. Thus, the present invention was completed by confirming that it ultimately contributes to the recovery of energy homeostasis by improving insulin resistance.

Accordingly, it is an object of the present invention to provide a composition for the prevention or treatment of metabolic diseases comprising an ORM gene or protein.

Another object of the present invention is to provide a method for screening a substance for preventing or treating metabolic diseases that causes up-regulation of the expression or protein amount of ORM having the prophylactic or therapeutic activity of the metabolic disease.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the invention is a pharmaceutically effective amount of an ORM (Orosomucoid) gene or protein; And it provides a pharmaceutical composition for the prevention or treatment of metabolic diseases comprising a pharmaceutically acceptable carrier.

The present inventors have tried to find in vivo molecules effective for the prevention and treatment of metabolic diseases. As a result, the present inventors have found that ORM derived from adipocytes inhibits inflammatory activity of adipocytes and monocytes / macrophages, reduces infiltration of macrophages into adipocytes, promotes insulin action, and ultimately insulin resistance. The findings suggest that ORM may be a molecular target for the prevention and treatment of metabolic diseases. The present invention is easy to manufacture and significantly reduce the possibility of side effects by using biomolecules rich in the body as a prophylactic or therapeutic agent for metabolic diseases.

Orosomucoid (ORM), also called α-1 acid glycoprotein, is one of the plasma proteins induced in stress-like conditions, reported to account for about 1% of all plasma proteins. (Fournier et al., 2000; Hochepied et al., 2003). ORM is a family of acute phase reactant proteins, expressed in hepatocytes and released into plasma under stress conditions such as tissue injury, infection and inflammation. Although the role of the ORM in relation to circulation is not well known, it is thought to be involved in at least three other functions: immunoregulatory, protective and carrier functions (Fournier et al., 2000; Hochepied et al., 2003).

The present inventors have uncovered new functions of ORM, an abundant immunomodulator in vivo, and at the same time, provide a new use of ORM as a therapeutic agent for metabolic diseases, and ORM is induced in response to metabolic and inflammatory signals in adipose tissue of obese subjects. Proved to protect against inflammation.

In the composition for preventing or treating metabolic diseases of the present invention, it can be used as an active ingredient ORM protein or ORM gene itself.

ORM encompassed by the present invention includes various types of isotypes, preferably at least one ORM isoform selected from the group consisting of ORM1, ORM2 and ORM3.

The ORM protein used in the present invention is preferably a human ORM protein, more preferably a human ORM protein having an amino acid sequence of SEQ ID NO: 1 (ORM1) or 2 (ORM2).

The ORM gene used in the present invention preferably comprises a nucleotide sequence encoding a human ORM, more preferably comprises a nucleotide sequence encoding a human ORM having an amino acid sequence of SEQ ID NO: 1 or 2 SEQ ID NO: And, most preferably, the nucleotide sequence of SEQ ID NO: 3 or 4 of the sequence listing.

It is apparent to those skilled in the art that the ORM used in the present invention is not limited to the amino acid sequence or nucleotide sequence described in the attached sequence list in the range having the prophylactic or therapeutic activity of the metabolic disease.

Variations in nucleotides do not result in changes in proteins. Such nucleic acids include functionally equivalent codons or codons that encode the same amino acid (eg, by degeneracy of the codon, six codons for arginine or serine), or codons that encode biologically equivalent amino acids. And nucleic acid molecules.

In addition, variations in nucleotides may result in changes in the ORM itself. Even in the case of mutations that bring about changes in the amino acids of the ORM, those exhibiting almost the same activity as the ORM used in the present invention can be obtained.

It is apparent to those skilled in the art that biological function equivalents that may be included in the ORM used in the present invention will be limited to variations in amino acid sequences that exert biological activity equivalent to ORM.

Such amino acid variations are made based on the relative similarity of amino acid side chain substituents such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.

In introducing variants, hydropathic idex of amino acids can be considered. Each amino acid is assigned a hydrophobicity index depending on its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

Hydrophobic amino acid indexes are very important in conferring the interactive biological function of proteins. It is known that substitution with an amino acid having a similar hydrophobicity index can retain similar biological activities. When introducing mutations with reference to the hydrophobicity index, substitutions are made between amino acids which exhibit a hydrophobicity index difference of preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

It is also well known that substitutions between amino acids with similar hydrophilicity values result in proteins with equivalent biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

When introducing mutations with reference to hydrophilicity values, substitutions are made between amino acids which exhibit a hydrophilicity value difference of preferably within ± 2, more preferably within ± 1 and even more preferably within ± 0.5.

Amino acid exchange in proteins that do not alter the activity of the molecule as a whole is known in the art (H. Neuroath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.

Considering the above-described variations with biologically equivalent activity, the ORM protein or gene used in the present invention is interpreted to include sequences that exhibit substantial identity with the sequences listed in the Sequence Listing. This substantial identity is at least 80% when the sequences of the present invention are aligned with each other as closely as possible and the aligned sequences are analyzed using algorithms commonly used in the art. By a sequence that shows homology, more preferably 90% homology, most preferably 98% homology. Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2: 482 (1981) Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994).

NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from the National Center for Biological Information (NBCI), etc. It can be used in conjunction with sequencing programs such as blastx, tblastn and tblastx. BLSAT is accessible at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

The ORM gene used in the present invention may preferably be contained in a gene delivery system and administered.

As used herein, the term “gene delivery system” refers to one designed to carry and express an ORM gene, and has the same meaning as intracellular transduction of a gene. At the tissue level, the term gene delivery has the same meaning as spread of a gene.

For preparing the gene delivery system of the invention, the ORM-encoding nucleotide sequence is preferably present in a suitable expression construct. In the expression construct, the ORM gene is preferably operably linked to the promoter. As used herein, the term “operably linked” refers to a functional binding between a nucleic acid expression control sequence (eg, an array of promoters, signal sequences, or transcriptional regulator binding sites) and other nucleic acid sequences, thereby The regulatory sequence will control the transcription and / or translation of said other nucleic acid sequence. In the present invention, the promoter coupled to the ORM gene is preferably capable of controlling transcription of the ORM gene by operating in animal cells, more preferably mammalian cells, and promoters and mammalian cells derived from mammalian viruses. Promoters derived from the genome of, for example, CMV (cytomegalo virus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, RSV promoter, EF1 alpha promoter, metallothionine Promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene, promoter of human GM-CSF gene, cancer cell specific promoter (e.g. , TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter and A FP promoters) and tissue specific promoters (eg, albumin promoters).

Preferably, the expression construct used in the present invention comprises a polyaninylation sequence (eg, a glial growth hormone terminator and a SV40 derived poly adenylation sequence).

According to a preferred embodiment of the present invention, the ORM-encoding nucleotide sequence used in the present invention has a structure of “promoter-ORM-encoding nucleotide sequence-poly adenylation sequence”.

The gene delivery system of the present invention can be manufactured in various forms, which include (i) naked recombinant DNA molecules, (ii) plasmids, (iii) viral vectors, and (iii) the Naked recombinant DNA molecules. Or in the form of liposomes or niosomes containing plasmids. ORM-encoding nucleotide sequences can be applied to all gene delivery systems used in conventional gene therapy, preferably plasmids, adenoviruses (Lockett LJ, et al., Clin. Cancer Res. 3: 2075-2080 (1997) ), Adeno-associated viruses (AAV, Lashford LS., Et al., Gene Therapy Technologies, Applications and Regulations Ed.A. Meager, 1999), retroviruses (Gunzburg WH, et al., Retroviral vectors) Gene Therapy Technologies, Applications and Regulations Ed.A. Meager, 1999), lentiviruses (Wang G. et al., J. Clin. Invest. 104 (11): R55-62 (1999)), herpes simplex virus (Chamber R., et al., Proc. Natl. Acad. Sci USA 92: 1411-1415 (1995)), basinian virus (Puhlmann M. et al., Human Gene Therapy 10: 649-657 (1999); Ridgeway, "Mammalian expression vectors," In: Vectors: A survey of molecular cloning vectors and their uses.Rodrigue and Denhardt, eds.Stoneham: Butterworth 467-492 (1988); Baichwal and Sugden, "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes," In: Kucherlapati R, ed. Gene transfer.New York: Plenum Press, 117- 148 (1986) and Coupar et al., Gene, 68: 1-10 (1988)), liposomes (Methods in Molecular Biology, Vol 199, SC Basu and M. Basu Eds. Human Press 2002; Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190 (1982) and Nicolau et al., Methods Enzymol., 149: 157-176 (1987)) or niosomes.

Meanwhile, the method of introducing the gene transfer system of the present invention into cells can be carried out through various methods known in the art.

In the present invention, when the gene delivery system is constructed based on a viral vector, it is carried out according to a virus infection method known in the art. Infection of host cells with viral vectors is described in the above cited documents.

In the present invention, when the gene delivery system is a naked recombinant DNA molecule or plasmid, microinjection (Capecchi, MR, Cell, 22: 479 (1980); and Harland and Weintraub, J. Cell Biol. 101: 1094 -1099 (1985)), calcium phosphate precipitation (Graham, FL et al., Virology, 52: 456 (1973); and Chen and Okayama, Mol. Cell. Biol. 7: 2745-2752 (1987)), electric Perforation (Neumann, E. et al., EMBO J., 1: 841 (1982); and Tur-Kaspa et al., Mol. Cell Biol., 6: 716-718 (1986)), liposome-mediated traits Infections (Wong, TK et al., Gene, 10:87 (1980); Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190 (1982); and Nicolau et al., Methods Enzymol., 149: 157-176 (1987)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci , 87: 9568-9572 (1990)) can be used to import genes into cells.

According to one embodiment of the present invention, the injection of the ORM gene carrier using the adenovirus of the present invention significantly reduced glucose and insulin resistance (see FIG. 14).

According to a preferred embodiment of the present invention, the metabolic disease is hyperlipidemia, fatty liver, diabetes or obesity.

As used herein, the term "hyperlipidemia" refers to a disease caused by a large amount of fat in the blood due to poor metabolism of triglycerides and cholesterol. More specifically, hyperlipidemia refers to high cholesterol hyperlipidemia, which occurs frequently with increased lipid components such as triglycerides, LDL cholesterol, phospholipids, and free fatty acids in the blood.

As used herein, the term “fatty liver” refers to a condition in which fat accumulates in hepatic cells due to adipose metabolism disorder of the liver, which causes various diseases such as angina, myocardial infarction, stroke, arteriosclerosis, fatty liver and pancreatitis. .

As used herein, the term “diabetes” means a chronic disease characterized by a relative or absolute lack of insulin resulting in glucose-intolerance. The term diabetes includes all kinds of diabetes and includes, for example, type 1 diabetes, type 2 diabetes and hereditary diabetes. Type 1 diabetes is insulin dependent diabetes mellitus, mainly caused by the destruction of β-cells. Type 2 diabetes is insulin-independent diabetes, caused by insufficient insulin secretion after meals or by insulin resistance.

According to a preferred embodiment of the present invention, the metabolic disease is caused by dysregulation of glucose or lipid homeostasis.

According to a preferred embodiment of the present invention, the composition for preventing or treating metabolic diseases comprising the ORM gene or protein of the present invention promotes the action of insulin.

The composition of the present invention induces enhanced energy metabolism of adipocytes, preferably reduced reactive oxygen species (ROS), increased glucose uptake activity, enhanced insulin signaling or adipocytes Decomposition leads to enhancement of energy metabolism. In addition, the composition of the present invention improves energy metabolism by lowering the glucose concentration in the body by increasing glucose uptake activity.

According to a preferred embodiment of the present invention, the composition of the present invention inhibits the inflammatory activity of adipocytes, macrophages or monocytes.

 According to a preferred embodiment of the present invention, the composition of the present invention reduces the infiltration of macrophages into adipocytes.

The composition of the present invention inhibits NF-κB or mitogen-activated protein kinase (MAPK) activity involved in the proinflammatory response of macrophages, and preferably IKK / NF-κB or MAPK (eg, JNK) by TNFα. , ERK or p38 MAPK), more preferably inhibits p38 MAPK in macrophages and inhibits phosphorylation / activation of IKK by TNFα, sequential degradation of IκB and nuclear accumulation of NF-κB.

In addition, the compositions of the present invention reduce the expression of inflammatory cytokine genes or oxidation promoting genes. According to one embodiment of the invention, the composition comprising the ORM of the present invention significantly improved insulin resistance without significant change in body weight in db / db mice, inflammatory cytokine genes (TNFα and IL-6 in adipose tissue) ) And expression of oxidation promoter genes (NOX2, p22Phox, p47Phox and p67Phox).

The composition of the present invention may be prepared as a pharmaceutical composition, a neutraceutical composition or a food composition.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the ORM gene or protein; And (b) a pharmaceutically acceptable carrier.

The term "pharmaceutically effective amount" as used herein means an amount sufficient to achieve the efficacy or activity of the ORM described above.

When the composition of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, mucosal administration, and eyedrop administration.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Preferably, the dosage of the pharmaceutical composition of the present invention is 0.001-100 mg / kg body weight per day, more preferably 0.01-80 mg / kg body weight, most preferably on an adult basis. 0.1-60 mg / kg body weight. In addition, depending on the judgment of the doctor or pharmacist may be divided administration once a day to several times.

The pharmaceutical and health food composition of the present invention may be formulated using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those skilled in the art. It may be prepared in a dosage form or incorporated into a multi-dose container.

According to a preferred embodiment of the invention, the formulations of the compositions of the invention are solutions, suspensions, syrups, emulsions, liposomes, extracts, powders, powders, granules, tablets, sustained-release preparations and capsules, dispersants or stabilizers It may further include.

Specifically, depending on the route of administration, solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is one or more inert pharmaceutically acceptable excipients or carriers (e.g. sodium citrate or dicalcium phosphate) and / or a) fillers or extenders (e.g. starch, lactose, sucrose, glucose) , Mannitol and silicic acid), b) binders (e.g. carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic), c) humectants (e.g. glycerol), d) disintegrants (e.g. Agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate), e) liquid retardants (e.g. paraffin), f) absorption accelerators (e.g. quaternary ammonium compounds), g) wetting agents (e.g. cetyl) Alcohols and glycerol monostearate), h) absorbents (e.g. kaolin and bentonite clay) and i) lubricants (e.g. talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and May be mixed with the mixture). In the case of capsules, tablets and pills the formulation may also comprise a buffer.

It can also be used as filler in soft and hard gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills, and granules may be prepared as coatings and shells, such as coatings, which are well known in the art of enteric and other pharmaceutical applications. They may optionally contain a clouding agent and may also be formulated to release only the active ingredient or preferentially release the active ingredient in a randomly delayed manner at certain sites of the intestine. The active compound may also be present in the form of microcapsules with one or more of the above-mentioned excipients if necessary.

Liquid formulations for oral administration include pharmaceutically acceptable emulsions, solvents, suspensions, syrups and elixirs. In addition to the active compounds, liquid formulations may be water or other solvents, solubilizers and emulsifiers (e.g. ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, Dimethyl formamide, oils (especially cottonseed oil, peanuts, corn oil, germ oil, olive oil, castor oil and sesame oil), fatty acid esters of glycerol, tetrahydrofuryl alcohol, polyethylene glycol and sorbitan and mixtures thereof Inert diluents commonly used in. In addition to inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifiers, suspending agents, sweetening agents, flavoring agents and fragrances.

Formulations for rectal or vaginal administration are preferably suitable non-irritating adjuvants or carriers (e.g. cocoa butter, polyethylene glycol or suppositories) which release the active compound by dissolving the compound of the invention in a solid or body temperature liquid at room temperature and thus in the rectum or vagina. And waxes that can be prepared by mixing.

Formulations suitable for parenteral injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, etc.), vegetable oils (olive oils), injectable organic esters (e.g. ethyl oleate) and these Suitable mixtures of are included.

In addition, the compositions of the present invention may contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Various antibacterial and antifungal agents (eg, parabens, chlorobutanol, phenol, sorbic acid, etc.) can inhibit the action of microorganisms. It may also be desirable to include osmotic agents such as sugars, sodium chloride and the like. Absorption delaying agents (eg, aluminum monostearate and gelatin) may be used to delay absorption of the injectable drug.

Suspending agents, in addition to the active compounds, suspending agents (e.g., ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth or mixtures thereof And the like).

In some cases, to sustain the effect of the drug, it may be desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material with low water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, will depend upon crystal size and crystalline form. On the other hand, delayed absorption of the parenterally administered drug form is achieved by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are prepared by forming the microcapsule matrix of the drug into a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of release of the drug can be controlled.

Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with the body tissue.

Injectable formulations can be sterilized, for example, by filtration through bacterial-retaining filters or by incorporation of the sterilizing agent in the form of a sterile solid composition which can be dissolved or dispersed in sterile water or other sterile injectable media immediately prior to use. .

According to another aspect of the present invention, the present invention provides a method for screening a substance for preventing or treating metabolic diseases, comprising the following steps:

(a) administering a test substance to be analyzed to the subject;

(b) measuring the expression level of ORM in adipose tissue and liver tissue of the subject;

(c) confirming whether the expression of the ORM is selectively induced in adipose tissue; And

(d) screening the test substance that caused the selective induction.

The present inventors endeavored to develop a method for effectively screening for the prevention and treatment of metabolic diseases. As a result, the present inventors have found that ORM derived from adipocytes inhibits inflammatory activity of adipocytes and monocytes / macrophages, reduces infiltration of macrophages into adipocytes, promotes insulin action, and ultimately insulin resistance. The findings suggest that ORM may be a molecular target for the prevention and treatment of metabolic diseases. The screening method of the present invention is a novel approach that can easily and efficiently screen effective ingredients effective for the prevention or treatment of metabolic diseases by measuring whether a test substance regulates the expression level of ORM in biological adipose tissue.

The method of the present invention will be described in detail in accordance with the respective steps.

Step (a): administering a test substance to be analyzed to an object

According to the method of the invention, the test substance to be analyzed is first administered to the subject. Preferably the object is a mammal, more preferably a human.

As used to refer to the screening method of the present invention, the term “test material” refers to an unknown material used in screening to examine whether it affects the expression level of ORM gene or the amount of ORM protein. The test substance includes, but is not limited to, chemicals, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts.

Test substances can be obtained from libraries of synthetic or natural compounds. Methods of obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA). ) And MycoSearch (USA).

Test materials can be obtained by a variety of combinatorial library methods known in the art, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution By the required synthetic library method, “1-bead 1-compound” library method, and synthetic library method using affinity chromatography screening. Methods of synthesizing molecular libraries are described in DeWitt et al., Proc . Natl. Acad . Sci . USA 90, 6909, 1993; Erb et al. Proc . Natl . Acad . Sci . USA 91, 11422, 1994; Zuckermann et al., J. Med . Chem . 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew . Chem . Int . Ed . Engl . 33, 2059, 1994; Carell et al., Angew . Chem . Int . Ed. Engl . 33, 2061; Gallop et al., J. Med . Chem . 37, 1233, 1994 and the like.

The object to be used in the method of the present invention is not limited as long as it includes the ORM gene in liver tissue and adipose tissue, and includes, for example, an ORM gene introduced from outside, as well as a tissue originally containing the ORM gene. If it can express even if it is included in the scope of the present invention. The method of introducing the ORM gene into the tissue can be introduced using a variety of methods known in the art. For example, it may be introduced into the tissue by the gene transfer method described above or microinjection method (Capecchi, MR, Cell , 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et al., Virology , 52: 456 (1973)), electroporation (Neumann, E. et al., EMBO J. , 1: 841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene , 10:87 (1980) )), DEAE-dextran treatment (Gopal, Mol . Cell Biol . , 5: 1188-1190 (1985)), and gene bombardment (Yang et al., Proc . Natl . Acad . Sci . , 87: 9568-9572 (1990)) and the like to directly inject ORM genes into cells. It may be.

Step (b): in adipose and liver tissue Measuring the expression level of a gene ORM

Subsequently, after administration of the test substance to the subject, the amount of ORM or ORM protein expressed in adipose tissue and liver tissue is measured up-regulated. As used herein, the term "up-regulation" refers to a condition in which the expression level of ORM or the amount of ORM protein is increased compared to before administration of a test substance. For example, the term "up-regulation" refers to a condition where the amount of expression of ORM as measured by RT-PCR, or the amount of ORM protein as determined by ELISA method is increased compared to before administration of the test substance.

As a result of the measurement, if the amount of expression of the ORM gene or the amount of the ORM protein is up-regulated in adipose tissue as compared to that in liver tissue, the test substance may be determined as a substance for preventing or treating metabolic diseases.

Measurement of the change in the expression level of the ORM gene can be carried out through various methods known in the art. For example, RT-PCR (Sambrook et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)), Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 102-108, CRC press ), hybridization (Sambrook using a cDNA microarray such as, Molecular Cloning. A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)) or in situ (in situ ) Hybridization reaction (Sambrook et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)).

Screening of the present invention can be achieved by examining the expression or protein amount of ORM isoforms, preferably by screening the expression or protein amount of ORM1, ORM2 or ORM3 isoforms, most preferably ORM1 isoforms (See FIGS. 1 and 10).

Step (c): Screening for Test Substances that Induced Up-regulation in Step (b)

Finally, the test substance is selected to cause the up-regulation of the expression level of ORM or the amount of ORM protein in adipose tissue. These substances can be very usefully applied as a prophylactic and therapeutic agent for metabolic diseases by ultimately up-regulating the expression or protein amount of ORM that regulates metabolic disease progression.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides a composition for the prevention or treatment of metabolic diseases comprising an ORM gene or protein.

(Ii) The present invention also provides a method for screening a substance for preventing or treating metabolic diseases.

(Iii) According to the present invention, the composition of the present invention comprising ORM, which is a natural substance present in abundance in vivo, has a selective regulating activity for various targets of metabolic diseases in vivo, thereby preventing all symptoms of the metabolic diseases. It can be effectively used to prevent or treat.

1 is a diagram showing the induction of ORM in the plasma and adipose tissue of obese subjects. Panel A is the northern blotting result of ORM expression in various mouse tissues. Panel B shows mRNA expression levels of ORM isoforms in adipocytes and SVCs extracted from epididymal adipose tissue of lean C57BL6 mice. Adiponectin (AdipoQ) was used as the adipocyte-specific marker gene. SEM (Standard errors of the mean) are indicated by error bars (n = 4). Panel C shows Northern blotting results of ORM expression in liver and adipose tissue of obese ob / ob and db / db mice. Panel D is the result of Western blotting of ORM expression in liver and adipose tissue. Since ORM is a large glycosylated protein (about 45% of its total mass constitutes carbohydrates), there were both glycosylated and non-glycosylated forms when western blotting using tissue samples. Panel E shows error bars of ORM protein levels in the plasma of lean and obese mice (n = 4 or 5). Panel F is the result of Western blotting of ORM expression in the plasma of obese and / or diabetic human patients.
Figure 2 is a diagram showing the result of ORM is selectively induced in adipose tissue during high fat diet (HFD). Panels A and B are Q-PCR analysis of ORM expression in epididymal adipose tissue (A) and liver (B) in C57BL6 mice fed with a normal chow diet (NCD) or HFD for 1, 3 or 7 days. The result is. ^* P <0.05; ^** P <0.01. SEM indicated error bars (n = 4 or 10). Panel C shows Western blotting results of ORM protein expression in epididymal adipose tissue of mice fed with NCD (control) and HFD for 3 days. Panel D shows Western blotting results of ORM protein expression in serum and epididymal adipose tissue of mice fed with NCD (control) and HFD for 3 months.
3 is a diagram showing the results of ORM induced by pro-inflammatory and metabolic signals and reduced by the diabetic rosiglitazone. Panel A shows that TNFα induces three ORM isoforms in 3T3-L1 adipocytes. Differentiated adipocytes were treated with TNFα (10 ng / ml) for 15 hours. MRNA levels of isoforms, TNFα and Adiponectin (AdipoQ) of each ORM were analyzed using Q-PCR. MRNA levels of TNFα and AdipoQ were used as controls for the effects of TNFα. Panel B shows that metabolic signals induce three ORM isoforms. Differentiated adipocytes were preincubated for 24 hours in low concentration (Glc; 5.5 mM) glucose medium and then treated with different concentrations of insulin (10 nM or 100 nM) in the presence or absence of high concentration (25 mM) glucose. It was. Panel C shows that secretion of ORM is promoted by metabolic and inflammatory signals in adipocytes. Differentiated adipocytes were precultured in low concentration (5.5 mM) glucose medium containing DMEN with 0.2% BSA added. After 24 hours, the culture medium was treated with insulin (100 nM), TNFα (10 ng / ml) or high glucose (25 mM) as fresh medium, which further passed for 24 hours. Western blotting analysis confirmed that the secretion of ORM was elevated. Panel D shows the effect of palmitate (Pal; 500 μM) on ORM mRNA expression in 3T3-L1 adipocytes. Panel E shows that the mRNA level of ORM isoforms is reduced by rosiglitazone (10 μM) treatment. TNFα and adiponectin mRNA expression were measured as positive and negative controls for the effect of rosiglitazone. ^* P <0.05; ^** P <0.01. SEM indicated error bars (n = 2).
4 is a diagram showing the result of inducing ORM in the liver during acute LPS treatment but not in adipose tissue. LPS was intraperitoneally injected into C57BL6 mice to analyze ORM expression in plasma, liver and adipose tissue. Panel A shows elevated plasma levels of ORM upon LPS injection (5 μg) in rin mice. Panel B shows Western blotting analysis for plasma ORM expression in mice 4 hours or 24 hours after LPS injection. Panels C and D show Qantral real-time RT- for three variants of ORM in epididymal adipose tissue (C) and liver (D) at 4 and 24 hours post-infusion in mice administered LPS. PCR) analysis.
5 shows that ORM inhibits the expression of inflammatory genes in adipocytes, monocytes and macrophages. Panel A shows ORM treatment effect and ORM1 overexpression effect by adenovirus in TNFα-induced inflammatory gene expression in 3T3-L1 adipocytes. Adenovirus-infected (50 moi, 2 days post injection) or uninfected adipocytes were incubated with TNFα (10 ng / ml) in the presence or absence of ORM (250 μg / ml). MRNA levels of inflammatory genes were analyzed using Q-PCR. ^* P <0.05; ^** P <0.01. SEM indicated error bars (n = 2 or 3). Panel B shows the time course results of ORM in the expression of TNFα-induced inflammatory genes in adipocytes. 3T3-L1 adipocytes were incubated with TNFα (10 ng / ml) in the presence or absence (250 μg / ml) or ORM for a designated period of time. MRNA levels of inflammatory genes were analyzed using Q-PCR. Panel C shows that knockdown of ORM1 induces inflammatory gene expression in adipocytes. 3T3-L1 stable cell lines overexpressing mock or mouse ORM1-specific siRNA were constructed by retroviral infection and selection with puromycin. At day 7 of adipocyte differentiation, the cells were treated with TNFα (10 ng / ml) and / or ORM (250 μg / ml) for 24 hours. MRNA levels of inflammatory genes were determined by Q-PCR analysis. Panel D shows that ORM in THP-1 monocytes inhibits the expression of certain genes involved in inflammation and cell adhesion. THP-1 monocytes were incubated for 6 hours with TNFα (10 ng / ml) in the presence (100 or 1000 μg / ml) or without ORM. MRNA levels of inflammatory genes were determined by Q-PCR analysis. Panel E shows that ORM (250 μg / ml) inhibits TNFα-induced inflammatory gene expression in peritoneal macrophages in mice. Panel F shows that ORM (250 μg / ml) inhibits palmitate-induced inflammatory gene expression in peritoneal macrophages in mice.
Figure 6 shows that ORM inhibits the interaction between adipocytes and macrophages. Panel A shows that ORM inhibits the effect of adipocyte-conditioning medium on adhesion of THP-1 monocytes to the bottom of the culture dish. Adipocyte-conditioned media was obtained from mock or ORM1 siRNA overexpressing 3T3-L1 adipocytes with or without ORM (250 μg / ml) or TNFα (10 ng / ml) for 24 hours. The cells were cultured in adipocyte-conditioned medium, washed with PBS and photographed for adhesion of THP-1 monocytes to the bottom of the culture dish. Panel B shows that ORM inhibits THP-1 monocytes infiltration into adipocytes. After 24 hours without or pretreatment of ORM (250 μg / ml) or sodium salicylate (5 mM) to 3T3-L1 adipocytes and THP-1 monocytes, THP-1 monocytes were subjected to transwell plates containing adipocytes. It was added to the gastric chamber and stimulated with TNFα. Infiltration of THP-1 cells into adipocytes was measured using immunostained monocytes using anti-CD68 antibody (red). Panel C shows that ORM binds to macrophages of adipose tissue. Epididymal adipose tissue extracted from lean mice or DIO (feed HFD for 3 months) is immunostained with FITC-binding BODIPY (green; adipocytes) or antibodies against ORM (red) or F4 / 80 (blue; macrophages) Whole tissues were sampled for multiphoton fluorescence confocal microscopy.
7 shows that ORM enhances energy metabolism in adipocytes. Panel A shows that 3T3-L1 adipocytes were cultured in low (5.5 mM) or high (25 mM) glucose medium in the presence (250 μg / ml) or absence of ORM for 24 hours. ROS levels accumulated in adipocytes were measured using DCFDA fluorescence staining. Panel B shows basal or insulin-dependent glucose uptake activity assays. 3T3-L1 adipocytes were incubated in low (5.5 mM) or high (25 mM) glucose medium with or without (250 μg / ml) ORM for 24 hours. Glucose uptake activity was measured 30 minutes after insulin treatment. Panel C shows that ORM enhances insulin signal. After 3T3-L1 adipocytes were left serum-free for 16 hours, ORM (250 μg / ml) was treated for 6 hours. After 15 minutes of insulin stimulation (10 nM), the cells were analyzed for Western blotting. Panels D to E were pretreated with 3T3-L1 adipocytes with ORM (250 μg / ml) or sodium salicylate (5 mM) in DMEM supplemented with 0.2% BSA, followed by TNFα (10 ng / ml) for 48 hours. Further stimulated. Panel D measures the mRNA levels of ferritin by Q-PCR analysis. Panel E is determined by measuring glycerol concentration in adipocyte-conditioned medium. ^* P <0.05; ^** P <0.01. SEM indicated error bars (n = 3).
8 shows that ORM inhibits MAPK and NF-κB activity in macrophages and adipocytes. Panel A shows pretreatment of RAW264.7 macrophages or 3T3-L1 adipocytes with vehicle, ORM (250 μg / ml) or sodium salicylate (5 mM) for 10 minutes and then TNFα (10 ng / ml). Stimulated). The level of phosphorylation or total MAPK protein was measured using Western blotting analysis. The result is representative of at least three independent experiments. Panels B to C show that ORM inhibits IKK activity in macrophages (B) and adipocytes (C). Panel D shows inhibition of intranuclear accumulation of NF-κB (p65) in macrophages. RAW264.7 macrophages were pretreated with excipient [ORM (250 μg / ml) for 6 hours and then stimulated for 15, 30 or 60 minutes with TNFα (10 ng / ml). Nuclei extracts were taken from the cells and analyzed for Western blotting for protein levels of p65 NF-κB.
9 is a diagram showing the in vivo effect of ORM in obese and diabetic db / db mice. PBS (◇)-or ORM protein (■) -containing osmotic pumps were implanted in db / db mice. After 2 weeks, the mice were analyzed. Panel A shows the insulin resistance test. Panel B measured mRNA levels of inflammatory genes in epididymal adipose tissue of db / db mice under ORM treatment or by no Q-PCR analysis. ^* P <0.05; ^** P <0.01; ^*** P <0.001. SEM indicated error bars (n = 6 or 8). Panel C shows Q-PCR analysis of inflammatory and metabolic genes in the liver of these mice. ^* P <0.05; ^** P <0.01. SEM indicated error bars (n = 6 or 8).
FIG. 10 shows that expression of three ORM isoforms is increased while 3T3-L1 cells differentiate into adipocytes. SEM indicated error bars (n = 2).
11 is a diagram showing the increase in ORM expression in adipocyte fragments extracted from adipose tissue of db / db mice. Total RNA was isolated from liver, adipocytes, SVCs and peritoneal macrophages and analyzed using quantitative RT-PCR for expression of ORM1, ORM2, ORM3, TNFα and Adiponectin (AdipoQ).
FIG. 12 shows detection of ORM proteins around adipocytes and nonvascular stromal cells of adipose tissue. Epididymal adipose tissue extracted from lean mice (control) and DIO mice (diet-induced obese mouse, fed HFD for 3 months) was subjected to FITC-binding BODIPY (green; adipocytes) and antibodies to ORM (red) and PECAM (left panel). Blue; blood vessels) were immunostained and whole tissue sampled for multiphoton fluorescence multifocal microscopic analysis.
FIG. 13 shows that ORM inhibits the development of ROS in THP-1 monocytes and RAW264.7 macrophages. Panel A shows that THP-1 cells were ORM treated for 24 hours and ROS levels were calculated using flow cytometry after DCFDA staining. Panel B shows ORM treatment of RAW264.7 macrophages for 6 hours in the presence or absence of LPS. ROS levels were measured after DCF-DA staining.
Figure 14 shows that overexpression of ORM1 with adenovirus improves insulin sensitivity in obese and diabetic db / db mice. Adenoviruses expressing GFP or mouse ORM1 were iv administered to db / db mice. Two weeks after the injection, the mice were analyzed. Panel A shows that mRNA levels of exogenous ORM were measured using Q-PCR analysis. Panel B shows that protein levels of ORM in plasma were measured using Western blotting analysis. Panel C shows the results of the oral glucose tolerance test. Panel D shows the results of the insulin resistance test.
Fig. 15 shows CCR5 and Siglec-E (mouse homolog of human Siglec-5) expressed in adipocytes and macrophages.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

1. Cell culture

3T3-L1 adipocytes were incubated in Dulbecco's modified Eagle's medium (DMEM) with 10% bovine calf serum until confluence. After two days of confluence, the cells were incubated for 48 hours in DMEM containing 10% FBS, MIX (methylisobutylxanthine, 500 μM), DEX (dexamethasone, 1 μM) and insulin (5 μg / ml). Medium was supplemented every two days using DMEM containing 10% FBS and insulin (1 μg / ml). THP-1 human monocytes and RAW264.7 macrophages were preserved in RPMI1640 with 10% FBS.

2. Q-PCR

cDNA was synthesized using dNTPs and oligo (dT) primers (Invitrogen, USA) and the reverse transcriptase of Moroni leukemia virus. dNTPs and Taq The cDNA was provided as a template with specific primers in the presence of an annealing temperature in the range of 54 ° C. to 60 ° C. in the presence of DNA polymerase. A final dose of 20 μl was obtained via real time RT-PCR amplification and included 20 ng of reverse-transcribed total RNA, 0.25 μM of forward and reverse primers and Cybergreen (Cybergreen, Bio-Rad, USA). PCR amplification was performed using a 96-well plate and a MyiQ real-time PCR detection system (Bio-Rad, USA). All reactions were repeated at least three times in three sets. The relative amount of each mRNA was calculated using comparative CT (threshold cycle) method. GAPDH mRNA or 18S rRNA was used as a control. The primer sequences used for PCR are as follows (18s rRNA--5 end AAC GCA GCT AGG AAT AAT GGA // end 3 GCC TCA GTT CCG AAA ACC A)

3. Human Serum Samples

Human serum samples were obtained from Samsung Medical Center and analyzed for ORM protein expression. Acquisition procedures for human serum samples were obtained with approval from Samsung Medical Center Institutional Review Board (IRB file no. 2006-03-053) and with written consent from volunteers. The volunteers were assigned to the obese and diabetic groups with a body mass index (BMI) of 30 or higher and fasting blood glucose level of 126 mg / dl or higher, respectively.

4. Experimental Animals and Treatment

Male C57BL / 6J, ob / ob , and db / db Mice were bred using cages at 12 h / 12 h dark cycles. All mice were sacrificed simultaneously at the end of the cancer cycle. Db / db mice were fasted for 16 and 6 hours respectively for glucose tolerance and insulin resistance testing, and after injection of glucose (1.5 g / kg) and insulin (2 U / kg, Novolin R, Novo Nordisk, Denmark) ip, basal Blood samples were obtained. Blood samples were taken at 15, 30, 60, 90 and 120 minutes or 15, 30, 45, 60, 90 and 120 minutes after the injection.

5. ORM processing on db / db mouse

Male db / db Mice were treated with purified ORM protein (Sigma, USA) by continuous systemic infusion using a micro-osmotic pump. Each mouse was inserted subcutaneously subcutaneously with an Alzet micro-osmotic pump (model 1004, Alza, USA). The pump released the purified ORM protein at 0.34 μg / min. The mice were subjected to insulin resistance and glucose tolerance tests and then sacrificed to prepare tissue samples.

6. Determination of Glycerol Concentration

Glycerol concentration released into 3T3-L1 adipocyte culture medium was used as an indicator of lipolysis and measured using a colorimetric assay kit (Sigma, USA).

7. Glucose Intake Analysis

Glucose uptake analysis was performed according to the previously described method (Lee et al., 2008).

8. Measurement of infiltration of monocytes into adipocytes (immunohistochemistry)

3T3-L1 cells differentiated into adipocytes on glass coverslips. Differentiated adipocytes (day 7) and THP-1 monocytes were pretreated for 6 hours with ORM (250 μg / ml) or sodium salicylate (500 μM), followed by the presence or absence of TNFα (10 ng / ml). Co-culture was further performed for 24 hours in a transwell plate (bottom plate: adipocytes, top plate: THP-1; pore size: 8 μm). After discarding the upper plate, the cells of the coverslip were washed three times with PBS, fixed with 3.7% paraformaldehyde, improved permeability with PBS (PBST, PBS with Triton X-100) containing 0.5% Triton X-100, and blocked. The reaction was performed for 1 hour using 0.1% PBST containing 3% BSA, followed by reaction with CD68 monoclonal antibody (DakoCytomation, USA) for 1 hour at room temperature, followed by washing with 0.1% PBST. The cells were then allowed to react at room temperature with tetramethylrhodamine isothiocyanate conjugated secondary antibodies. The coverslips were washed and then placed on a slide glass containing a mounting solution containing 4 ', 6-diamino-2-phenylindole. Then, after visualizing using a fluorescence microscope (Olympus, Japan), the quantity of the CD68-positive cells was measured using an image analysis program (LSM510 version 3.5; Carl Zeiss, Germany).

9. Whole-tissue immunohistochemistry

Anesthesia was mixed (rumoon 1: ketamine 2), anesthetized by intramuscular injection into the mouse, and fixed with PBS vascular perfusion containing 1% paraformaldehyde, followed by whole-mounted adipose tissue of the mouse (whole-mounted). It was. The specimen was then reacted for 1 hour at room temperature with a blocking solution containing 5% goat serum (Jackson ImmunoResearch Laboratories Inc., USA) in 3% PBST. After blocking the specimens were reacted overnight at 4 ° C. using antibodies against F4 / 80 (clone Cl: A3-1, 1: 1,000 dilution; Serotec, UK) and AGP (1: 500 dilution; DakoCytomation, USA). . After washing several times with PBST, the specimens were reacted with secondary antibodies, ie, Cy3- or Cy5-binding anti-retret antibodies or anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, USA) for 1 hour at room temperature. The adipose tissue was stained using FITC-bound BODIPY. For control experiments, primary antibodies were removed or replaced with full-surface serum. Signals were visualized using a Zeiss ApoTome microscope and Zeiss LSM 510 confocal microscope (Carl Zeiss, Germany) equipped with an argon and helium-neon laser to obtain digital images.

Experiment result

1. ORM is selectively induced in adipose tissue of obese and / or diabetic mice.

To identify new adipocytokines, we used surface-enhanced laser desorption ionization time-of-flight (SELDTOF) mass spectrometry coupled to RNA microarrays in adipose tissue and plasma of normal and obese / diabetic mice. In addition, we analyzed gene expression characteristics in 3T3-L1 adipocytes and adipocytes. Among the new adipocytokine candidates, they expressed much higher ORMs in adipocytes than adipose tissue stromal vascular cells (SVCs), and also found increased ORMs in adipose tissue and plasma in obese and / or diabetic mice. Could. To investigate the tissue distribution of ORM mRNA in mice, we performed Northern blotting using several mouse tissues. In the prior literature it has been reported that ORM is produced mainly in the liver and expressed in abundant amounts in adipose tissue (Fournier et al., 2000; Hochepied et al., 2003; Lin et al., 2001). As reported above, it was confirmed that ORM was expressed at high levels in liver, white adipose tissue (WAT) and brown adipose tissue (BAT) (FIG. 1A). In mice, the ORM has three variants (ORM1, ORM2 and ORM3). In order to investigate which ORM isoforms are mainly expressed in adipocytes, we performed real-time quantitative RT-PCR (Q-PCR) on adipocytes and SVCs extracted from epididymal adipose tissue. As shown in FIG. 1B, all three ORM variants were abundantly expressed in adipocytes than SVCs, and ORM1 was predominantly expressed in adipocytes compared to other ORM variants (FIG. 1B). Consistently, expression of ORM1 mRNA was induced while 3T3-L1 cells differentiated into adipocytes (FIG. 10).

We then tested whether ORM expression is elevated in adipose tissue of obese mice using Northern and Western blotting assays. ob / ob in obese and / or diabetic mice at the mRNA and protein levels And a significant increase in ORM expression in adipose tissue of db / db mice was observed (FIGS. 1C, 1D and 11). In addition, in obese mice, it was observed that induction of ORM occurs selectively in adipose tissue, not in the liver, and coincides with an increase in plasma ORM levels (FIGS. 1C-1E and 11). Similar to the above results obtained in mice, it was also found that plasma ORM levels were elevated in obese and diabetic human patients (FIG. 1F). When ORM expression was measured in a diet-induced obese (DIO) mouse model, ORM mRNA expression was rapidly increased in epididymal adipose tissue of the high-fat diet (HFD) group (FIGS. 2A-2C). ORM induction was significant at 3 months of high fat diet (FIG. 2D). On the other hand, ORM levels in the liver of obese mice did not change significantly (FIGS. 1 and 2), and the results indicate that fat ORM is more in metabolic stress and / or change in obese and / or diabetic subjects than liver ORM. It suggests that you will react sensitively.

2. ORM Is induced by both inflammatory and metabolic signals in adipocytes.

Non-ideal regulation of adipocyte-specific gene expression in obese subjects is associated with proinflammatory signals and elevated metabolic stress in adipose tissue. In order to determine whether ORM induction is associated with these signals in adipose tissue of obese mice, we treated 3T3-L1 adipocytes with TNFα, insulin, palmitate (FFA) or high glucose. As shown in FIGS. 3A-3D, ORM isoforms were induced with TNFα, insulin, palmitate and high glucose treatment. Thus, the stimuli markedly increased secretion of ORM protein from adipocytes (FIG. 3C), suggesting that adipose ORM levels tend to rise when they receive both proinflammatory and metabolic signals. In contrast, the famous diabetic rosiglitazone, which acts as a ligand for PPARγ and possesses anti-inflammatory activity (Pascual et al., 2005), significantly reduces the expression of ORM mRNA (FIG. 3E). As such, these results indicate that adipose ORM expression is sensitively upregulated by increased pro-inflammatory and metabolic signals in adipose tissue of obese and / or diabetic subjects.

To investigate whether adipose ORM responds to acute systemic inflammation, lean B6 mice were administered LPS (lipopolysaccharide) at 4 and 24 hours to monitor ORM mRNA expression in both liver and adipose tissue. It was. As shown in Figures 4A and 4B, plasma ORM levels increased at 6 hours by LPS administration and increased up to 48 hours. However, with a significant increase in plasma ORM expression after LPS administration, there was a strong induction of liver ORM at the 4 hour time point where the expression level of fat ORM did not change (FIGS. 4C and 4D). In view of the selective and rapid induction of fat ORM in HFD, the above results suggest that the fat ORM is more sensitive to metabolic stress than in acute systemic inflammation caused by LPS.

3. ORM Inhibits the inflammatory activity of adipocytes and monocytes / macrophages.

In obese subjects, adipose tissue inflammation not only induces inflammatory gene expression of adipocytes, but also contributes to increased infiltration, adhesion and activity of monocytes / macrophages, resulting in increased local inflammation and ultimately breaking adipocyte homeostasis. To investigate the role of ORMs in adipose tissue, we tested the effects of ORMs in both adipocytes and monocytes / macrophages in the presence and absence of inflammatory stimuli using TNFα to induce the expression of ORMs. TNFα-induced expression of proinflammatory genes such as IL-6, iNOS and MCP-1 in 3T3-L1 adipocytes and mouse primary adipocytes when ORM levels are elevated by treatment of ORM proteins or infection with ORM1 adenovirus Was reduced (FIG. 5A). In addition, mRNA levels of NADPH oxidase subunits, such as NOX2, p47phox and p67phox, associated with increased ROS production from adipose tissue of obese subjects were significantly reduced by ORM treatment. In adipocytes, time course analysis showed a delay in the induction of inflammatory gene expression by TNFα and a decrease in mRNA expression level (FIG. 5).

On the other hand, knocking down ORM1 promoted both basal and TNFα-induced expression of proinflammatory genes in adipocytes and was inhibited upon ORM protein treatment (FIG. 5C). Similarly, increased ORM levels in THP-1 monocytes and mouse intraperitoneal macrophages downregulated pro-inflammatory gene expression in the presence of TNFα (FIGS. 5D and 5E). In addition, ORM inhibited basal and TNFα dependent expression of adhesion molecules such as CD11a, CD11b, CD11c and CD49d in THP-1 monocytes, which would be required when monocytes adhere and infiltrate into adipose tissue (FIG. 5D). In addition, ORM reduces FFA (palmitate) -induced activation of inflammatory gene expression in primary macrophages (FIG. 5F). This indicates that ORM has the ability to inhibit the inflammatory response by breaking the link between interactions between adipocytes and macrophages. Taken together, these results indicate that pro-inflammatory and metabolic stress-induced ORM expression can inhibit excessive inflammatory responses in both adipocytes and monocytes / macrophages to inhibit further progression of inflammatory responses in adipose tissue. .

4. ORM Reduces the infiltration of macrophages into adipocytes.

In obese subjects, elevated proinflammatory cytokines and chemokines of adipocytes play a crucial role in inducing adipose tissue infiltration of macrophages, which requires the acquisition of monocyte / macrophage adhesion (Kanda et al., 2006). ). In order to investigate whether ORM can affect the physical interactions between adipocytes and monocytes / macrophages, the inventors of the present invention were directed to the preparation of adipocyte-conditioned media expressing non-ORM1 (mock) and ORM1 siRNAs for monocyte adhesion. The impact was tested. Compared to the control, THP-1 monocytes were cultured using the adipocyte-conditioning medium expressing ORM1 siRNA to improve cell adhesion to the cell culture dish bottom (FIGS. 6A, VIII and VIII). However, pretreatment of ORM with adipocytes reduced the adhesion of monocytes to the bottom of the culture dish under the same conditions (FIGS. 6A, VII and VII). In addition, monocyte adhesion was induced in TNFα-treated adipocyte-condition medium (FIGS. 6A, VIII and VIII). Then, to investigate whether ORM influences monocyte infiltration into adipocytes, we incubated 3T3-L1 (lower chamber) and THP-1 (upper chamber) in transwell plates and TNFα, salicylic acid or ORM Was examined to see if THP-1 infiltrated and adhered through the polycarbonate membrane to adipocytes in the presence or absence of. As previously reported (Cai et al., 2005; Suganami et al., 2005), we have increased the extent of TNFα infiltration and adhesion of monocytes to adipocytes (FIGS. 6B, VII and ii), It was confirmed that it was inhibited by salicylic acid treatment (FIGS. 6B, ii and iii). Like salicylic acid, ORM inhibited TNFα-induced increase in monocyte infiltration into adipocytes (FIGS. 6B, II and VIII), indicating that ORM inhibits physical interactions between adipocytes and macrophages in inflammatory signal contact. Indicates.

Since most circulating signal molecules bind to the surface of target cells and transmit signals in a stepwise manner, the present inventors have found that if ORM is actively involved in regulating macrophage function in adipose tissue, ORM binds to the macrophage surface of adipose tissue. I hypothesized that I would do it. To investigate whether ORM interacts with adipose macrophages, we performed whole-mount immunohistochemistry using epididymal WAT in lean and DIO mice. The highest ORM expression was detected around fat droplets (BODIPY positive, green) (red), indicating that ORM is expressed in monolocular adipocytes (FIG. 6C). In addition, ORM signals were higher in adipose tissue of DIO mice than in lean mice, which indicates that the degree of ORM expression is greater in the adipose tissue of obese subjects. Interestingly, high ORM levels were frequently observed around non-vascular stromal cells, including F4 / 80 positive macrophages, forming crown-like structures, especially observed around dead adipocytes (FIGS. 6C and 12). The above data suggest that ORM can inhibit inflammation in adipose tissue by inhibiting the interaction between adipocytes and macrophages.

5. ORM Promotes the action of insulin in adipocytes.

Elevated inflammatory response to chronic metabolic stress in adipose tissue causes, at least in part, reactive oxygen species to disrupt steady-state lipid and glucose metabolism (Furukawa et al., 2004; Houstis et al., 2006; Lin et al. , 2005). In order to evaluate whether the anti-inflammatory activity of ORM affects glucose metabolism, we investigated the effect of ORM on high glucose-induced ROS development and insulin resistance in 3T3-L1 adipocytes. Consistent with previously reported (Lin et al., 2005), hyperglycemia increased ROS incidence (FIG. 7A) and inhibited insulin-stimulated glucose uptake activity by 45% (FIG. 7A). 7B, lane 3 and lane 4). In contrast, ORM treatment significantly reduced hyperglycemic-induced ROS incidence (FIG. 7A) and improved insulin resistance (FIG. 7B, lane 7 and lane 8). In addition, ORM inhibited LPS-induced ROS development from THP-1 monocytes and RAW264.7 macrophages as in adipocytes (FIG. 13). Consistent with the above results, ORM delayed the phosphorylation of serine at IRS-1 while insulin-stimulated tyrosine phosphorylation was accelerated (FIG. 7C), indicating that ORM stimulates insulin signaling in adipocytes.

TNFα inhibits perilipin expression, increases hormone sensitive lipase activity, induces lipolysis and facilitates release of FFA (Souza et al., 1998; Souza et al., 2003; Zhang et al., 2002), reported by the anti-inflammatory agent salicylic acid (Zu et al., 2008). In order to test whether ORM delays TNFα-induced lipolysis in adipocytes, we examined the ferritin mRNA and lipolysis levels after ORM treatment. As reported above (Zu et al., 2008), administration of salicylic acid reversed the effect of TNFα on ferriripine expression and glycerol release (FIGS. 7D and 7E). Similarly, ORM inhibited TNFα-induced lipolysis while continuously expressing periripine (FIGS. 7D and 7E). The results indicate that ORM mitigates the dysregulation of glucose and lipid homeostasis mediated by the chronic accumulation of inflammatory and metabolic stress in adipose tissue.

6. ORM In adipocytes and macrophages NF -κB and MAPKs of TNF inhibits α-induced activity.

The proinflammatory response of macrophages is primarily mediated by IκB kinase (IKK) / NF-κB and / or MAPKs (eg, JNK, ERK and p38 MAPK) pathway activity. In adipocytes, the activity of the IKK / NF-κB or MAPKs pathway by TNFα is implicated in the regulation of lipid and glucose metabolism as well as proinflammatory responses (Hotamisligil, 2006; Hotamisligil et al., 1993; Souza et al. , 2003; Suganami et al., 2007; Zhang et al., 2002). Since ORM inhibits TNFα-induced inflammatory gene expression in adipocytes and macrophages (FIG. 5), we investigated whether ORM alters NFα-induced phosphorylation / activation of TMAPKs and IKK pathways in these cells. In both RAW264.7 macrophages and 3T3-L1 adipocytes, ORM strongly reduced the TNFα-dependent activity of JNK and ERK like salicylic acid. Unlike JNK and ERK, ORM effectively inhibited p38 MAPK in macrophages but not adipocytes. In addition, ORM administration inhibited phosphorylation / activation of IKK by TNFα, sequential degradation of IκB and nucleus accumulation of NF-κB (FIGS. 8B and 8D), suggesting that the anti-inflammatory activity of ORM was associated with IKK / NF-κB and MAPK activity. Related to inhibition.

7. ORM Has systemic insulin resistance Relax .

To determine whether ORM inhibits inflammatory gene expression in vivo and alleviates insulin resistance, we administered ORM to fat and diabetic db / db mice . In order to increase the level of ORM circulating at a constant rate without the periodic fluctuations caused by daily injections, we adopted an automatic osmotic pump system that sustains ORM levels (347 ng / min). Insulin resistance assays were then performed two weeks after transplantation to avoid artifacts from the activity of endogenous ORM after treatment and to stabilize the release of anaerobic ORM from the pump. As shown in Figure 9A, ORM significantly improved insulin resistance without significant change in body weight in db / db mice. In addition, consistent with in vitro results (FIG. 5), expression of inflammatory cytokine genes (TNFα and IL-6) and oxidant genes (NOX2, p22Phox, p47Phox and p67Phox) in adipose tissue upon ORM treatment in db / db mice And the above results coincided with a decrease in macrophage marker gene expression such as F4 / 80 and CD11c) (FIG. 9B). On the other hand, ORM increased the expression level of Glut4, an insulin-sensitive glucose transporter in adipose tissue (FIG. 9B). These results indicate that ORM can enhance insulin resistance by inhibiting macrophage activity and / or infiltration in in vivo adipose tissue. Interestingly, in the liver, elevated ORM inhibited the expression of glucose gluconeogenic genes (PEPCK and G6Pase) and promoted the expression of fatty acid oxidation genes (CPT-1 and ACO) in db / db mice ( Figure 9C). These results are believed to contribute to the restoration of glucose and lipid metabolism. Similar to the above results, adenovirus overexpression of ORM1 in db / db mice improved glucose and insulin resistance (FIG. 14). Taken together, the data indicate that increased plasma ORM can restore energy homeostasis by limiting inflammatory responses in insulin-sensitive tissues such as fat and liver.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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Claims (22)

Pharmaceutically effective amount of an ORM (Orosomucoid) gene or protein; And a pharmaceutically acceptable carrier. A pharmaceutical composition for preventing or treating hyperlipidemia, fatty liver, or obesity.
The composition of claim 1, wherein the ORM comprises at least one ORM isoform selected from the group consisting of ORM1, ORM2 and ORM3.
The composition of claim 1, wherein the metabolic disease is caused by dysregulation of glucose or lipid homeostasis.
delete The composition of claim 1, wherein the composition induces enhanced energy metabolism of adipocytes.
6. The composition of claim 5, wherein the energy metabolism improvement is a reduction in reactive oxygen species (ROS), an increase in glucose uptake activity, an enhancement of insulin signaling, or degradation of adipocytes. .
The composition of claim 1, wherein the composition inhibits the inflammatory activity of adipocytes, macrophages or monocytes.
The composition of claim 1, wherein the composition reduces the infiltration of macrophages into adipocytes.
The composition of claim 1, wherein the composition promotes the action of insulin.
The composition of claim 1, wherein the composition lowers the glucose concentration in the body.
The composition of claim 1, wherein the composition inhibits NF-κB or mitogen-activated protein kinase (MAPK) activity.
12. The composition of claim 11, wherein the MAPK is JNK, ERK or p38 MAPK.
The composition of claim 1, wherein the composition reduces the expression of an inflammatory cytokine gene or an oxidation promoting gene.
The composition of claim 13, wherein the inflammatory cytokine gene is TNFα or IL-6.
The composition of claim 13, wherein the oxidation promoting gene is NOX2, p22Phox, p47Phox or p67Phox.
The composition of claim 1, wherein the composition improves insulin resistance.
The composition of claim 1, wherein the ORM gene is included in a gene carrier.
18. The method of claim 17, wherein said gene carrier is a plasmid, recombinant adenovirus, Adeno-associated viruses (AVA), retrovirus, lentivirus, herpes simplex virus, basinia virus, liposomes or niosomes. Characterized by a composition.
19. The composition of claim 18, wherein said gene carrier is a recombinant adenovirus.
delete A method for screening a substance for preventing or treating metabolic diseases comprising the following steps:
(a) administering a test substance to be analyzed to a subject other than a human;
(b) measuring whether the expression level of ORM in the subject or the amount of protein is up-regulated in adipose tissue compared to liver tissue;
(c) selecting the test substance that caused the up-regulation.
22. The method of claim 21 wherein the ORM is ORM1.

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