WO2005073398A1

WO2005073398A1 - Method of assaying capability of inhibiting fat level increase

Info

Publication number: WO2005073398A1
Application number: PCT/JP2004/000976
Authority: WO
Inventors: Yasushi Hiramine; Syunsuke Takasuga; Hiroko Murakami
Original assignee: Sumitomo Chemical Company, Limited; Dainippon Sumitomo Pharma Co., Ltd.
Priority date: 2004-01-30
Filing date: 2004-01-30
Publication date: 2005-08-11

Abstract

A simple method of assaying the capability of inhibiting fat level increase that is indispensable for searching for a substance capable of inhibiting fat level increase in fat tissue. In particular, a method of assaying the capability of inhibiting fat level increase of a test substance, characterized in that the method comprises: step (1) of measuring the activity of monoacylglycerol acyltransferase in a system of monoacylglycerol acyltransferase and test substance brought into contact with each other, and step (2) of estimating the capability of inhibiting fat level increase of the substance on the basis of difference obtained by comparing the activity measured in the step (1) with reference activity.

Description

Method for testing ability to suppress fat mass increase

The present invention relates to a method for testing the ability to suppress an increase in fat mass, and the like. Light

Background art

Fat in the body can be broadly classified into fat, which is accumulated finely in cells of body tissues, and fat, which is present in blood.

For example, in the body of a mammal or the like, the former fat is used for (1) subcutaneous adipose tissue such as the abdomen, thigh, buttocks or chest, (2) fat tissue or intestine in contact with the kidney or epididymis, etc. It accumulates in various tissues such as membrane adipose tissue or abdominal adipose tissue such as omental adipose tissue, (3) adipose tissue contained in the liver, or (4) pleural adipose tissue. An increase in the amount of such tissue due to accumulation of fat in adipose tissue, for example, abdominal adipose tissue (especially, adipose tissue existing around blood vessels flowing into the portal vein such as mesenteric adipose tissue and omental adipose tissue) occurs. Closely related to the development of metabolic diseases such as impaired glucose tolerance, diabetes, hyperlipidemia, hypertension, and atherosclerosis, and cardiovascular disorders such as coronary artery disease, angina pectoris, and myocardial infarction, etc. Has been revealed. Therefore, by suppressing the accumulation of fat in adipose tissue, for example, in the thigh hollow fiber, and suppressing the increase in the amount of fat thread, the treatment or prevention of the above-mentioned diseases closely related to the accumulation of fat in adipose tissue can be achieved. It becomes possible. Fat in the blood is the release of fat synthesized in the cells into the blood. Specifically, the fat in the blood forms complex particles (lipoproteins) with a protein core called apoprotein. Circulating. In recent years, an increase in the amount of fat in the blood, that is, hypertriglyceridemia, is considered to be one of the risk factors for atherosclerotic diseases such as ischemic heart disease and cerebral infarction, as well as diabetes, hypertension or smoking. It became so. Thus, by suppressing the synthesis of fat in the organs that supply fat to the blood, specifically, the small intestine and liver, and suppressing the increase in the amount of fat in the blood, the amount of fat in the blood can be reduced. The treatment or prevention of the diseases closely related to the increase is enabled. To date, several methods have been known for finding substances having the ability to suppress the increase in fat mass (for example, WO04475454), It was not something.

Meanwhile, the following reaction:

2 or 1—Monoacyl's Receptor Port + Acil Coa

→ 1,2 or 1,3-Diacylglycerol + CoA

An enzyme that catalyzes the reaction of monoacylglycerol-lacyltransferase

The contribution of (hereinafter sometimes abbreviated to TMGAT) to fat accumulation and increase in blood fat mass by lipoprotein synthesis was unknown. The sequence of monoacylglycerol acyltransferase has been reported to date.

(RV Farese Jr. et al., Proceedings of National Academy of Science USA, P8512-8517, 2002) .These sequences are mostly expressed in the liver and small intestine, which are involved in lipoprotein synthesis and release of triglycerides into the blood. The sequence of the enzyme that works effectively in human organisms was not known. Disclosure of the invention

For this reason, there has been a demand for the development of a simple and excellent test method for the ability to inhibit the increase in fat mass, which is essential for searching for a substance having the ability to inhibit the increase in fat mass.

The present inventors have conducted intensive studies in such a situation, and found that a substance having a monoacylglycerose / retransferase (MGAT) P and harmful activity is capable of accumulating fat in adipocytes and liposome in the small intestine and liver. It has an effect of suppressing protein synthesis and has been found to be effective as a substance that suppresses an increase in fat mass. In addition, by simultaneously identifying the nucleotide sequence and amino acid sequence of a novel human, mouse, and rat MGAT, by inhibiting the MGAT enzyme activity comprising the sequence identified in the present invention, They have found that they can suppress an increase in fat mass in adipocytes or lipoprotein synthesis in small intestine or liver cells, and have completed the present invention. That is, the present invention

1. A method for testing the ability of the test substance to increase fat mass 0 inhibition,

(1) Contact system between monoacylglycerol lucyltransferase and test substance A first step of measuring the activity of the monoacylglycerol lucyltransferase in the

(2) a second step of evaluating the ability of the substance to suppress an increase in fat mass based on a difference obtained by comparing the activity measured in the first step with the activity in a control. Method;

2. A method for searching for a fat mass increase inhibiting substance, which comprises selecting a substance having a fat mass increase inhibiting ability based on the fat mass increasing inhibiting ability tested by the assay method described in Item 1;

3. An agent for suppressing an increase in fat mass, characterized in that the substance selected by the search method described in Item 2 or a pharmaceutically acceptable salt thereof is used as an active ingredient;

4. An agent for suppressing an increase in fat mass, characterized by comprising a substance having a monoacylglycerose lucyltransferase inhibiting ability or a pharmaceutically acceptable salt thereof as an active ingredient;

5. The assay method according to item 1, wherein the monoacylglycerol lucyltransferase is derived from a mammal;

6. The assay method according to item 1, wherein the monoacylglycerol lucyltransferase is any of the following proteins:

(a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, 3 or 11; (a-1) a protein consisting of the amino acid sequence represented by SEQ ID NO: 13, 15 or 17

(b) an amino acid sequence represented by SEQ ID NO: 1, 3 or 11 wherein one or more or a plurality of amino acids have been deleted, added or substituted, and have a monoacylglycerol luacyltransferase. Active protein

(b-1) an amino acid sequence represented by SEQ ID NO: 13, 15, 15 or 17, comprising one or more amino acids deleted, added or substituted, and a monosaccharide Protein with lurasyltransferase activity

(c) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, 3 or 11; Proteins with 'Shuella enzyme activity'

(c-1) a protein comprising an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, 15, or 17, and having a monoglycylglyceryltransferase activity

(d) a protein consisting of an amino acid sequence encoded by a DNA having the nucleotide sequence of SEQ ID NO: 2, 4, or 12

(d-1) a protein consisting of an amino acid sequence encoded by a DNA having the nucleotide sequence of SEQ ID NO: 14, 16, or 18

(e) an amino acid sequence encoded by a DNA having a nucleotide sequence having 80% or more sequence identity with a DNA having the nucleotide sequence of SEQ ID NO: 2, 4 or 12, and a monoglycylglycerol "Siltransferase activity" | A protein with life

(e-1) an amino acid sequence encoded by a DNA having a nucleotide sequence having 80% or more sequence identity with a DNA having a nucleotide sequence represented by SEQ ID NO: 14, 16 or 18, and Protein with lucyltransferase activity

(f) a DNA comprising a nucleotide sequence complementary to a DNA having a nucleotide sequence represented by SEQ ID NO: 2, 4 or 12, and an amino acid sequence encoded by a DNA that hybridizes under stringent conditions, And a protein having monoglycosyltransylase activity

(f-1) consists of a DNA consisting of a base sequence complementary to a DNA having the base sequence of SEQ ID NO: 14, 16 or 18, and an amino acid sequence encoded by a DNA that hybridizes under stringent conditions , And a protein having a monoacylglycerol acyltransferase activity ·

7. The test method according to any one of Items 1, 5 and 6, wherein the fat mass increase suppressing ability is a fat accumulation suppressing ability;

8. A method for searching for a fat accumulation inhibiting substance, which comprises selecting a substance having a fat accumulation inhibiting ability based on the fat accumulation inhibiting ability tested by the assay method described in Item 7; 9. A fat accumulation inhibitor characterized by comprising, as an active ingredient, a substance selected by the search method described in Item 8 or a pharmaceutically acceptable salt thereof;

10. A fat accumulation inhibitor comprising a substance having a monoacylglycerolacyltransferase inhibitory ability or a pharmaceutically acceptable salt thereof as an active ingredient;

11. The assay method according to any one of Items 1, 5, and 6, wherein the ability to suppress an increase in fat mass is an ability to suppress lipoprotein synthesis;

12. A method for searching for a lipoprotein synthesis-inhibiting substance, which comprises selecting a substance having lipoprotein synthesis-inhibiting ability based on the lipoprotein synthesis-inhibiting ability tested by the assay method described in Item 11;

13. A lipoprotein synthesis inhibitor comprising, as an active ingredient, a substance selected by the search method described in Item 12 or a pharmaceutically acceptable salt thereof;

14. A lipoprotein synthesis inhibitor comprising, as an active ingredient, a substance having an ability to inhibit monoacylglycerol luasyltransferase or a pharmaceutically acceptable salt thereof;

15. The substance having the ability to inhibit monoacylglycerolacyltransferase is N- [2- (4-benzinole-1-ethyl / lephenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [ [15] The lipoprotein synthesis inhibitor according to [14], which is 1,5-a] pyrimidine-17-amine;

16. N- [2- (4-benzyl-1-2-ethynolephenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1,5-a] pyrimidin-1-7-amine A monoacylglycerol lucyltransferase inhibitor, which is contained as an active ingredient;

17. A protein characterized by having one of the following amino acid sequences;

(a) the amino acid sequence represented by SEQ ID NO: 1, 3, or 11

(a-1) the amino acid sequence represented by SEQ ID NO: 13, 15, or 17

(b) an amino acid sequence represented by SEQ ID NO: 1, 3 or 11, wherein one or more amino acids or a plurality of amino acids are deleted, added or substituted; W

6

The amino acid sequence of a protein having monoasinoglycerol transferase activity

(b-1) an amino acid sequence represented by SEQ ID NO: 13, 15 or 17, wherein one or more amino acids are deleted, added or substituted, and Glycerol acyltransferase activity

(c) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, 3, or 11, and an amino acid sequence of a protein having a mono-acylglycerol-l-acyltransferase activity

(c-1) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, 15, or 17, and having a monosylglycerose lucyltransferase activity; Array

(d) the amino acid sequence encoded by the DNA having the nucleotide sequence represented by SEQ ID NO: 2, 4 or 12.

(d-1) an amino acid sequence encoded by a DNA having the nucleotide sequence of SEQ ID NO: 14, 16, or 18

(e) an amino acid sequence encoded by a DNA having a nucleotide sequence having a sequence identity of 80% or more with a DNA having a nucleotide sequence represented by SEQ ID NO: 2, 4 or 12; Amino acid sequence of a protein with oral luucyltransferase activity

(e-1) an amino acid sequence encoded by a DNA having a nucleotide sequence having 80% or more sequence identity with DNA having a nucleotide sequence represented by SEQ ID NO: 14, 16 or 18; Amino acid sequence of proteins having oral-lucyltransferase activity

(f) a DNA consisting of a nucleotide sequence complementary to a DNA having the nucleotide sequence shown in SEQ ID NO: 2, 4, or 12, and an amino acid sequence encoded by a DNA that hybridizes under stringent conditions, Amino acid sequence of a protein having monoacylglycerolacyltransferase activity

(f-1) a DNA having the base sequence of SEQ ID NO: 14, 16 or 18; Amino acid sequence of a protein comprising a DNA consisting of a complementary nucleotide sequence and an amino acid sequence encoded by DNA that hybridizes under stringent conditions and having monoacylglycerol lucyltransferase activity

18. A gene characterized by having a nucleotide sequence encoding any of the following amino acid sequences;

Amino acid sequence group>

(a) the amino acid sequence represented by SEQ ID NO: 1, 3 or 11

(a-1) the amino acid sequence represented by SEQ ID NO: 13, 15 or 17

(b) an amino acid sequence represented by SEQ ID NO: 1, 3 or 11 in which one or more or a plurality of amino acids have been deleted, added or substituted; Amino acid sequence of active protein

(b-1) an amino acid sequence represented by SEQ ID NO: 13, 15, or 17, wherein one or several or more amino acids are deleted, added or substituted, and Amino acid sequence of protein having monoacylglycerol luacyltransferase activity

(c) Amino acid of a protein which is an amino acid sequence having an amino acid sequence represented by SEQ ID NO: 1, 3 or 11 and a sequence having an H "sequence of 80 ° / ₀ or more, and which has a monoacylglycerol luciferase activity. Acid sequence

(c-1) an amino acid sequence having a sequence identity of 80% or more to the amino acid sequence represented by SEQ ID NOS: 13, 15, or 17, wherein Amino acid sequence of a protein having

(d) the amino acid sequence encoded by the DNA having the nucleotide sequence represented by SEQ ID NO: 2, 4 or 12 (I

(d-1) Amino acid sequence encoded by DNA having the nucleotide sequence shown in SEQ ID NO: 14, 16 or 18! 1

(e) an amino acid sequence encoded by a DNA having a nucleotide sequence having a sequence identity of 80% or more with the DNA having the nucleotide sequence represented by SEQ ID NO: 2, 4 or 12, and Has monoacylglycerol lucyltransferase activity Amino acid sequence of protein

(e-1) an amino acid sequence encoded by a DNA having a nucleotide sequence having 80% or more sequence identity with the DNA having the nucleotide sequence represented by SEQ ID NO: 14, 16, or 18, and Amino acid sequence of protein having activity of silglycerol asinotransferase

(f) an amino acid sequence encoded by a DNA having a nucleotide sequence complementary to the DNA having the nucleotide sequence of SEQ ID NO: 2, 4 or 12, and a DNA that hybridizes under stringent conditions. Amino acid sequence of a protein having a monosacylglycerol acyltransferase activity

(f-1) an amino acid sequence encoded by a DNA having a nucleotide sequence complementary to the DNA having the nucleotide sequence of SEQ ID NO: 14, 16, or 18, and a DNA that hybridizes under stringent conditions. Amino acid sequence of a protein having monoacylglycerol lucinoletransferase activity

19. A catalyst for the following reaction, comprising the protein according to item 17.

2 or 1—monoacylglycerol + acyl-one CoA → 1,2-diacylglycerol + CoA;

And so on. Brief Description of Drawings

Figure 1: Using the mouse AT12 sequence as a probe, Northern hybridization was performed on mouse Adu1tNormal1Tissue mRNA NorthernBlot. From left lane, brain, heart, kidney, liver, lung. Tissue RNA of skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus.

Figure 2: Using the mouse AT14 sequence as a probe, northern predication was performed on mouse Adu1tNormal1Tissue mRNA NorthernBlot. From left lane, brain, heart, kidney, liver, lung. Tissue RNA of skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus.

Figure 3: Using the human AT12 sequence as a probe, Northern against human Adult Norma 1 Tissue Total RNA No rthern B lot. A hybridization was performed. In order from the left lane, tissue RNA of esophagus, stomach, small intestine, colon, uterus, placenta, bladder, and fat. On the left of the figure, the gender (male = M, female-F) and age of the specimen from each tissue are shown.

Figure 4: Using the human AT14 sequence as a prop, Northern hybridization was performed on human AdlntNoRmTlssueTotalRNANorthernBlOT. Starting from the left lane, they are tissue RNA from the esophagus, stomach, small intestine, colon, ova, placenta, bladder, and fat. On the left of the figure, the gender (male-M, female-F) and age of the specimen from each tissue are shown.

Figure 5: The mice were divided into two groups (group of 8), the first group was administered only olive oil and the second group was administered olive oil and Compound A. Approximately 101 blood samples were collected from the tail vein every hour from immediately after administration to 6 hours, and the amount of triglyceride in the blood was determined. The quantification results are shown in a graph in which the vertical axis plots the blood triglyceride concentration (mg / dL) and the horizontal axis plots time.

Figure 6: Divide mice into 3 groups (8 / group), group 1 only olive oil, group 2 olive oil + compound A: 1 Omg / kg, group 3 olive About 10 t1 of blood was collected from the tail vein before administration (Ohr) and 3 hours (3 hr) after administration for each group, and trig in the blood was administered. The amount of reseride was determined The measured triglyceride concentration (mg / dL) is shown on the vertical axis.

As used herein, the term “fat” refers to fat contained in any cell, tissue or body fluid in a living body of a mammal, and specifically refers to a glycerol fatty acid ester such as triglyceride.

The term “fat mass increase suppression ability” is a concept that includes both fat accumulation suppression ability and fat synthesis suppression ability, and specifically, the ability to suppress fat accumulation in fat cells, adipose tissue or hepatocytes, or It means the ability to suppress fat synthesis in the small intestine or liver.

“Adipose tissue” refers to any adipose tissue of a mammal. Subcutaneous adipose tissue such as the side, thigh, buttocks, or chest; (2) adipose tissue in contact with the kidney or epididymis, mesenteric adipose tissue, or abdominal adipose tissue such as the omentum adipose tissue; 3) Adipose tissue contained in the liver, or (4) Intrathoracic adipose tissue. Fat cells refer to the individual cells that make up adipose tissue.

In adipose tissue, glycerol fatty acid esters such as triglyceride accumulate as fat.

On the other hand, fats in blood exist in the form of lipoproteins in which glycerol fatty acid esters such as triglycerides form complexes with apoproteins. The lipoproteins are classified according to their densities into chylomicron, VLDL (very low density lipoprotein), LDL (low density lipoprotein), HDL (high density lipoprotein) and the like.

The tissues supplying triglyceride to the blood are the small intestine and the liver. The small intestine controls triglyceride absorption from food, and the liver regulates the amount of triglyceride in the fluid by collecting and re-secreting lipoproteins in the blood. In the small intestine, tridaliseride in food is decomposed into monoacylglycerol and fatty acids in the intestinal tract by the action of knee lipase, then absorbed by small intestinal epithelial cells, and recombined into triglyceride in small intestinal cells . The resynthesized triglyceride is incorporated into chylomicron, a type of lipoprotein, in small intestinal cells and secreted into the blood. Chylomicron is converted to particles called chiral micron remnants while supplying triglyceride to peripheral tissues, and is eventually absorbed by the liver. In the moon dried pancreas, triglyceride synthesis is performed using fatty acids in liver cells, and the synthesized tridalicelide is incorporated into VLDL, a type of lipoprotein, and secreted into the blood. VLDL, like chylomicron, circulates through the body while supplying triglyceride to peripheral tissues, and is finally recovered in the liver in the form of LDL.

That is, the above-mentioned ability to inhibit fat synthesis preferably refers to the ability to inhibit lipoprotein synthesis in the small intestine or liver.

The monoacylglycerol lysyltransferase used in the assay method of the present invention is an enzyme that catalyzes the following reaction (hereinafter sometimes referred to as the present enzyme):

2 or 1—Mono-Asino-Leg Sereno-Lauren + Asino-La Co o = 1, 2, Noreglycerum + CoA.

The activity of such an enzyme can be measured by the following method, for example, the method of S.C. Jamdar et al. (Arch. Biochem. Biophys. 1992; 296 (2): 419-25).

Enzyme sample containing this enzyme was mixed with 150 / i L of reaction buffer (24 mM Tris-hydrochloric acid (H7.5), 50 mM potassium chloride, 8 mM magnesium sulfate, 1.25 mg / mL ゥ serum albumin, lmM DTT , 0.5 mM 2-monooleoylglycerol), and add 5 μL of 900 (0.2 MB q / μL) ⁴ C] palmitolucoenzyme Α aqueous solution to the suspension. Incubate the mixture at room temperature for 10 minutes. The reaction is stopped by adding 300 L of heptane / 2-propanol / water (= 80: 20: 2) to the mixture and stirring. After standing for 10 minutes, 200 μL of heptane and 100 μL of water are further added to the mixture, followed by stirring. After that, the reaction product contained in the organic layer obtained by centrifuging the mixture is separated by thin-layer chromatography, and detected and quantified using a bio-imaging analyzer system BAS-2000 (Fuji Film Co., Ltd.) or the like. I do.

As the present enzyme, monoacylglycerolacyltransferase derived from mammals such as humans, mice, and rats is preferred. Also, for example, (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, 3 or 11, (b1) an amino acid sequence S represented by SEQ ID NO: 1 (single letter code:

In MAVTVEEAPWLGWIVA ALMRF Keio VAmVAIPSYICYVIILQPLRVLDSKRFWYIEGL thigh WLLG thigh Sff JidaburyuwaieijiwaitibuiemuidaburyujiidiaikeieiaieikeidiieibuiemuLVNHQATGDVCTLMMCLQDKGPWAQ orchid LMDHIFKYTNFGIVSLIH GDFFIRQG YRDQQLLVLKKHLEHNYRSRDR IVLFPEGGFLRKRRETSQAFAKK LPFLTHVTLPRFG ATNIILKALVARQENGSPAGGDARGLECKSRGLQWIIDTTIAYPKAEPIDIQTflLGYRKPTVTHVHYRIFP IGDVPLETEDLTSWLYQRFIEKEDLLSHFYKTGAFPPPQGQKEAVCREMTLSNMWIFLIQSFAFLSGYLWYH IIQYFYHCLF), 1 or several or more amino acids are deleted, added if Ku consists Amino acid sequence replaced, the force one Monoashi (B2) an amino acid sequence represented by SEQ ID NO: 3 (single-letter code: In MAITLEEAPWLGWLLVKALMRFAF WmVAIPSYICYVIILQPLRVLDSKRFWYIEGI YKWLLG fragility SW Goruden'wikuYAGYTVMEWGEDIKAVSKDEAVML image QATGDVCTLMMCLQDKGLWAQMMWL s Awakening IFKYTNFGIVSLVH GDFFIRQGRSYRDQQLLLL thigh LE anonymous RSRD 丽 IVLFPEGGFL belly RETSQAFAKKNNl jNVTLPRSG AT IimLVAQQKNGSPAGGDAKELDSKSKGLQWIIDTTIAYPKAEPIDIQTWILGYRKPTVTHVHYRIFP IKDVPLETDDLTTWLYQRFVEKEDLLSHFYETGAFPPSKGHKEAVSREMTLSNLWIFLIQSFAFLSGYMWY IIQYFYHCLF), from one or several or more amino acids are deleted, added if Ku is Amino acid sequence replaced And (b3) an amino acid sequence represented by SEQ ID NO: 11 (single-letter code:

In VTVEEAPWLGWIVAKALMRFAFMVAmVAIPSYICYAIILQPLRVLDSKRF YIEGLMYKWLLGMVASW GWYAGYTVMEWGEDIKAIAKDEAVML 丽 QATGDVCTLMMCLQDKGPWAQMMWL negation IFKYT FGIVSLIH GDFFIRQG YRDQQLLVLKKHLEHNYRSRDRKWIVLFPEGGFLRKRlffiTSQAFAKKmPFLTHVTLPRFR ATNIILKALVARQENGSPAGGDARGLECKSRGLQ IIDTTIAYPKAEPIDIQTWILGY PTnHVHY¾IFP IADVPLETEDLTNWLYQRFIEKEDLLSHFYKTGAFPPPQGQKEAVSRAMTLSNVWILLIQSFAFLSGYLWYH VIQYFYHCLF), 1 or several or more amino acids are deleted, added if Ku consists Amino acid sequence replaced, and Monoashirugurise port Ruashirutora transferase activity (B4) an amino acid sequence represented by SEQ ID NO: 13;

In MVSWKGIYFILFLFAGSFFGSIFMLGPILPLMFINLSWYRWISSRLVATWLTLPVALLETMFGVRWITGDA FVPGERSVIIMNHRTRVDWMFLWNCLMRYSYLRVEKICLKSSLKSVPGFGWAMQVMFIFIHRKWKDDKSHF EDMIDYFCAIHEPLQLLIFPEGTDLTENmRSlWFAEKNGLQKYEWLHPRTTGFTFVVDRLREGKNLDAV HDITVAYPYNIPQTEKHLLLGDFPKEIHFHVQRYPADSLPTSKEDLQLWCHRRWEEKEERLRSFYQGEKNFH FTGQSTVPPCKSELRVLWKLLSIVYWALFCSAMCLLIYLYSPVRWYFIISIVFFVLQERIFGGLEI IELAC YRFLHKHPHLNSKKNE), 1 or several or a plurality of amino acids are deleted, with pressurized or substituted Amino acid sequences, and protein having a Monoashirugurise port ^ "Ruashi transferase activity, (b 5 ) Amino acid sequence represented by SEQ ID NO: 15 (single letter code:

MVSMGIYFILTLFWGSFFGSIFMLSPFLPL FVNPSWYRWINNRLVATWLTLPVALLETMFGVKVIITGDA FVPGE SVIIM HRT DWMFLWCLMRYSYL LEKICL SLKGWGFGWA QAMYIFIH1¾WKDDKSHF In ED IDYFCDIHEPLQLLIFPEGTDLTENSKSRSNAFAEK GLQ YEYVLHPRTTGFTFVVDRLREGKNLDAV HDlTVAYPH IPQSEmLQGDFPREIHFHVHRYPIDTLPTSKEDLQLWCHKRWEEKEERLRSFYQGEKNFY FTGQSVIPPCKSELRVLWKLLSILYWTLFSPAMCLLIYLYSLVKWYFIITIVIFVLQERIFGGLEIIELAC YRLLHKQPHLNSKKNE), 1 or several or more amino acids is deleted, with pressurized or substituted Amino acid sequences, proteins with Monoashirugurise port Ruashi transferase activity Tsu force, (b 6 ) Amino acid sequence represented by SEQ ID NO: 17 (single letter code:

MS GIYFILFLFAGSFFGSIFMLGPILPLMFINLSWYRWISSRLVAMWLTLPVALLETMFGVKWITGDA FVPGERSVIIMraffiTRVDW FLMCLMRYSYLRLEKICLKSSLKSVPGFG AMQVAAFIFIHRKWKDD SHF EDWDYFCAIHEPLQLLIFPEGTDLTE KARS ^ FAEKNGLQ YEWLHPRTTGFTFVVDRLRERRNLDAV HDITVAYPYNIPQTEKHLLLGDFPKEIHFHVHRYPVDTLPTSKEDLQLWCHKRWEEKEERLRSFYQGEK FH FTGQSTVPPCKSELRVLWKLLSIVYWALFCSAMCLLIYLYSPVRWYFIISIVFFVLQE IFGRLEIIELAC in YRFLHKHPHLNSKKNE), consists of one or several or more amino acids are deleted, with pressurized or substituted Amino acid sequence and Monoashirugurise port Ruashi transferase activity (Cl) a protein consisting of an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 1 and having a monoglycylglycerol lucyltransferase activity; c 2) Amino acid sequence having 80 ° / ₀ or more sequence identity with the amino acid sequence represented by SEQ ID NO: 3 (C3) a protein having an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 11; and (C4) a protein having an amino acid sequence having a sequence identity of 80% or more with the amino acid sequence represented by SEQ ID NO: 13 and having a monoacylglycerolacyltransferase activity A protein comprising ( _C 5) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 15, and having a strong monoacylglycerol lucyltransferase activity. ) Consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence represented by SEQ ID NO: 17, And (d) a DNA having a nucleotide sequence represented by SEQ ID NO: 2 (D 2) a protein consisting of an amino acid sequence encoded by a DNA having the nucleotide sequence represented by SEQ ID NO: 4, (d 3) a protein consisting of the amino acid sequence represented by SEQ ID NO: 12 A protein consisting of an amino acid sequence encoded by DNA; (d4) a protein consisting of an amino acid sequence encoded by DNA having the nucleotide sequence shown by SEQ ID NO: 14; (d5) a nucleotide sequence shown by SEQ ID NO: 16 A protein consisting of an amino acid sequence encoded by DNA having the following sequence: (d6) a protein consisting of an amino acid sequence encoded by a DNA having the nucleotide sequence shown by SEQ ID NO: 18, (e1) a protein consisting of an amino acid sequence represented by SEQ ID NO: 2 Consisting of an amino acid sequence encoded by DNA having a nucleotide sequence having 80% or more sequence identity with DNA having a nucleotide sequence of A protein having a retransferase activity, (e2) an amino acid sequence encoded by a DNA having a nucleotide sequence having 80% or more sequence identity with a DNA having a nucleotide sequence represented by SEQ ID NO: 4, (E3) a DNA having a nucleotide sequence having 80% or more sequence identity with a DNA having a nucleotide sequence represented by SEQ ID NO: 12; (E4) a protein having a strong monoacylglycerol lysyltransferase activity and comprising a nucleotide sequence represented by SEQ ID NO: 14 and having a sequence identity of 80% or more A protein consisting of an amino acid sequence coded by a DNA having a base sequence having No. 16 consisting of an amino acid sequence encoded by a DNA having a nucleotide sequence having 80% or more sequence identity to a DNA having the nucleotide sequence shown in No. 16 A protein having an activity; (e6) an amino acid sequence encoded by a DNA having a nucleotide sequence having 80% or more sequence identity with DNA having a nucleotide sequence represented by SEQ ID NO: 18; A protein having an oral lucyltransferase activity, (f1) encoded by a DNA having a nucleotide sequence complementary to the DNA having the nucleotide sequence of SEQ ID NO: 2, and a DNA that hybridizes under stringent conditions A protein consisting of an amino acid sequence and having an activity of monoacylglycerolacyltransferase; DNA consisting of a base sequence complementary to D having the base sequence shown in No. 4 and an amino acid sequence encoded by DNA that hybridizes under stringent conditions. A protein having a transferase activity, (ί3) a DNA having a nucleotide sequence complementary to DΝ having a nucleotide sequence represented by SEQ ID NO: 12, and an amino acid encoded by a DNA that hybridizes under stringent conditions (F4) a DNA having a nucleotide sequence complementary to the DNA having the nucleotide sequence shown in SEQ ID NO: 14, and a protein having a nucleotide sequence complementary to the DNA having the nucleotide sequence of SEQ ID NO: 14. Consists of an amino acid sequence encoded by DNA that hybridizes with A protein having a transferase activity; (f5) an amino acid encoded by a DNA having a base sequence complementary to a DNA having a base sequence represented by SEQ ID NO: 16, and a DNA hybridizing under stringent conditions (F6) a DNA having a base sequence complementary to a DNA having a base sequence represented by SEQ ID NO: 18, and a protein having a monosaccharide glycerol acyltransferase activity; A protein consisting of an amino acid sequence encoded by a DNA hybridizing with the above and having a monoacylglycerol lucyltransferase activity can also be mentioned as a preferable example.

Here, the "deletion, addition or substitution of amino acid" in (b), (bl), (b2), (b3), (b4), (b5). c), (c 1),

(c 2), (c 3), (c 4), (c 5), (c 6) and (e), (el), (e 2), (e 3), (e 4), (e 5) The sequence identity of Γ80% or more in (e 6) includes, for example, a protein having an amino acid sequence represented by SEQ ID NO: 1, 3, 11, 13, 15, or 17 It includes mutations that occur naturally due to processing, species differences, individual differences, differences between tissues, etc. of the organism from which the protein is derived, and artificial amino acid mutations.

In the above (b), (bl), (b2), (b3), (b4), (b5), and (b6), the "deletion, addition or substitution J of amino acid (hereinafter referred to as amino acid (Also referred to as modification) as a method of artificially performing, for example, SEQ ID NO: 1, 3, There is a method in which conventional site-specific mutagenesis is performed on DM encoding the amino acid sequence represented by 11, 13, 15, or 17 and then this DNA is expressed by a conventional method. Examples of the site-directed mutagenesis method include a method using amber mutation (gapped 'duplex method, Nucleic Acids Res., 12, 9441-9456 (1984)), and a method using PCR using mutagenesis primers. And the like. The number of amino acids modified as described above is at least one residue, specifically, one or several, or more. The number of such modifications may be within a range in which monoacylglycerol transacylase activity can be found. Of the above-mentioned deletions, additions or substitutions, modifications relating to amino acid substitutions are particularly preferred. The substitution is more preferably an amino acid having similar properties such as hydrophobicity, charge, p, and steric structure. Such substitutions include, for example, ① glycine, alanine; ② parin, isoleucine, leucine; ③ aspartic acid, glutamic acid, asparagine, glutamine; ② serine, threoyun; ② lysine, arginine; Substitution within the group of Yun, Tyrosine.

In the present invention, “sequence identity 14” refers to sequence identity and homology between two DNAs or two proteins. The “sequence identity” is determined by comparing two sequences that are optimally aligned over the region of the sequence to be compared. Here, the DNA or protein to be compared may have an addition or deletion (for example, a gap or the like) in the optimal alignment of the two sequences. Such sequence identity can be calculated, for example, by creating an alignment using ClustalWalgorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)) using Vector NTI. Can be. The sequence identity is measured using sequence analysis software, specifically, an analysis tool provided by Vector NTI, GENET II, or a public database. The public database is generally available, for example, at homepage address http: \ ww.ddbj.nig.ac.jp.

The sequence identity in the present invention may be 80% or more, preferably 90% or more, and more preferably 95% or more. (F), (f 1). (F 2), (f 3), (f 4), (f 5), (f 6) “Hybridize under stringent conditions” The hybridization used in, for example, Sambrook J "., Frisch EF, Maniatis T., Molecular Cloning 2nd edition, published by Cold Spring Harper Laboratory (Cold Spring)

Harbor Laboratory press) and the like. "Stringent conditions" refers to, for example, a solution containing 6XSSC (a solution containing 1.5M NaCl, 0.15M trisodium citrate is 10XSSC) and a solution containing 50% formamide at 45 ° C. After forming a hybrid at, conditions such as washing at 50 ° C with 2X SSC (Molecular Biology, John Wiley & Sons,

N. Y. (1989), 6.3.1-6.3.6). For example, the salt concentration in the washing step ranges from 50 ° C (low stringency conditions) at 2 XSSC to 50 at 0.2 XSSC. You can select from conditions up to C (high stringency conditions). The temperature in the washing step can be selected, for example, from room temperature (low stringency conditions) to 65 ° C (high stringency conditions). It is also possible to change both the salt concentration and.

The method for preparing the present enzyme will be described below.

When the present enzyme is in the form of an enzyme sample containing the present enzyme, for example, a microsomal fraction, the present enzyme is prepared by a normal cell fractionation method using an adipose tissue containing the present enzyme as a biomaterial. do it. Specifically, for example, the method described in Wang Fang Cao et al. (Archives of Biochemistry and Biophysics, 296 (2), 419-425 (1992)) can be mentioned.

Also, using a conventional genetic engineering method, a gene consisting of a nucleotide sequence encoding the amino acid sequence of the present enzyme (hereinafter sometimes referred to as the present gene), for example, (I) SEQ ID NO: 1, 3 , A nucleotide sequence encoding the amino acid sequence represented by 11, 13, 15 or 17; (II) a nucleotide sequence represented by SEQ ID NO: 2, 4, 12, 14, 16 or 18; The offspring can be prepared using conventional genetic engineering techniques (eg, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition, Cold Springno, One Bar). Laboratory acquisition (method described in Cold Spring Harbor Laboratory press). Next, using the obtained present gene, the present enzyme is produced and obtained according to a usual genetic engineering method. Thus, the present enzyme can be prepared.

For example, a culture obtained by preparing a plasmid capable of expressing the present gene in a host cell, introducing it into a host cell, transforming the cell, and culturing the transformed host cell (transformant). The enzyme may be obtained from a product. The above-mentioned plasmid contains, for example, genetic information that can be replicated in a host cell and can be propagated autonomously, is easily isolated and purified from the host cell, and can function in the host cell Preferably, an expression vector having a simple promoter and a detectable marker into which a gene comprising a base sequence encoding the amino acid sequence of the present enzyme has been introduced. Various types of expression vectors are commercially available. For example, vectors used for expression in mammalian cells include the SV40 virus promoter, cytomegalovirus promoter (CMV promoter), Raus Sarcoma Virus promoter (HSV promoter), actin gene promoter ^ ", aP This is an effort vector containing a promoter such as a two-gene promoter, which is commercially available from Toyobo, Takara Shuzo, or the like.

Examples of the host cell include a prokaryotic or eukaryotic microbial cell, an insect cell, a mammalian cell, and the like. For example, mammalian cells and the like can be preferably mentioned from the viewpoint that the original structure of the present enzyme can be expected to be maintained.

The plasmid obtained as described above can be introduced into the host cell by a conventional genetic engineering method.

The transformant can be cultured by a conventional method used for culturing microorganisms, insect cells or mammalian cells. For example, in the case of mammalian cells, culture is performed in a medium containing an appropriate carbon source, a nitrogen source, micronutrients such as vitamins, and cell growth factors such as blood serum. The culture method may be any of solid culture and liquid culture, and preferably includes liquid culture such as aeration and agitation culture. This enzyme may be obtained by combining methods generally used for isolation and purification of general proteins. For example, transformants obtained by culturing tins are collected by centrifugation or the like, and the transformants are crushed or dissolved, proteins are solubilized if necessary, and various types of ion exchange, hydrophobicity, gel filtration, etc. are performed. Purification may be performed by using the steps using chromatography alone or in combination. In addition, affinity purification may be performed by adding an amino acid sequence suitable for affinity purification to the N-terminus or C-terminus of the present enzyme by ordinary genetic engineering techniques. If necessary, an operation for restoring the higher-order structure of the purified protein may be further performed.

The assay method of the present invention comprises: (1) a first step of measuring the activity of monoacylglycerol acyltransferase in a contact system between monoacylglycerol acyltransferase and a test substance; and (2) a first step. A second step of evaluating the ability of the substance to suppress an increase in fat mass based on the difference obtained by comparing the activity measured by the above with the activity in the control.

In the assay method of the present invention, without directly measuring the amount of fat, for example, by calculating an inhibition rate (which will be described in detail later) as an index for evaluating the enzyme inhibitory ability, the fat of the test substance can be calculated. It is simple and suitable for primary screening, etc., because the ability to suppress the amount of B intensification can be evaluated.

In order to enhance the sensitivity of the assay method of the present invention, for example, a divalent metal cation such as magnesium ion, dithiothreitol (hereinafter referred to as DTT), etc., is added to the contact system in the first step. Good. For example, in the case of magnesium ions, the added concentration is, for example, 2 mM or more, and preferably 5 niM to 10 mM. In the case of DTT, the added concentration is, for example, 50 μM or more, and preferably 0.2 mM to 2 mM. In order to increase the reactivity of the substrate, phosphatidicolin (hereinafter sometimes referred to as PC) ゃ phosphatidinoreserin (hereinafter sometimes referred to as PS) and the like are added to the above contact system. Is also good. For example, in the case of PC or PS, the added concentration is, for example, 5 μg or more, preferably 10 / mL to 50 μg / 111).

The first step of the assay method of the present invention comprises: (A) two kinds of substrates (for example, 2 or 1-mono A form of contacting the test substance with a test substance, such as a glycylglycerol (preferably, 2-monoacinoreglycerol) and acetyl-CoA); and (B) two types of substrates (for example, 2 or 1-Monoacylglycerol and / or acyl-CoA) and the test substance are in contact with the enzyme (i.e., the test substance is used as the other of the above two substrates). When handling), either of the forms.

Furthermore, in the first step of the present Eimei assay method, the order in which the enzyme and the test substance are brought into contact with the enzyme is as follows: (a) Contact the enzyme with the test substance first, incubate for a certain period of time, A form in which one kind of substrate is added, (b) a form in which the enzyme, the test substance and two or one kind of substrate are contacted at the same time, and (c) a form in which this enzyme and two kinds or one kind of substrate are used. Any of the following forms may be used: first, contact, incubation for a certain period of time, and then addition of a test substance, but the form (a) is preferred.

In the case of the above (a), the contact time between the present enzyme and the test substance may be, for example, 1 minute or more, preferably 1 minute to 1 hour. As the heat retention temperature in the contact system, for example, 0 ° C to 70 ° C, preferably 4 ° C to 40 ° C can be mentioned.

In the case of the above (c), as the heat retaining temperature in the contact system, for example, 0 ° C to 70 ° C, preferably 4 ° C to 40 ° C can be mentioned.

The concentration of the test substance in the first step of the assay method of the present invention is, for example, 1 ηΜ to 10 mM, and preferably 0.01 / zM to 5 mM. The concentration of the substrate is, for example, 1 nM to 1 mM, preferably 5 ^ M to 100 M.

The form of the enzyme to be brought into contact with the test substance in the first step of the assay method of the present invention may be (A) a purified product or a crudely purified product of the enzyme, or (B) an intracellular form. Contacting a test substance with a transformed cell (hereinafter, sometimes referred to as a transformed cell) into which a gene consisting of a nucleotide sequence encoding the amino acid sequence of the present enzyme is introduced. Or the like). Further, the cells may be cells separated from the tissue or cells forming a group having the same functional form. For example, in the case of the above (A), the concentration of the enzyme to be brought into contact with the test substance is, for example, 1 ng / m 1 or more, and preferably 10 ng / m 1 or more.

In the case of the above (B), the concentration of the present enzyme to be brought into contact with the test substance is, for example, 1 × 10 ³ cells or more, preferably 1 × 10 ⁴ cells or more, as the concentration of the transformed cells. l or more.

This transformed cell can be prepared as follows.

Plasmids are prepared by inserting the present gene into a vector that can be used in cells into which the present gene is to be introduced in such a manner that it can be expressed and connected to a promoter, using ordinary genetic engineering techniques. The promoter used here may be any promoter that can function in the cell into which the present gene is to be introduced. When the cell is an animal cell, the SV40 virus promoter, the cytomegalovirus promoter-1 (CMV Promoter, Raus Sarcoma Virus promoter (RSV promoter), J3 actin gene promoter, aP2 gene promoter and the like. A commercially available vector containing such a promoter upstream of the multiple cloning site may be used.

Next, the plasmid is introduced into cells. Examples of the method of introduction into cells include the canoleic acid phosphate method, the electroporation method, the DAE dextran method, and the Myseno 1 ^ synthesis method. The calcium phosphate method is described in Grimm, S. et al., Proc. Natl. Acad. Sci. USA, 93, 10923-10927, etc., and the electroporation introduction method and the DEAE dextran method are Ting, AT et al. , EMBO J., 15,

Examples of the method described in 6189-6196 and the method for producing miseno include the method described in Hawkins, C. J. et al., Proc. Natl. Acad. Sci. USA, 93, 13786-13790. When a micelle formation method is used, a commercially available reagent such as lipofectamine (manufactured by Gibco) or Fugene (manufactured by Behringer) may be used.

The transformed cells can be obtained by culturing the cells into which the plasmid has been introduced, for example, by using a selection marker gene previously contained in the vector and culturing them in a medium under selection conditions according to the selection marker gene. Can be selected. The selection may be continued to obtain the present transformed cell which has become a stable transformant in which the present gene has been introduced into the chromosome. The introduced gene is located on the chromosome To confirm that the gene has been integrated, prepare a genomic DNA of the cell in accordance with a conventional genetic engineering method, and perform PCR using a DNA having a partial nucleotide sequence of the gene as a primer or PCR of the gene. The presence of the present gene in genomic DNA may be detected and confirmed using a method such as Southern hybridization using DNA having a partial base sequence as a probe.

In the first step of the assay method of the present invention, a method for measuring the activity of monoacylglycerol lucyltransferase (ie, the present enzyme) includes, for example, a method in which a mixed solution containing the present enzyme, a test substance, and a substrate is fixed. After reacting for a period of time, a method of analyzing the amount of reduction of the raw materials such as substrates in the reaction or a method of analyzing the amount of addition of the product in the reaction can be exemplified. These methods may be any known activity measuring method. Specific examples include the method described in Arch. Biochem. Biophys. 1992; 296 (2): 419-25 described above.

The reaction temperature at the time of activity measurement in the first step of the assay method of the present invention may be, for example, 15 ° C to 70 ° C, and preferably 20 ° C to 40 ° C. The reaction time is, for example, 1 minute or more, preferably 15 minutes to 1 hour. The reaction pH is, for example, from 6.0 to 9.5, preferably ρΗ7.0 to 8.0.

In order to detect or measure the amount of a raw material or a product such as a substrate in an enzyme reaction, or to measure the amount thereof, the reaction solution is separated by, for example, HPLC, thin-layer chromatography, paper chromatography, or the like, and then the raw material or the product is separated. A method such as detecting the ultraviolet absorption or radioactivity of the product [4 (when the raw material previously labeled with a radioisotope is used)), or measuring the ultraviolet absorption or radioactivity, etc. If it is necessary to remove the protein from the reaction solution during the above analysis, the protein may be removed from the reaction solution by the method described in, for example, Methods Enzyraol. 280, 211-221. Should be removed.

Based on the difference obtained by comparing the activity measured as described above with the activity of a control (ie, reference substance, negative control, etc.), the ability of the IB substance to suppress an increase in fat mass is evaluated. That is, when the activity measured as described above is lower than the activity in the control, it may be evaluated that the substance has an ability to suppress an increase in fat mass. In this manner, the test for the ability of the test substance to inhibit the increase in fat mass (the test method of the present invention) can be performed.

In the assay method of the present invention, when comparing the measured activity with the activity in a control, at least one of the two or more different substances does not have the ability to inhibit fat mass increase (for example, A negative control such as a solvent or a test system solution serving as a background may be used) to evaluate the ability of the other test substance to suppress an increase in the amount of fat, Of the above substances, the fat mass increase suppression ability of the other test substance may be evaluated based on the fat mass increase suppression ability of at least one substance (for example, a reference substance). Of course, both may evaluate.

More specifically, for example, the first step of the assay method of the present invention comprises the step (A) wherein the two substrates (for example, 2- or 1-monoacylglycerol (preferably, 2-monoacylglycerol)) are used. If the negative contact is used as a control when the enzyme and the test substance are in contact with the enzyme, the inhibition rate should be determined according to the following formula:

Inhibition rate (%) = {control (negative control) value-measured (test substance) value}

X100 / control (negative control / re) value.

Then, the fat mass increase suppression ability may be evaluated based on the calculated inhibition rate. That is, the inhibition rates of the various test substances calculated as described above are compared, and as a result, the test substance having a high inhibition rate has a higher P than the test substance having a low damage rate. What is necessary is just to evaluate that it has the capacity | capacitance suppression amount.

On the other hand, the first step of the assay method of the present invention comprises the step (A) wherein the two kinds of substrates (for example, 2- or 1-monoacylglycerol (preferably, 2-monoacylglycerol) and asinole-CoA) are used. And the test substance are in contact with this enzyme, and the control (reference substance) value is measured when a substance (reference substance) having the ability to suppress fat mass increase is used as a control. (Test substance) By comparing the values, the ability to suppress the increase in fat mass may be evaluated. In this case, if the measured (test substance) value is lower than the control (reference substance) value, the test substance is evaluated as having a higher ability to suppress the increase in fat mass than the reference substance. 4 000976

twenty four

Further, for example, the first step of the assay method of the present invention may comprise the step (B) of one of the two substrates (for example, 2- or 1-monoacylglycerol (preferably, 2-monoacylglycerol)) If the test substance is in the form of contact with the enzyme (i.e., diasyl-CoA) and the test substance (that is, when the test substance is treated as the other of the two substrates), A substance having a fat mass increase suppressing ability (reference substance) may be used as a control, and the fat mass increase suppressing ability may be evaluated by comparing the control (reference substance) value and the measured (test substance) value. In this case, if the measured (test substance) value is higher than the control (reference substance) value, the test substance is superior to the reference substance as a substrate for the enzyme. This means that this enzyme is superior to the reference substance as a competitive inhibitor of this enzyme.

In order to search for a substance that inhibits an increase in fat mass, a substance having the ability to inhibit the increase in fat mass may be selected based on the ability to inhibit the increase in fat mass, which has been assayed by the assay method of the present invention (the present invention search method).

For example, in the case of (A) above, when negative control is used as a control, the inhibition rate, which is an index for evaluating the ability of the test substance to suppress the increase in fat mass, is a statistically significant value. And more preferably, for example, a substance having an inhibition rate of 30% or more in the above formula, more preferably a substance having an inhibition rate of 50% or more, is selected as a substance having an ability to suppress an increase in fat mass. . On the other hand, in the case of (i) above, when a substance (reference substance) having the ability to suppress the increase in fat mass is used as a control, the measured (test substance) value is higher than the control (reference substance) value. Substances exhibiting low values are selected as substances having the ability to suppress fat addition. Further, for example, in the case of the above (物質), a substance whose measured (test substance) value is higher than the control (reference substance) value is selected as a substance having an ability to suppress the increase in fat amount. The substance may be any substance such as a low molecular weight compound, a protein or a peptide as long as it has an ability to suppress an increase in fat mass.

The substance selected by the search method of the present invention has an ability to suppress an increase in fat mass, and may be used as an active ingredient of a fat mass increase inhibitor.

Examples of such a substance (that is, a substance having an ability to inhibit monoacylglycerolacyltransferase) include, for example, Ν— [2 -— (4-benzyl-2-ethyl) Phenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1,5-a] pyrimidin-1 7-amine.

The monoacylglycerol lysyltransferase inhibitor (hereinafter referred to as P and harmful agent) selected by the search method of the present invention has the ability to suppress the increase in the amount of fat, as described in Examples in the present specification. Alternatively, it can be confirmed by a known method such as the method described in WO 00/47475.

In addition, the fact that the inhibitor selected by the search method of the present invention has the ability to inhibit lipoprotein synthesis can be confirmed by the following known method.

That is, cells having lipoprotein synthesis ability are cultured in a test tube in the presence of a substance having an ability to inhibit monoacylglycerol lucyltransferase (hereinafter sometimes referred to as the present inhibitor), and the amount of triglyceride is determined. Is measured and compared with the amount of triglyceride in the absence of the present inhibitor. As the cells, small intestine cells or liver cells prepared from living tissue, cultured cells derived from the small intestine or liver, or a lipoprotein synthesizing ability obtained by externally introducing a monoacylglycerol lucyltransferase gene were obtained. Cells and the like can be mentioned. Preparation of small intestinal cells from tissues is described, for example, in The Journal of Biological Chemistry, Milton M.

A method described in Weiser, p2536-2541, 1973, that is, a method in which recovered small intestine cells are suspended in a medium, a method described in a certain Poma Experimental Cell Research, Jean-Francois Beaulieu et al., P34-42, pl998, That is, it can be carried out by a method in which the collected small intestine cells are adhered to the plate vessel wall and cultured. The preparation of liver cells can be performed, for example, by the method described in Endocrinology, Jonathan R. Seckl et al., P4754-4761, 1995, that is, a method of preparing liver cells by treating an isolated liver with collagenase, or a method of preparing liver cells. It can be carried out by, for example, a method in which cells are adhered to the plate wall and cultured. No cultured cells derived from the small intestine have been established as a general cell line, but cells derived from human colon cancer, such as CaR-1, 2, 3 and CaCo-2 cells. Alternatively, it can be used as a capable cell. The cultured cell derived from liver,: ΤΗΗ-1,2, although 3 ^^ ηιΗ-1, 2, 4, He P G2 and many others is generally possible for utilization, usually the HepG2 as human liver model Often used.

As a method for quantifying the amount of triglyceride in the above liver and small intestine cells, fatty acids Alternatively, there is a method in which glycerol is added to cells and cultured for a certain period of time, and the amount of triglyceride contained in the supernatant fraction or cells is quantified. There are two methods for quantifying triglyceride: a method using a radiolabel, and a method for directly quantifying tridarylide without using a radiolabel. Examples of the former method include the method described in Journal of Lipid Research, Joan A. Higgins et al., Pl728-1739, 2000,

There is a method described in The Journal of Biological Chemistry, M. Mahmood Hussian et al. Pl9565-19572, 1999. The latter method is, for example, a method of culturing small intestine-like cells such as Caco2 or cultured liver cells in a test tube, and extracting and quantifying triglyceride from the culture supernatant with an organic solvent such as chloroform / methanol. I can do it. In any method, it is preferable to fractionate the culture supernatant cultured by the above method according to the density, and to quantify triglyceride contained in VLDL for liver cells and chylomicron for small intestine cells. . The evaluation of the ability of the inhibitor to inhibit lipoprotein synthesis can be carried out by allowing the above-mentioned tridaliceride synthesis to be carried out in contact with the inhibitor and comparing it with the ability of synthesizing triglyceride in the absence of P. The VLDL and chylomicron can be isolated by methods well known to those skilled in the art, such as centrifugation.

Alternatively, the inhibitor is administered to mice and the like, and the amount of triglyceride (= lipoprotein) in the blood is measured, and compared with the amount of triglyceride in the blood when the inhibitor is not administered. The ability to inhibit protein synthesis can also be confirmed. Specifically, blood triglyceride concentration was reduced by fasting, and a method of measuring an increase in blood triglyceride concentration when lipid was administered thereto was used (International Journal of Obesity, K. Cianflone et al., P705- 713, 2001).

A fat mass increase inhibitor (namely, the fat mass increase inhibitor of the present invention) characterized by comprising a substance having a monoglycol glycerol lusyltransferase inhibitory ability or a pharmaceutically acceptable salt thereof as an active ingredient. An effective amount thereof can be orally or parenterally administered to mammals such as humans. For example, when administered orally, the fat mass increase inhibitor of the present invention can be used in a usual form such as a tablet, a capsule, a syrup, a suspension and the like. In addition, when administered parenterally, the present invention The fat mass increase inhibitor can be used in the form of ordinary liquids such as solutions, emulsions, suspensions and the like. Examples of the method for parenterally administering the above-mentioned fat-increasing inhibitor of the present invention in the form of injection include a method of injection, a method of rectum administration in the form of suppositories, and the like. The above-mentioned suitable dosage form is a substance having a monoacylglycerol lucyltransferase inhibitory activity or an acceptable pharmaceutically acceptable carrier, excipient, binder, stabilizer, diluent or the like. It can be produced by blending a salt. When used in the form of an injection, an acceptable buffer, solubilizing agent, isotonic agent and the like can be added.

The dosage varies depending on the age, sex, body weight, degree of disease, type of fat-increasing U-suppressant, dosage form, etc., of the mammal to be administered. About 1 mg to about 2 g, preferably about 5 mg to about 1 g, of the active ingredient may be administered per injection.In the case of injection, about 0.1 mg to about 50 mg of the active ingredient is used for an adult. 0 mg may be administered. In addition, the above-mentioned dose can be administered once or in several divided doses.

Examples of the disease to which the fat mass increase inhibitor of the present invention can be applied include metabolic diseases such as decreased glucose tolerance due to insulin resistance, type II diabetes, hyperlipidemia, hypertension, and atherosclerosis, coronary artery disease, and stenosis. Diseases such as cardiovascular disorders such as heart disease and myocardial infarction can be mentioned.

As described above, in the present invention, a pharmacologically effective amount of a substance having an ability to inhibit monoacylglycerol acyltransferase for inhibiting the activity of monoacylglycerol acyltransferase is administered to the living body. However, an increase in fat mass can be suppressed.

Further, in the present invention, the use of monoacylglycerolacyltransferase as a reagent for providing an index for evaluating the fat mass increase suppressing ability, the use of fat mass increase [1. The use of the monoacylglycerol transferase gene as a reagent to provide the enzyme is also ^^. Examples of the former include the use of monoacylglycerol acyltransferase associated with the assay method of the present invention, the search method of the present invention, and the like. Another example is the regulation of the expression of monoacylglycerol lucyltransferase to reduce fat content. It can be used as a reagent for providing an index for evaluating the ability to suppress the increase in fat mass when suppressing the increase. More specifically, use as a primer or probe in known methods for specifically detecting a specific gene, such as the Northern blot method, RT-PCR method, in situ hybridization method, and DNA chip. And the like. Example

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

Example 1

Preparation of Miku Mouthsome Fraction (one form of enzyme sample containing this enzyme) from adipose tissue

(1-1) Removal of adipose tissue

Thirty 14-week-old male Wistar rats (three SLC) were sacrificed and laparotomized to remove adipose tissue attached to the mesentery (hereinafter referred to as mesenteric fatty thread II). The issued hysterectomy, phosphate buffer (0. 20 g / L KC 1 , 0. 20 g / LH 2 P0 4, 8. 00 g / L Na C l, 2. 1 6 g / LN a 2 HPO 4 · 7 H ₂ 0, 100 Interview - Tsu door Zm 1 penicillin (Gibco), 100 μ g / m 1 Streptomyces Ma leucine (Gibukone soil), 250 ng / m 1 amphotericin (Gibukone soil)) to about 10 Om 1 Soaked and washed at room temperature.

(1-2) Preparation of microsomal fraction (a form of enzyme sample containing this enzyme) from adipose tissue

First, three times the volume of the adipose tissue of MediumA (0.25M Sucrose, 1 mM Tris monohydrochloride (H

7.5) ImM tetrasodium ethylenediaminetetraacetate (hereinafter referred to as EDTA'4Na) and ImM dithiothreitol (hereinafter referred to as DTT)) were added, and the fat was added using a homogenizer (Wheaton). The tissue was homogenized by grinding. The homogenized adipose tissue was centrifuged at 600 g × 15 min, 4 for 5 min. PT / JP2004 / 000976

29

The middle layer that did not contain atCake) and the lower layer (H) was separated. The separated intermediate layer was further centrifuged at 4 ° C. at 16,000 g × 15 minutes. The resulting supernatant was centrifuged at 105,000 g × 60 minutes at 4 ° C. to collect a precipitate (microsomal fraction). After suspending the recovered precipitate in the Medium A, 105,000 g × 60 minutes, 4. Washing was performed by centrifugation at C. The washed precipitate was resuspended in the above Medium A to prepare a microsomal fraction having a protein concentration of 1 mg Zm1. Example 2

Measurement of the activity of monoacinolegrecephalus lucyltransferase in the microsomal fraction (one form of the enzyme sample containing this enzyme)

Atsushi solution (24 mM Tris-hydrochloric acid (pH 7.5), 5 Om chloride, 8 mM magnesium sulfate, 1.S SmgZml ゥ serum albumin, 1 mM DTT) 800 gZm 1 test substance (1% dimers hoki) Cid (hereinafter referred to as DMSO) solution) 0, 75 ^ 1, lmgZml of a microsomal fraction of 5 µl was added. 0.71 of 2-Omole oil glycerol (Sigma) (0.71 M) was further added to the Atsey solution and mixed. To this mixture was added 0.45 mM ⁴ C] palmitoylcoenzyme A (Amersham) 10 μl (0.03 μθ ί Ζ total amount of reaction solution 15 Oju 1) and incubated at room temperature for 5 minutes. . In the test substance-free group, the above-mentioned test substance was replaced with a DMSO solution1. /. The same method as described above was used except that only 0.75 μl of DMS O was added. In the background plot, the same method as in the test plot with 2-monooleoylglycerol was used, except that only DMS II was added instead of the test substance and 2-monooleoylglycerol. Was used.

Next, after 5 minutes of incubation, add isopropanol / heptane / water to the reaction mixture.

(80: 20: 2, ν / ν) The reaction was stopped by the addition of 3001, and 200 μl of heptane and 100/1 of water were added, followed by suspension. This suspension was centrifuged at room temperature for 3, OOO rpmXl at room temperature to remove the lower layer, and the remaining upper layer (organic solvent) was evaporated to dryness. The resulting dryness The suspension was suspended in 30 µl of black-mouthed form / "methanol (2: 1, VNOV). This suspension was then placed on a thin-layer chromatography glass plate (Κ5 silica gel, 150 angstroms, Whatman). , Which is sometimes referred to as a TLC plate below) In a closed container, add a developing solvent of hexane Ζethyl ether / acetic acid (75: 25: 1, ν / ν) and use the developing solvent. After the TLC plate was developed, the TLC plate was dried at room temperature, and the dried TLC plate was exposed to an imaging plate (Fuji Film Co., Ltd.) for 16 hours. BAS2500, and analyzed by Fuji off Irumu Co.), the one on the TLC plate, 2-di § sill glycerol Contact and triglyceride L ^{1 4} C of a portion corresponding to the deployed position of the radiation Katsusei were measured (hereinafter, 1, 2-di § sills glycerol moiety ⁴ C of the portion corresponding to the deployed position] radioactivity measurements A, the portion corresponding to the deployed position of the bird § sill glycerol ^'4 C ] The radioactivity is referred to as measured value B. From these measured values, the activity of monoacyl and lysine lucyltransferase when the test substance is added (measured value A + measured value ΒΖ2, hereinafter referred to as activity value 1) Using the same method as above, the sections corresponding to the development positions of 1,2-diacylglycerol and triglyceride on the TLC plate were also calculated for the test substance-free group and the background group. I: ¹⁴ C] radioactivity was measured (hereinafter, ⁴ C] of the portion corresponding to the development position of 1,2-diasylglycerol in the test substance-free area was measured. Addition trier Measured value D of ⁴ C] radioactivity of the portion corresponding to the deployed position Le glycerol, 1 background Ward, 2-di § sill measurements E radioactivity of a portion corresponding to the deployed position of glycerol, Bakkuguraundo The radioactivity of the portion corresponding to the development position of triacylglycerol in the test group is referred to as measured value F.) From these measured values, the activity of monoacylglycerose lucyltransferase in the test group without test substance (measured value C + The measured value D / 2, hereinafter referred to as activity value 2) and the activity of monoacylglycerol lucyltransferase in the background (measured value E + measured value FZ ² , hereinafter referred to as activity value 3) were calculated. From the obtained activity values, the test substance's ability to inhibit monoacylglycerol lucyltransferase was expressed as the inhibition rate ((activity value 2-activity value 1) XI 00 (activity value 2-activity value 3)). Calculated.

As a result, the known compound N- [2- (4-benzyl-2-ethylphenoxy) ethyl] -1,5-dimethyl [1,2,4] triazolo [1,5-a] pyrimidine-1 The inhibition rate of 7-amine (hereinafter sometimes referred to as compound A) was 44 to 50%. Furthermore, while changing the test concentration of the test substance, the inhibition rate at each test concentration was calculated using the same method as described above. Table 1 shows the results together with the results of the former. table

** Values based on the results of Reference Example 2 Also, when the test substance was another known substance having an ability to suppress an increase in fat mass, similar results were obtained.

Based on the above, the inhibition rate, which is an index for evaluating the enzyme inhibitory ability, and the accumulated rate The relationship with the inhibition rate in Reference Examples 1 and 2 described below, which is based on the direct measurement of fat mass, shows at least dose dependence in a specific concentration range. It was confirmed that the correlation was positive. Reference example 1

(1-1) Removal of adipose tissue

Thirty-two 14-week-old male Wistar rats (Japan SLC) were sacrificed and laparotomized to remove mesenteric adipose tissue. The excised tissue, phosphate buffer (0. 20 g / L KC 1 , 0. 20 g / L KH 2 P0 4, 8. 00 g / L. Na C l, 2. 1 6 g / L Na 2 HP0 ₄ · 7H ₂ 0, 100 units Roh m 1 penicillin (Gibco), 100 mu g / 1 streptomycin (Gipuko Co.), soaked in 250 ng / m 1 § Nfoterishin (Gibukone earth)) and washed at room temperature.

(1-2) Preparation and culture of fat cells from adipose tissue

The following treatment was performed on the mesenteric adipose tissue after washing.

First, the adipose tissue extracted in (1-1) above was treated with collagenase (Type I or Vin, Sigma), penicillin (Gibco), streptomycin (GIB VIIIC) and amphotericin (Gibco), respectively. Dulbecco's modified eigland (4.5 g LD-glucose and 584 mg / l) added to a final concentration of lmgZm1, 100 cut_: 011, 100 / X g / m1 and 250 ngZm1 (LL-glutamine-containing, GIPCO) The pieces were cut into about 5 mm squares using scissors in about 3 O Oml. Next, this was shaken at 37 ° C for 60 minutes (about 170 rpm), filtered through a nylon mesh (80 S [mesh size 250 µm], Sanshin Kogyo Co., Ltd.), and the filtrate (cell suspension) Liquid) was recovered. After the filtrate was centrifuged at room temperature at 1800 rpm for 5 minutes, the liquid layer was gently removed by decantation to obtain a sediment. The sedimentation was analyzed using fetal calf serum (hereinafter referred to as FBS) (Gibco), ascorbic acid (Wako Pure Chemical Industries), pecillin (Gipco), streptomycin (Gibco) and amphotericin (Gibco). ) Were added to a final concentration of 10%, 200 μΜ, 100 units / ml, 100 g / m1 and 250 ng / m1, respectively. Dulbecco's modified Eagle's medium (4.5 g / LD-glucose) Medium and 584 mg / LL—containing glutamine, gibconed earth, hereafter sometimes referred to as FBS-containing medium) and suspended in 50 ml of nylon mesh (420 S [mesh size 25 μΐη], Sangen) (Industry). The filtrate was collected and centrifuged at room temperature at 1800 rpm for 5 minutes. The liquid layer was gently removed by decantation, and the precipitate was suspended again in 5 Oml of an FBS-containing medium. The operation of centrifuging, removing the liquid layer, and suspending in a FBS-containing medium was further performed twice on the suspension in the same manner as described above to prepare a suspension (120 ml). The suspension was dispensed into cell culture flasks (T150 for adherent cells, Iwaki Glass Co., Ltd.) in an amount of 3 Oml each and cultured at 37 ° C. in the presence of 5% CO ₂ . Two to three hours after the start of the culture, the medium was removed, and the vessel wall of the flask was washed with 15 ml of the above-mentioned phosphate buffer. Except for washings, after the cleaning operation was again Gyotsu, except phosphate buffer, was added FBS-containing media 30 m 1 flask, and cultured at 3 7 ° C, 5% C 0 2 presence . One day after the start of the culture, the medium was removed, the wall of the flask was washed once with 15 ml of a phosphate buffer solution, and then the trypsin-ethylenediaminetetraacetic acid (hereinafter referred to as EDTA) solution (0.05%) was added to the flask. Trypsin, 0.53 mM EDTA '4Na, Gibco) was added to the cell

It was left at 7 ° C for 5 minutes. Thereto, the 83-containing medium was added to about 10 times the amount of trypsin over £ 0 Ding solution to obtain a cell suspension _P

(1-3) Measurement of test substance's ability to suppress fat mass and calorie increase (Part 1)

Using a hemocytometer, measure the number of cells in the cell suspension prepared from the mesenteric adipose tissue in (1-2) above, and set it to 1.4 x 10 ⁵ cells _ m1. The cell suspension was diluted by adding FBS containing medium. Thus a 96-well plate and the resulting diluted solution (for adherent cell culture, Iwaki Glass Co.) dispensed at 10 Q / X 1 min per Ueru, 5% C 0 ₂ presence, at 37 ° C After culturing for 2 to 3 days, remove the medium from each well of the 96-well plate, and add 10 μg Zm1 insulin (Sigma), 0.25 / ζΜdexamethasone (Wako Pure Chemical Industries), 0.5 mM Isobutinole

1-methyl-one xanthine (Sigma) and 5Myumyu 15-Dokishi one厶¹² ^'14 - The FBS-containing medium 100 mu 1 containing prostaglandin J ₂ (Ca yma n, Inc.) was added to each Ueru with 5% C0 ₂ presence, and cultured for 2 days at 37 ° C. Then remove 10 μg of Zm1 insulin and 5 / xM 15 Dokishi one Δ ¹² '"-. The FBS-containing medium 100 mu 1 containing prostaglandin J ₂ were added to each Ueru additional 2 days, 5% C0 ₂ presence after incubation at 37 ° C, each Ueru the medium was removed, 10 μ g / m 1 insulin, 5 M 15-Dokishi - FBS-containing containing prostaglandin J _2, a test substance 50 M, 0. 5% DMSO (the Wako pure drug Industries, Ltd.) - Δ ¹² ^'14 medium was added to 100 1 each Ueru, it was similarly cultured in the test substance untreated silage, 10 μ gZni l incidents Yurin instead of the culture medium, 5 μΜ 1 5- Dokishi one Δ ¹² ^'14 -. prostacyclin The culture was carried out in the same manner as described above except that 100 / i 1 of FBS-containing medium containing glandin J ₂ and 0.5% DMSO was added, and after culturing for 2 days, oil red O (Sudan II, Wako) The intracellular fat was stained using Junyaku Kogyo Co., Ltd., and the amount of stained fat was measured by a colorimetric method. First, directly to each containing a cell culture Ueru, after leaving 0. 07 5 ° / ₀ Oiruretsudo O staining solution was added Z 60% phosphoric acid Toryechiru solution 30 mu 1. 30 minutes at room temperature, the Oiruretsudo Ο staining solution The medium containing the solution was removed, and 1 μl of a 20% aqueous solution of triethyl phosphate was added to each well, after which the 20% aqueous solution of triethyl phosphate was removed from each well, and 100 1 of the same aqueous solution was newly added to each well. After the above operation was repeated, a cell lysate (2% SDS, 0.2N NaOH) lO Ojul was added to each well, and the mixture was incubated at 37 ° C for 3 hours or more. The absorbance at a wavelength of 490 nm was measured for each well using a plate reader (Vmax, Beckman) (hereinafter referred to as measurement value 1). Cell 脂肪 fat was stained and colorimetrically stained The amount of fat was measured (hereinafter referred to as measured value 2.) These measured values were calculated according to the following formula, using the test substance's ability to suppress the increase in fat mass as the inhibition rate: Inhibition rate (%) = { (Measured value 2—Measured value 1) No Measured value 2} XI 00

As a result, the known test compound N- [2- (4-benzinole-2-ethylenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1,5

-a] The inhibition of pyrimidine-17-amine (ie, compound A) was 72%. Reference example 2

Measurement of ability of test substance to suppress fat mass increase (Part 2) (2-1) Preparation of adipose tissue piece

Thirty-two 14-week-old Wistar strains (Japan SLC) were sacrificed and laparotomized, and all adipose tissue attached to the mesentery (ie, mesenteric adipose tissue) was removed. In addition, subcutaneous adipose tissue (hereinafter referred to as abdominal adipose tissue) over the abdominal force thigh was removed from only one animal per animal. The extracted fibers were washed with 2 ml of Dulbecco's modified Igno ground (containing 4.5 g / LD-glucose and 584 mg / LL-glutamine, Gibco), and then scissors were used in the same medium. To about lmm square.

(2-2) Measurement of ability of test substance to suppress increase in fat mass (Part 2)

First, Dulbecco's Modified Eagle Medium-Low Darco ^ "S (1 g containing Og / LD-glucose and 584 mg / LL-glutamine, Gibco) was added to a 48-well plate (for adhesion cell culture, Sumitomo Belite). The test substance dissolved in DMSΟ was added so that the final concentration of the test substance was 50 iΜ and the final concentration of DMSO was 0.5 ° / ₀ . . each Ueru, the (2 1) were placed the prepared fat Ojen 1:50 to 0 Omg with, 5% C0 ₂ presence 3

Incubated at 7 ° C for 30 minutes. In the test substance-free group, the same method as described above was used except that only DMSO was added to a final concentration of 0.5% instead of the DMSO solution of the test substance. 30 minutes after the start of the incubation, a radioisotope-labeled Darcos solution (D— [U- ^14C ] glucose, 7.4 MB q / m 1, Amersham) was added to each well. % C 0 ₂ presence, and incubated for 7 hours at 37 ° C. Next, after the incubation, each adipose tissue piece was transferred into 750 μl of heptane Z isopropanol (heptane: isopropanol = 3: 2) and left at room temperature for about 15 hours. Next, the adipose tissue pieces were removed from the above solution, heptane and isopropanol were volatilized, and 6 / il of the obtained residue was subjected to 120 μl of black-mouthed formnomethanol (black-mouthed form: methanol 1 = 2: 1). .1, and spotted on a glass plate for thin layer chromatography (# 5 silica gel 150 Angstrom, Whatman, sometimes referred to as a TLC plate hereinafter). A developing solvent of hexane: ethyl ether: acetic acid (75: 25: 1) is placed in a closed container, and the TLC plate is developed using the developing solvent. The pieces were dried at room temperature. An imaging plate (Fuji Film Co., Ltd.) was exposed to the dried TLC plate for 4 to 5 hours. After the exposure, the imaging plates Bio Image Analyzer (BAS 2000, Fuji Film Co.) was analyzed, the said measured ⁴ C] radioactivity of the portion corresponding to the deployed position of Toridariseraido on TLC plates (hereinafter, measurements 5) For even test substance untreated silage was measured ⁴ C] radioactivity of the portion corresponding to the deployed position of the triglyceride on TLC plates using the same method (hereinafter, referred to as the measurement value 6). From these measured values, the ability of the test substance to suppress the increase in fat mass was calculated as the inhibition rate of fat (triglyceride) accumulation per unit volume of adipose tissue according to the following formula. Inhibition rate of increase in fat mass per unit volume of adipose tissue (%) = {(measured value 6—measured value 5) Z measured value 6} XI 00

As a result, the test compound, a known compound N- [2- (4-benzyl-2-ethylphenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1,5-a] pyrimidine-1 The inhibition for 7-amine (ie, compound A) was 84%. Furthermore, while changing the test concentration of the test substance, the inhibition rate at each test concentration was calculated using the same method as described above. The results are shown in Table 2 together with the results of the former.

Table 2

Example 3

Cloning of this mouse-derived gene (Part 1) In order to amplify a DNA fragment containing the mouse-derived gene (mAT12) by PCR, it must first consist of 1 L of Mouse Norma 1 Adiposec DNA (Biochain soil) and 1 L of the base sequence represented by SEQ ID NO: 5. Primer 5 2

0 pmo 1, a primer consisting of the nucleotide sequence represented by SEQ ID NO: 6 20 pmo 1, Takara Ex-Taq polymerase (Takara Shuzo) 2U, Takara

Prepare a 50 μΐ ^ PCR reaction solution containing 5 μL of buffer attached to ExTaq polymerase and 4 / iL of dNTP mixture (2.5 mM) attached to Takara Ex-Taq polymerase, and use it for PCR. did. The PCR was carried out under the conditions that the heat cycle consisting of 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute was repeated 50 times, and finally the temperature was kept at 72 ° C for 5 minutes. I was After PCR, a PCR product showing about 1.2 Kbp was recovered by agarose electrophoresis. A plasmid prepared by subcloning the recovered PCR product into ρT7—B1 uevector (Novagen soil) was used to transform E.coli JM10 9 strain competent cells (Toyobo). From the cultured cells obtained by culturing the transformed cells in 10 OmL of LB medium containing 50 g / niL ampicillin, the plasmid was Q I AGEN P 1 a sm i d Ma X i Kit (Q

The DNA was isolated and purified using AGEN (1AGEN) to obtain a plasmid containing DNA derived from a mouse and having the nucleotide sequence of the present gene (mAT12). Example 4

Determination of the nucleotide sequence of this gene derived from mouse (Part 1)

The plasmid containing the PCR product (approximately 1.2 kbp) obtained in Example 3 was converted into a type III plasmid, and then converted to a Thermo Sequenase II dye kit (Amersham Pharmacia Biotech). ) Using the ABI 373DN A sequence reader (PE Ap 1 ied Biosystems), Sanger's method (F. Sanger, S. Nicklen, AR Cou1sonA, roceedingsoi Natio nal Academyof Science USA (1977), 74, 5463-5467), a mouse-derived book consisting of a DNA having the nucleotide sequence of SEQ ID NO: 2. The nucleotide sequence of the gene (mAT12) was determined. Example 5,

Cloung of this gene derived from human (Part 1)

In order to amplify the DNA fragment containing the human-derived gene (hAT12) by PCR, first, 1 L of hum an Adipocyte Marat hon-ready cDNA (CLONTEC H), SEQ ID NO: 7 Primer 7 20 pmo 1 consisting of the base sequence shown, primer 20 20 pmo 1 consisting of the base sequence shown in SEQ ID NO: 8, Takara ExTaq polymerase (Takara Shuzo) 2U, Takara ExTaq polymerase attached Buffer of 5 iL and

Fifty PCR reaction solutions containing dNTPmixTre (2.5 mM) 4; L attached to TakaraEx-Taq polymerase were prepared and subjected to PCR. The PCR is performed under the conditions that the heat cycle consisting of 94 ° C for 30 seconds, then 60 ° C for 30 seconds, 72 ° C for 1 minute is repeated 50 times, and finally the temperature is kept at 72 ° C for 5 minutes. Was. After PCR, a PCR product showing about 1.2 Kbp was recovered by agarose gel electrophoresis. Collected PCR products! ) E.co1i JM109 strain competent cell (Toyobo) was transformed with a plasmid prepared by subcloning into T7—B1 uevector (Novagen). QI AGEN Plasmid Maxi Kit (QIAGEN) from the cultured cells obtained by culturing the transformed cells in 10 OmL of LB medium containing 5 O / ^ g / mL ampicillin By separating and purifying the plasmid, a plasmid containing DNA having the nucleotide sequence of the present gene (hATl2) derived from human was obtained. Example 6

Determination of the nucleotide sequence of this gene derived from human (Part 1)

Plasmid containing the PCR product (about 1.2 kbp) obtained in Example 5 was designated as type III, and then the Thermo Sequenase II gui terminator kit (Amersh Am Pharma cia Biotech) and ABI 373DN Using an A array reader (PEAp lied Biosystems ¾h), 6

39

According to the method of Sanger (F. Sanger, S. Nicklen, AR Cou 1 son A, Proceeding sof Na ti lion Ac ode of Science USA (1977), 74, 5463-5467). Thus, the nucleotide sequence of the present human-derived gene (hAT12) consisting of the DNA having the nucleotide sequence of SEQ ID NO: 4 was determined. Example 7

Cloning of the rat-derived gene and determination of its nucleotide sequence (Part 1) A plasmid containing a DNA having the rat-derived gene (rAT12) nucleotide sequence according to the method described in Example 3 ^ and 4. The nucleotide sequence of this rat-derived gene (rATI2) consisting of DNA having the nucleotide sequence of SEQ ID NO: 12 was determined. Example 8

Preparation of a plasmid that expresses a fusion protein in which FLAG is added to the amino terminal of the amino acid sequence represented by SEQ ID NO: 1

0.05 g of the plasmid separated and purified in Example 3, primer 9 20 pmo 1 consisting of the base sequence shown in SEQ ID NO: 9 and primer 10 20 pmol consisting of the base sequence shown in SEQ ID NO: 10 20 pmol, Takara ExTa q polymerase (Takara Shuzo) 0.5 U, 5 l containing 5 μL of buffer attached to Takara ExTa q polymerase and 4 μL of Takara ExTaq polymerase and dNTP mixture (2.5 mM) A PCR reaction mixture was prepared and used for PCR. The PCR is performed under the conditions that the heat-retention cycle consisting of 94 ° C for 1 minute, then 60 ° C for 30 seconds, and 72 ° C for 1 minute is repeated 30 times, and finally, the temperature is kept at 72 for 5 minutes. Was. After PCR, agarose gel electrophoresis

A PCR product showing 1 Kbp was recovered. The E. coli JM109 strain competent cell (Toyobo) was transformed with a plasmid prepared by subcloning the recovered PCR product into pCMV-Tag2B (Stratagene). Transfer the transformed cells to LB medium containing 50 μg / mL The plasmid was separated and purified from the culture cells obtained by culturing with OL using QI AGE NQI Ailter Megait (QI AG EN), so that the plasmid was added to the amino terminal of the amino acid sequence represented by SEQ ID NO: 1. A plasmid expressing a fusion protein to which FLAG (amino acid sequence in one-letter code: MDYKDDDDKSPGGSPGLQEF) was added (hereinafter, also referred to as FLAG fusion mAT12 protein) was obtained. Furthermore, the absence of mutation in the plasmid was confirmed by the method described in Example 4. Example 9

Preparation of enucleated cell extract containing this enzyme (Part 1): Preparation of enucleated cell extract containing FLAG-fused mAT12 protein

The FLAG-fused mATl2 protein expression plasmid obtained in Example 8 was transferred to a CHO-K1 cell (Odaimotomoto) using the Lipofec tAMI NE PLUS reagent kit (Invitrogen) according to the procedure attached to the reagent kit. Pharmaceutical company). The CHO-K1 cells into which the expression plasmid was introduced were cultured for 24 hours according to the procedure manual distributed when the cells were purchased. After the culture, the cells were washed twice with D-PBS (Gibco), scraped with a cell scraper, and collected by centrifugation H H. The recovered cells were treated with 50 mM Tris-HCl (H7.5), 0.25 M sucrose, 1 mM ethylene glycol bis 2 aminoethyl ether tetraacetic acid (hereinafter referred to as EGTA), ImM dithiothreitol (hereinafter referred to as DTT). The suspension was suspended in a buffer solution containing 1/100 volume of the protease inhibitor Kaktenore (Sigma) (hereinafter, referred to as "solution A"). The suspension was sonicated (x 6 times for 5 seconds under ice-cooling) to crush the cells in the suspension. By centrifugation at, uncrushed cells and nuclear fraction were removed as a precipitate. The protein concentration of the obtained supernatant was measured by the Bradford method using serum albumin as a standard, and the dilution obtained by adding buffer 液 to 2 / X g ΖμL each was removed. A nuclear cell extract was used. Example 10

Preparation of control solution for enucleated cells (Part 1)

As control (negative control), as in Example 9 except that CMV-Tag 2B (without insert) was used instead of the plasmid for expressing FLAG fusion mATl2 protein According to the method described above, an enucleated cell extract (control) was obtained. Example 11

Separation detection of this enzyme (Part 1)

When 5 μL of the enucleated cell extract obtained in Examples 9 and 10 was used for immunoblotting with an anti-FLAG tag M2 antibody (Sigma), a control (negative control) was used. In the case of the enucleated cell extract, nothing was detected. On the other hand, in the case of the enucleated cell extract prepared from the cells into which the plasmid for expressing the FLAG-fused mAT12 protein was introduced, a single band protein having a molecular weight of about 45 kDa was detected. Example 12

Measurement of the activity of monoacylglycerol lucyltransferase (Part 1) The enucleated cell extract lO / ^ L obtained in Examples 9 and 10 was treated with a 150 / L reaction buffer (24 mM Tris-hydrochloride ( H 7.5), 50 mM salted potassium, 8 mM magnesium sulfate, 1.25 mg / mL serum albumin, 1 mM DTT, 0.5 mM 2-monooleoylglycerol) To the suspension was added 5 / L of 900 AtM (0.2 MB q // L) I: ¹⁴ C] palmitoyl-coenzyme A aqueous solution. The mixture was kept at room temperature for 10 minutes. In addition, a test system in which the same operation as above was performed using a reaction buffer having the same composition as described above except that 2-monooleoylglycerol was not used was used as a background section.

Next, 300 L of heptane / 2-propanol / water (= 80: 20: 2) was added to the mixture, and the mixture was stirred and allowed to stand for 10 minutes. After that, the mixture was further added with 200 μL of heptane and 100 μL of Water was added and stirred. After stirring, centrifuge the mixture. After the lower layer was removed by centrifugation, the remaining upper layer was dried using a centrifugal evaporator. The dried matter (fat fraction) thus obtained was redissolved in black hole form. Next, this solution was spotted on a silica plate for silica gel 150 thin-layer chromatography (K5 silica gel Λ50 Å, Whatman), and hexane / getyl ether acetic acid (75:25: By developing using the developing solvent of 1), ⁴ C] 1,2-diacylglycerol contained in the solution was separated. Separated C ^{1 4} C] ^1, 2-Jiashirugurise opening one les were quantitated have use the Roh I O Imaging Analyzer System BAS- 2 0 0 0 (Fuji Film Co.). The value obtained by subtracting the value measured in the packed ground section from the value measured in the reaction buffer containing 2-monooleoylglycerol was calculated as the enzyme activity. In the test system in which the plasmid for the control (negative control) was introduced, and in the test system in which the plasmid for expressing the FLAG-fused mAT12 protein was introduced, the monoacylglyceryl luciferase in each enucleated cell extract was used. Table 3 shows the activity of the transferase.

Table 3

As described above, the test system in which the FLAG-fused mAT12 protein-enhancing plasmid was introduced had a higher concentration in the enucleated cell extract than the test system in which the control (negative control) plasmid had been introduced. Since the activity of the monoacylglycerol lucinoletransferase was increased, it was confirmed that the protein comprising the amino acid sequence shown in SEQ ID NO: 1 exhibited the activity of monoacylglycerol luciryltransferase. Example 13 Cloung of this gene from mouse (Part 2)

In order to amplify the DNA fragment containing the mouse-derived gene (mAT14) by PCR, first use a primer consisting of the nucleotide sequence represented by Mouse Normal Adiosec DNA (Biochain) l / xL, SEQ ID NO: 19 19 20 pmo 1, comprising the nucleotide sequence represented by SEQ ID NO: 20 primer _{20 20 pmo 1 N T akara Ex} -Ta q polymerase (Takara Shuzo Co., Ltd.) 2 U, T akara E xT aq polymerase buffer attached 5 mu L and T Akara A 50 iL PCR reaction solution containing 4 μL of the dNTP mixture (2.5 mM) attached to ExTaq polymerase was prepared, and subjected to PCR. The PCR was first incubated at 94 ° C for 60 seconds, followed by 40 cycles of incubation at 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute, and finally at 72 ° C for 4 seconds. The test was performed under the condition of keeping the temperature for one minute. After PCR, a PCR product showing about 1.3 Kbp was recovered by agarose electrophoresis. The E.coli JM109 strain competent cell (Toyobo) was transformed with a plasmid prepared by subcloning the recovered PCR product into pT7-B1uevector (Novagen). From the cultured cells obtained by culturing the transformed cells in 10 OmL of LB medium containing 50 g / mL ampicillin, the above plasmid was transformed into QI AGEN Plasmid Ma Xi Kit (QI AG EN). By separating and purifying the plasmid, a plasmid containing DNA having the nucleotide sequence of the mouse-derived present gene (1HAT14) was obtained. Example 14

Determination of the nucleotide sequence of the mouse-derived gene (Part 2)

The plasmid containing the PCR product (approximately 1.3 kbp) obtained in Example 13 was used as type I, and the ThermoSequenase II dye terminator kit (Amersham Pharmacia Biotech) The Sanga method (F. Sanger, S. Nick 1 en, AR Cou 1 son A ^, Proceedingsof) was performed using an ABI 373D NA sequence reader (PE Ap 1 ied Biosystems). National A cade Based on my of Science USA (1977), 74, 5463-546 7), the nucleotide sequence of this gene (mAT14) derived from mouse consisting of DNA having the nucleotide sequence shown in SEQ ID NO: 14 was determined. Example 15

Cloning of this gene derived from human (Part 2)

A DNA fragment having the human-derived gene (hAT1) is amplified by PCR.Human Adipocyte Marat hon-ready DNA (CLONTECH) 1 / z L, represented by SEQ ID NO: 21 Primer consisting of base sequence 21 20 pmo 1, Primer consisting of base sequence shown in SEQ ID NO: 22 20pmol, Takara Ex-TaII polymerase (Takara Shuzo) 2U, buffer attached to Takara ExTaq polymerase Fifty PCR reaction solutions containing 5 μL and 4 μL of dNTP mixture (2.5 mM) attached to Takara Ex-Taq polymerase were prepared, and subjected to PCR. PCR was first incubated at 94 ° C for 60 seconds, then at 94 ° C for 30 seconds.

At 60 for 30 seconds, 72. Insulation cycle consisting of 1 minute at C was repeated 40 times, and finally the condition was kept at 72 at 5 minutes. After PCR, a PCR product showing about 1.4 Kbp was recovered by agarose electrophoresis. A plasmid prepared by subcloning the recovered PCR product into pT 7 — Bluevector (No vage ηΫ soil) was used to transform E.co1i JM109 strain Combinent Cell (Toyobo) into a plasmid. Transformed. Using QI AGEN P 1 a smid Ma Xi Kit (QI AGEN), the above plasmid was obtained from cultured cells obtained by culturing the transformed cells in 10 OmL of LB medium containing 50 g / mL ampicillin. By separation and purification, a plasmid containing a DNA having the nucleotide sequence of the present human-derived gene (hAT14) was obtained. Example 16

Determination of the nucleotide sequence of this gene derived from human (Part 2)

The plasmid containing the PCR product (about 1.4 kbp) obtained in Example 15 was designated as type I. Then, using a Thermo Sequenase II dye terminator kit (Amersham Pharmacia Biotech) and an ABI 373 DNA sequence reader (PEApplied Biosystems), By the method (F. Sanger, S. Nick 1 en, AR Cou 1 son A, Proceedings of National Academy of Science USA (1977), 74, 5463-5467), the base represented by SEQ ID NO: 16 is obtained. The nucleotide sequence of the human-derived gene (hAT14) consisting of the DNA having the sequence was determined. Example 1

Cloning of this gene from rat (Part 2)

In order to amplify a rat-derived DNA fragment having this gene (rAT14) by PCR, first, the rat base sequence represented by Rat Adipocyte Marat hon-ready cDNA (CLONTECH) L, SEQ ID NO: 23, was used. primer 23 20 pmo 1 that, SEQ ID NO primer 24 having the nucleotide sequence represented by _{24 20 pmo 1 N T akara Ex} -Ta q polymerase (Takara Shuzo) 2U, Ta kara E xT aq polymerase buffer attached 5 mu L Contact A 50 / L PCR reaction solution containing 4 μL of dNTP mixture (2.5 mM) attached to the PCR-Takara Ex-Taq polymerase was prepared and subjected to PCR. The PCR was first incubated at 94 ° C for 60 seconds, followed by 40 cycles of incubation at 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute, and finally at Ί2 ° C for 5 seconds. The test was performed under the condition of keeping the temperature for minutes. After PCR, a PCR product showing about 1.3 Kbp was recovered by agarose electrophoresis. The E.coli JM109 strain competent cell (Toyobo) was transformed with the plasmid prepared by subcloning the recovered PCR product into pT7-Bluevector (Novagen). Using a plasmid obtained by culturing the transformed cells in 10 OmL of LB medium containing 50 g / mL ampicillin using the QI AGEN P 1 a smid Ma Xi Kit (QI AGEN), It has the nucleotide sequence of this rat-derived gene (rAT14) A plasmid containing DNA was obtained. Example 18

Determination of the nucleotide sequence of this gene from rat (Part 2)

The plasmid containing the PCR product (approximately 1.3 kbp) obtained in Example 17 was used as type I, and then converted to a Thermo Sequenase II dye terminator kit (Amersham Pharmacia Biotech). Using the ABI 373D NA sequence reader (PEAp 1 ied Biosystems), Sanger's method (F. Sanger, S. Nicklen, AR Cou 1 son A, Proceedings of National Ac a The nucleotide sequence of this rat-derived gene (rAT14) consisting of DNA having the nucleotide sequence of SEQ ID NO: 18 was determined by emyof Science USA (1977), 74, 5463-5467). Example 19

Preparation of a plasmid for expressing a fusion protein in which FLAG is added to the amino terminal of the amino acid sequence represented by SEQ ID NO: 13

0.05 g of the plasmid separated and purified in Example 13; a primer 25 20 pmol consisting of the nucleotide sequence represented by SEQ ID NO: 25; and a primer 26 20 pmo 1 consisting of the nucleotide sequence represented by SEQ ID NO: 26, Takara ExTaq polymerase (Takara Shuzo) 0.5 U, Takara ExTaq polymerase 50 LL PCR reaction containing 5 AZL buffer and 4 d dNTP mixture (2.5 mM) attached to Takara ExTaq polymerase A liquid was prepared and subjected to PCR. The PCR was carried out under the conditions of first incubating at 94 ° C for 1 minute, then at 60 ° C for 30 seconds, then at 72 ° C for 1 minute 30 times, and finally at 72 ° C for 5 minutes. I was After PCR, an agarose electrophoresis was used to recover a PCR product exhibiting about 1.1 Kbp. A plasmid prepared by subcloning the recovered CR product into pCMV-.Tag2B (Stratagene) was used to construct E.co1i JM109 strain competent cells (Toyo Was transformed. From the cultured cells obtained by culturing the transformed cells in 50 OmL of an LB medium containing 5 O zg / mL kanamycin, the plasmid was used using QI AGEN Q IAfi i ter Mega Kit (QI AGEN). By separating and purifying, a fusion protein in which FLAG (an amino acid sequence by letter notation: MDYKDDDDKSPGGS) is added to the amino terminal of the amino acid sequence represented by SEQ ID NO: 13 (hereinafter, referred to as FLAG fusion mAT14 protein) ) Was obtained. Furthermore, it was confirmed by the method described in Example 14 that there was no mutation in the plasmid. Example 20

Preparation of enucleated cell extract containing this enzyme (Part 2): Preparation of enucleated cell extract containing FLAG fusion mAT14 protein

Using the plasmid for expressing the FLAG-fused mAT14 protein obtained in Example 19, the Lipofect AMI NE PLUS reagent kit (Invitrogen) was used to prepare CHO-K1 cells (Dainippon, Japan) according to the procedure described in the reagent kit. Pharmaceutical company). The CHO-K1 cells into which the expression plasmid was introduced were cultured for 24 hours according to the procedure manual distributed when the cells were purchased. After the culture, the cells were washed twice with D-PBS (Gipco), scraped with a cell scraper, and collected by centrifugation. Collect the collected cells in 50 mM Tris-hydrochloric acid (pH 7.5), 0.25 M sucrose, 1 mM ethylene glycol bis

Buffer solution containing 2 aminoethyl ether tetraacetic acid (hereinafter referred to as EGTA), ImM dithiothreitol (hereinafter referred to as DTT), and 1/100 volume of protease inhibitor cocktail (Sigma) (hereinafter referred to as buffer solution A) ). The suspension is sonicated (6 times x 5 seconds under ice-cooling) to crush the cells in the suspension, and the lysate obtained is then lysed at 1000 X g for 30 seconds at 4 ° C. By centrifugation at, unbroken cells and the nuclear fraction were removed as a precipitate. For the obtained supernatant, the protein concentration was measured by the Bradford method using serum albumin as a standard, and buffer A was added and diluted to the respective concentrations to obtain the enucleated cell extract. . Example 21

Preparation of control enucleated cell extract (Part 2)

As a control (negative control), pCMV-Tag 2B (without insert) was used in place of the plasmid for expressing FLAG-fused mAT14 protein, except that the method described in Example 20 was used. Nuclear cell extract

(Control) was obtained.

Example 22 (Separation and detection of this enzyme (part 2))

When 5 mL of the enucleated cell extract obtained in Examples 20 and 21 was used for immunoblotting using an anti-FLAG-tagged M2 antibody (Sigma), the control (negative control) was removed. In the case of nuclear cell extracts, nothing was detected. On the other hand, in the case of the enucleated cell extract prepared from the cells into which the plasmid for expressing the FLAG fusion mAT14 protein was introduced, a single band protein having a molecular weight of about 46 kDa was detected. Example 23

Measurement of the activity of monoacylglycerol lucyltransferase (Part 2) The enucleated cell extract l Oiz L obtained in Examples 20 and 21 was added to a 150 / zL reaction buffer (24 mM Tris-HCl (pH 7.5), 50 mM potassium chloride, 8 mM magnesium sulfate, 1.25 mg / mL serum albumin, 1 mM

DTT, 0.5 mM 2—monooleoylglycerol), and then add 5 μL of 900 / iM (0.2 MB qL) I: ¹⁴ C] Palmi Toluy Coenzyme A aqueous solution to the suspension. added. The mixture was kept at room temperature for 10 minutes. The test system was the same as above except that it did not contain 2-monooleoylglycerol! Next, add 300 μL of heptane 2-propanol / water (= 80: 20: 2) to the mixture, stir and let stand for 10 minutes, then add 20 L of heptane 100 μL to the mixture. After stirring, the mixture was centrifuged to remove the lower layer, and the remaining upper layer was dried using a centrifugal evaporator. (Fat fraction) was redissolved in black-mouthed form, and this solution was spotted on a glass plate for silica gel 150 thin-layer chromatography (Κ5 silica gel, 150 Å, Whatman). And, hexane Roh GETS chill ether Honoré Z acetate to by developing using a developing solvent of (75:: 25 1), 4 C] 1 contained in the solution was separated 2-Jiashirugurise roll. Isolated ⁴ C] 1, 2-di § sill glycerol was quantified have use bus I O Imaging Analyzer System BAS- 2000 a (Fuji Film Co.). The value obtained by subtracting the value measured in the background section from the value measured in the reaction buffer containing 2-oleoylglycerol was calculated as the enzyme activity, and a control (negative control) plasmid was introduced. Table 4 shows the activity of monoacylglycerolacyltransferase in each enucleated cell extract in the test system in which the plasmid for expressing FLAG-fused mAT14 protein was introduced. Was. Table 3

As described above, the test system in which the plasmid for expressing the FLAG-fused mAT14 protein was introduced was compared with the test system in which the control (negative control) plasmid was introduced, in comparison with the test system in which the plasmid of the control (negative control) was introduced. Since the activity of the transferase was increased, it was confirmed that the protein consisting of the amino acid sequence shown in SEQ ID NO: 13 exhibited the activity of monoacyl / regulise lucyltransferase. Example 24 (Preparation of a plasmid for expressing a fusion protein in which the amino acid sequence represented by SEQ ID NO: 3 and SEQ ID NO: 15 is added with FLAG at the amino terminus)

As in Example 8, a PCR method using the hAT-12 plasmid isolated and purified in Example 5, using primers consisting of the nucleotide sequences of SEQ ID NOS: 27 and 28 As a result, a plasmid expressing a fusion protein in which FLAG was added to the amino terminal of the amino acid sequence represented by SEQ ID NO: 3 (hereinafter, also referred to as FLAG fusion hAT-12) was obtained. Furthermore, the absence of mutation in the plasmid was confirmed by the method described in Example 4.

Similarly to Example 8, the hAT-14 plasmid isolated and purified in Example 15 was subjected to PCR using a primer consisting of the nucleotide sequences of SEQ ID NOS: 29 and 30, and was represented by SEQ ID NO: 15. A plasmid expressing a fusion protein in which FLAG was added to the amino acid end of the amino acid sequence (hereinafter sometimes referred to as FLAG fusion hAT-14) was obtained. Further, the absence of mutation in the plasmid was confirmed by the method described in Example 16. Example 25

Preparation of enucleated cell extract containing this enzyme (Part 3): Preparation of enucleated cell extract containing FLAG-fused hATl 2 protein and FLAG-fused hAT14 protein

The FLAG fusion protein expression plasmid obtained in Example 24 was introduced into CHO-K1 cells, and an enucleated cell extract was prepared in the same manner as in Example 9. Example 26

Preparation of control enucleated cell extract (Part 3)

The control (negative control) is described in Example 9 except that pCMV-Tag2B (without insert) is used instead of the plasmid for expressing FLAG-fused hAT-12 protein or the plasmid for expressing FLAG-fused hAT-14 protein. An enucleated cell extract (control) was obtained in the same manner as described above. Example 27

Separation and detection of this enzyme (Part 3)

Using 5 μL of the enucleated cell extract obtained in Examples 25 and 26 and performing immunoblotting using an anti-FLAG tag M2 antibody (Sigma), a control (negative control) was obtained. In the case of the enucleated cell extract of It was. On the other hand, in the case of an enucleated cell extract prepared from cells into which the plasmid for expressing the FLAG-fused hAT-12 protein or the plasmid for expressing the FLAG-fused hAT-14 protein has been introduced, Single band proteins with molecular weights of 45 kD and 46 kD were detected. Example 2 8

Measurement of the activity of monoacyl'lysyl lucyltransferase.

In the same manner as in Example 12, the activity of monoacylglycerol lucyltransferase in the cell extract prepared in Examples 25 and 26 was measured. The results are shown in Table 5. Table 4

Based on the above, the test system in which the plasmid for expressing FLAG-fused hAT12 protein and the plasmid for expressing FLAG-fused hAT14 protein were compared with the control system (in comparison with the test system in which the negative control was introduced). The protein comprising the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 15 was converted to monoacyl, 'reseguchi, because of the increased activity of monoacylglycerol lucyltransferase in the enucleated cell extract. It was confirmed that it exhibited the activity of lucasyltransferase. Example 29

Expression distribution of monoacylglycerol lucyltransferase gene

Plasmids obtained in Examples 8, 19 and 24 (FLAG-mATl2, mAT14, hAT12, hAT14) were treated with restriction enzymes so as to cut at both ends of the gene coding sequence excluding the FLAG sequence. A band of the desired length was cut out from the gel on which the DNA was electrophoresed, and the DNA was purified using a GENECLEANII kit (Takara Shuzo). And the DNA of about 10- 100 ng to铸型were ^{3 3} P- dCTP radiolabeled by random priming method. The reaction was performed using a random prime kit (manufactured by Takara Shuzo) according to the protocol of the kit. After removing unreacted dNTPs from the reaction solution by a spin column, the specific activity of the probe was measured by a liquid scintillation counter to confirm that the probe was sufficiently radiolabeled. A portion of the probe was heat-denatured and quenched to form a single strand, and then added to 8 ml of DIGE asy-Hyb solution (Boehringer Mannheim) to obtain a hybrid solution. In this hybridized solution, the tissue RNA plot membrane (Mouse Adult No rmal l Tissue mRNA No rthern B lot, Human Adu) previously prehybridized in DIGE as y-Hyb solution lt No rmal l T issue T ota 1 RNA No r tern B lot (all manufactured by BIOCHAIN) was immersed in a hybridization bag to start hybridization. Hybridization conditions are: pre-hybridization: 50 ° C, 3 hours or more, hybridization: 50. C

— 晚 Shaking. After hybridization, wash the membrane under the conditions of 2X SSC—O. 1% SDS, 2 times at room temperature, 0.1X SSC—0.1% SDS, 1 time at 50 ° C, and place the membrane on the imaging plate.晚 Exposure. Figures 1, 2, 3, and 4 show the results of reading the imaging plate using the Bio Imaging Analyzer System BAS-2500 (Fuji Film Co., Ltd.).

These results indicate that mouse AT12 and AT14 mRNA are significantly expressed in mouse liver and small intestine, and that human ATI2 and AT14 mRNA are significantly expressed in human small intestine. . Summarizing the above results, ATI 2 and AT 14 have monoacylglycerol lucyltransferase activity in cells, have significant expression in the liver and small intestine in mice, and have significant expression in the small intestine in humans. The expression of AT12 and AT14 suggests that AT12 and AT14 are monoacylglycerol acyltransferases involved in triglyceride synthesis in the liver and small intestine. Example 30

Establishment of cell line stably expressing FLAG fusion mAT12 protein

CHO-K1 cells (Dainippon Pharmaceutical Co., Ltd.) were obtained from the plasmid for expressing the FLAG fusion mAT12 protein obtained in Example 8 using Lipofec tAMINE PLUS reagent kit (Invitrogen) according to the procedure attached to the reagent kit. ). After culturing the CHO-K1 cells into which the expression plasmid has been introduced for 72 hours according to the procedure manual distributed at the time of purchase of the cells, Ham, containing 400 μg Zm1 of G ENET IC IN (Gibco) and 10% FCS, The culture was continued for 7 days while replacing the medium with sF-12 medium (hereinafter referred to as selection medium) and replacing with a new medium as appropriate. After the culture, the stable expression strain was cloned by the ultra-dilution method. A part of each of the obtained cell lines was partially dissolved in 5 OmM Tris-hydrochloric acid (pH 7.5), 0.25 M sucrose, ImM ethylene glycol bis 2 aminoethynol ether tetraacetic acid (hereinafter referred to as EGTA), ImM Dithiothreitol (hereinafter, referred to as DTT) was suspended in a buffer solution (hereinafter, referred to as buffer solution A) containing 1/100 volume of a protease inhibitor cocktail (Sigma). The cells in the suspension were disrupted by sonicating the suspension (x 6 times under ice-cooling for 5 seconds), and the resulting cells were crushed at 1000 Xg for 30 seconds at 4 ° C. By centrifugation, unbroken cells and the nuclear fraction were removed as a precipitate. The obtained supernatant was subjected to immunoblotting using an anti-FLAG-tagged M2 antibody (Sigma), and cells expressing the most single protein with a molecular weight of about 45 kDa were most expressed. A strain was selected to obtain a stable expression strain of mAT-12. 2004/000976

54

Example 31

Purification of FLAG fusion mAT12 protein

The cell line stably expressing the FLAG fusion mAT12 protein obtained in Example 30 was expanded in a selective medium. After the culture, the cells were washed twice with D-PBS (Gibco), scraped with a cell scraper, and collected by centrifugation. The collected cells were subjected to 5 OmM Tris monohydrochloride (H7.5), 15 OmM sodium chloride, 0.1% {3-[(3-Cholam idopropy 1) dime tny la mm on lio] These suspensions were suspended in a buffer solution containing —1—propanesulfonate} (hereinafter, referred to as CHAPS), 1 mM EGTA, 1 mM DTT, and a 1/1000 volume of protease inhibitor Kakutenore (Sigma). The suspension is sonicated (x 6 times for 6 seconds under ice-cooling) to disrupt the cells in the suspension, and the resulting cell lysate is crushed at 15000Xg for 5 minutes at 4 ° C. By centrifuging at C, uncrushed cells and the nuclear fraction were removed as a precipitate, and the supernatant was obtained as a crude cell extract. 1 ml of Anti-FLAG M2 affinity reagent was equilibrated with 5 OmM tris-hydrochloric acid (pH 7.5), 15 OmM sodium chloride, 0.1% CHAPS (hereinafter referred to as buffer B). The crude cell extract was added to the column to adsorb the FLAG-fused mAT12 protein. After washing the column with 10 volumes of buffer B, the effluent was stopped, and buffer B containing FLAG peptide was added and incubated. After 10 minutes, re-flow was started to obtain a FLAG-fused mAT12 protein purified fraction. Imunoblotting using an anti-FLAG tag M2 antibody (Sigma) was performed, and it was confirmed that this fraction contained a protein, which was a single band with a molecular weight of about 45 kDa. Example 32

Measurement of monoacylglycero ^ "lucyltransferase activity of purified FLAG-fused mAT12 protein

The purified FLAG fusion mAT12 protein purified fraction 101 obtained in Example 31 was combined with a reaction buffer (24 mM Tris-hydrochloric acid (pH 7.5), 5 OmM salted potassium, 8 mM magnesium sulfate, 1. 25 mg / m 1 ゥ serum albumin, 1 mM DTT, 0.5 mM 2-monooleoylglycerol) 1 After suspending in 30 μ 溶解, dissolve in the test substance (dimethylsulfoxide (hereinafter referred to as DMSO)) and add to a final concentration of 1% DMSΟ ) Was added and mixed. To this mixture was added 10 Shino ^{450 ^ 1 ^ C 1 4 C} ] Norumitoiru one Koenzaimu A solution was incubated at room temperature for 20 minutes. In the test substance-free group, the same method as described above was used except that 1.5 μl of only DMSO was added instead of the DMSO solution of the test substance. In the background group, the same method as that in the group to which the test substance and 2-monooleoylglycerol were added was used, except that only DMSO was added instead of the test substance and 2-monooleoylglycerol. After completion of the incubation for 20 minutes, the reaction was stopped by adding 30 O / i L of heptane / 2-propanol / water (= 80: 20: 2) to the reaction mixture, and further heptane 200 / X After adding 1 and water 100, the mixture was suspended, and after stirring, the mixture was centrifuged to remove the lower layer, and the remaining upper layer was dried using a centrifugal evaporator. The dried product (fat fraction) was redissolved in 30 μl of form / methanol (2: 1, v / v), and this solution was then dissolved in a silica gel 150 thin-layer chromatography glass plate ( Κ 5 Silica Genome 1 50 o Hexane, dimethyl ether / acetic acid (75: 25: 1, v / v) in a sealed container, and use the developing solvent to prepare the TLC plate. After the development, the TLC plate was dried at room temperature, and the dried TLC plate was exposed with an imaging plate (Fuji Film Co., Ltd.) for 16 hours. BAS 2500, and analyzed by Fuji film Co.), 1 on the TLC plate, 2 - C ¹ of a portion corresponding to the deployed position of the di § sill glycerol Contact Yopi triglyceride ⁴ C] radioactivity was measured (hereinafter, 1 , 2-di § sill glycerol moiety C ^{1 4} C] exhibition of a portion corresponding to the open position of the radioactivity measurements G, the portion corresponding to the deployed position of the bird § sill triglycidyl Theroux Le I: ^{1 4} C] radiation active The property is described as a measured value H). From these measurements it was calculated activity Monoashirugurise port Ruashirutoran Sufueraze of adding a test substance (measured value G + measurements H Bruno ^2, hereinafter referred to as activity value 4). The same applies as above for the test substance-free and packed ground areas. On TLC plates using such methods 1, 2-di § sill glycerol Contact Yopi C ^{1 4} C] radioactivity of the portion corresponding to the deployed position of the bird glycerides were measured (hereinafter, the test substance untreated silage E ¹⁴ C] radioactivity measured at the portion corresponding to the development position of 1,2-diasylglycerol I, ⁴ C] radioactivity at the portion corresponding to the development position of triacylglycerol in the test substance-free area Is the measured value J, the radioactivity of the portion corresponding to the development position of 1,2-diasylglycerol in the background plot was measured K, and the radioactivity of the portion corresponding to the development position of triacylglycerol in the background plot was measured Measured value L). From these measured values, the activity (measured value I + measured value J 2; hereinafter, referred to as activity value 5) of the monoacylglycerol acyltransferase in the test substance-free group and the background group The activity (measured value + measured value L / 2, hereinafter, referred to as activity value 6) of monoacylglycerol lucyltransferase was calculated. From the obtained activity values, the monoacylglycerol acyltransferase inhibitory ability of the test substance was calculated as an inhibition rate ((activity value 5—activity value 4) × 100 / (activity value 5—activity value 6)).

As a result, the known test compound N- [2- (4-benzyl-1-2-ethylphenoxy) ethyl] —5,6-dimethyl [1,2,4] triazolo [1,5-a] pyrimidine-1 The inhibition rate of 7-amine (hereinafter sometimes referred to as compound A) was about 65% at 10 μΜ. Further, while changing the test concentration of the test substance, the inhibition rate at each test concentration was calculated using the same method as described above. Table 6 shows the results.

From the above, it was found that the known compound 効果 effectively inhibits the enzyme activities of monoacyl and lysacyl lucyltransferase of mAT12 or FLAG-fused mAT12. Table 5 Test substance Examples

Test concentration (βΜ) Inhibition rate

Compound A-1 0

Compound A 1 0 65 Compound A 3 29

Compound A 120 Example 33

Inhibitory effect of monoacylglycerolacyltransferase inhibitor on triglyceride synthesis in human liver cells

After culturing human hepatoma-derived cell line HepG2 cells (Dainippon Pharmaceutical Co., Ltd.) in a 6-well plate until the cells are in a confluent state, the cells are cultured in serum-free D-MEM medium (GIBCO, containing high glucose) for 1 晚. Starved by treatment. Replacing the medium of the cells, the final concentration ^{12. 9 μΜ [U- 1 4 C} ] D- glucose (Amersham, radioactive AmC iZL) D- MEM medium containing (GI BCO Co., low glucose containing) the Immediately afterwards, it was dissolved in a test substance (dimethylsulfoxide (hereinafter referred to as DMSO)), added to a final concentration of 0.5% DMSO, and cultured at 37 ° C for 4 hours. In the test substance-free group, the same method as described above was used except that only DMS O was added instead of the DM SO solution of the test substance. After washing the cells after the culture once with a PBS buffer, the cells were treated with 1 ml of heptane'isopropanol (2: 1) for 10 minutes. The heptane-isopropanol solution containing triglyceride in the cells was collected with a pit, and the collected solution was dried using a centrifugal evaporator. The dried product (fat fraction) thus obtained was redissolved in 30 μl of form / methanol (2: 1, ν / ν). Next, this solution was spotted on a glass plate for silica gel 150 thin-layer chromatography (Κ5 silica gel, 150 Angstrom, Whatman). Hexane Ζethyl ether was placed in a closed container.

A developing solvent of acetic acid (75: 25: 1, v / v) was added, and the TLC plate was developed using the developing solvent, and then the TLC plate was dried at room temperature. The dried TLC plate was exposed to an imaging plate (Fuji Film Co., Ltd.) for 16 hours. After the exposure was completed, the imaging plate was analyzed with a bio-image analyzer (BAS 2500, Fujifinorem Co., Ltd.). The ⁴ C] radioactivity was measured at the portion corresponding to the development position of triacylglycerol in the TLC plate using the same method as above for the test substance-free area. The ⁴ C] radioactivity of the portion corresponding to the development position of the above triacylglycerol was measured.

As a result, the test compound known compound N- [2- (4-benzyl-2-ethylphenyloxy) ethyl] —5,6-dimethyl [1,2,4] triazolo [1,5—a] pyrimidine-1 7-Amine (hereinafter sometimes referred to as Compound A) The amount of triglyceride synthesized at the time of addition 1 is about 31 ° at 10 / ΛΜ, assuming that the amount of synthesis in the test substance-free group is 100%. / ₀ . Furthermore, while varying the test concentration of the test substance, the results of calculating the amount of synthesis at each test concentration using the same method as described above are shown in Table 7.

As a result, it was found that the known compound 効果 effectively inhibited triglyceride synthesis in human liver cells. Therefore, it is considered that the monoasinoleglycerol noreacinoletransferase activity inhibitor can inhibit lipoprotein (VLDL) synthesis by inhibiting hepatic triglyceride synthesis. Table 6

Example 34

Effects of monoacylglycerol inhibitors on blood triglyceride elevation after administration of fats and oils to mice

I CR mice (12-week-old, female, Japan SLC) 8 animals / group were fasted for 1 hour, then N- [2- (4-benzyl-1-2-ethylfurenoxy) ethyl:] — 5,6-dimethyl [ 1,2,4] Triazolo [1,5-a] Olive oil (Nacalai Tesque, Inc.) suspended with pyrimidine-17-amine (hereinafter referred to as Compound A): 5 ml / kg Oral administration. Olive oil containing no compound A was orally administered to eight mice as a non-treated substance. 1 Ο L of blood was collected over time from the tail vein of the mouse and dissolved in 1 ml of isopropanol. The amount of toridariside in the isopropanol extract was measured using Toridariseride Test Kit Co. (Wako Pure Chemical Industries). Figure 5 shows the time course of triglyceride levels in the blood when Compound A was administered at a dose of 30 mg Zkg, and 3 hours when the dose of Compound A was changed to 0, 10 and 30 mg / kg. FIG. 6 shows the results of measuring the amount of triglyceride in blood after time.

As a result, it was confirmed that when the known compound A was administered to mice together with lipids, the blood triglyceride increasing power S was effectively inhibited. Therefore, monoacylglycerol lucyltransferase inhibitors effectively inhibit intestinal cell lipoprotein (chylomicron) synthesis' secretion by inhibiting intestinal cell tridaliseride synthesis, and consequently blood triglyceride concentration. It is considered possible to reduce Industrial potential

The present invention provides a simple method for testing the ability to suppress an increase in fat mass. According to the present invention, by suppressing fat accumulation in the fat system and suppressing an increase in adipose tissue mass, glucose tolerance decreased closely related to fat accumulation in fat tissues, diabetes, hyperlipidemia, hypertension, It is possible to treat or prevent metabolic diseases such as arteriosclerosis and diseases such as coronary artery disease, angina pectoris, and cardiovascular disorders such as myocardial infarction. In addition, it suppresses fat synthesis in the supply of fat to the blood and suppresses the increase in fat mass in the blood. It is possible to treat or prevent arteriosclerotic diseases such as blood heart disease and cerebral infarction.

[Sequence list free text] '

SEQ ID NO: 5

Primers designed for PCR

SEQ ID NO: 6

Primers designed for PCR SEQ ID NO: 7

Primers designed for PCR SEQ ID NO: 8

Primers designed for PCR SEQ ID NO: 9

Primers designed for PCR SEQ ID NO: 10

Primers designed for PCR SEQ ID NO: 19

Primers designed for PCR SEQ ID NO: 20

Primers designed for PCR SEQ ID NO: 21

Primer designed for PCR SEQ ID NO: 22

Primers designed for PCR SEQ ID NO: 23

Primers designed for PCR SEQ ID NO: 24

Primers designed for PCR SEQ ID NO: 25

Primers designed for PCR SEQ ID NO: 26

Primers designed for PCR SEQ ID NO: 27

Primer designed for PCR ^ "SEQ ID NO: 28

Primer designed for PCR SEQ ID NO: 29 Primers designed for PCR SEQ ID NO: 30

Primers designed for PCR

Claims

The scope of the claims

1. A method for testing the ability of a test substance to suppress the increase in fat mass,

(1) a first step of measuring the activity of the monoacylglycerol acyltransferase in the contact system between the monoacylglycerolacyltransferase and the test substance; and

(2) a second step of evaluating the ability of the substance to suppress an increase in fat mass based on a difference obtained by comparing the activity measured in the first step with the activity in a control. Method.

2. A method for searching for a substance having an increased amount of fat, which comprises selecting a substance having an ability to inhibit an increase in fat mass based on the ability to inhibit an increase in fat mass tested by the assay method according to claim 1.

3. A fat mass increase inhibitor comprising a substance selected by the search method according to claim 2 or a pharmaceutically acceptable salt thereof as an active ingredient.

4. An agent for suppressing an increase in fat mass, comprising a substance having a monoacylglycerol luacyltransferase inhibitory ability or a pharmaceutically acceptable salt thereof as an active ingredient.

5. The assay method according to claim 1, wherein the monoacylglycerol luassyltransferase is derived from a mammal.

6. The assay method according to claim 1, wherein the monoacylglycerol-lacyltransferase is any of the following proteins:

(a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, 3 or 11;

(a-1) a protein consisting of the amino acid sequence represented by SEQ ID NO: 13, 15, or 17;

(b) an amino acid sequence represented by SEQ ID NO: 1, 3, or 11, wherein one or more amino acids have been deleted, added or substituted, and the amino acid sequence has been deleted. An active protein,

(b-1) an amino acid sequence represented by SEQ ID NO: 13, 15, or 17, comprising one or more amino acid deletion, addition or substitution amino acid sequences; A protein having an activity of monoacylglycerol acyltransferase;

(c) a protein consisting of an amino acid sequence having at least 80% of the amino acid sequence represented by SEQ ID NO: 1, 3 or 11 and having an amino acid sequence, and having monosylglycerol lucyltransferase activity;

(c-11) a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, 15, or 17, and having a monoacylglycerol-l-acyltransferase activity;

(d) a protein consisting of an amino acid sequence encoded by a DNA having the nucleotide sequence of SEQ ID NO: 2, 4, or 12,

(d-1) a protein comprising an amino acid sequence encoded by DNA having a nucleotide sequence represented by SEQ ID NO: 14, 16 or 18;

(e) an amino acid sequence encoded by a DNA having a nucleotide sequence having a sequence identity of 80% or more with DNA having a nucleotide sequence represented by SEQ ID NO: 2, 4, or 12, and A protein having lucasyltransferase activity,

(e-1) an amino acid sequence encoded by DNA having a nucleotide sequence having 80% or more sequence identity with DNA having the nucleotide sequence of SEQ ID NO: 14, 16 or 18, and A protein having lurasyltransferase activity,

(f) a DNA comprising a nucleotide sequence complementary to the DNA having the nucleotide sequence of SEQ ID NO: 2, 4 or 12, and an amino acid sequence encoded by a DNA that hybridizes under stringent conditions A protein having a potent monoacylglycerol lucyltransferase activity, and

(f-1) DNA comprising a nucleotide sequence complementary to DNA having the nucleotide sequence of SEQ ID NO: 14, 16 or 18, and an amino acid sequence encoded by a DNA that hybridizes under stringent conditions A protein having an activity of monoasinoleglycerose lucyltransferase.

7. The test method according to any one of claims 1, 5 or 6, wherein the fat mass increase suppressing ability is a fat accumulation suppressing ability.

8. A method of searching for a fat accumulation inhibiting substance, which comprises selecting a substance having a fat accumulation inhibiting ability based on the fat accumulation inhibiting ability tested by the assay method according to claim 7.

9. A fat accumulation inhibitor comprising, as an active ingredient, a substance selected by the search method according to claim 8 or a pharmaceutically acceptable salt thereof.

10. A fat accumulation inhibitor comprising, as an active ingredient, a substance having a monoacylglycerolacyltransferase inhibitory ability or a pharmaceutically acceptable salt thereof.

1. The assay method according to claim 1, wherein the ability to inhibit an increase in fat mass is an ability to inhibit lipoprotein synthesis.

12. A method for searching for a lipoprotein synthesis inhibitor, which comprises selecting a substance having lipoprotein synthesis inhibitory ability based on the lipoprotein synthesis inhibitory ability assayed by the assay method according to claim 11.

13. A lipoprotein synthesis inhibitor comprising, as an active ingredient, a substance selected by the search method according to claim 12 or a pharmaceutically acceptable salt thereof.

14. A lipoprotein synthesis inhibitor comprising, as an active ingredient, a substance having a monoacylglycerol luasyltransferase inhibitory ability or a pharmaceutically acceptable salt thereof.

15. The substance having the ability to inhibit monoacylglycerol lucyltransferase is N- [2- (4-benzyl-1-ethylphenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1, 15. The lipoprotein synthesis inhibitor according to claim 14, which is 5-a] pyrimidine-17-amine.

16. Contains N- [2- (4-benzyl-1-2-ethylphenoxy) ethyl] -5,6-dimethyl [1,2,4] triazolo [1,5-a] pyrimidine-17-amine as an active ingredient Monoacylglycerose ^ "Lucyltransferase inhibition.

17. A protein having any one of the following amino acid sequences: (a) an amino acid sequence represented by SEQ ID NO: 1, 3 or 11;

(a-1) an amino acid sequence represented by SEQ ID NO: 13, 15, or 17; (b) an amino acid sequence in which one or more amino acids have been deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 1, 3 or 11, wherein the amino acid sequence is aminoglycoside; Amino acid sequence of a protein having transferase activity,

(b-1) an amino acid sequence represented by SEQ ID NO: 13, 15, or 17 in which one or more amino acids are deleted, added, or substituted, and wherein the amino acid sequence is mono-acylglycerol-luacyltransferase; An amino acid sequence of a protein having activity;

(c) an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 1, 3 or 11, which is an amino acid sequence of a protein having a strong mono-acylglycerol-lusyltransferase activity;

(c-1) an amino acid sequence which is an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, 15 or 17, and which has a monoacylglycerol lucyl transpuerase activity; Acid sequence,

(d) an amino acid sequence IJ encoded by a DNA having a nucleotide sequence represented by SEQ ID NO: 2, 4, or 12,

(d-1) an amino acid sequence encoded by a DNA having the nucleotide sequence of SEQ ID NO: 14, 16, or 18,

(e) an amino acid sequence encoded by a DNA having a nucleotide sequence having 80% or more sequence identity with DNA having a nucleotide sequence represented by SEQ ID NO: 2, 4 or 12; and Amino acid sequence of a protein having lucyltransferase activity,

(e-1) an amino acid sequence S encoded by DNA having a nucleotide sequence having 80% or more sequence identity with DNA having a nucleotide sequence represented by SEQ ID NO: 14, 16, or 18, An amino acid sequence of a protein having an activity of monoacyloleglyceronoresyltransferase;

(f) a DNA consisting of a nucleotide sequence complementary to a DNA having the nucleotide sequence of SEQ ID NO: 2, 4 or 12, and an amino acid sequence encoded by a DNA that hybridizes under stringent conditions , Powerful monoglycerose An amino acid sequence of a protein having siltransferase activity, and

(f-1) An amino acid sequence encoded by a DNA having a nucleotide sequence represented by SEQ ID NO: 14, 16 or 18 and a DNA consisting of a complementary nucleotide sequence and a DNA hybridizing under stringent conditions And an amino acid sequence of a protein having monoacynoregulose oral lucyltransferase activity.

18. A gene characterized by having a nucleotide sequence encoding any of the following amino acid sequences:

(a) the amino acid sequence represented by SEQ ID NO: 1, 3 or 11,

(a-1) an amino acid sequence represented by SEQ ID NO: 13, 15, or 17;

(b) an amino acid sequence represented by SEQ ID NO: 1, 3 or 11, wherein one or more amino acids are deleted, added or substituted, and the protein has a monoacylglycerose luciferase activity Amino acid sequence,

(b-1) an amino acid sequence in which one or more amino acids have been deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 13, 15, or 17, and An amino acid sequence of a protein having

(c) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 1, 3 or 11, and having a strong monoacylglycerol acyltransferase activity; ,

(c-1) an amino acid sequence having 80% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 13, 15, or 17, and having a monoacylglycine-to-lucyltransferase activity; ,

(d) the amino acid sequence I encoded by the DNA having the nucleotide sequence of SEQ ID NO: 2, 4, or 12

(d-1) an amino acid system IJ encoded by a DNA having a nucleotide sequence represented by SEQ ID NO: 14, 16 or 18;

(e) an amino acid sequence encoded by DNA having a nucleotide sequence having 80% or more sequence identity with DNA having the nucleotide sequence of SEQ ID NO: 2, 4 or 12 An amino acid sequence of a protein having a monoasinoregylglycerol acyltransferase activity;

(e-1) an amino acid sequence encoded by a DNA having a nucleotide sequence having 80% or more sequence identity with the DNA having the nucleotide sequence shown by SEQ ID NO: 14, 16, or 18; An amino acid sequence of a protein having monoacylglycerol lucyltransferase activity;

(f) a DNA consisting of a nucleotide sequence complementary to a DNA having the nucleotide sequence of SEQ ID NO: 2, 4 or 12, and an amino acid sequence encoded by the DNA that hybridizes under stringent conditions. An amino acid sequence of a protein having a strong monoacylglycerol lucyltransferase activity; and

(f-1) a DNA consisting of a base sequence complementary to the DNA having the base sequence of SEQ ID NO: 14, 16, or 18, and an amino acid encoded by a DNA that hybridizes under stringent conditions An amino acid sequence of a protein having a monosacylglycerolacyltransferase activity.

19. The catalyst of the following reaction, comprising the protein of claim 17.

2 or 1-monoacylglycerol + acyl-CoA → 1,2-diacylglycerolone + CoA.