JP6514282B2 - Evaluation or selection method of GIP elevation inhibitor - Google Patents

Description

  The present invention relates to a method for evaluating or selecting a GIP elevation inhibitor.

  GIP (glucose-dependent insulinotropic polypeptide) is an incretin which is secreted from secretory cells (K cells) of gastrointestinal epithelia by ingesting lipids, carbohydrates and amino acids. GIP promotes insulin secretion from pancreatic β cells in a glucose dependent manner and contributes to regulation of blood glucose level. In recent years, it has been reported that mice artificially increased blood GIP concentration suppress lipid burning under high-fat intake. In addition, it has been revealed that GIP receptor deficient mice are inhibited from accumulation of visceral fat and subcutaneous fat due to high fat diet loading. From these findings, postprandial GIP regulation is considered to be effective for the prevention and improvement of obesity. Furthermore, since GIP is known to have a gastric acid secretion suppressive action and a gastric motility suppressive action, it is considered that the suppression of the elevation of GIP is effective for the promotion of postprandial digestion and the improvement of stomach upset. Therefore, development of a substance that suppresses the elevation of GIP is desired, and for that purpose, development of a method capable of rapidly evaluating the ability to suppress the elevation of GIP of a substance with high sensitivity is required.

  As a conventional method for evaluating or selecting a GIP elevation inhibitor, a method using expression of CPT1 gene or CPT1 protein in cells or activity of CPT1 protein as an indicator (Patent Document 1), FAT / CD36 gene or FAT / CD36 protein The method (patent document 2) etc. which make expression an index are known.

  FABPs (fatty acid-binding proteins) are a family of fatty acid binding proteins in cells, and are 14-15 kDa proteins having high binding to fatty acids (Non-patent Documents 1 and 2). Fatty acids have diverse functions in cells such as energy sources and signal molecules for metabolic control. FABPs play an important role in the functional expression of fatty acids by binding to insoluble fatty acids to enable transport to various organs in cells.

  The isoforms of FABPs, FABP4 and FABP5, are highly homologous to amino acid sequences and three-dimensional structures, and are co-expressed in adipocytes and macrophages (non-patent documents 2 to 4). Functional analysis using knockout mice has been performed for FABP4 and FABP5 (Non-patent Documents 2 to 15). FABP4 accounts for 1 to 3% of cytoplasmic proteins of fat cells, and is widely used as a fat cell differentiation marker (Non-patent Document 5). In addition to its function as a molecular chaperone in adipocytes, FABP4 has also been reported to be involved in lipid signal transduction and organelle responses, and in macrophages in inflammatory reactions ( Non Patent Literatures 2, 6, 9). FABP5 has been suggested to be involved in the pathogenesis of psoriasis in keratinocytes (Non-patent Document 7) and reported to be involved in the control of cytokine (IL-12p70), which is a key molecule for innate immune response, in the pancreas. (Non-Patent Document 8).

  In the ob / ob mouse which is a type 2 diabetes model, a mouse in which FABP4 was knocked out was found to suppress the reduction in insulin sensitivity (Non-patent Document 11). In addition, feeding a high-fat diet to FABP4 knockout mice suppresses the decrease in insulin sensitivity compared to wild-type, but no effect is observed on weight gain or fatty liver (Non-patent Documents 4 and 10). Since the expression of FABP5 is elevated in adipocytes of FABP4 knockout mice, it is believed that FABP5 works as a compensator (Non-patent Documents 16 and 17). The same tendency as FABP4 knockout mice is also observed in FABP5 knockout mice (Non-patent Document 12). Based on the results in single knockout mice, analysis with FABP 4/5 double knockout was performed, and diet-induced obesity, insulin resistance, type 2 diabetes, when fed a high fat diet compared to wild type It has been reported that the induction of fatty liver was suppressed (Non-patent Documents 13, 14, 15).

  Oral administration of BMS309403, which is an inhibitor of FABP, to a model mouse of type 2 diabetes or arteriosclerosis improved the pathological condition (Non-patent Document 18). Administration of a FABP 4/5 inhibitor to diet-induced obese mice resulted in the improvement of dyslipidemia, such as the reduction of blood triglyceride levels and free fatty acid levels (Non-patent Document 19).

  However, FABP that is mainly expressed in the intestine is FABP2, and thus, the role of lipid metabolism in the intestine was conventionally thought to be played by FABP2.

JP, 2011-080804, A JP, 2011-080803, A

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  The present invention relates to a method for evaluating or selecting a GIP elevation inhibitor.

  The present inventors have expressed that FABP4 and FABP5 (these may be collectively referred to as FABP4 / 5 in the present specification) in GIP-secreting enterocytes, and inhibit the FABP4 / 5. It has been found that the concentration of GIP in the blood is lowered by the use of the substance, and therefore a substance that inhibits FABP4 / 5 is useful as a GIP elevation inhibitor. From these findings, the present inventors have found that it is possible to evaluate or select GIP elevation inhibitors using the inhibitory action of FABP4 / 5 expression or activity as an indicator.

Accordingly, the present invention comprises the following steps (A) to (D):
(A) contacting a test substance with a FABP4 gene or FABP5 gene, or a tissue or cell derived from a mammal capable of expressing FABP4 protein or FABP5 protein, (B) a FABP4 gene or a gene or the like in the tissue or cell derived from the mammal Measuring the expression level of FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein,
(C) comparing the expression level or activity measured in (B) with the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein in a control group, or the activity of FABP4 protein or FABP5 protein,
(D) Based on the results of (C) above, a GIP elevation inhibitor is a test substance that decreases the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or FABP4 protein or FABP5 protein activity Evaluating or selecting as
Provided a method for evaluating or selecting a GIP elevation inhibitor comprising

In the present invention, the following steps (A ′) to (D ′):
(A ') administering a test substance to a non-human mammal,
(B ') measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal,
(C ') The expression level or activity measured in the above (B'), the expression level of the FABP4 gene or FABP5 gene, the expression level of the FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal of the control group, or FABP4 Comparing with the activity of the protein or FABP5 protein,
(D ') Based on the result of the above (C'), GIP elevation of the test substance which decreases the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or FABP4 protein or FABP5 protein activity Evaluating or selecting as an inhibitor,
Provided a method for evaluating or selecting a GIP elevation inhibitor comprising

  ADVANTAGE OF THE INVENTION According to this invention, the GIP raise inhibitory effect of various substances can be evaluated more simply and correctly, and it becomes possible to select the outstanding GIP raise inhibitor. Moreover, the GIP elevation inhibitor selected by the method of the present invention reduces the possibility of onset of obesity or the like, prevents or improves weight gain, or reduces body weight, or promotes digestion after meals and improves stomach upset. It is useful as an active ingredient for

Co-immunostaining of FABP5 and GIP in enterocytes. Blood TG and blood GIP concentrations in mice after single administration of lipid emulsion. A: Time-dependent changes in blood ΔTG and blood ΔGIP, B: iAUC (0-1 h) and Cmax of blood GIP concentration. Effects of FABP4 / 5 inhibitors on lipid-induced GIP secretion in small intestinal primary cultured cells. A: Influence by BMS 309403, B: Influence by Compound 2. Principle of FABP5 activity measurement method used in Example 4. FABP5 inhibitory activity by GIP elevation inhibitor. Fish oil GIP rise suppressive action. A: change in blood GIP concentration (ΔGIP), B: iAUC (0-4 h) of blood GIP concentration. *: P <0.05.

  Conventionally, it is known that FABPs are expressed in the intestine, and by immunostaining of mouse intestinal tissue sections, it is known that FABP4 is localized on the cell membrane of intestinal epithelial cells ([ www.rndsystems.com/ihc_molecule_images.aspx?m=1416]). On the other hand, conventionally, FABP which is mainly expressed in the intestine is known to be FABP2, and therefore, lipid metabolism in the intestine was considered to be borne by FABP2.

  However, when we performed co-immunostaining of GIP and FABP5 in intestinal tissue sections, a strong signal of FABP5 was observed in cells in which a GIP signal was detected (FIG. 1). From these results, it was revealed that FABP4 / 5 is a protein expressed in intestinal epithelial cells. In addition, as shown in Examples described later, it was found that FABP4 / 5 was involved in GIP secretion because the GIP concentration was lowered by inhibiting FABP4 / 5. Therefore, substances that inhibit FABP4 / 5 are useful as GIP elevation inhibitors, and by measuring the expression level of FABP4 / 5 gene or the expression level or activity of FABP4 / 5 protein, GIP elevation suppression of various substances is suppressed. The effects can be assessed and GIP elevation inhibitors can be selected.

  Therefore, in the present invention, the GIP elevation inhibitory action of various substances is evaluated using the FABP 4/5 inhibitory action as an index, and a GIP elevation inhibitor is selected based on the evaluation result. As used herein, the term "GIP elevation suppression" refers to a diet containing lipids and carbohydrates, particularly a diet rich in lipids, and more preferably a diet rich in triacylglycerol, from K cells present in the small intestine. To suppress the elevation of secreted GIP. That is, "suppressing GIP elevation" in the present specification refers to suppressing GIP elevation that mainly occurs after eating. And the "GIP elevation suppression action" in this specification suppresses the GIP secretion suppression action by suppressing GIP secretion from K cells, and suppresses the GIP elevation by lowering blood GIP concentration. It is a concept that includes any of the GIP lowering effects.

  As used herein, “expression of FABP4 / 5” refers to expression of FABP4 gene or FABP5 gene, or expression of FABP4 protein or FABP5 protein. Further, in the present specification, "the activity of FABP4 / 5" refers to the activity of FABP4 protein or FABP5 protein.

  The method for evaluating or selecting a GIP elevation inhibitor of the present invention can be performed in vitro or ex vivo, or in vivo.

When performed in vitro or ex vivo, the method for evaluating or selecting a GIP elevation inhibitor of the present invention comprises the following steps (A) to (D).
(A) contacting a test substance with a tissue or cell derived from a mammal capable of expressing FABP4 / 5,
(B) measuring the expression level or activity of FABP4 / 5 in tissues or cells derived from said mammal,
(C) comparing the expression level or activity measured in (B) with the expression level or activity of FABP4 / 5 in a control group,
(D) A step of evaluating or selecting a test substance which decreases the expression level or activity of FABP 4/5 as a GIP elevation inhibitor based on the result of the above (C).

  The test substance used in the method of the present invention is not particularly limited as long as it is a substance desired to be used as a GIP elevation inhibitor. The test substance may be a naturally occurring substance, a substance artificially synthesized by a chemical or biological method or the like, and whether it is a compound or a composition or a mixture. Good.

  Examples of tissues or cells derived from a mammal capable of expressing FABP4 / 5 used in the above step (A) include a FABP4 gene or FABP5 gene, or a mammal alone expressing FABP4 protein or FABP5 protein. It includes separated tissues or cells, or cultures thereof. The isolated tissue or cell, or a culture thereof, small intestine tissue or cell such as duodenum or jejunum collected from a mammal, adipose tissue or cell, thymic epithelial tissue or cell, skin epithelial tissue or cell, Cultures of them; small intestine primary cultured cells; small intestinal cultured cells such as Caco-2 cells, IEC-6 cells, IEC-18 cells, STC-1 cells, and GLUTag cells; 3T3-L1 cells, and the like.

  Alternatively, as an example of a tissue or cell derived from a mammal capable of expressing FABP4 / 5, which is used in the above step (A), genetically expressing the FABP4 gene or the FABP5 gene, or the FABP4 protein or the FABP5 protein Included are modified mammalian tissues or cells, or cultures thereof. The genetically modified mammalian tissue or cell, and a culture thereof, for example, introduce the FABP4 protein or the gene encoding the FABP5 protein into any tissue or cell of the mammal, and the tissue or cell It can be produced by transforming to express FABP4 / 5, or to enhance expression of FABP4 / 5. Methods of introducing genes into cells include, but are not limited to, vector introduction by electroporation, lipofection, and the like.

  The mammals from which tissues or cells capable of expressing FABP4 / 5 used in the method of the present invention are derived are not particularly limited, and examples include humans, mice, rats, hamsters, rabbits and the like.

  The contact of the test substance with a tissue or cell derived from a mammal capable of expressing the FABP 4/5 and the test substance may be carried out, for example, by adding the test substance to the culture solution in advance to a predetermined concentration, and then culturing the tissue or cell Alternatively, the test substance can be added to a culture solution on which the tissue or cells are placed so as to have a predetermined concentration. The tissue or cells after contact are preferably cultured, for example, at room temperature (25 ° C.) to 37 ° C. for usually about 3 to 48 hours, preferably about 6 to 24 hours.

  The concentration of the tissue or cells at the time of seeding to the culture medium is not particularly limited as long as the cells can grow. Further, the concentration of the test substance to be added is preferably 0.00001 to 10% by mass (dry residue), and particularly preferably 0.0001 to 3% by mass (dry residue).

  As the culture medium for culturing the above-mentioned tissues or cells, a common culture medium can be used, and examples thereof include Dulbecco's Modified Eagle's Medium containing 10% FBS. At the time of cell passaging and growth, it is preferable to add a growth additive such as serum, growth factor and insulin, an antibacterial agent and the like to these media.

  Next, in the step (B), the tissue or cells are recovered, and the expression level of the FABP4 gene or FABP5 gene, the expression level of the FABP4 protein or FABP5 protein, or the activity of the FABP4 protein or FABP5 protein is measured. When measuring the expression level of the gene, when detecting at the mRNA level, for example, total RNA is extracted from cells, and using real time RT-PCR, RNAse protection assay, Northern blot analysis, etc. This can be performed by detecting and quantifying mRNA transcribed from the FABP4 / 5 gene.

  The expression level of FABP4 / 5 protein can be measured by a conventional immunoassay, such as RIA method, EIA method, ELISA, bioassay method, western blot, etc. However, western blot is preferable because of its inexpensiveness, simplicity and convenience. The activity of FABP4 / 5 protein can be measured, for example, by measuring the amount of bound substrate bound to FABP4 / 5 protein.

  In steps (C) to (D), expression of FABP 4/5 in a tissue or cell (test group) derived from a mammal capable of expressing FABP 4/5 contacted with the test substance measured in step (B) above The amount or activity is compared with the expression amount or activity of FABP4 / 5 in the control group, and based on the comparison result, a test substance that can be used as a GIP elevation inhibitor is selected.

  The control group includes tissues or cells derived from a mammal capable of expressing the same FABP 4/5 as the test group and which has not been contacted with the test substance. Alternatively, as a control group, a tissue or cell derived from a mammal which naturally has no or little ability to express FABP4 / 5; the same tissue or cell as the test group is modified to not express FABP4 / 5; Furthermore, those obtained by contacting these tissues or cells with a test substance, and the like can be mentioned. Tissues or cells modified so as not to express FABP4 / 5 include FABP4 / 5 knockdown cells by siRNA, tissues or cells derived from FABP4 / 5 knockout mice, and the like. The measurement of the expression level or activity of FABP4 / 5 in the control group can be performed by the same procedure as in the case of the test group described in relation to step (B) above.

  Next, the expression level or activity of FABP4 / 5 in the test group is compared with the expression level or activity of FABP4 / 5 in the control group. When the expression level or activity in the test group is reduced as compared to the control group, the test substance is evaluated as having a GIP elevation inhibitory effect, and the test substance is selected as a GIP elevation inhibitor. For example, when the expression level or activity in the test group is statistically significantly decreased relative to the expression level or activity in the control group, the test substance is evaluated to have a GIP elevation inhibitory effect. As another example, when the expression level or activity in the control group is 100%, the expression level or activity in the test group is 90% or less, preferably 80% or less, more preferably 60% or less. It is evaluated that the test substance has a GIP elevation inhibitory effect. The test substance evaluated as having a GIP elevation inhibitory effect is selected as a GIP elevation inhibitor.

When performed in vivo, the method for evaluating or selecting a GIP elevation inhibitor of the present invention comprises the following steps (A ′) to (D ′).
(A ') administering a test substance to a non-human mammal,
(B ') measuring the expression level or activity of FABP4 / 5 in the small intestine collected from the non-human mammal,
(C ′) comparing the expression level or activity measured in (B ′) above with the expression level or activity of FABP 4/5 in the small intestine collected from a non-human mammal of the control group,
(D ′) A step of evaluating or selecting a test substance that decreases the expression level or activity of FABP 4/5 as a GIP elevation inhibitor based on the result of the above (C ′).

  The non-human mammal used in the step (A ′) may be any type of animal regardless of gender or age. For example, primates such as mice, rats, hamsters, guinea pigs, rabbits, cats, dogs and monkeys can be mentioned, but rodents such as rats and mice are preferable in terms of easy availability and handling.

  Examples of the method of administering the test substance to the non-human mammal include oral administration, gastrointestinal administration, intraperitoneal administration, intravascular administration, intradermal administration, subcutaneous administration and the like. From the viewpoint of simplicity and low invasiveness, oral administration is preferred. Alternatively, since GIP is secreted from K cells in the duodenum and jejunum, a method of refluxing directly into the duodenum and jejunum using cannula or the like is also preferable.

  The dose of the test substance is 0.0004 mg / g body weight or more, preferably 0.04 to 2 mg / g body weight. The number of times of administration may be single or divided into several times at intervals. Administration for each diet is preferable, and administration between 60 minutes before and 60 minutes after meals is more preferable.

  Next, in step (B ′), the expression level or activity of FABP4 / 5 in the small intestine of the non-human mammal to which the test substance has been administered is measured. The method of measurement may be an invasive method or a non-invasive method, and is not particularly limited.

  For example, the small intestine is collected from the non-human mammal after 1 to 360 minutes, preferably 5 to 120 minutes after administration of the test substance. The small intestine is collected under anesthesia or immediately after euthanasia, and the lower part of the stomach is collected from the pylorus. Then, the expression level or activity of FABP4 / 5 in the collected cells is measured.

  In steps (C ') to (D'), the test substance is not administered but the expression amount or activity of FABP 4/5 in the small intestine (test group) of the non-human mammal to which the test substance obtained above is administered The expression level or activity of the small intestine (control group) collected from the same non-human mammal is compared. When the expression level or activity in the test group is reduced as compared to the control group, the test substance is evaluated as having a GIP elevation inhibitory effect, and the test substance is selected as a GIP elevation inhibitor.

  Other methods in this method, such as types of test substances that can be used, procedures for measuring the expression level or activity of FABP 4/5, procedures for comparing test groups and control groups, procedures for evaluation or selection of test substances are described above. The same as in the in vitro or ex vivo method.

  The selected substance may be further screened if necessary. For example, for test substances evaluated or selected to have GIP elevation suppression effect by the above-mentioned method, secretion stimulation test using cultured cell line of mammalian small intestine K cell model or single administration experiment using experimental animal By directly measuring the ability to suppress GIP elevation, it is possible to further sort out substances having a stronger GIP elevation inhibitory action.

  The GIP elevation inhibitor selected according to the present invention in the above procedure reduces postprandial GIP, and for the reduction, prevention or amelioration of the onset of obesity, for weight loss and weight gain suppression, or for promoting digestion It is used as an active ingredient to improve stomach upset.

Therefore, in another aspect, the present invention provides a method for evaluating or selecting an anorectic agent comprising the above steps (A) to (D) or (A ') to (D'). In still another aspect, the present invention provides a method for evaluating or selecting an agent for preventing and / or improving obesity, comprising the steps (A) to (D) or (A ′) to (D ′) above. In yet another aspect, the present invention provides a method for evaluating or selecting a weight gain inhibitor comprising the above steps (A) to (D) or (A ') to (D'). In still another aspect, the present invention provides a method for evaluating or selecting a digestive aid or a stomach ameliorating agent, which comprises the above steps (A) to (D) or (A ') to (D').
In step (D) or (D ') of these methods, a test substance that decreases the expression level or activity of FABP 4/5 based on the result of step (C) or (C') is an anorectic agent, obesity It is evaluated or selected as a preventive and / or ameliorating agent, a weight gain inhibitor, or a digestive aid or a stomach ameliorating agent.

  The following compositions, methods of manufacture, applications or methods are further disclosed herein as exemplary embodiments of the present invention. However, the present invention is not limited to these embodiments.

<1> Steps (A) to (D) below:
(A) contacting a test substance with a FABP4 gene or FABP5 gene, or a tissue or cell derived from a mammal capable of expressing FABP4 protein or FABP5 protein, (B) a FABP4 gene or a gene or the like in the tissue or cell derived from the mammal Measuring the expression level of FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein,
(C) comparing the expression level or activity measured in (B) with the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein in a control group, or the activity of FABP4 protein or FABP5 protein,
(D) Based on the results of (C) above, a GIP elevation inhibitor is a test substance that decreases the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or FABP4 protein or FABP5 protein activity Evaluating or selecting as
A method for evaluating or selecting a GIP elevation inhibitor, comprising

<2> the following steps (A) to (D):
(A) contacting a test substance with tissues or cells derived from a mammal capable of expressing FABP4 gene or FABP5 gene, or FABP4 protein or FABP5 protein,
(B) measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in a tissue or cell derived from said mammal,
(C) comparing the expression level or activity measured in (B) with the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein in a control group, or the activity of FABP4 protein or FABP5 protein,
(D) Based on the results of (C) above, a test substance that decreases the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein as an appetite suppressant Evaluating or selecting process,
A method of evaluating or selecting an appetite suppressant, comprising

<3> The following steps (A) to (D):
(A) contacting a test substance with tissues or cells derived from a mammal capable of expressing FABP4 gene or FABP5 gene, or FABP4 protein or FABP5 protein,
(B) measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in a tissue or cell derived from said mammal,
(C) comparing the expression level or activity measured in (B) with the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein in a control group, or the activity of FABP4 protein or FABP5 protein,
(D) Based on the results of the above (C), a test substance that reduces the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or FABP4 protein or FABP5 protein activity can be used to prevent and / or reduce obesity. Or a step of evaluating or selecting as a remedy,
A method for evaluating or selecting an agent for preventing and / or improving obesity, including

<4> The following steps (A) to (D):
(A) contacting a test substance with tissues or cells derived from a mammal capable of expressing FABP4 gene or FABP5 gene, or FABP4 protein or FABP5 protein,
(B) measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in a tissue or cell derived from said mammal,
(C) comparing the expression level or activity measured in (B) with the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein in a control group, or the activity of FABP4 protein or FABP5 protein,
(D) Based on the results of the above (C), a test substance that decreases the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein is a weight gain inhibitor Evaluating or selecting as
A method of evaluating or selecting a weight gain inhibitor, comprising

<5> Steps (A) to (D) below:
(A) contacting a test substance with tissues or cells derived from a mammal capable of expressing FABP4 gene or FABP5 gene, or FABP4 protein or FABP5 protein,
(B) measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in a tissue or cell derived from said mammal,
(C) comparing the expression level or activity measured in (B) with the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein in a control group, or the activity of FABP4 protein or FABP5 protein,
(D) Based on the result of the above (C), a test substance which decreases the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or FABP4 protein or FABP5 protein activity is used as a digestion promoter Evaluating or selecting process,
A method of evaluating or selecting a digestive enhancer, which comprises

<6> The following steps (A) to (D):
(A) contacting a test substance with tissues or cells derived from a mammal capable of expressing FABP4 gene or FABP5 gene, or FABP4 protein or FABP5 protein,
(B) measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in a tissue or cell derived from said mammal,
(C) comparing the expression level or activity measured in (B) with the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein in a control group, or the activity of FABP4 protein or FABP5 protein,
(D) Based on the results of the above (C), the agent for improving the stomach is a test substance that reduces the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein Evaluating or selecting as
A method for evaluating or selecting an agent for improving stomach upset, including

<7> The method according to any one of <1> to <6>, wherein preferably the above-mentioned FABP4 gene or FABP5 gene, or a tissue or cell derived from a mammal capable of expressing FABP4 protein or FABP5 protein is the following: :
(1) Small intestine tissue or cells collected from a mammal, or a culture thereof
(2) adipose tissue or cells collected from a mammal, thymic epithelial tissue or cells, skin epithelial tissue or cells, or a culture thereof;
(3) FABP4 gene or FABP5 gene, or mammalian tissue or cell genetically modified to express FABP4 protein or FABP5 protein, or a culture thereof;
(4) Small intestine primary cultured cells; or
(5) Caco-2 cells, IEC-6 cells, IEC-18 cells, STC-1 cells, GLUTag cells, or 3T3-L1 cells.

<8> Preferably, the method according to any one of <1> to <7>, wherein the control group is the following:
(1) A tissue or cell derived from a mammal capable of expressing the above FABP4 gene or FABP5 gene, or FABP4 protein or FABP5 protein, which has not been contacted with the above-mentioned test substance;
(2) A tissue or cell derived from a mammal which has no or little ability to express FABP4 gene or FABP5 gene, or FABP4 protein or FABP5 protein by nature;
(3) The above-mentioned FABP4 gene or FABP5 gene, or a tissue or cell derived from a mammal capable of expressing FABP4 protein or FABP5 protein is modified such that the gene or protein is not expressed; or (4) the (2) Or (3) in which the tissue or cells are brought into contact with a test substance.

<9> Preferably, when the expression level or activity measured in (B) above is statistically significantly reduced relative to the expression level or activity in the control group, the test substance suppresses GIP elevation The method according to any one of <1> to <8>, which is evaluated to have an effect, an appetite suppressive effect, an obesity preventing and / or improving effect, a weight gain suppressing effect, a digestive promotion effect, or a stomach upset improving effect .

<10> Preferably, the expression level or activity measured in the above (B) is 90% or less, preferably 80% or less, more preferably 60% or less when the expression level or activity in the control group is 100% If it is, the above-mentioned test substance is evaluated as having a GIP elevation suppressing effect, anorectic suppressing effect, an obesity preventing and / or improving effect, a weight gain suppressing effect, a digestive promotion effect, or a stomach upset improving effect, <1> The method according to any one of <8>.

<11> The following steps (A ') to (D'):
(A ') administering a test substance to a non-human mammal,
(B ') measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal,
(C ') The expression level or activity measured in the above (B'), the expression level of the FABP4 gene or FABP5 gene, the expression level of the FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal of the control group, or FABP4 Comparing with the activity of the protein or FABP5 protein,
(D ') Based on the result of the above (C'), GIP elevation of the test substance which decreases the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or FABP4 protein or FABP5 protein activity Evaluating or selecting as an inhibitor,
A method for evaluating or selecting a GIP elevation inhibitor, comprising

<12> The following steps (A ') to (D'):
(A ') administering a test substance to a non-human mammal,
(B ') measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal,
(C ') The expression level or activity measured in the above (B'), the expression level of the FABP4 gene or FABP5 gene, the expression level of the FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal of the control group, or FABP4 Comparing with the activity of the protein or FABP5 protein,
(D ') Based on the result of the above (C'), the test substance which decreases the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein is appetite-suppressed Evaluating or selecting as an agent,
A method of evaluating or selecting an appetite suppressant, comprising

<13> The following steps (A ') to (D'):
(A ') administering a test substance to a non-human mammal,
(B ') measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal,
(C ') The expression level or activity measured in the above (B'), the expression level of the FABP4 gene or FABP5 gene, the expression level of the FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal of the control group, or FABP4 Comparing with the activity of the protein or FABP5 protein,
(D ') Based on the result of the above (C'), a test substance which decreases the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein is used for obesity prevention And / or evaluating or selecting as a remedy
A method for evaluating or selecting an agent for preventing and / or improving obesity, including

<14> The following steps (A ') to (D'):
(A ') administering a test substance to a non-human mammal,
(B ') measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal,
(C ') The expression level or activity measured in the above (B'), the expression level of the FABP4 gene or FABP5 gene, the expression level of the FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal of the control group, or FABP4 Comparing with the activity of the protein or FABP5 protein,
(D ') Based on the result of the above (C'), the test substance which decreases the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein is increased in weight Evaluating or selecting as an inhibitor,
A method of evaluating or selecting a weight gain inhibitor, comprising

<15> Steps (A ') to (D') below:
(A ') administering a test substance to a non-human mammal,
(B ') measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal,
(C ') The expression level or activity measured in the above (B'), the expression level of the FABP4 gene or FABP5 gene, the expression level of the FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal of the control group, or FABP4 Comparing with the activity of the protein or FABP5 protein,
(D ') Based on the result of (C') above, digestion of a test substance that reduces the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein is promoted Evaluating or selecting as an agent,
A method of evaluating or selecting a digestive enhancer, which comprises

<16> The following steps (A ') to (D'):
(A ') administering a test substance to a non-human mammal,
(B ') measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal,
(C ') The expression level or activity measured in the above (B'), the expression level of the FABP4 gene or FABP5 gene, the expression level of the FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal of the control group, or FABP4 Comparing with the activity of the protein or FABP5 protein,
(D ') Based on the result of the above (C'), the test substance which decreases the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein, Evaluating or selecting as a remedy,
A method for evaluating or selecting an agent for improving stomach upset, including

<17> The method according to any one of <11> to <16>, preferably, the control group is a small intestine collected from the non-human mammal to which no test substance has been administered.

<18> Preferably, when the expression level or activity measured in the above (B ′) is statistically significantly decreased relative to the expression level or activity in the control group, the test substance is increased in GIP An evaluation according to any one of <11> to <17>, which is evaluated to have an inhibitory effect, anorectic effect, an obesity preventing and / or improving effect, a weight gain suppressing effect, a digestive promotion effect, or a stomach upset improving effect. Method.

<19> Preferably, the expression level or activity measured in the above (B ′) is 90% or less, preferably 80% or less, more preferably 60% when the expression level or activity in the control group is 100% In the following cases, the test substance is evaluated as having a GIP elevation suppressing effect, an appetite suppressing effect, an obesity preventing and / or improving effect, a weight gain suppressing effect, a digestive promotion effect, or a stomach upset improving effect, <11 The method according to any one of> to <17>.

  Hereinafter, the present invention will be more specifically described with reference to examples.

(FABP 4/5 inhibitor)
In the following examples, BMS309403 (AstaTech, Inc.) or Compound 2 (J Lipid Res. 2011, 52, 646-656) described below was used as a FABP 4/5 inhibitor.

Example 1 Expression of FABP 4/5 in enterocytes (immunostaining)
After fixing the duodenum collected from C57BL / 6J mice (male) (CLEA Japan, Inc.) with 4% aqueous solution of Paraformaldehyde, frozen sections were prepared and immunostained. Double fluorescence staining was performed using antibodies in GIP and FABP5 to examine the expression of FABP5 in enterocytes. As a primary antibody, T-4340 (manufactured by Peninsula) was used as a GIP antibody. Moreover, AF1476 (made by R & D systems) was used for the antibody of FABP5. As secondary antibodies, Alexa Fluor 488 Donkey Anti-Rabbit IgG (manufactured by Invitrogen) and Alexa Fluor 568 Dnkey Anti-goat IgG (manufactured by Invitrogen) were used. After fixing in a 4% Paraformaldehyde aqueous solution for 10 minutes at 4 ° C. according to a conventional method, and washing with PBS, they were reacted in a primary antibody (100 × blocking solution: 10% Donkey Serum in PBS) for 3 hours at room temperature. After washing again with PBS, the secondary antibody (500 × blocking solution) was reacted at room temperature for 1 hour, washed with PBS, nuclear stained and encapsulated with Prolong Gold antifade reagent with DAPI (manufactured by Invitrogen), and observed under a microscope (Using a 405 nm, 488 nm, 568 nm laser).

(result)
The stained image is shown in FIG. In the mouse small intestine, expression of FABP5 was continuously observed on the luminal side of the cells present in the villi (arrow). In addition, FABP5 expression was also observed in K cells (within the white dotted line) that are GIP positive. In addition, it is already known that FABP4 is localized on the cell membrane of intestinal glandular epithelium ([www. Rndsystems. Com / ihc_molecule_images. Aspx? M = 1416]). Therefore, it was revealed from the results of this example that both FABP4 / 5 were expressed in intestinal epithelial cells.

Example 2 Effects of FABP 4/5 Inhibition on GIP Secretion (Mouse Single Dose Study)
A solvent (10% 1%) containing FABP 4/5 inhibitor BMS 309 403 (30 mg / kg body weight) under anesthesia in mice fasted for 18 hours (C57BL / 6J, male, 9 weeks old, n = 4-5, CLEA Japan) -Methyl-2-pyrrolidone, 5% cremophor, 2% ethanol) or solvent alone (control) was orally administered as an oral probe. Thirty minutes later, the lipid emulsion (triolein; 2 mg / g body weight) was administered intragastrically. Blood was collected by orbital venous plexus blood collection before lipid emulsion administration and at 10, 30, and 60 minutes after administration. The collected blood was centrifuged at 11,000 rpm and 4 ° C. for 10 minutes to prepare plasma. The blood GIP concentration and the blood triglyceride (TG) concentration were measured using a GIP ELISA kit (for rat / mouse) (Millipore) and triglyceride E-Test Wako (Wako Pure Chemical Industries), respectively.

(result)
The time-course changes of TG concentration change (ΔTG) and GIP concentration change (ΔGIP) in blood after single administration of lipid emulsion, and iAUC (0-1 h) and Cmax of blood GIP concentration are shown in FIG. . The elevation of blood TG concentration and blood GIP concentration after lipid emulsion administration was statistically significantly reduced in the inhibitor addition group as compared to the control group (FIG. 2A). In addition, iAUC and Cmax of blood GIP concentrations were also statistically significantly reduced in the inhibitor addition group compared to the control group (FIG. 2B).

Example 3 FABP 4/5 inhibition test in small intestine primary cultured cells (preparation of small intestinal primary cultured cells)
After euthanasia by 13- to 17-week-old C57BL / 6J mice by cervical dislocation under anesthesia, the upper small intestine (10 cm from just under the pylorus of the stomach) was isolated and penetrated in ice-cold L-15 medium (Sigma) . This is transferred into ice cold PBS, fat and blood vessels adhering to the intestinal tract are carefully removed, and after the lumen is washed with ice cold PBS, the tissue is finely dusted using a scalpel (small piece of 2 mm ² or less) I made it. The pieces were washed 3 to 4 times with PBS, then 10 mL of DMEM (Sigma) in which collagenase XI (Sigma; 0.4 mg / mL) was dissolved was added, shaken vigorously for 10 seconds, and then incubated at 37 ° C. for 5 minutes . After removing the supernatant, 10 mL of the collagenase solution was again added, vigorously shaken for 10 seconds, and incubated at 37 ° C. for 5 minutes. Thereafter, the supernatant was removed, and 10 mL of the collagenase solution was again added, shaken vigorously for 10 seconds, and incubated at 37 ° C. for 15 minutes. During that time, shaking was performed for 10 seconds every 5 minutes. After incubation, the supernatant was collected (supernatant 1), similarly, the collagenase solution was added and incubated for 15 minutes, and the supernatant was collected (supernatant 2). The supernatant 1 and the supernatant 2 were centrifuged at 100 rcf (about 800 rpm) for 3 minutes (room temperature). The supernatant was removed and suspended in 10 mL of DMEM, and then combined and centrifuged at 100 rcf for 3 minutes (room temperature). The supernatant was removed and suspended in 7 mL of DMEM (containing 10% FBS (Invitrogen), 1% Glutamax (Invitrogen), 1% penicillin / streptomycin (Invitrogen)). This was seeded in 48-well plates coated with matrigel ^TM (BD Biosciences) (1 animal Atari 24-well), 37 ° C., and incubated under 5% CO _2.

(GIP secretion stimulation)
24-hr cultured intestinal primary cell stimulation medium (4.5mM KCl, 138mM NaCl, 4.2mM NaHCO 3, 1.2mM NaH 2 PO 4, 2.6mM CaCl 2, 1.2mM MgCl 2, 10mM HEPES, NaOH After gently washing once with (adjusted to pH 7.4), the stimulation medium alone or the stimulation medium containing FABP 4/5 inhibitor (10 or 25 μM) was added and incubated. After 30 minutes, wash gently once with stimulation medium, stimulation medium only (control), lipid micelles (PPM: stimulation medium + 500 μM Na taurocholate, 200 μM oleic acid, 50 μM 2-monoolein), or PPM + FABP 4/5 inhibitor (10 or 25 μM) was added and after 30 minutes the medium was harvested. As a FABP 4/5 inhibitor, BMS309403 or Compound 2 was used.

(Measurement of GIP secretion rate)
The collected medium was centrifuged at 8,000 rpm for 5 minutes at 4 ° C. to obtain a supernatant (Secretion sample). Cell Lysis Buffer (0.5% (w / v) Sodium Deoxycholate Monohydrate, 1% (v / v) Igepal CA-630, 50 mmo L / L Tris-HCl (pH 7.4), 150 mmol / l) on the plate after supernatant collection. L NaCl, 1 tablet EDTA-free protease inhibitor cocktail tablet (Roche) was added, and cells were recovered after freezing and thawing. This was centrifuged at 12,000 rpm and 4 ° C. for 5 minutes to obtain a supernatant (Lysis sample). These samples were subjected to GIP quantification using a GIP ELISA kit (for rat / mouse) (Millipore) to determine the GIP secretion rate. The calculation method of the GIP secretion rate is shown below.

(result)
The GIP secretion rate in PPM-stimulated primary small intestinal cultured cells is shown in FIG. In the PPM-stimulated cells (PPM group), the GIP secretion rate at 30 minutes after stimulation was statistically significantly increased as compared to the control group. On the other hand, in the cells to which the FABP 4/5 inhibitor was added in addition to PPM stimulation (PPM + inhibitor group), the increase in the GIP secretion rate was statistically significantly suppressed with respect to the PPM group. In the PPM-stimulated + BMS309403-added group, a dose-dependent tendency to suppress the elevation of GIP secretion was observed (FIG. 3A).

Example 4 Screening of GIP elevation inhibitor based on FABP5 activity A GIP elevation inhibitor was screened based on FABP5 activity. The influence of the test substance on FABP5 activity was examined by measuring FABP5 activity using the antagonistic binding of the test substance to the FABP5 protein. The principle of the method for measuring FABP5 activity used in this example is shown in FIG. 1-anilinonaphthalene-8-sulfonic acid (1, 8-ANS) is a substance that is reported to emit fluorescence upon binding to FABP 5 (Hum. Mol. Genet., 2014, 23 (24): 6495-6511). 1, 8-ANS is a substance that fluoresces only in a hydrophobic environment, and does not fluoresce in a hydrophilic environment (FIG. 4A). On the other hand, in the presence of FABP5, 1,8-ANS emits fluorescence because it becomes a hydrophobic environment around the molecule by binding to FABP5 (FIG. 4B). However, if a substance having a smaller binding constant to FABP5 is further present, 1,8-ANS will not fluoresce because the substance preferentially binds to FABP5 over 1,8-ANS (FIG. 4C). The test substance whose fluorescence intensity has been suppressed by the above method is a substance that binds to the FABP5 protein and inhibits its activity.

(Measurement of FABP5 inhibitory activity by test substance)
E. coli-derived recombinant FABP5 was expressed and purified by a conventional method. The purified FABP5 protein, 1,8-ANS and test substance are added to a 96-well plate and allowed to stand at room temperature for 5 minutes, after which the fluorescence intensity (excitation wavelength: 355 nm, fluorescence wavelength: 480 nm) is measured with a plate reader. It was measured. As a test substance, FABP5 inhibitor (Compound 2) which has GIP rise inhibitory effect used in Example 3, and fish oil which is a fat and oil which has GIP elevation inhibitory action as shown in the below-mentioned reference example 1 were used. The final concentration of the test substance in the evaluation system was 0.002% to 0.1% (w / v).

(result)
The evaluation results are shown in FIG. Due to the coexistence of FABP5 and 1,8-ANS, a significant increase in fluorescence intensity was observed as compared with only buffer or FABP5 or 1,8-ANS alone. This increase in fluorescence intensity was statistically significantly suppressed by the addition of the FABP5 inhibitor having a GIP elevation inhibitory action (t-test, p <0.05). Moreover, also when the fish oil which is a fat and oil which has GIP rise inhibitory effect is added, the fluorescence intensity was statistically significantly reduced (t-test, p <0.05). On the other hand, when a test substance having no GIP elevation inhibitory effect was added, the fluorescence intensity did not decrease (data not shown). These results show that the GIP elevation inhibitory effect of the test substance can be evaluated using the activity of FABP5 as an index.

Reference Example 1 GIP elevation inhibitory activity of fish oil The GIP elevation inhibitory activity of fish oil was examined by a single mouse diet test. High-fat diet (HF) diet (25% rapeseed oil + 5% palm oil) or fish oil diet (15 minutes) to mice fasted for 18 hours (C57BL / 6J, male, 8 weeks old, n = 10, CLEA Japan) for 60 minutes .4% rapeseed oil + 5% palm oil + 9.6% fish oil (10% DHA)) was fed freely as a powder feed. Blood was collected from the orbital venous plexus of each mouse before free feeding and at 60, 120 and 240 minutes after feeding. The collected blood was centrifuged at 11,000 rpm and 4 ° C. for 10 minutes to prepare plasma, and the blood GIP concentration was measured using a GIP ELISA kit (for rat / mouse) (Millipore). The time-course changes in blood GIP concentration change amount (ΔGIP) in mice after test food feeding and the blood GIP concentration iAUC (0-4 h) are shown in FIGS. 6A and 6B. Elevated blood GIP concentrations were statistically significantly reduced in the fish oil diet group compared to the HF diet group.

Claims (8)

The following steps (A) to (D):
(A) contacting a test substance with a FABP4 gene or FABP5 gene, or a tissue or cell derived from a mammal capable of expressing FABP4 protein or FABP5 protein, (B) a FABP4 gene or a gene or the like in the tissue or cell derived from the mammal Measuring the expression level of FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein,
(C) comparing the expression level or activity measured in (B) with the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein in a control group, or the activity of FABP4 protein or FABP5 protein,
(D) Based on the results of the above (C), a test substance which decreases the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in the stomach Evaluating or selecting as an accelerator,
A method for evaluating or selecting an agent for promoting digestion in the stomach, comprising The following steps (A) to (D):
(A) contacting a test substance with a FABP4 gene or FABP5 gene, or a tissue or cell derived from a mammal capable of expressing FABP4 protein or FABP5 protein, (B) a FABP4 gene or a gene or the like in the tissue or cell derived from the mammal Measuring the expression level of FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein,
(C) comparing the expression level or activity measured in (B) with the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein in a control group, or the activity of FABP4 protein or FABP5 protein,
(D) Based on the result of the above (C), an agent for improving the stomach, a test substance which reduces the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or FABP4 protein or FABP5 protein activity Evaluating or selecting as
A method for evaluating or selecting an agent for improving stomach upset, including   If the expression level or activity measured in (B) is statistically significantly reduced relative to the expression level or activity in the control group, the test substance has an effect of promoting digestion in the stomach, or the stomach The method according to claim 1 or 2, which is evaluated as having the effect of improving the feeling of falling. The expression level or activity measured in (B) is, if the it is the expression amount or activity of 90% or less is taken as 100% in the control group, the test substance of digestion in the stomach promoting effect or stomach, The method according to claim 1 or 2, which is evaluated as having the effect of improving the feeling of falling. The following steps (A ') to (D'):
(A ') administering a test substance to a non-human mammal,
(B ') measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal,
(C ') The expression level or activity measured in the above (B'), the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal of the control group, or FABP4 Comparing with the activity of the protein or FABP5 protein,
(D ') Based on the result of (C') above, a test substance which decreases the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in the stomach Evaluating or selecting as a digestive enhancer,
A method for evaluating or selecting an agent for promoting digestion in the stomach, comprising The following steps (A ') to (D'):
(A ') administering a test substance to a non-human mammal,
(B ') measuring the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal,
(C ') The expression level or activity measured in the above (B'), the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein in the small intestine collected from the non-human mammal of the control group, or FABP4 Comparing with the activity of the protein or FABP5 protein,
(D ') Based on the result of the above (C'), the test substance which decreases the expression level of FABP4 gene or FABP5 gene, the expression level of FABP4 protein or FABP5 protein, or the activity of FABP4 protein or FABP5 protein, Evaluating or selecting as a remedy,
A method for evaluating or selecting an agent for improving stomach upset, including   When the expression level or activity measured in (B ′) above is statistically significantly reduced relative to the expression level or activity in the control group, the test substance has an effect of promoting digestion in the stomach, or The method according to claim 5 or 6, which is evaluated to have an effect to improve stomach upset.   When the expression level or activity measured in (B ′) is 90% or less when the expression level or activity in the control group is 100%, the test substance has an effect of promoting digestion in the stomach, or the stomach The method according to claim 5 or 6, which is evaluated as having the effect of improving the feeling of falling.