JP2020097531A - GLP-1 secretagogue - Google Patents

Abstract

The present invention relates to a GLP-1 secretagogue. The GLP-1 secretagogue of the present invention is propionic acid, butyric acid, valeric acid, pantothenic acid, cholic acid, 3-aminoisobutyric acid, γ-aminobutyric acid, 2-aminobutyric acid, pipecolic acid, ornithine, citrulline. , Tyrosine methyl ester, piperidine, taurine, pyridoxal, pyridoxamine, pyridoxine, sarcosine, N,N-dimethylglycine, cytosine, 1,3-diaminopropane, nicotinic acid, indole-3-acetamide, D-alanine, 5-hydroxylysine. , 1-methylnicotinamide, thiamine, carnitine, gluconic acid, prostaglandin E2, methionine sulfoxide, spermidine and spermine, and one or more compounds selected from the group consisting of: [Selection diagram] None

Description

The present invention relates to a GLP-1 secretagogue, a composition containing the GLP-1 secretagogue, and a food or drink containing the GLP-1 secretagogue.

GLP-1
In the intestinal tract, there are endocrine cells that secrete gastrointestinal hormones and regulate the functions of other organs. Some of the gastrointestinal hormones secreted from intestinal endocrine cells, also called incretins, are secreted into the blood with food intake, act on pancreatic β cells (hereinafter “pancreatic β cells”), and secrete insulin. Promote. This incretin not only promotes insulin secretion, but also promotes pancreatic β-cell regeneration and also acts on the central nervous system via the vagus nerve to suppress feeding behavior, and thus, it is a novel disease of diabetes and obesity. It is attracting attention as a target for therapeutic drug development.

There are two types of incretins, a glucose-dependent insulin secretion stimulating polypeptide (glucose-dependent insulinotropic peptide; GIP) and a glucagon-like peptide-1 (GLP-1).

GLP-1 is secreted by small intestinal endocrine L cells (hereinafter, “L cells”) existing in the lower small intestine. L cells face both the basal lamina and luminal side of the small intestinal mucosa and are referred to as "open" endocrine cells. A sodium-dependent glucose transporter exists in L cells and co-transports glucose and sodium extracellularly, that is, in the lumen, into the cells. As a result, the membrane potential is depolarized and the voltage-dependent L-type Ca ²⁺ channel is activated. The inflow of Ca ²⁺ from the voltage-dependent L-type Ca ²⁺ channel into cells causes GLP-1 to be released in a glucose-dependent manner. Furthermore, fatty acid receptors, bile acid receptors and sweet taste receptors of G protein-coupled receptors (GPCRs) are expressed in L cells, and lipids, bile acids and glucose in the lumen are expressed. Upon sensitization, it is believed to exocytose GLP-1. However, the details are unknown.

Diabetes Diabetes refers to a condition in which the blood glucose level and hemoglobin A1c (HbA1c) level exceed a certain standard, and is one of the diseases that the country places particular importance on in medical measures. In type 1 diabetes, β cells of the pancreas are destroyed for some reason, and insulin, which is one of the hormones that regulates blood glucose level, is depleted, leading to hyperglycemia and diabetes. In type 2 diabetes, due to obesity and the like, the amount of insulin secreted from β cells in the islets of Langerhans (pancreatic islets) of the pancreas is decreased, and the ability of glucose uptake into muscle and adipose tissue is decreased (insulin resistance is increased). However, as a result, blood glucose is not stored as glycogen in the liver and adipose tissue, and the blood concentration deviates from the normal range to a high blood glucose level, which is characterized by hyperglycemia, insulin resistance, and relative insulin deficiency. It is a long-term metabolic disorder.

Diabetes may cause symptoms due to hyperglycemia itself such as thirst and polydipsia, but various complications truly cause problems. Diabetes neuropathy, diabetic retinopathy, and diabetic nephropathy are known as complications, and it can be said that the main purpose of diabetes treatment is to prevent these complications.

Insulin injection is known as a treatment method for diabetes, but it is highly invasive and burdens the patient. Therefore, various oral antidiabetic agents have been developed that have a low administration burden. So far, drugs such as insulin secretagogues such as GLP-1, sulfonylureas, glucose transporter (GLUT) inhibitors and the like have been developed. However, there have been problems such as side effects and a decrease in the effects of regular use. Further, hypoglycemia due to excessive secretion of insulin has become a problem, and it has been desired to develop a medicine that solves such a problem.

JP-A-2016-138045 describes GLP-1 secretagogues containing compounds having the specific structure of formula (I).

JP, 2016-138045, A

Experimental Medicine Vol. 35, No. 2 (Special Issue) 2017, p. 106-110 Scientific Reports 2:233, 2012

An object of the present invention is to provide various GLP-1 secretagogues, compositions containing the GLP-1 secretagogues, and foods and drinks containing the GLP-1 secretagogues.

Since small intestinal endocrine L cells are present in only about a few% of cells constituting the small intestine, it is difficult to isolate and purify them and perform primary culture. The present inventors used GLUTag cells, which are a mouse-derived small intestinal endocrine L cell line having GLP-1 secreting ability, and STC-1 cells derived from a mouse small intestinal endocrine cell carcinoma, for analysis, and used GLP -1 secretion promotion was investigated, various GLP-1 secretion promoters were discovered, and this invention was conceived.

The present invention includes, but is not limited to, the following aspects.
[Aspect 1]
Propionic acid, butyric acid, valeric acid, pantothenic acid, cholic acid, 3-aminoisobutyric acid, γ-aminobutyric acid, 2-aminobutyric acid, pipecolic acid, ornithine, citrulline, tyrosine methyl ester, piperidine, taurine, pyridoxal, pyridoxamine, pyridoxine, Sarcosine, N,N-dimethylglycine, cytosine, 1,3-diaminopropane, nicotinic acid, indole-3-acetamide, D-alanine, 5-hydroxylysine, 1-methylnicotinamide, thiamine, carnitine, gluconic acid, A GLP-1 secretagogue comprising one or more compounds selected from the group consisting of prostaglandin E2, methionine sulfoxide, spermidine and spermine.
[Aspect 2]
A composition for promoting insulin secretion, which comprises the GLP-1 secretagogue according to aspect 1.
[Aspect 3]
A composition for lowering blood glucose level, which comprises the GLP-1 secretagogue according to aspect 1.
[Mode 4]
A composition for preventing or treating diabetes, which comprises the GLP-1 secretagogue according to aspect 1.
[Aspect 5]
A composition for preventing or improving obesity, which comprises the GLP-1 secretagogue according to aspect 1.
[Aspect 6]
A composition for suppressing appetite, which comprises the GLP-1 secretagogue according to aspect 1.
[Aspect 7]
A food or drink comprising the GLP-1 secretagogue according to aspect 1.

The substance found in the present invention exerts a GLP-1 secretory action at, but not limited to, about μM to about mM.

1. GLP-1 secretagogue In one aspect, the present invention relates to a GLP-1 secretagogue.

The GLP-1 secretagogue includes, but is not limited to, propionic acid, butyric acid, valeric acid, pantothenic acid, cholic acid, 3-aminoisobutyric acid, γ-aminobutyric acid, 2-aminobutyric acid, pipecolic acid, ornithine, citrulline, Tyrosine methyl ester, piperidine, taurine, pyridoxal, pyridoxamine, pyridoxine, sarcosine, N,N-dimethylglycine, cytosine, 1,3-diaminopropane, nicotinic acid, indole-3-acetamide, D-alanine, 5-hydroxylysine, Includes one or more compounds selected from the group consisting of 1-methylnicotinamide, thiamine, carnitine, gluconic acid, prostaglandin E2, methionine sulfoxide, spermidine and spermine. A mode in which these substances are orally administered to a living body by inserting a carrier such as a capsule, or a mode in which a precursor of these substances is orally administered to a living body and enterobacteria produce these substances, -1 Included in secretagogues.

The GLP-1 secretagogue may consist of one of the above compounds or a mixture of two or more. Each of the above compounds alone has the ability to promote GLP-1 secretion. It also includes those produced by enterobacteria.

"GLP-1" is an abbreviation for "glucagon-like peptide-1". GLP-1 is a gastrointestinal peptide hormone produced primarily by small intestinal endocrine L cells. In production cells, it is excised from preproglucagon as GLP-1(1-37). The amino acid on the N-terminal side is cleaved to become GLP-1(7-37) and GLP-1(7-36)amide, which have strong physiological activity. When the term "GLP-1" is used in the present specification, all of GLP-1(1-37), GLP-1(7-37) and GLP-1(7-36) having physiological activity, or Means any one. GLP-1 acts on pancreatic β cells to increase insulin secretion and decrease glucagon secretion. Increased insulin secretion inhibits gastric acid secretion and gastric emptying, and increases satiety to reduce food intake.

"GLP-1 secretion-promoting" means that the secretion of GLP-1 from GLP-1 secreting cells is promoted in the presence of the GLP-1 secretagogue, as compared with the absence thereof. To do. In the absence of the GLP-1 secretagogue, secretion of GLP-1 from GLP-1 secreting cells was not detected, but it also includes the case where it becomes detectable due to the presence of the GLP-1 secretagogue. Preferably, without limitation, the secretion of GLP-1 is 1.05 times or more, 1.1 times or more, 1.2 times or more, 1.5, as compared with the case where a GLP-1 secretion promoting agent is not present. Double or more, 1.8 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 5 or more.

2. Compositions Containing GLP-1 Secretagogues The present invention, in one aspect, relates to compositions comprising the GLP-1 secretagogues of the present invention. The composition of the present invention includes, but is not limited to, one of "promoting insulin secretion", "decreasing blood glucose level", "preventing or treating diabetes", "preventing or improving obesity", "suppressing appetite" or It can have multiple uses. The meaning of “GLP-1 secretagogue” is as described in the item of “1. GLP-1 secretagogue”. The composition of the present invention may be a pharmaceutical composition or a food composition. In addition to ordinary foods and drinks, the food composition also includes modes such as seasonings and supplements.

In one aspect, the present invention relates to a composition for promoting insulin secretion, which comprises the GLP-1 secretagogue of the present invention.

The term "insulin" is a polypeptide hormone that regulates glucose metabolism. Insulin binds to the insulin receptor on insulin-sensitive cells and mediates glucose uptake. Insulin is used to treat type 1 diabetes and may also be used to treat type 2 diabetes.

"Insulin secretagogue" means that the secretion of insulin from β cells in the islets of Langerhans (pancreatic islets) of the pancreas is promoted when the GLP-1 secretagogue is present, rather than when it is not present. Means In the absence of the GLP-1 secretagogue, the secretion of insulin from β cells was not detected, but it also includes the case where the presence of the GLP-1 secretagogue makes it possible to detect it. Preferably, without limitation, the secretion of insulin is 1.05 times or more, 1.1 times or more, 1.2 times or more, 1.5 times or more, as compared with the case where no GLP-1 secretagogue is present. 1.8 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 5 times or more.

GLP-1 is known to promote glucose-dependent insulin secretion and reduce blood glucose levels. The GLP-1 secretagogues are various types associated with insufficient insulin secretion, insufficient GLP-1 secretion or hyperglycemia. It can be used as a medicine for the above diseases. Examples of the disease include diabetes, obesity, hypertension, hyperlipidemia and the like.

In one aspect, the present invention relates to a composition for lowering blood glucose level, which comprises the GLP-1 secretagogue of the present invention.

The “composition for lowering blood glucose level” refers to a composition having the action of lowering (or suppressing the elevation of) the blood glucose level when the composition is administered, as compared to when the composition is not administered. The blood glucose level is the concentration of glucose (glucose) in the blood. In the case of humans, the fasting blood glucose level is approximately 80-120 mg/dL, but it slightly increases due to food intake, and after about 20-40 minutes, it rises to a maximum of 120-200 mg/dL in healthy subjects. .. Then, in the case of a healthy person, the blood glucose level becomes fasting within 120 minutes after food intake. In the present invention, "the blood sugar level decreases (or suppresses the increase)" means that the maximum blood sugar level increase due to food intake decreases, and the difference between the maximum blood sugar level after intake and the fasting blood sugar level ( The maximum blood sugar level rise due to food intake-fasting blood sugar level) decreases, the blood sugar level rises slowly, the time until the blood sugar level drops to fasting after the blood sugar level rises, etc. means.

"The maximum increase in blood glucose level due to food intake is reduced" means that, when the composition is administered, the maximum blood glucose level is decreased as compared with the case where the composition is not administered, Preferably, it means 1 mg/dL or more, 5 mg/dL or more, 10 mg/dL or more, 20 mg/dL or more, 30 mg/dL, 40 mg/dL or more.

"The difference between the maximum blood glucose level after ingestion and the fasting blood glucose level (the maximum blood glucose level increase due to food intake-fasting blood glucose level) is reduced" means that the composition was administered without limitation. In this case, the "maximum blood glucose level increase due to food intake-fasting blood glucose level" is decreased, preferably 1 mg/dL or more, 5 mg/dL or more, 10 mg/dL or more, 20 mg/ It means decrease by dL or more, 30 mg/dL, 40 mg/dL or more.

The phrase "the time until the blood glucose level on an empty stomach is lowered after the blood glucose level rises is shortened" means that, when the composition is administered, it is shorter than that when the composition is not administered, preferably 1 Minutes or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, 30 minutes or more, or 60 minutes or more.

In one aspect, the present invention relates to a composition for preventing or treating diabetes, which comprises the GLP-1 secretagogue of the present invention. Diabetes can be either type 1 diabetes or type 2 diabetes. Diabetes can be diagnosed, for example, by the diabetes diagnostic criteria defined in the Diabetes Standard Practice Manual issued by the National Center for Global Health and Medicine. The GLP-1 secretion-promoting agent can be administered in the preventive sense not only to patients diagnosed with diabetes but also to patients suspected of having diabetes due to high blood sugar level.

In one aspect, the present invention relates to a composition for preventing or improving obesity, which comprises the GLP-1 secretagogue of the present invention. Obesity generally means a situation in which the body weight is higher than in a normal state, or a state in which body fat is excessively accumulated. It does not matter whether there are symptoms associated with an increase in body weight or body fat.

In one aspect, the present invention relates to a composition for suppressing appetite, which comprises the GLP-1 secretagogue of the present invention.

The dosage form of the composition containing the GLP-1 secretagogue is not particularly limited and can be appropriately selected depending on the therapeutic purpose. Specific examples include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, syrups, suppositories, injections and patches.

The composition containing the GLP-1 secretagogue may contain an active ingredient other than the substances specified above. For example, an insulin secretagogue such as SU agent, a DPP-4 inhibitor, a sugar absorption delaying agent, or an insulin sensitizer may be contained in one agent together with the above-identified substance, or both may be separated. What was mixed with the agent may be one kit.

In formulating the composition containing the GLP-1 secretagogue, the excipient, the binder, the disintegrant, the lubricant, the stabilizer, the preservative, the flavoring agent, and the diluent are not limited to the above. You may add the additive required for formulation such as.

Compositions containing the GLP-1 secretagogues may be formulated, for example, according to standard techniques in the pharmaceutical field. For example, Alphanso Gennaro, ed. , Remington's Pharmaceutical Sciences, 18th Ed. , (1990) Mack Publishing Co. , Easton, Pa.

The administration method of the composition containing the GLP-1 secretagogue is not particularly limited and may be oral administration or parenteral administration. The dose of the agent of the present invention is determined according to the symptoms, weight, age, sex, etc. of the patient, but generally, the amount of the active ingredient is about 1 μg/kg to 1000 mg/kg per day for an adult. The range is preferably 10 μg/kg to 100 mg/kg, more preferably 100 μg/kg to 100 mg/kg. 3. Food-drinks TECHNICAL FIELD In one aspect, this invention relates to food-drinks containing the GLP-1 secretion promoter of this invention.

The GLP-1 secretagogue can be added to any food or beverage, and the type of food or beverage is not particularly limited.

The “food” can be used for all foods such as Japanese food, Western food, Chinese food, Asian food and the like. For example, it is seasoned, stir-fried, stewed, rice, soup, pasta sauce, pot, soup, stew and the like.

"Beverages" include, for example, milk drinks, soups for drinks such as canned products, yogurt, soy milk, sports drinks, fruit juice drinks, alcoholic beverages, tea, coffee, green tea, cocoa and the like.

Without limitation, the GLP-1 secretagogue may be included in seasonings such as soy sauce, sauce, soup, mirin, and cooking sake.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples. Those skilled in the art can easily make modifications and changes to the present invention based on the description of the present specification, and those are included in the technical scope of the present invention.

Unless otherwise stated, the examples were made with the following materials and procedures.

GLUTag cells, a small intestinal endocrine L cell line derived from cell culture mice, were obtained from Daniel J. I got it from Professor Drucker. GLUTag cells were cultured in a 100 mm dish, and Dulbecco's modified Eagle medium containing 1000 mg/L glucose, 584 mg/LL-glutamine, 3.7 g/L sodium hydrogen carbonate and 110 mg/L sodium pyruvate (Sigma Aldrich Japan GK, Tokyo, (Japan) with inactivated 10% fetal bovine serum (Sigma-Aldrich Japan GK, Tokyo, Japan), 100 U/mL penicillin/0.1 mg/mL streptomycin (Sigma Aldrich Japan GK, Tokyo, Japan) added. Was used as the culture medium. The cells were cultured at 37° C. under 5% CO ₂ , and trypsinized when the cells grew to 80% to 90% of a 100 mm dish, and then passaged.

STC-1 cells, which are a mouse-derived GLP-1-producing endocrine cell line, were kindly donated by Dr. Douglas Hanahan of the University of California (now Swiss Cancer Institute). STC-1 cells were cultured in a 100 mm dish, and Dulbecco's modified Eagle medium containing 4500 mg/L glucose, 584 mg/L L-glutamine, 3.7 g/L sodium hydrogen carbonate and 110 mg/L sodium pyruvate (Sigma Aldrich Japan GK). , Tokyo, Japan), inactivated 10% fetal bovine serum (Sigma Aldrich Japan GK, Tokyo, Japan), 100 U/mL penicillin, 0.1 mg/mL streptomycin (Sigma Aldrich Japan GK, Tokyo, Japan). Was added as a culture solution. The cells were cultured at 37° C. under 5% CO ₂ , and trypsinized when the cells grew to 80% to 90% of a 100 mm dish, and passaged.

When carrying out an experiment with the reagent GLUTag cells, various substances to be tested were dissolved in the glucose-containing imaging recording solution described below.

When carrying out the experiment with STC-1 cells, various substances to be tested were dissolved in a glucose-containing imaging recording solution described later.

All of the various substances used in the test are substances confirmed to be produced by intestinal bacteria (Matsumoto et al., Scientific Reports 2:233, 2012).

Example 1 Measurement of GLP-1 Secretion by Calcium ²⁺ Imaging In this example, GLUTag cells or STC-1 cells were cultured in a culture medium containing a stimulating solution containing various substances, and GLP-1 was measured by calcium ²⁺ imaging. Secretion was observed.

(1) Introduction of Calcium Indicator In order to fix the cells on the cover glass of the glass bottom dish, the cover glass was coated with poly L-lysine (Sigma Aldrich Japan GK, Tokyo, Japan). 200 μL of 1 mg/mL P poly L-lysine (Sigma Aldrich Japan LLC, Tokyo, Japan) was dropped on the cover glass of a 35 mm glass bottom dish that had been UV projected for 15 minutes, and allowed to stand for 30 minutes, and then phosphate buffered saline. It was washed three times with water (Phosphate Buffered Saline: PBS).

Then, a suspension of GLUTag cells was dropped on the cover glass and cultured in 2 mL of culture medium for 2 days. Similarly, the suspension of STC-1 cells was dropped on the cover glass, and then cultured in 2 mL of culture medium for 2 days, and was replaced with 2 mL of culture medium containing 4500 mg/L glucose on the day before imaging.

After culturing GLUTag cells, an imaging recording solution containing 5 mM glucose (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH ₂ PO ₄ , 0.5 mM MgSO ₄ , 1.5 mM CaCl ₂ , 10 mM HEPES, 2 mM NaHCO ₃ ) The cell culture dish was washed twice with. Then, to 1 mL of an imaging recording solution containing 5 mM glucose, a fluorescent indicator Fluo4-AM (Dojindo, Kumamoto, Japan) for visualization analysis of intracellular calcium ²⁺ concentration change as fluorescence intensity change was added so as to be 250 nM, Incubated for 20 minutes at 37° C., 5% CO ₂ . After culturing the STC-1 cells, the cell culture dish was washed twice with an imaging recording solution containing 2.2 mM glucose. Then, Fluo4-AM was added to 750 μL of an imaging recording solution containing 2.2 mM glucose at 2.5 μM, and the mixture was incubated at 37° C. under 5% CO ₂ for 20 minutes.

(2) Imaging by Inverted Microscope The culture dish of GLUTag cells introduced with Fluo4-AM was washed twice with an imaging recording solution containing 0.1 mM glucose. Then, the solution was replaced with 1.9 mL of an imaging recording solution containing 0.1 mM glucose, and observation was performed. The culture dish of STC-1 cells introduced with Fluo4-AM was washed twice with a recording solution containing 2.2 mM glucose, replaced with 2 mL of a recording solution containing 2.2 mM glucose, and observed.

The photography when stimulating with putrescine, spermidine, and spermine was performed every 5 seconds for 20 minutes, and the stimulation solution was refluxed 3 minutes after the start of photography. Imaging for stimulation with other substances was performed every 5 seconds for 10 minutes, 100 μL of the stimulation solution was directly dropped 3 minutes after the start of imaging, and Fluo4-AM-derived fluorescence was measured with an inverted microscope. The stimulating solution was adjusted to a concentration 20 times higher than the condition so that the concentration became the condition after the dropping.

In the observation, an inverted microscope (Axio Observer D1, Carl Zeiss, Oberkochen, Germany) or an IX71 inverted microscope (Olympus Corporation, Tokyo, Japan) was used. In the former microscope, a mercury lamp (HBO100, Carl Zeiss) is used for excitation light, a BP470/40 excitation filter and a set of FT495 dichroic mirrors (B38-HE, Carl Zeiss) are used for blue excitation, and a camera (ORCA-) is used. Flash 4.0V2, C11440, Hamamatsu Photonics KK, Shizuoka, Japan). As the objective lens, an oil immersion 40× lens (UPlanApo, Olympus) having a numerical aperture of 1.00 was used. The IX71 inverted microscope was excited with a xenon lamp (Olympus), photographed with an Evolve EM-CCD camera (Photometrics, Tuscon, Arizona, USA) through a fluorescence filter, and the objective lens was an oil immersion lens with a numerical aperture of 1.35. Double (Olympus) was used.

(result)
As a result of imaging with an inverted microscope, the maximum value of the intracellular calcium ²⁺ concentration change rate of each cell after administration of the stimulation solution and the maximum value of the intracellular calcium ²⁺ concentration change rate of each cell after administration of the recording solution were compared by statistical analysis. It was judged that substances with a significant difference induced GLP-1 secretion (+), and substances with no significant difference did not induce GLP-1 secretion (-). did. The results are shown in Table 1.

As shown in Table 1, propionic acid, butyric acid, valeric acid, pantothenic acid, cholic acid, 3-aminoisobutyric acid, γ-aminobutyric acid, 2-aminobutyric acid, pipecolic acid, ornithine, citrulline, tyrosine methyl ester, piperidine, taurine. , Pyridoxal, pyridoxamine, pyridoxine, sarcosine, N,N-dimethylglycine, cytosine, 1,3-diaminopropane, nicotinic acid, indole-3-acetamide, D-alanine, 5-hydroxylysine, 1-methylnicotinamide, Various substances such as thiamine, carnitine, gluconic acid, prostaglandin E2, methionine sulfoxide, spermidine and spermine were found to increase intracellular Ca ²⁺ concentration showing a GLP-1 secretion-inducing effect at a concentration of about μM to mM. It was

Example 2 Measurement of GLP-1 Secretion Amount by Enzyme Immunoassay In this example, GLUTag cells or STC-1 cells were cultured in a culture medium to which a stimulating solution containing various substances was added, and an enzyme immunoassay method (ELISA) was used. ), the GLP-1 concentration was measured and the promotion of GLP-1 secretion was quantified.

(Example 2-1) Measurement of GLP-1 secretion in GLUTag cells When GLUTag cells were used, the cells were seeded in a 24-well plate at 1.0×10 ⁵ cells per well, and 37° C. in the culture medium. Incubated for 2 days under 5% CO ₂ . After 2 days, each well was washed twice with an imaging recording solution containing 0.1 mM glucose, and among the substances used in Example 1, an increase in intracellular Ca ²⁺ concentration showing a GLP-1 secretion-inducing effect was observed. 500 μL of a stimulating solution containing the above substance, that is, the substance of (+) in Table 1, was added and incubated in the culture medium at 37° C. under 5% CO ₂ for 30 minutes.

Then, the total amount of the cell culture solution was transferred to a 1.5 mL tube, centrifuged at 1000×g and 4° C. for 10 minutes, and the supernatant was diluted 2-fold with an imaging recording solution containing 0.1 mM glucose. The concentration of GLP-1 in the diluted solution was measured using GLP-1 (7-36) Active ELISA Kit (Merck Millipore, Darmstadt, Germany) and Varioskan LUX (Thermo Fisher SCIENTIFIC, Mass., USA).

(Example 2-1) Measurement of GLP-1 secretion in STC-1 cells When using STC-1 cells, the cells were seeded in a 6-well plate at 1.0×10 ⁶ cells per well, and in the culture medium. Incubated at 37° C., 5% CO ₂ for 2 days. The day before, the medium was replaced with a culture solution containing 1000 mg/L glucose. Each well was washed twice with an imaging recording solution containing 2.2 mM glucose, 1 mL of a stimulating solution corresponding to each condition was added, and the mixture was incubated in a culture medium at 37° C. under 5% CO ₂ for 120 minutes. Then, the total amount of the cell culture solution was collected in a 1.5 mL tube, and after centrifugation at 1000 xg and 4°C for 10 minutes, the GLP-1 concentration of the GLP-1 ELISA kit Wako, a high-sensitivity product (Fuji Film Wako Pure Chemical Industries, Ltd. (Company, Osaka, Japan) and Varioskan LUX.

Results GLUTag cells or STC-1 cells were cultured in a culture medium to which a stimulation solution containing various substances was added, and the GLP-1 concentration was measured by enzyme-linked immunosorbent assay (ELISA). Various substances such as propionic acid, butyric acid, valeric acid, pantothenic acid, cholic acid, taurine, pyridoxamine, spermidine, and spermine induced or promoted GLP-1 secretion at a concentration of about μM to mM.

Claims (7)

Propionic acid, butyric acid, valeric acid, pantothenic acid, cholic acid, 3-aminoisobutyric acid, γ-aminobutyric acid, 2-aminobutyric acid, pipecolic acid, ornithine, citrulline, tyrosine methyl ester, piperidine, taurine, pyridoxal, pyridoxamine, pyridoxine, Sarcosine, N,N-dimethylglycine, cytosine, 1,3-diaminopropane, nicotinic acid, indole-3-acetamide, D-alanine, 5-hydroxylysine, 1-methylnicotinamide, thiamine, carnitine, gluconic acid, A GLP-1 secretagogue comprising one or more compounds selected from the group consisting of prostaglandin E2, methionine sulfoxide, spermidine and spermine. A composition for promoting insulin secretion, comprising the GLP-1 secretagogue according to claim 1. A composition for lowering blood glucose level, comprising the GLP-1 secretagogue according to claim 1. A composition for preventing or treating diabetes, which comprises the GLP-1 secretagogue according to claim 1. A composition for preventing or improving obesity, comprising the GLP-1 secretagogue according to claim 1. A composition for suppressing appetite, comprising the GLP-1 secretagogue according to claim 1. A food or drink comprising the GLP-1 secretagogue according to claim 1.