KR20160107610A - Compositions for Preventing or Treating Metabolic Diseases - Google Patents
Compositions for Preventing or Treating Metabolic Diseases Download PDFInfo
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- KR20160107610A KR20160107610A KR1020150030573A KR20150030573A KR20160107610A KR 20160107610 A KR20160107610 A KR 20160107610A KR 1020150030573 A KR1020150030573 A KR 1020150030573A KR 20150030573 A KR20150030573 A KR 20150030573A KR 20160107610 A KR20160107610 A KR 20160107610A
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
- A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
Description
The present invention relates to a composition for preventing or treating metabolic diseases, particularly obesity.
Due to the rapid development of modern society and the abundance of nutritional status, enormous environmental changes have occurred in the metabolic organ of the human body, which is not different from primitive age. The prevalence of metabolic diseases in which two or more diseases such as obesity,
It is known as insulin resistance syndrome, which is caused by loss of metabolic waste and toxins caused by inadequate balance of metabolism and waste of human body due to loss of toxins. It develops into Metabolic Syndrome. Metabolic Syndrome gives damage to the coronary artery and provides the cause of heart disease or paralysis. It is not capable of removing salt from the kidneys, causing hypertension, providing the cause of cardiovascular disease It is known that it increases the triglyceride ratio, increases the risk of blood clotting, and also decreases insulin production by
Diabetes mellitus is a disease in which glucose is released into the urine due to its higher blood sugar concentration than normal people. It is caused by abnormal metabolic processes or abnormal physiological activity of insulin in β-cells of Langerhans islets in the pancreas. Diabetes mellitus is a chronic metabolic disease, and it causes diseases such as vascular disorders, nerves, kidneys and retinas as a result of a long time, and it causes the loss of life. Diabetes is divided into
Continuous insulin resistance leads to beta cell dysfunction leading to relative hypothyroidism. In particular, decreased insulin ratio to glucagon increases your life in the liver. In addition, an increase in free fatty acids in the blood is suggested as a cause of insulin resistance. The increase in free fatty acids in the blood suppresses insulin - induced glucose utilization in peripheral tissues and increases blood glucose levels by inhibiting glycation inhibition in liver tissue. In the non-insulin-dependent diabetes mellitus, not only the increase of free fatty acid in the blood but also the blood cholesterol and triglyceride increase phenomenon and the decrease of HDL-cholesterol are shown, and the onset of the dyslipidemia is 2 to 4 times higher than that of the normal person. It also causes obesity. Diabetes mellitus has been reported to be closely related to oxidative stress in diabetes and diabetic complications. Chronic hyperglycemia in diabetes increases the production of free radicals by various pathways such as autoxidation of glucose and protein saccharification, and oxidative stress is increased by these highly reactive substances. Moreover, antioxidant enzyme expression and activity are inadequate to prevent oxidative stress induced by hyperglycemia, resulting in an abnormal increase in antioxidant enzyme activity, thereby destroying the balance state maintained between these enzymes. In addition, diabetes mellitus causes high osmotic stress in the body's hyperglycemic environment, leading to complications such as cataract and kidney disease (Campbell, RK and Steil, CF 1988. Diabetes, clinical pharmacy and therapeutics. William & Wilkins. . Diabetes is characterized by hormonal imbalances such as insulin, glucagon and glucocorticoid, which are characteristic of symptoms such as hyperglycemia and diabetes mellitus due to abnormal regulation of physiological metabolism such as carbohydrate, protein, lipid and electrolyte metabolism , And chronic complications such as arteriosclerosis, vascular disorders, neurological disorders, osteopenia, and infectious diseases are frequently observed in diabetes mellitus.
As a composition for preventing and treating diabetes or insulin resistance syndrome, Korean Patent No. 0721508 discloses a composition for prevention and treatment of diabetes mellitus, diabetic complication, insulin resistance, and the like, which contains extract of Radix Clematidis (ß% ß% Korean Patent No. 1269208 discloses a composition for preventing or treating insulin resistance comprising, as an active ingredient, sauchinone isolated from a Saururus chinensis extract fraction, and a composition for preventing or treating insulin resistance syndrome, Korean Patent Laid-Open Publication No. 2012-0122970 discloses a composition for prevention and treatment of diabetes and diabetic complications including powder of Rhynchosia volubilis Lour. Or extract thereof. However, the development of new therapeutic agents useful for the treatment of diabetes or insulin resistance syndrome is still required.
Serotonin is also known as 5-HT (5-hydroxytryptamine). Serotonin is a neurotransmitter and is involved in controlling emotions related to eating, sleeping, awakening, pain control, and dreaming. Serotonin is synthesized from the amino acid tryptophan, and the enzyme tryptophan
A tryptophan hydroxylase attaches -OH to the tryptophan to produce the intermediate 5-hydroxytryptophan (5-HTP). Another enzyme 5-HTP decarboxylase then removes -COOH from 5-HTP to complete 5-HT, or serotonin. P-chlorophenylalanine (PCPA) is a synthetic amino acid that induces systemic inhibition of serotonin synthesis by an irreversible inhibitor of tryptophan hydroxylase, a rate-limiting enzyme for serotonin synthesis.
The present inventors have made extensive efforts to develop new strategies for the prevention or treatment of metabolic diseases, particularly obesity. As a result, the present inventors have found that TPH1 (Trypotophan hydroxylase 1), HTR2A (5-hydroxytryptamine 2A receptor) and HTR3 (5-hydroxytryptamine 3 receptor) are potential therapeutic targets for metabolic diseases, particularly obesity.
Accordingly, an object of the present invention is to provide a composition for preventing or treating metabolic diseases.
It is another object of the present invention to provide a method for screening a therapeutic agent for a metabolic disease.
According to one aspect of the present invention, there is provided a composition for preventing or treating metabolic diseases comprising TPH1 (Trypotophan hydroxylase 1), HTR2A (5-hydroxytryptamine 2A receptor) or HTR3 (5-hydroxytryptamine 3 receptor) .
The present inventors have made extensive efforts to develop a novel strategy for the prevention or treatment of metabolic diseases, particularly obesity. As a result, the present inventors have found that TPH1 (Trypotophan hydroxylase 1), HTR2A (5-hydroxytryptamine 2A receptor) and HTR3 (5-hydroxytryptamine 3 receptor) are potential therapeutic targets for metabolic diseases, particularly obesity.
According to one embodiment of the invention, the prevention or treatment of metabolic diseases is achieved through inhibition of TPH1, HTR2A or HTR3.
HTR3 acts as a cation channel that is activated by serotonin as a heteropentamer of HTR3A and HTR3B. 23,24
The inhibition of TPH1, HTR2A or HTR3 can be done in a variety of ways.
According to one embodiment of the present invention, the TPH1, HTR2A or HTR3 inhibitor is an inhibitor of expression of TPH1, HTR2A or HTR3A.
Preferably, the expression inhibitor of TPH1, HTR2A or HTR3A is an antisense oligonucleotide or siRNA oligonucleotide that specifically binds to the TPH1, HTR2A or HTR3A gene.
The term "antisense oligonucleotide " as used herein refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and binds to a complementary sequence in mRNA to inhibit translation of mRNA into a protein The antisense sequence may be selected from the group consisting of TPH1 mRNA (for example, GenBank Accession No. NM_004179.2), HTR2A mRNA (for example, GenBank Accession Nos. NM_000621.4 and NM_001165947.2) or HTR3A mRNA (for example, GenBank Accession No.
The antisense nucleic acid may be modified at one or more base, sugar or backbone locations to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol. , 5 (3): 343-55 (1995)). The nucleic acid backbone can be modified with phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic biantennary bond, and the like. In addition, the antisense nucleic acid may comprise one or more substituted sugar moieties. The antisense nucleic acid may comprise a modified base. Modified bases include, but are not limited to, hypoxanthin, 6-methyladenine, 5-me pyrimidine (especially 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, -Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine . In addition, the antisense nucleic acid of the present invention may be chemically combined with one or more moieties or conjugates that enhance the activity and cytotoxicity of the antisense nucleic acid. A cholesterol moiety, a cholesteryl moiety, a cholic acid, a thioether, a thiocholesterol, an aliphatic chain, a phospholipid, a polyamine, a polyethylene glycol chain, adamantane acetic acid, a palmityl moiety, octadecylamine, hexylamino- And liposoluble moieties such as Cole sterol moieties. Oligonucleotides, including liposoluble moieties, and methods of preparation are well known in the art (U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid may increase the stability to nuclease and increase the binding affinity of the antisense nucleic acid with the target mRNA.
In the case of antisense oligonucleotides, they can be synthesized in vitro in a conventional manner and administered in vivo or in vivo to synthesize antisense oligonucleotides. One example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose recognition site (MCS) origin is in the opposite direction. Such antisense RNAs are preferably made such that translation stop codons are present in the sequence so that they are not translated into the peptide sequence.
The design of antisense oligonucleotides that can be used in the present invention can be readily made by methods known in the art with reference to nucleotide sequences of TPH1 mRNA, HTR2A mRNA or HTR3A mRNA known in GenBank (Weiss, B. . ed): Antisense Oligodeoxynucleotides and Antisense RNA:... Novel Pharmacological and Therapeutic Agents, CRC Press, Boca Raton, FL, 1997; Weiss, B., et al, Antisense RNA gene therapy for studying and modulating biological processes Cell Mol. Life Sci . , 55: 334-358 (1999).
As used herein, the term "siRNA" refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99 / 07409 and WO 00/44914) siRNA is provided as an efficient gene knockdown method or as a gene therapy method because it can inhibit the expression of a target gene. SiRNA was first found in plants, insects, fruit flies and parasites, siRNA was developed and applied to mammalian cell research.
When the siRNA molecule is used in the present invention, the sense strand (sequence corresponding to TPH1 mRNA, HTR2A mRNA or HTR3A mRNA sequence) and antisense strand (sequence complementary to TPH1 mRNA, HTR2A mRNA or HTR3A mRNA sequence) May have a double-stranded structure located on the opposite side, or may have a single-stranded structure having self-complementary sense and antisense strands.
The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases.
The siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit the expression of the TPH1, HTR2A or HTR3A gene by the RNAi effect. The sticky end structure can be a 3'-end protruding structure and a 5'-end protruding structure.
In the present invention, the siRNA molecule may have a form in which a short nucleotide sequence (e.g., about 5-15 nt) is inserted between self-complementary sense and antisense strands, in which case by expression of the nucleotide sequence The formed siRNA molecules form a hairpin structure by intramolecular hybridization and form a stem-and-loop structure as a whole. This stem-and-loop structure is processed in vitro or in vivo to produce an active siRNA molecule capable of mediating RNAi.
According to one embodiment of the present invention, the TPH1, HTR2A or HTR3 inhibitor is an antagonist of the TPH1, HTR2A or HTR3A.
Many antagonists of TPH1 are known. According to one embodiment of the present invention, the antagonist of TPH1 is p-chlorophenylalanine, p-ethanylphenylalanine, AGN-2979 [3- (3-dimethylaminopropyl) -3- (3-methoxyphenyl) piperidine-2,6-dione] or LX1031 [(2S) -2-amino-3- [4- [2-amino- 6- (1R) -2,2,2- 3-methoxyphenyl) phenyl] ethoxy] pyrimidin-4-yl] phenyl] propanoic acid).
Many HTR2A antagonists are known. According to one embodiment of the invention, the antagonist of HTR2A is selected from the group consisting of ketanserin [3- {2- [4- (4-fluorobenzoyl) piperidin- 1- yl] ethyl} quinazoline- -dione], ritanserin [6- [2- [4- [bis (4-fluorophenyl) methylidene] piperidin- 1 -yl] ethyl] -7-methyl- [1,3] thiazolo [ -b] pyrimidin-5-one], nefazodone [1- (3- [4- (3-chlorophenyl) piperazin-1-yl] propyl) -3- 1H-1,2,4-triazol-5 (4H) -one], clozapine [8-Chloro-11- (4-methylpiperazin- 4] diazepine], olanzapine [2-Methyl-4- (4-methyl-1-piperazinyl) -10H-thieno [2,3- b] [1,5] benzodiazepine], quetiapine [2- (2- (4-dibenzo [b, f] [1,4] thiazepine-11-yl-1-piperazinyl) ethoxy] ethanol], risperidone [4- [2- [4- -fluorobenzo [d] isoxazol-3-yl) -1-piperidyl] ethyl] -3-methyl-2,6-diazabicyclo [4.4.0] deca-1,3-dien- [(3aRS, 12bS) -rel-5-Chloro-2,3,3a, 12b-tetrahydro-2-methyl-1H-dibenz [2,3: 6,7] oxepino [4,5- c] pyrrole ], Aminomethyl-9,10-diazabicyclo [2.2. ≪ RTI ID = 0.0 > (3-dimethoxyphenyl) dihydroanthracene).
Many HTR3A antagonists are known. According to one embodiment of the invention, the antagonist of HTR3A is selected from the group consisting of ondansetron [(RS) -9-methyl-3- [(2-methyl-1H-imidazol- -9H-carbazol-4 (9H) -one], granisetron [1-methyl-N - ((1R, 3R, 5S) 3-yl) -1H-indazole-3-carboxamide], tropisetron [(1R, 5S) -8-methyl-8-azabicyclo [3.2.1] octan- carboxylate], dolasetron [(3R) -10-oxo-8-azatricyclo [5.3.1.03,8] undec-5-yl 1H- indole-3-carboxylate], palonosetron [ 3aS) -2 - [(3S) -1-Azabicyclo [2.2.2] oct-3-yl] -2,3,3a, 4,5,6-hexahydro-1H-benz [de] isoquinolin- , Ramosetron [(1-methyl-1H-indol-3-yl) [(5R) -4,5,6,7-tetrahydro-1H-benzimidazol- 5- yl] methanone] Methyl-2-methyl-1H-imidazol-5-yl) methyl] -2,3,4,5-tetrahydro-1H-pyrido [4,3-b] indole- 1-one], batanopride [4-amino-5-chloro-N- (2-diethylaminoethyl) -2- (3-oxobutan-2-yloxy) Amino-N - [(4S, 5S) -1-azabicyclo [3.3.1] non-4-yl] -5-chloro-2-methoxybenzamide] or zacopride [ -chloro-2-methoxy-N- (quinuclidin-3-yl) benzamide].
According to one embodiment of the present invention, the composition of the present invention further comprises an activator of? 3-adrenergic receptor. As demonstrated in the following examples, activating the? 3-adrenergic receptor with inhibition of HTR2A or HTR3 increases energy expenditure in white adipose tissue (WAT) and brown adipose tissue (BAT) Or prevention of the disease.
Activators of multiple [beta] 3-adrenergic receptors are known. According to one embodiment of the present invention, the activator of the? 3-adrenergic receptor is according to claim 7, wherein the activator of the? 3-adrenergic receptor is CL-316243 [5 - [(2R) -2- [ 2 - [(2R) -2- [3-chlorophenyl] -2-hydroxyethyl] amino] propyl] -1, 3- benzodioxol- [(2R) -2- (3-Chlorophenyl) -2-hydroxyethyl] amino] propyl] -1,3-benzodioxole-2,2-dicarboxylic acid]; Amibegron [Ethyl ([(7S) -7 - [[(2R) -2- (3-chlorophenyl) -2-hydroxyethyl] amino) -5,6,7,8-tetrahydronaphthalen- ] oxy) acetate]; (2R) -2-hydroxy-2-phenylethyl] amino} ethyl < / RTI > ) phenyl] acetamide]; Solabegron [3 '- [(2 - {[(2R) -2- (3-chlorophenyl) -2-hydroxyethyl] amino} ethyl] amino] biphenyl-3-carboxylic acid; Amino] ethyl] phenyl] -4-iodobenzenesulfonamide [N- [4-Hydroxyphenoxy) - [2 - [[(2S) -2-hydroxy-3- (4-hydroxyphenoxy) propyl] amino] ethyl] phenyl] -4-iodobenzenesulfonamide; Or L-742,791 [(R) -N- [4- [2- [[2-Hydroxy- 2- (3- pyridinyl) ethyl] amino] ethyl] (3-pyridinyl) ethyl] amino] - < / RTI > ethyl] -phenyl] -4- [4- [4- (trifluoromethyl) phenyl] thiazol-2-yl] -benzenesulfonamide]].
According to one embodiment of the present invention, the inhibitor of TPH1, HTR2A or HTR3 is an antibody that specifically binds to TPH1, HTR2A or HTR3A.
An antibody that specifically binds to TPH1, HTR2A, or HTR3A protein, which can be used in the present invention and inhibits activity, is a polyclonal or monoclonal antibody, preferably a monoclonal antibody. Antibodies against TPH1, HTR2A or HTR3A proteins can be prepared using methods commonly practiced in the art, for example, the fusion method (Kohler and Milstein, European Journal of (Clackson et al., Nature , 352: 624-628 (1991) and Marks et al., J. Immunology , 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) or phage antibody library methods . Mol Biol, 222:.. 58, can be prepared by 1-597 (1991)). The general process for manufacturing antibodies is Harlow, E. and Lane, D., Using Antibodies : A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies : A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley / Greene, NY, 1991, the disclosures of which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art. Polyclonal antibodies can be obtained by injecting a TPH1, HTR2A or HTR3A protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.
Peptides capable of specifically inhibiting the activity of TPH1, HTR2A or HTR3A by specifically binding to TPH1, HTR2A or HTR3A can be obtained by conventional methods known in the art, for example phage display methods (Smith GP, "Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface ". Science 228 (4705): 13151317 (1985); Smith GP, Petrenko VA,"... phage display "Chem Rev 97 (2): 391410 (1997 )).
TPH1, HTR2A or HTR3 inhibitors are natural extracts as antagonists of TPH1, HTR2A or HTR3A.
As used herein, natural extract means an extract obtained from various organs or parts (e.g., leaves, flowers, roots, stems, branches, bark, fruits, etc.) of natural products. Natural extracts can be prepared by conventional extraction solvents known in the art, such as (a) water, (b) anhydrous or hydric lower alcohol having 1-4 carbons such as methanol, ethanol, propanol, butanol, n-propanol, iso- (E) ethyl acetate, (f) chloroform, (g) 1,3-butylene glycol, and (h) n-butanol. Hexane, (i) diethyl ether, or (j) butyl acetate.
The extract of the present invention includes not only those obtained by using the above-mentioned solvents, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the above extract through an ultrafiltration membrane having a constant molecular weight cut-off value, and a separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) The fraction obtained by the purification method is also included in the extract of the present invention.
According to one embodiment of the invention, the TPH1, HTR2A or HTR3 inhibitor is a peptide as an antagonist of TPH1, HTR2A or HTR3A.
As used herein, the term " peptide " means a linear molecule formed by peptide bonds and formed by bonding amino acid residues to one another, and is composed of 4-40 amino acid residues, preferably 4-30, most preferably 4-20 have.
The TPH1, HTR2A or HTR3A inhibitor peptide of the present invention is prepared by a solid-phase synthesis method commonly used in the art (Merrifield, RB, J. Am . Chem . Soc . , 85: 2149-2154 1963), Kaiser, E., Colescot, RL, Bossinger, CD, Cook, PI, Anal . Biochem . , 34: 595-598 (1970)). That is, after the amino acid in which the? -Amino and side chain functional groups are protected is bound to the resin, the? -Amino protecting group is removed, and the remaining? -Amino and the amino acid protected with the side chain functional group are stepwise coupled in a desired order to obtain an intermediate . The amino acid sequences for producing the TPH1, HTR2A or HTR3A inhibitor peptides of the present invention were determined by the conventional method (Chen L, Hahn H, Wu G, Chen CH, Liron T, Schechtman D, Cavallaro G, Banci L, Guo .. Y, Bolli R, Dorn GW, Mochly-Rosen D., Proc Natl Acad Sci, 98, 11114-9 (2001);.. Phillipson A, Peterman EE, Taormina P Jr, Harvey M, Brue RJ, Atkinson N , Omiyi D, Chukwui, Young LH., Am . J. Physiol . Heart Circ . Physiol ., 289, 898-907 (2005); and Wang J, Bright R, Mochly-Rosen D, Giffard RG., Neuropharmacology ., 47, 136-145 (2004)).
According to one embodiment of the invention, the TPHl, HTR2A or HTR3 inhibitor is an aptamer as a antagonist of TPHl, HTR2A or HTR3A. Aptamers are oligonucleic acid (e.g., RNA) or peptide materials, details of which are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library ". Proc Natl Acad Sci USA. 95 (24): 142727 (1998).
According to embodiments of the present invention, the composition of the present invention may be manufactured from a pharmaceutical composition and a food composition.
When the composition of the present invention is a pharmaceutical composition, the composition of the present invention comprises (i) an effective amount of a TPH1, HTR2A or HTR3 inhibitor of the present invention; And (ii) a pharmaceutically acceptable carrier. As used herein, the term " effective amount " means an amount sufficient to exert the therapeutic effect of the present invention described above.
The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include carbohydrate-based compounds such as lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, etc. ), Acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, gum arabic, vegetable oil But are not limited to, vegetable oils, soybean oil, soybean oil, olive oil, coconut oil), polyethylene glycol, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations include, but are not limited to, Remington's Pharmaceutical Sciences (19th ed., 1995).
The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, or the like.
The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the invention, a suitable daily dosage is 0.0001-100 mg / kg body weight. The administration may be carried out once a day or divided into several doses.
The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
The compositions of the present invention may be prepared with foods, especially functional food compositions. The functional food composition of the present invention includes components that are ordinarily added during the manufacture of food, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared from a drink, a flavoring agent or a natural carbohydrate may be included as an additional ingredient in addition to the TPH1, HTR2A or HTR3 inhibitor as an active ingredient. For example, natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.); Disaccharides (e.g., maltose, sucrose, etc.); oligosaccharide; Polysaccharides (e.g., dextrin, cyclodextrin and the like); And sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.). Natural flavoring agents (e.g., tau martin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) may be used as flavorings.
According to one embodiment of the present invention, HTR2A and HTR3 are present in white adipose tissue (WAT) and brown adipose tissue (BAT). As shown in the examples and Figures 3A-3P, inhibition of HTR2A in white adipose tissue (WAT) contributes to lipolysis and inhibition of HTR3A in brown adipose tissue (BAT) contributes to heat generation.
According to one embodiment of the present invention, the metabolic diseases to be prevented or treated by the present invention are obesity, diabetes, insulin resistance, hyperlipidemia or hypercholesterolemia, especially obesity.
As shown in the examples and Figures 3A-3P, inhibition of HTR3A (e.g., rust-out of the HTR3A gene and treatment of the HTR3A antagonist) results in resistance to obesity. In addition, inhibition of HTR3A is a reduction in fat droplet size in the BAT, increased expression of heat generated gene (e.g., Ucp1 and Dio2) in BAT (Fig. 3c and 3k), the increase of carbon dioxide produced (VCO 2) (Fig. 3d), and insulin Resulting in increased susceptibility (Figure 3g). Interestingly, inhibition of HTR3A increases phosphorylation of the cAMP level and PKA pathway in BAT only in the presence of an agonist of the? 3-adrenergic receptor (Fig. 3h, 3i, 3j) Inhibition of HTR3A with an agonist of BAT intensively increases oxygen consumption in BAT (Fig. 31). In addition, inhibition of HTR3A decreases LDL cholesterol and leptin and increases free fatty acid levels (Fig. 3r). Inhibition of HTR3A increases the size and number of mitochondria in BAT (Fig. 3t). As described in the Examples and Figures 3m, 3n and 3o, HTR2A is involved in fat production.
Collectively, TPHl, HTR2A or HTR3 inhibition is a useful strategy for the prevention or treatment of metabolic diseases, particularly obesity, including obesity, diabetes, insulin resistance, hyperlipidemia or hypercholesterolemia.
According to another embodiment of the present invention, the present invention provides a method for screening a therapeutic agent for a metabolic disease comprising the steps of:
(a) contacting a test substance with TPH1 (Trypotophan hydroxylase 1), HTR2A (5-hydroxytryptamine 2A receptor) or HTR3 (5-hydroxytryptamine 3 receptor);
(b) analyzing whether the test substance inhibits TPH1, HTR2A or HTR3; When the test substance inhibits TPH1, HTR2A or HTR3, the test substance is determined to be a therapeutic agent for a metabolic disease.
According to the present invention, TPH1 (tryptophan hydroxylase 1), HTR2A (5-hydroxytryptamine 2A receptor) or HTR3A is first brought into contact with the test substance to be analyzed.
According to the present invention, TPH1, HTR2A or HTR3A is either in the cell or separated or purified from the cell. According to one embodiment of the present invention, HTR2A and HTR3A are present in white adipose tissue (WAT) and brown adipose tissue (BAT).
The term " test substance " used in reference to the screening method of the present invention means an unknown substance used in screening to check whether it affects the activity of TPH1, HTR2A or HTR3A. Such samples include, but are not limited to, chemicals, peptides, antibodies, aptamers, siRNA, antisense oligonucleotides, and natural extracts. The sample analyzed by the screening method of the present invention is a single compound or a mixture of compounds (e.g., a natural extract or a cell or tissue culture). Samples can be obtained from a library of synthetic or natural compounds. Methods for obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co., Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA) ) And MycoSearch (USA). Samples can be obtained by various combinatorial library methods known in the art and include, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, , The " 1-bead 1-compound " library method, and the synthetic library method using affinity chromatography screening. Methods for synthesis of molecular libraries are described in DeWitt et al., Proc . Natl . Acad . Sci .
The screening method of the present invention can be carried out in various ways and can be carried out in a high throughput manner according to various binding assay, expression analysis or activity analysis techniques known in the art.
According to one embodiment of the present invention, said TPH1, HTR2A or HTR3 inhibition is inhibition of the expression of TPH1, HTR2A or HTR3A.
The expression of TPH1, HTR2A or HTR3A can be analyzed by measuring the expression of the TPH1, HTR2A or HTR3A gene. The measurement of the expression of the TPH1, HTR2A or HTR3A gene can be carried out through various methods known in the art. For example, such as RT-PCR (Sambrook, 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 reaction using a cDNA microarray (Sambrook et al., 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)).
The expression of TPH1, HTR2A or HTR3A can be analyzed by measuring the expression of the TPH1, HTR2A or HTR3A protein. The measurement of the expression of the TPH1, HTR2A or HTR3A protein can be carried out through a variety of methods known in the art including, for example, Western blotting, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay, capture-ELISA, inhibition or hard-ware analysis, and sandwich analysis.
Alternatively, analyzing whether the test substance inhibits TPH1, HTR2A or HTR3A is to analyze inhibiting the function of TPH1, HTR2A or HTR3A.
According to one embodiment of the invention, the contacting of the test substance with HTR3 present in brown adipose tissue (BAT) in step (a) is carried out with activation of the? 3-adrenergic receptor.
According to one embodiment of the present invention, the metabolic disease is obesity, diabetes, insulin resistance, hyperlipidemia or hypercholesterolemia.
The features and advantages of the present invention are summarized as follows:
(a) The present invention provides a novel approach for the prevention or treatment of metabolic diseases, particularly obesity, including obesity, diabetes, insulin resistance, hyperlipidemia or hypercholesterolemia.
(b) The present invention provides TPH1, HTR2A or HTR3, particularly HTR3A, as a therapeutic target for metabolic diseases, particularly obesity.
(c) inhibition of HTR3A results in a variety of beneficial effects on the treatment of metabolic diseases, including resistance to obesity, decreased fat drop size, increased expression of heat-producing genes, increased metabolic activity, increased insulin sensitivity, and LDL Cholesterol and leptin.
(d) Interestingly, inhibition of HTR3A increases phosphorylation of the cAMP level and PKA pathway in BAT only in the presence of an agonist of the? 3-adrenergic receptor and inhibits the phosphorylation of the? 3-adenylate receptor agonist with HTR3A Inhibition of intensely increases oxygen consumption in BAT. This fact, which has been identified by the present invention, demonstrates that the concomitant administration of an antagonist of HTR3A and an agonist of the? 3-adrenergic receptor is very effective in the treatment of metabolic diseases, particularly obesity.
Figures 1a-1h show that serotonin deficiency protects mice from obesity. Figure 1 A is the serotonin level of adipose tissue in C57BL / 6J male mice after 16 weeks of high-fat diet (HFD) feeding as measured by LC-MS; N = 4 mice per group. HFD increased cephotonin levels in eWAT and iWAT. Figure 1B shows the expression of Tph1 mRNA in adipose tissue by RT-PCR after 2 weeks of HFD administration. N = 4 mice per group. Figure 1c shows the growth curves of control and PCPA-treated mice fed standard diet (SCD) or HFD. N = 4 mice per group. The HFD fed PCPA-treated mice showed a decrease in weight gain and a decrease in eWAT as compared to HFD fed control mice. Figure 1d shows an overall image of an SCD-fed or HFD-fed control mouse and a PCPA-treated mouse after 10 weeks of HFD ingestion. HFD fed PCPA-treated mice showed reduced eWAT compared to HFD fed control mice. In Figures 1e and 1f, HFD fed PCPA-treated mice showed increased glucose tolerance (e) and improved insulin sensitivity (f) over HFD fed control mice. Figures 1g and 1h show the metabolic rates of HFD feeding controls and PCPA-treated mice for 6 weeks. Metabolic parameters were measured using an 8-chamber Oxymax system. Mice were adapted in a cage for 24 hours and data were collected for an additional 48 hours. The PCPA-treated mice showed increased carbon dioxide production (VCO 2 ) and heat production. N = 4 mice per group. All data are expressed as mean ± standard deviation. * P < 0.05. LC-MS: Liquid chromatography-mass spectrophotometer (Li).
Figure 1I shows detection of Tph1 and 5-HT receptor mRNAs in mouse adipose tissue. Adipose tissue was isolated from epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT) and interscapular brown adipose tissue (BAT) of 8 week old C57BL / 6J mice. Other genes involved in Tph1 mRNA, 5-HT receptor subtype and 5-HT metabolism were amplified from mouse adipose tissue RNA by RT-PCR.
Figure 1J shows the effect of 5-HT deficiency in lipid profiles, leptin and adiponectin. 8 week old mice were treated with vehicle or PCPA (300 mg / kg) for 6 weeks. During the treatment period, the mice fed SCD or HFD. After 6 weeks of treatment, blood was obtained from the tail vein. Serum levels of total cholesterol (a), LDL cholesterol (b), FFA (c) and leptin (d) were measured by ELISA. HFD fed PCPA-treated mice showed reduced levels of cholesterol, leptin and FFA compared to HFD fed control mice. All data are expressed as mean ± standard deviation. Statistical significance vs. Untreated controls were analyzed by Student's t test: *, P < 0.05. Excipient:
Figure 1k shows the effect of peripheral Tph1 inhibitors on body weight and glucose metabolism. 8-week-old mice were orally treated with vehicle or LP-53340 (30 mg / kg) for 10 weeks. During the treatment period, the mice fed SCD or HFD. (a) Growth curves of SCD or HFD feeding control mice and LP-533401-treated mice. (b) After overnight fasting, IPGTT was performed. Blood glucose levels were measured after intraperitoneal injection of 2 g / kg glucose. HFD eating LP-533401-treated mice showed improved glucose tolerance compared to HFD fed control mice. (c) IPITT was performed after fasting for 4 hours. Their blood glucose levels were measured after 0.75 U / kg intraperitoneal injection. N = 4 mice per group. (d) Representative BAT compartments of control mice and LP-533401-treated mice stained with hematoxylin and eosin. The scale bar represents 20 μm. All data are expressed as mean ± standard deviation. Statistical significance vs. Untreated controls were analyzed by Student's t test: *, P < 0.05. LP-533401: small molecule inhibitor of peripheral Tph; Excipients:
FIG. 11 shows the food intake and physical activity of HFD fed PCPA-treated mice. After 6 weeks of feeding HFD, the metabolic profile of 14-week old control and PCPA-treated mice was measured using the Oxymax system. The mice were adapted for 24 hours and data were collected for 48 hours. Daily food intake (a) and physical activity (b) were similar in both groups. (c) O 2 consumption of PCPA-treated mice was increased. N = 4 mice per group. All data are expressed as mean ± standard deviation. Statistical significance vs. Untreated controls were analyzed by Student's t test: *, P < 0.05. Excipients: PBS; VO 2 : O 2 consumption; RER: The respiratory exchange ratio.
Figure 1m shows the food intake and metabolic rate of PCPA-treated mice during SCD feeding. Metabolic profiles of 14-week-old SCD-treated mice were measured using the Oxymax system after feeding for 6 weeks with excipients or PCPA (300 mg / kg). Metabolic cage data show no significant difference between excipients and PCPA-treated mice. The metabolic cage data includes daily food intake (a), physical activity (b), heat production (c), oxygen consumption (d), VCO 2 removal (e), and respiratory exchange rate (f). N = 4 mice per group. All data are expressed as mean ± standard deviation. Statistical significance vs. Untreated controls were analyzed by Student's t test: *, P < 0.05. Excipients: PBS; VCO 2 : CO 2 removal.
Figures 2a-2i show serotonin deficiency in adipose tissue of mice. (Figure 2a) Representative images of eWAT of control mice and PCPA-treated
Figure 2J shows representative compartments of PCPA-treated mouse eWAT stained with Plin1 antibody. Histology of control and PCPA-treated
Figure 2K shows serotonin deficiency effect in mouse eWAT. 8 week old mice were treated with vehicle or PCPA (300 mg / kg) for 6 weeks. During treatment the mice were fed either SCD or HFD. After 6 weeks of treatment, eWAT was isolated and mRNA levels of genes associated with lipid metabolism and heat generation were measured by real-time RT-PCR. N = 4 mice per group. All data are expressed as mean ± standard deviation. Statistical significance vs. Untreated controls were analyzed by Student's t test: *, P < 0.05. Excipient:
Figure 21 shows the fat cell size of iWAT. Image J software was used to calculate the mean fat cell size of H & E stained images. IWAT of 18-week-old PCPA-treated mice fed HFD for 10 weeks showed a decrease in adipocyte size compared to PCPA-treated mice fed SCD. N = 4 mice per group. All data are expressed as mean ± standard deviation. Statistical significance v s. Untreated controls were analyzed by Student's t test: *, P < 0.05.
Figure 2m-2o shows the effect of serotonin deficiency in mouse iWAT. 8 week old mice were treated with vehicle or PCPA (300 mg / kg) for 6 weeks. During treatment the mice were fed either SCD or HFD. After 6 weeks of treatment, iWAT was isolated and mRNA levels of genes associated with lipid metabolism and heat generation were measured by real-time RT-PCR. N = 4 mice per group. All data are expressed as mean ± standard deviation. Statistical significance vs. Untreated controls were analyzed by Student's t test: *, P < 0.05.
Figures 2p-2r show the serotonin deficiency effect in mouse BAT. 8 week old mice were treated with vehicle or PCPA (300 mg / kg) for 6 weeks. During treatment the mice were fed either SCD or HFD. After 6 weeks of treatment, BAT was isolated and mRNA levels of genes associated with lipid metabolism and heat generation were measured by real-time RT-PCR. N = 4 mice per group. All data are expressed as mean ± standard deviation. Statistical significance vs. Untreated controls were analyzed by Student's t test: *, P < 0.05.
Figure 2S shows representative images of PET-CT. BAT (triangle) and cardiac tissue (arrow) are highlighted. 18 F-FDG uptake was increased in BAT and heart. BAT in WT mice showed a decrease in 18 F-FDG uptake after HFD feeding, whereas BAT in HFD fed PCPA-treated mice showed increased glucose uptake compared to HFD fed mice.
Figure 2T shows a TEM image of the mitochondria of PCPA-treated mouse BAT. Interscapular BAT was isolated from HFD-fed mice treated with SCD fed mice, HFD-fed mice and PCPA for 6 weeks. BAT of PCPA-treated HFD-fed mice showed increased size and number of mitochondria. The scale bar represents 1 m. TEM: transmission electron microscopy.
Figures 3a-3p show that Htr3 regulates heat generation in BAT and Htr2a regulates lipogenesis in WAT. (Fig. 3a) Growth curves of SCD or HFD-fed Htr3a KO mice and their WT literate . Htr3a KO mice were resistant to obesity. N = 4 mice per group. (Figure 3b) H & E staining of WT and Htr3a KO mouse BAT after 6 weeks of SCD or HFD feeding. BAT of KO mice showed a decrease in lipid droplet size compared to WT literate. The scale bar represents 20 μm. (Fig. 3c) Heat Generation Gene Expression Levels in BAT. N = 4 per group. (Figures 3d, 3e) Metabolic rate of HFD fed WT and Htr3a KO mice. Htr3a KO mice showed increased carbon dioxide production (VCO 2 ) (FIG. 3d) and heat production (FIG. 3e). N = 4 mice per group. (Fig. 3f, 3g) Glucose resistance and insulin resistance test after 6 weeks of HFD administration. N = 4 per group. (Fig. 3h). Changes in cAMP levels following treatment with Htr3 agonist / antagonist in IBA (immortalized brown adipocytes) measured using cAMP ELISA. In the absence of β3 agonist, the Htr3 agonist / antagonist did not affect cAMP in brown adipocytes. However, after β3 stimulation, Htr3 antagonist-pretreatment IBAs showed an increase in cAMP levels. Ondan: ondansetron; CL:
Figure 3q shows the HFD feeding Htr3a KO This shows the total image of the mouse. 8-week-old mice were fed SCD or HFD for 6 weeks. Although HFD increased visceral fat, HFD-fed Htr3a KO mice had less visceral fat than WT literate.
Figure 3r shows Htr3a KO mouse lipid profile and serum leptin levels. Htr3a KO mice were fed SCD or HFD for 6 weeks and blood was obtained from the tail vein. Serum levels of total cholesterol (a), LDL cholesterol (b), FFA (c) and leptin (d) were measured by ELISA. HFD feeding Htr3a KO mice decreased LDL and leptin levels but increased FFA levels. N = 4 mice per group. All data are expressed as mean ± standard deviation. Statistical significance vs. Untreated controls were analyzed by Student's t test: *, P < 0.05.
3s are also Htr3a KO mice and representative WT littermate eWAT and iWAT compartments. After 6 weeks of HFD feeding, Htr3a KO mice and WT literate. The tissue compartment was stained with H & E. Htr3a In eWAT or iWAT of KO mice, mean fat cell size did not decrease significantly. The scale bar represents 20 μm.
Figure 3t shows Htr3a KO BAT Representative TEM images of mitochondria. After 6 weeks of HFD feeding, Htr3a KO mouse and WT littermate. Htr3a KO BAT showed increased size and number of mitochondria. The scale bar represents 1 μm.
Figure 3u shows Htr3a Expresses an up-regulated mitochondrial gene in KO BAT. After 6 weeks of HFD feeding, Htr3a KO mice and their WT litemates. Real-time RT-PCR in BAT was used to measure the expression of mature brown adipocytes associated with mitochondrial biogenesis. N = 4 per group. All data are expressed as mean ± standard deviation. Statistical significance vs. Untreated controls were analyzed by Student's t test: *, P < 0.05.
Figure 3v shows a comparison between the control during HFD feeding and Htr3a KO mouse food absorption and metabolic rate. After 6 weeks of feeding HFD, the mice were treated with Oxymax system (Columbus instrument) at 14 weeks of age with Htr3a KO mice and their WT literate were measured. Htr3a KO mice showed an increase in metabolic rate compared to WT literate. All data are expressed as mean ± standard deviation. Statistical significance vs. Untreated controls were analyzed by Student's t test: *, P < 0.05.
Figure 3w shows 5-HT induction during differentiation of 3T3-L1 adipose precursor cells. 5-HT immunohistochemical staining in 3T3-L1 adipocytes during differentiation. Bodipy staining for lipids was green and 5-HT fluorescence was red. Overlapping images show lipid droplets where the green and red lipid bodies co-localize after 4 days of differentiation (D4). The scale bar represents 20 μm.
Figures 4A-4M show cell autonomous function of 5-HT in adipocytes. (Figure 4a) HFD feeding Tph1 Weight of FKO mice and their WT literate. N = 4 per group. (Fig. 4b) After 6 weeks of HFD feeding, Tph1 Representative images of FKO mice and WT littermate adipose tissue. The compartment was stained with H & E. (Fig. 4c) Immunohistochemistry for Ucp1 (silver dye). Tph1 In the iWAT of FKO mice, Ucp1-positive multipotent fat cells were increased. (Fig. 4d) Glucose resistance test. HFD-feeding Tph1 FKO mice showed improved glucose tolerance as compared to HFD-fed WT literate. (Fig. 4E) Tph1 X-Bibo Study Using SVF of FKO Mouse and WT Literate BAT. After 8 days of differentiation, Tph1 FKO SV cells were more likely to express Ucp1 mRNA expression was increased. 5-HT is Tph1 In SV cells of FKO mice, Ucp1 mRNA levels were suppressed. (Fig. 4f) Growth curves of HFD fed Tph1 AFKO mice and their WT literate. N = 4 per group. (Fig. 4g, 4h) After 6 weeks of HFD feeding, HFD fed Tph1 AFKO mice showed improved glucose tolerance (Fig. 4g) and reduced insulin resistance (Fig. 4h) compared to control mice. (Fig. 4i) Representative images of adipose tissue of WT litmate and Tph1 AFKO mice after 6 weeks of HFD feeding. The compartment was stained with H & E. (Fig. 4j). Average fat cell sizes were measured in H & E staining images. (Fig. 4k) Immunohistochemistry for Ucp1 (silver dye). (Fig. 4L, 4m) Proposed model for energy metabolism regulation by serotonin in adipocytes. The red arrow indicates activity and the blue arrow indicates inhibition. All data are expressed as mean ± standard deviation. * P < 0.05.
Figure 4n shows that Tph1 The growth curve of FKO mice is shown. SCD feeding Eight weeks old Tph1 FKO The mice showed a decrease in body weight than the WT literate, but this difference was not statistically significant.
Figure 4o shows SCD feeding Tph1 FKO mice and their wild-type litemates, eWAT, iWAT and BAT compartments. The compartment was stained with H & E. In the SCD feeding state, eWAT and BAT are Tph1 There was no significant difference between FKO mice and their WT littermates. The scale bar represents 20 μm.
Figure 4p shows SCD feeding Tph1 AFKO mice and their wild-type litemates, eWAT, iWAT and BAT compartments. The compartment was stained with H & E. Tph1 at 6 weeks of age by IP injection of tamoxifen Cre-recombination of AFKO mice was induced. Thereafter, adipose tissue of male mice was isolated at 14 weeks of age. The scale bar represents 20 μm.
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
Experimental Method
reagent
PCPA, D-glucose, insulin, CL-316243 (3-adrenergic receptor agonist), ondansetron, m-CPBG, triiodothyronine, 3-isobutyl- (IBMX: 3-isobutyl-1-methylxanthine), indomethacin, dexamethasone, Oil Red O dye, ketanserin, DOI, isopropyl alcohol alcohol, formalin, ascorbic acid, perchloric acid, tamoxifen, and polyethylene glycol 400 (PEG-400) were purchased from Sigma, USA. LP-533401 was purchased from Dalton Pharma Services, Canada. TRIzol reagent, Dulbecco's modified Eagle's medium, calf serum, fetal bovine serum (FBS) and penicillin-streptomycin (P / S) were purchased from Invitrogen, ). All antibodies were purchased from Cell Signaling Technologies (USA) unless otherwise noted.
Experimental animals and diets
Tph1flox / flox mice, Adipoq-cre mice and aP2-CreERT2 mouse generations were previously reported 17,30,31. C57BL / 6J mice, Htr3a-targeted KO mice (B6.129X1-Htr3atm1jul / J, Htr3a KO) and leptin-deficient ob / ob mice (B6.V-Lepob / J) were purchased from Jackson Laboratories Respectively. Tph1flox / flox mice were mated with Adipoq-Cre mice (Tph1 FKO mice) and aP2-CreERT2 mice (Tph1 AFKO mice) to produce adipose tissue-specific Tph1 KO mice. The mice were housed in a climate-conditioned SPF (specific pathogen-free) isolation facility on a 12 h dark cycle and randomly provided food and water. The Institutional Animal Care and Use Committee of the Korea Advanced Institute of Science and Technology approved the experimental protocol for this study. Htr3a KO mice and Tph1flox / flox mice were backcrossed with C57BL / 6J mice for more than 10 generations. Cre-recombination of 6-week-old Tph1 AFKO mice was induced by intraperitoneal injection of 2 mg tamoxifen (Sigma) 5 times per week. The present inventors fed SCD (standard chow diet; 12% fat calories, Purina Laboratory Rodent Diets 38057) or HFD (60% fat calories, Research Diets D12492) to male mice (8-10 weeks old). PBS or 300 mg / kg PCPA was administered daily by intraperitoneal injection. LP-533401 was dissolved in PEG-400 and 5% dextrose (40:60 ratio). The vehicle or 30 mg / kg LP-533401 was administered daily with feeding needles. The present inventors randomly divided C57BL / 6J mice into 2-4 groups. For the transgenic mice, KO mice and their WT littermates were compared. No blinding was done.
Cell culture
Murine 3T3-L1 cells (American Type Culture Collection) were cultured in DMEM supplemented with 10% fetal calf serum and 100 μg / mL P / S in a humidified atmosphere of 37 °
IBAs were cultured in DMEM supplemented with 10% FBS and P / S in a humidified atmosphere at 37 ° C and 5% CO2. After 95% confluence was reached, cells were cultured in DMEM supplemented with 10% FBS, 0.5 μg / mL insulin, 1 nM T3, 0.125 mM indomethacin, 2 μg / mL dexamethasone, 0.5 μM IBMX and P / (Day 0). ≪ / RTI > Two days later, the medium was replaced with DMEM supplemented with 10% FBS, 0.5 μg / mL insulin, 1 nM T3 and P / S (day 2) and replaced every two days from
SVF detach
Epididymal, inguinal and stromal vascular fraction of BAT were isolated from 6-7 week old mice by collagenase digestion. Briefly, the adipose tissue was dissected and chopped and then shaken with 0.2% collagenase A (Roche) in Hank's balanced salt solution (Sigma) at 37 ° C for 45 minutes with constant shaking. Adipocyte and connective tissue were separated from the cell pellet by centrifugation at 800 xg for 10 min at 4 < 0 > C. The cell pellet was then suspended in RBC lysis buffer (Sigma) and filtered through a 40-μm mesh filter (BD bioscience). The pelleted stromal vascular cells (SVCs) were resuspended in DMEM containing 10% FBS and inoculated into 6-well plates for adipocyte differentiation.
oil Red Five( Oil Red O) dyeing
After pre-differentiation (day 8), 3T3-L1 adipocytes were fixed with 3.7% (w / v) formaldehyde in PBS for 15 min at room temperature and washed three times with PBS. The cells were then stained with filtered Oiledo's solution (1.5 mg /
Metabolism analysis
To determine the metabolic rate, the mice were each housed in an 8-chamber, open circuit Oxymax / CLAMS (Columbus Instruments Comprehensive Lab Animal Monitoring System) system as previously described 33. To determine their metabolic rate, each mouse was fed for 72 hours with feeding. PET imaging was performed as previously described using a microPET R4 scanner (Concorde Microsystems, Siemens).
Glucose resistance test and insulin resistance test
For the glucose tolerance test, 2 g / kg D-glucose in PBS was administered to the mice after over night fasting. For the insulin resistance test, insulin (0.75 U / kg) was intraperitoneally injected after fasting for 4 hours. Blood glucose levels in the blood samples obtained from the tail vein at 0, 15, 30, 45, 60, 90 and 120 minutes after injection were measured using Gluco DR Plus glucometer (Allmedicus).
Blood chemistry analysis
Tissue serotonin was extracted by homogenization in extraction buffer containing 0.02% ascorbic acid in 0.1 M perchloric acid and centrifuged. The serotonin levels in the supernatant were determined by liquid chromatography-mass spectrometry method. Serum leptin (Enzo Life Science, USA), LDL-cholesterol (Waco) and FFA (Biovision, USA) were measured using an enzyme-linked immunosorbent assay (ELISA) kit.
cAMP analysis
1 [mu] M ondansetron, 100 nM m-CPBG or 1 [mu] M CL-316243 were treated for 15 minutes with differentiated IBAs. 1 [mu] M CL-316243 was used as a positive control. The cAMP competitive ELISA (Promega) was performed according to the manufacturer's instructions. Briefly, cAMP was extracted by adding 0.1 M HCl containing 0.5% Triton X-100 to the cells. After centrifugation at 600 g for 10 min, the supernatant was used for cAMP level determination by cAMP competitive ELISA.
OCR analysis
Cellular OCRs were measured using a Seahorse XF analyzer (Seahorse Bioscience, USA). After inoculating the XF-24 plate with IBAs, the cells were incubated and differentiated using the protocol described above. Pre-differentiated IBAs were pretreated with PBS and ondansetron for 30 min. The? 3 agonist (CL-316243) and the mitochondrial inhibitor oligomycin and rotenone / antimycin were then treated with the IBAs. OCRs were calculated and recorded with the sensor cartridge and Seahorse XF-24 software.
real time PCR ( Real - time PCR ) analysis
Total RNA was extracted from mouse tissues or cell lines using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. After TURBO DNase (Invitrogen) treatment, 2 μg of total RNA was used to generate complementary DNA with Superscript III reverse transcriptase (Invitrogen). Real-time PCR was performed with the ViiA 7 Real-Time PCR system (Applied Biosystems) and Power SYBR Green PCR Mastermix (Applied Biosystems) to analyze gene expression. Relative quantification was based on the ddCt method and ActB was used as an endogenous control (internal control). The primer sequences are shown in Table 1.
Histological analysis
The inguinal, epididymal and scandalous adipose tissues were harvested, fixed with 4% (w / v) paraformaldehyde in PBS and embedded in paraffin. Subsequently, tissue sections of 5 μm thickness were deparaffinized and rehydrated and used for hematoxylin and eosin (H & E) staining, immunohistochemistry and immunofluorescence. For antigen recovery, the slides were immersed in 10 mM sodium citrate (pH 6.0) and heated to 95 캜 for 20 minutes. Visualization of Ucp1 and Plin1 was performed using the VECTASTAIN ABC kit (PK-4001, Vector Laboratories, USA) according to the manufacturer's instructions. Briefly, the slides were incubated in BLOXALL blocking solution (SP-6000, Vector Laboratories) and then incubated in 2% normal goat serum for 30 minutes at room temperature to block non-specific binding. The compartment was incubated with the primary antibody against Ucp1 (ab10983, Abcam) or Plin1 (ab3526, Abcam) for 1 hour at room temperature and then incubated with the species-specific biotinylated secondary antibody for 30 minutes. The slides were incubated with Vectastain ABC-AP reagent for 30 minutes and then incubated with alkaline phosphatase substrate (DAB, SK-4100, Vector Laboratories) for visualization. Dyes and antibodies used in immunofluorescent staining include BODIPY (BODIPY®493 / 503, Invitrogen), anti-5HT (ab10385, Abcam) and DAPI (D9542, Sigma).
An electron microscope image of BAT was obtained by transmission electron microscopy (Tecnai Spirit TEM) as previously described. Briefly, BAT was first fixed with 2.5% glutaraldehyde, and after further fixation, the ultra-thin section was cut, stained with uranyl acetate, and citric acid was read and examined by electron microscopy.
Western Blot analysis
Cells incubated with RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor (Roche) Whole cell lysates were extracted. The supernatant was collected by simple centrifugation and protein concentration in the supernatant was determined using a BCA protein assay kit (Thermo Scientific, USA). The cell lysate was then mixed with an equal volume of 2x Laemmli buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.01% Bromophenol Blue and 120 mM Tris-HCl, pH 6.8) And boiled at 95 ° C for 5 minutes. Protein samples were then separated by SDS-PAGE and transferred to a PVDF membrane (Millipore). After blocking in a 5% skim milk solution (Sigma), the membrane was incubated with a specific primary antibody, anti-phospho Hsl antibody (ser660), anti-phospho (Ser / Thr) PKA substrate antibody and anti- ≪ / RTI > The membrane was then washed with 1x TBST and incubated with anti-rabbit IgG horseradish peroxidase-linked antibody or anti-mouse IgG antibody. Detection of each protein was carried out using Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific) according to the manufacturer's instructions. Signals were collected by the ChemiDoc MP system (Bio-Rad).
Glycerol release assay
Glycerol release rate was measured using a free glycerol reagent (Sigma) according to the manufacturer's protocol. Briefly, 3T3-L1 adipocytes differentiated in 24-well plates were incubated with Krebs Ringer phosphate buffer (136 mM NaCl, 4.7 mM KCl, 10 mM NaPO4, 0.9 mM) containing 4% fatty acid-free serum albumin MgSO4 and 0.9 mM < RTI ID = 0.0 > CaCl2) < / RTI > for 24 hours. Isoproterenol was used as a positive control. After incubation, 10 μL of the cell culture supernatant was mixed with 0.8 mL of free glycerol reagent, and then the mixture was incubated at 37 ° C. for 5 minutes. The absorbance of the sample was measured at 540 nm using a spectrophotometer (DU730 Life Science UV / Vis, Beckman Coulter, USA). The amount of released glycerol relative to cellular protein content was expressed.
Data Analysis and Statistics
All values are expressed as mean and standard error of the mean (SEMs). Comparisons between groups were performed by Student t test or one-way ANOVA. Normal distribution was tested by f-test. The P-value < 0.05 was considered statistically significant.
Experiment result
5-HT exists in WAT (white adipose tissue) and BAT (brown adipose tissue) and is known to promote adipogenesis in 3T3-L1 preadipocytes 15-17. Indeed, 5-HT was detected in adipose tissue and serotonin-operable genes, except Tph2, were expressed in these tissues (FIGS. 1A and 1I). Interestingly, administration of HFD increased the 5-HT and Tph1 mRNA levels of eWAT (epididymal white adipose tissue) and iWAT (inguinal white adipose tissue) (Figs. Ia and b) Suggesting a potential role for 5-HT. To investigate the role of peripheral 5-HT in energy homeostasis, PCPA (p-chlorophenylalanine) was injected by intraperitoneal injection with HFD for 10 weeks. Systemic inhibition of 5-HT synthesis resulted in decreased weight gain in HFD fed PCPA-treated mice and reduced eWAT mass compared to HFD fed WT controls (Fig. 1c and d). Thus, PCPA enhanced glucose tolerance and insulin sensitivity in HFD fed mice (Fig. 1e, f). Blood levels of low density lipoprotein (LDL) cholesterol, free fatty acid (FFA) and leptin were lowered with decreasing eWAT in PCPA-treated mice (Fig. 1j). The anti-obesity effect of PCPA was also observed in mice treated with the peripheral Tph inhibitor LP-533401, which does not pass the blood-brain barrier (Fig. 1k).
The decrease in eWAT mass observed following PCPA treatment suggests that an increase in energy consumption or a decrease in energy storage may be caused by inhibition of 5-HT synthesis. To address this problem, we analyzed the energy expenditure of mice using
In order to explore the cell type-specific effects of 5-HT in adipose tissue, we analyzed eWAT, iWAT and BAT. In eWAT, PCPA administration caused a reduction in the size of adipocytes, not normal cell structures (Figs. 2a, 2b and 2j). Real-time RT-PCR analysis of eWAT shows that most gene expression involved in triglyceride storage is inhibited in PCPA-treated mice, regardless of HFD consumption (FIGS. 2c and 2k), as compared to control mice. In iWAT of PCPA-treated mice, brown adipocyte-like multipotent cells with reduced adipocyte size and expressing Ucp1 were observed, indicating iWAT browning (Fig. 2d, e and 2l). Consistent with the Ucp1 immunostaining results, Ucp1 and Dio2 mRNA levels were increased in iWAT with PCPA treatment (Fig. 2f and 2m-2o). These results suggest that serotonin plays a role in lipid production and maintenance of WAT.
The intake of HFD results in a dynamic change in BAT, which includes the expansion of monotonic lipid droplets and the reduction of mitochondrial content21. In the present invention, BAT of the control mice showed monotonic lipid droplet formation in brown adipose cells after HFD feeding, but BAT of PCPA-treated mice showed a decrease in lipid droplet size and an increase in polyarticular adipocytes (Fig. 2g) . Real-time RT-PCR analysis showed that PCPA treatment increased expression of thermogenic genes in BAT, with the highest increase in Dio2 mRNA levels (FIG. 2h and 2p-2r). Since brown adipose cells use glucose as well as lipid for heat generation, the absorption of 18 fluorodeoxyglucose (18F-FDG: 18 fluorodeoxyglucose) by positron emission tomography (PET-CT) And the metabolic activity of the cells was measured. Glucose uptake to BAT is reduced after consumption of HFD; However, inhibition of 5-HT synthesis by PCPA significantly increased glucose uptake into BAT (Figs. 2i and 2s). In addition, the number and size of mitochondria and the density of cristae were increased in PCPA-treated mouse BAT (Fig. 2t).
Since the anti-obesity effect of PCPA in BAT was due to the enhancement of adaptive heat generation, the present inventors have attempted to identify receptors responsible for the activation of BAT. Of the 5-HT receptors (Htr) in BAT, we focused on Htr3a, which acts as a functional serotonin-gated cation channel, and Htr3, a heteropentamer of Htr3b. In pancreatic islets, Htr3 activation depolarizes the β-cell membrane and thus increases glucose-stimulated
To exclude the effect of Htr3 in the central nervous system, the present inventors explored the direct action of Htr3 in BAT using immortalized brown adipocytes (IBAs). IBA pretreated with the Htr3 antagonist ondansetron showed increased phosphorylation of cAMP levels, hormone-sensitive lipase (HSL) and PKA substrates in the presence of the? 3-adrenergic receptor agonist (Fig. 3h, i). Ondansetron also increased mRNA expression of heat-producing genes such as Ucp1 and Ppargc1a in IBA (Fig. 3j). Conversely, the Htr3 agonist, 1- (m-chlorophenyl) -biguanide (m-CPBG: 1- (m-chlorophenyl) -biguanide) phosphorylates Ucp1 mRNA as well as HSL and PKA substrates in IBA (Fig. 3i, k). The inventors then measured the oxygen consumption rate (OCR) of IBA. Ondansetron increased OCR synergistically to a? 3-adrenergic receptor agonist (Fig. 31), indicating through Htr3 that local 5-HT can directly regulate heat production in BAT.
Htr3a KO mice showed a decrease in eWAT mass, but this decrease was not as severe as PCPA-treated mice (Fig. 1j, 3q). These results suggest that Htr3 inhibitory effect is more selective in BAT. In order to elucidate an additional mechanism to account for the severe loss of eWAT upon PCPA administration, we performed in vitro assays using 3T3-L1 adipose precursor cells, which express Tph1 and express it in adipocytes It gradually increases during
To confirm the cell autonomic function of 5-HT in adipose tissue, we generated adipocyte-specific Tph1 KO (Adipoq-Cre + / - / Tph1floxlflox, Tph1 FKO) mice. Tph1 FKO mice appeared to be extremely normal and no histological differences were observed in their adipose tissues (Supplementary Figures 20 and 21). However, Tph1 FKO mice were resistant to HFD-induced obesity and showed similar histological changes in both WAT and BAT as in PCPA-treated mice (Fig. 4a, b). Ucp1 expression was significantly increased in the pluripotent cells of Tph1-deficient iWAT (Fig. 4c) and improved glucose resistance in Tph1 FKO mice (Fig. 4d). Then, we isolated the stromal vascular fraction (SVF) from Tph1 FKO BAT and tested the possibility of differentiation into brown adipocytes. After 8 days of differentiation in differentiation medium, Ucp1 mRNA expression in Tph1 null cells was upregulated, which was eliminated by 5-HT treatment (Fig. 4e). In addition, β3 adrenergic functional stimulation markedly increased Ucp1 mRNA expression in SVF of Tph1 FKO BAT (FIG. 4e). These data suggest that the ability of brown adipocytes is greater in Tph1 FKO mice 29. To test the 5-HT role in mature adipocytes, we further analyzed the phenotype of the induced Tph1 FKO (aP2-CreERT2 + / - / Tph1floxlflox, Tph1 AFKO) mice. HFD-fed Tph1 AFKO mice also showed reduced weight gain and increased glucose tolerance and insulin sensitivity (Fig. 4f, g, h), which showed similar histological changes in adipose tissue as PCPA-treated mice , 4j, 4k and 4p). These results demonstrate the cell-autonomic role of 5-HT in adipose tissue.
In the present invention, we provide a complex model for energy metabolism regulation in other adipose tissues (FIG. 4I). In the over-feeding state, 5-HT levels in WAT were increased and increased lipogenesis through Htr2a. The basal 5-HT level also inhibited heat generation in BAT through Htr3a. When 5-HT signaling was blocked, lipolysis in WAT was increased and heat generation was increased in both iWAT and BAT (FIG. 4m). HFD stimulated β3 adrenergic signaling coupled with unrestrained heat generation induced by blockade of serotonin-Htr3 signaling has resulted in increased energy consumption in both iWAT and BAT. Thus, inhibition of 5-HT production in adipose tissue may suggest a novel strategy for anti-obesity treatment.
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. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
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<110> Korea Advanced Institute of Science and Technology <120> Abc <130> PN150000 <160> 92 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Acaca <400> 1 cagtaacctg gtgaagctgg a 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Acaca <400> 2 gccagacatg ctggatctca t 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Acly <400> 3 ccctcttcag ccgacatacc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Acly <400> 4 ctgcttgtga tccccagtga 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Actb <400> 5 cagcttcttt gcagctcctt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Actb <400> 6 cttctccatg tcgtcccagt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Adipoq <400> 7 ctccacccaa gggaacttgt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Adipoq <400> 8 ggaccaagaa gacctgcatc 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Cidea <400> 9 gccgtgttaa ggaatctgct g 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Cidea <400> 10 tgctcttctg tatcgcccag t 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Cox8b <400> 11 gaaccatgaa gccaacgact 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Cox8b <400> 12 gcgaagttca cagtggttcc 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Cptla <400> 13 agctcgcaca ttacaaggac a 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Cptla <400> 14 ccagcacaaa gttgcaggac 20 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Cycs <400> 15 gcaagcataa gactggacca aa 22 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Cycs <400> 16 ttgttggcat ctgtgtaaga gaatc 25 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Dgat1 <400> 17 ggatctgagg tgccatcgtc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Dgat1 <400> 18 atcagcatca ccacacacca 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Dgat2 <400> 19 catcatcgtg gtgggaggtg 20 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Dgat2 <400> 20 tgggaaccag atcagctcca t 21 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Dio2 <400> 21 ttggggtagg gaatgttggc 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Dio2 <400> 22 tccgtttcct ctttccggtg 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Fabp4 <400> 23 aacaccgaga tttccttcaa 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Fabp4 <400> 24 tcacgccttt cataacacat 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Fasn <400> 25 aagcggtctg gaaagctgaa 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Fasn <400> 26 aggctgggtt gatacctcca 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Gpam <400> 27 ccacagagct gggaaaggtt 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Gpam <400> 28 gtgccttgtg tgcgtttcat 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Hsl <400> 29 aacgagacag gcctcagtgt 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Hsl <400> 30 gaatcggcca ccggtaaaga 20 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Htr1a <400> 31 tcagctacca agtgatcacc tct 23 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Htr1a <400> 32 gtccacttgt tgagcacctg 20 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Htr1b <400> 33 tgctcctcat cgccctctat g 21 <210> 34 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Htr1b <400> 34 ctagcggcca tgagtttctt ctt 23 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Htr1d <400> 35 cctccaacag atccctgaat g 21 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Htr1d <400> 36 cagagcaatg acacaga gat gca 23 <210> 37 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Htr1f <400> 37 tgtgagagag agctggatta tgg 23 <210> 38 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Htr1f <400> 38 tagttccttg gtgcctccag aa 22 <210> 39 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Htr2a <400> 39 agctgcagaa tgccaccaac tat 23 <210> 40 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Htr2a <400> 40 gggattggca tggatatacc tac 23 <210> 41 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Htr2b <400> 41 aaataagcca cctcaacgcc t 21 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Htr2b <400> 42 tcccgaaatg tcttattgaa gag 23 <210> 43 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Htr2c <400> 43 ttcttaatgt ccctagccat tgc 23 <210> 44 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Htr2c <400> 44 gcaatcttca tgatggcctt agt 23 <210> 45 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Htr3a <400> 45 aaatcagggc gagtgggagc tg 22 <210> 46 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Htr3a <400> 46 gacacgatga tgaggaagac tg 22 <210> 47 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Htr3b <400> 47 cgtgtggtac cgagaggttt 20 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Htr3b <400> 48 ggatgggctt gtggtttcta 20 <210> 49 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Htr4 <400> 49 atggacaaac ttgatgctaa tgtga 25 <210> 50 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Htr4 <400> 50 tcaccagcac cgaaaccagc a 21 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Htr5a <400> 51 gattgacttc agtgggctcg 20 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Htr5a <400> 52 aaagtcagga ctagcactcg 20 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Htr7 <400> 53 ctcggtgtgc tttgtcaaga 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Htr7 <400> 54 ttggccatac atttcccatt 20 <210> 55 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Lep <400> 55 acacacgcag tcggtatcc 19 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Lep <400> 56 gcagcacatt ttgggaaggc 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Lpin1 <400> 57 catacaaagg cagccacacg 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Lpin1 <400> 58 catacaaagg cagccacacg 20 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Maoa <400> 59 gcggtacaag ggtctgttcc 20 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Maoa <400> 60 cagccaatcc tgagatgccg 20 <210> 61 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Maob <400> 61 gggcggcatc tcaggtatgg 20 <210> 62 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Maob <400> 62 aagtcctgcc tcctacacgg 20 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Me1 <400> 63 gacccgcatc tcaacaagga 20 <210> 64 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Me1 <400> 64 caggagatac ctgtcgaagt ca 22 <210> 65 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Nrf1 <400> 65 cagcaaccct gatggcaccg tgtcg 25 <210> 66 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Nrf1 <400> 66 ggcctctgat gcttgcgtcg tctgg 25 <210> 67 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Plin1 <400> 67 ggtgttacag cgtggagagt a 21 <210> 68 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Plin1 <400> 68 tctggaagca ctcacaggtc 20 <210> 69 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Pparg <400> 69 ggtgtgatct taactgccgg a 21 <210> 70 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Pparg <400> 70 gcccaaacct gatggcattg 20 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Ppargc1a <400> 71 gcccaggtac gacagctatg 20 <210> 72 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Ppargc1a <400> 72 acggcgctct tcaattgctt 20 <210> 73 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Prdm16 <400> 73 agccctcgcc cacaacttgc 20 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Prdm16 <400> 74 tgacccccgg cttccgttca 20 <210> 75 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Scd1 <400> 75 agagtcagga gggcaggttt 20 <210> 76 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Scd1 <400> 76 gaactggaga tctcttggag ca 22 <210> 77 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Slc6a4 <400> 77 cgcagttccc agtacaagc 19 <210> 78 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Slc6a4 <400> 78 cgtgaaggag gagatgagg 19 <210> 79 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Srebf1 <400> 79 gtgggcctag tccgaagc 18 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Srebf1 <400> 80 ctggagcatg tcttcgatgt 20 <210> 81 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Tfam <400> 81 agttcccacg ctggtagtgt 20 <210> 82 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Tfam <400> 82 gcgcacatct cgaccc 16 <210> 83 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Tmem26 <400> 83 accctgtcat cccacagag 19 <210> 84 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Tmem26 <400> 84 tgtttggtgg agtcctaagg tc 22 <210> 85 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Tph1 <400> 85 accatgattg aagacaacaa ggag 24 <210> 86 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Tph1 <400> 86 tcaactgttc tcggctgatg 20 <210> 87 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Tph2 <400> 87 gccatgcagc ccgcaatgat gatg 24 <210> 88 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Tph2 <400> 88 caactgctgt cttgctgctc 20 <210> 89 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Ucp1 <400> 89 ctttgcctca ctcaggattg g 21 <210> 90 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Ucp1 <400> 90 ctttgcctca ctcaggattg g 21 <210> 91 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Ucp2 <400> 91 gtggtcggag ataccagagc 20 <210> 92 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Ucp2 <400> 92 gaggttggct ttcaggagag 20
Claims (16)
(a) contacting a test substance with TPH1 (Trypotophan hydroxylase 1), HTR2A (5-hydroxytryptamine 2A receptor) or HTR3 (5-hydroxytryptamine 3 receptor);
(b) analyzing whether the test substance inhibits TPH1, HTR2A or HTR3; When the test substance inhibits TPH1, HTR2A or HTR3, the test substance is determined to be a therapeutic agent for a metabolic disease.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20190061782A (en) * | 2017-11-28 | 2019-06-05 | 한국과학기술원 | A novel pharmaceutical composition for treating dystonia |
| US10898449B2 (en) | 2016-12-20 | 2021-01-26 | Lts Lohmann Therapie-Systeme Ag | Transdermal therapeutic system containing asenapine |
| US11033512B2 (en) | 2017-06-26 | 2021-06-15 | Lts Lohmann Therapie-Systeme Ag | Transdermal therapeutic system containing asenapine and silicone acrylic hybrid polymer |
| CN113967416A (en) * | 2021-10-29 | 2022-01-25 | 新疆医科大学第四附属医院 | Dissolving method and application of high-concentration p-chlorophenylalanine |
| US11278549B2 (en) | 2013-02-15 | 2022-03-22 | Mcmaster University | Method of treating obesity |
| US11337932B2 (en) | 2016-12-20 | 2022-05-24 | Lts Lohmann Therapie-Systeme Ag | Transdermal therapeutic system containing asenapine and polysiloxane or polyisobutylene |
| US11648213B2 (en) | 2018-06-20 | 2023-05-16 | Lts Lohmann Therapie-Systeme Ag | Transdermal therapeutic system containing asenapine |
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2015
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11278549B2 (en) | 2013-02-15 | 2022-03-22 | Mcmaster University | Method of treating obesity |
| US10898449B2 (en) | 2016-12-20 | 2021-01-26 | Lts Lohmann Therapie-Systeme Ag | Transdermal therapeutic system containing asenapine |
| US10980753B2 (en) | 2016-12-20 | 2021-04-20 | Lts Lohmann Therapie-Systeme Ag | Transdermal therapeutic system containing asenapine |
| US11337932B2 (en) | 2016-12-20 | 2022-05-24 | Lts Lohmann Therapie-Systeme Ag | Transdermal therapeutic system containing asenapine and polysiloxane or polyisobutylene |
| US11033512B2 (en) | 2017-06-26 | 2021-06-15 | Lts Lohmann Therapie-Systeme Ag | Transdermal therapeutic system containing asenapine and silicone acrylic hybrid polymer |
| KR20190061782A (en) * | 2017-11-28 | 2019-06-05 | 한국과학기술원 | A novel pharmaceutical composition for treating dystonia |
| US11648213B2 (en) | 2018-06-20 | 2023-05-16 | Lts Lohmann Therapie-Systeme Ag | Transdermal therapeutic system containing asenapine |
| CN113967416A (en) * | 2021-10-29 | 2022-01-25 | 新疆医科大学第四附属医院 | Dissolving method and application of high-concentration p-chlorophenylalanine |
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