Evaluation methods for functional ingredients that support dieting, supplements, and diet foods.

Abstract

To provide a functional component having a dieting effect corresponding to a genotype diet type identified by an obesity-related genetic test.SOLUTION: A method for evaluating a diet supporting functional component includes the steps of: (1) identifying a genotype diet type by an obesity-related genetic test; (2) constructing an iPS cell-derived adipocyte evaluation system for the identified genotype diet type; and (3) evaluating a diet supporting functional component by the iPS cell-derived adipocyte evaluation system.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a method for evaluating a functional ingredient that supports dieting, a supplement (nutritional supplement) containing the functional ingredient, and a food product containing the functional ingredient as an active ingredient.

[0002] The present invention provides a method for evaluating diet support functional ingredients, which involves constructing a human iPS cell (induced pluripotent stem cell)-derived adipocyte evaluation system that reflects an individual's obesity-related genes based on genetic test results, and using this human iPS cell-derived adipocyte evaluation system to detect diet support functional ingredients that are suitable for an individual's genetic constitution. Furthermore, the present invention helps individuals effectively achieve their diet goals by providing supplements containing such diet support functional ingredients or foods containing these functional ingredients as active ingredients. [Background technology]

[0003] It is generally said that approximately 30% of disease onset is due to congenital genetic factors, and approximately 70% is due to acquired lifestyle and environmental factors. Therefore, it is considered beneficial to understand the risks from congenital genetic factors in advance in order to live a healthy life and reach a full lifespan. There are mainly three types of obesity-related genes in Japanese people: the β3AR gene, the UCP1 gene, and the β2AR gene. These are classified into four gene mutation-related types (genotype diet types): apple type, pear type, banana type, and Adam and Eve type. The apple type mainly has mutations (SNPs (single nucleotide polymorphisms)) in the β3AR gene (β3AR mutation type). This leads to a decrease in carbohydrate metabolism, causing visceral fat obesity. Approximately 34% of Japanese people have a mutation in the β3AR gene. The pear-shaped body type mainly has a mutation (SNP) in the UCP1 gene (UCP1 mutant). When the UCP1 gene is mutated, lipid metabolism decreases, and if you eat too many foods high in lipids (fried foods, cakes, snacks, etc.), fat tends to accumulate in the lower body. Approximately 35% of Japanese people have a mutation in the UCP1 gene. The banana-shaped body type mainly has a mutation (SNP) in the β2AR gene (β2AR mutant). When the β2AR gene is mutated, it becomes easier to metabolize proteins quickly. Because protein metabolism improves, it is known that it is difficult to build muscle, and once you gain weight, it is difficult to lose it. Finally, the Adam and Eve type is a type in which there are no mutations in any of the β3AR, UCP1, or β2AR genes (unmutated type). This is a type in which obesity is thought to be influenced more by lifestyle factors than by genetic factors. In the following description, the β3AR mutant, UCP1 mutant, β2AR mutant, and unmutated types may be referred to as apple type, pear type, banana type, and Adam & Eve type, respectively. Furthermore, the gene mutation-related type may be referred to as the genotype diet type. Furthermore, apple-shaped, pear-shaped, banana-shaped, Adam & Eve-shaped, and genotype diets type All of these are registered trademarks. The designation of registered trademarks may be omitted.

[0004] Furthermore, there are two types of fat cells: so-called white fat cells and brown fat cells. White fat cells are distributed under the skin and around the internal organs, and store excess energy in the body as fat. Brown fat cells, on the other hand, are mainly distributed around the collarbone and chest, and are responsible for burning fat and producing heat. Furthermore, in addition to the classical brown adipocytes mentioned above, another type of adipocyte is known. These cells are called beige adipocytes, and unlike white and brown adipocytes, they are induced to produce heat in response to stimuli such as cold stimulation or the administration of certain diabetes medications. They also disappear when the stimulation ceases. This inducibility is the most distinctive feature that distinguishes them from white and brown adipocytes.

[0005] While research into foods and medicines to prevent lifestyle-related diseases caused by obesity has been actively conducted, to the best of the inventors' knowledge, methods for preventing and improving obesity caused by congenital genetic factors have yet to be established.

[0006] However, in August 2006, Professor Yamanaka of Kyoto University established iPS cells with pluripotency similar to embryonic stem cells (ES cells) by introducing four factors (Oct3 / 4, Klf4, Sox2, c-Myc) into somatic cells, and then in 2007, he established human iPS cells. They succeeded in this. In addition to regenerative medicine, iPS cells are expected to be applied to personalized medicine, where iPS cells created from patients with specific diseases can be used to develop drugs and treatments tailored to each patient by understanding the differences in the functions of brown, beige, and white adipose tissue. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Takeshi Yoneshiro, Shingo Kajimura, "Regulatory Mechanisms and Clinical Significance of Brown and Beige Adipocytes," Biochemistry, Vol. 89, No. 6, pp. 917-920 (2017) [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] This invention focuses on the heat-producing function of brown adipocytes and beige adipocytes, and their inducibility into beige adipocytes. The problem it aims to solve is to provide an evaluation system for diet support functional ingredients that can identify diet types that are precisely suited to each genotype diet type by identifying the genotype diet type through genetic testing of the subject, establishing iPS cells from the subject, and then differentiating the iPS cells into adipocytes. Since the iPS cells of this invention are derived from tested human bodies, unless otherwise specified, they all refer to human iPS cells. [Means for solving the problem]

[0009] As a result of diligent research, the inventors have completed a method for evaluating diet support functional components using iPS cell-derived adipocytes of a identified genotype diet type. In other words, the present invention encompasses the following: 1. A method for evaluating diet support functional components using an iPS cell-derived adipocyte evaluation system based on obesity-related genes, wherein in this method, (1) Steps to identify the genotype diet type through obesity-related gene testing, (2) The steps of establishing iPS cells of the identified genotype diet type and constructing an iPS cell-derived adipocyte evaluation system, (3) The present invention relates to a method for evaluating diet support functional components, which includes the step of identifying diet support functional components using an iPS cell-derived adipocyte evaluation system that has been constructed. 2. The present invention relates to a method for evaluating the diet support functional ingredient described in paragraph 1 above, wherein the genotype diet type is pear-shaped, apple-shaped, or Adam & Eve-shaped. 3. The present invention relates to a method for evaluating a diet support functional ingredient according to item 1 or 2, wherein the diet support functional ingredient contains at least a beige-coloring component. 4. The method for evaluating the diet support functional ingredient described in 3 above, wherein the beige-coloring component contains at least one selected from the group consisting of forskolin, γ-tocopherol, and α-lipoic acid. 5. The present invention relates to a method for evaluating a diet support functional component according to any one of 1 to 4 above, wherein in step (3) above, the diet support functional component is added to the iPS cell-derived adipocyte evaluation system to increase the relative expression level of at least one selected from the group consisting of brown adipocyte markers UCP1, PGC1α, and beige adipocyte marker CD137. 6. A supplement containing a diet support functional component identified by the method for evaluating a diet support functional component according to any one of 1 to 5 above. 7. A food containing, as an active ingredient, a diet support functional component identified by the method for evaluating a diet support functional component according to any one of 1 to 5 above.

Effects of the Invention

[0010] According to the present invention, a diet support functional component evaluation method can be established for identifying a diet support functional component that constructs an iPS cell-derived adipocyte evaluation system of a genotype diet type identified by genetic testing and induces the conversion (beiging) from white fat progenitor cells to beige fat cells. Further, according to the present invention, a supplement containing such a diet support functional component and a food containing the diet support functional component as an active ingredient can be provided.

Brief Description of the Drawings

[0011] [Figure 1] FIG. 1 is a diagram showing a process of establishing human iPS cells from prepared monocytes. [Figure 2] FIG. 2 is a process showing the induction of differentiation from human iPS cells to white adipocytes. [Figure 3] FIG. 3 is a photograph of the cell morphology at each time point during the induction of differentiation from human iPS cells of the 1231A3 strain, the no-apple type, the apple type, and the Adam and Eve type to white adipocytes. [Figure 4] FIG. 4 is a photograph of Oil Red O staining after the induction of differentiation from human iPS cells of the 1231A3 strain, the no-apple type, the apple type, and the Adam and Eve type to white adipocytes (on the 30th day of culture). [Figure 5] FIG. 5 is a graph showing the expression analysis of various adipocyte differentiation markers by RT-PCR (reverse transcription PCR) of human iPS cells of the 1231A3 strain, the no-apple type, the apple type, and the Adam and Eve type (on the 0th day of culture) and after the induction of differentiation to white adipocytes (on the 30th day of culture). [Figure 6] Figure 6 is a diagram showing the evaluation process of functional components by a human iPS cell-derived adipocyte evaluation system. [Figure 7] Figure 7 shows the evaluation of functional components (untreated, γ-tocopherol, forskolin) in banana-shaped adipocytes. The photograph is a photograph of Oil Red O-stained adipocytes on the 30th day of culture initiation, and the graph is a graph showing the expression analysis of various adipocyte differentiation markers by RT-PCR on the 30th day of culture initiation. [Figure 8] Figure 8 shows the evaluation of functional components (untreated, γ-tocopherol, forskolin) in apple-shaped adipocytes. The photograph is a photograph of Oil Red O-stained adipocytes on the 30th day of culture initiation, and the graph is a graph showing the expression analysis of various adipocyte differentiation markers by RT-PCR on the 30th day of culture initiation. [Figure 9] Figure 9 shows the evaluation of functional components (untreated, forskolin) in Adam and Eve-shaped adipocytes. The photograph is a photograph of Oil Red O-stained adipocytes on the 30th day of culture initiation, and the graph is a graph showing the expression analysis of various adipocyte differentiation markers by RT-PCR on the 30th day of culture initiation. [Figure 10] Figure 10 shows the comparison of gene expression levels by genotype diet type. Each genotype shows the expression levels of genes UCP1 and PGC1α under forskolin treatment conditions when the gene expression level under untreated conditions is set to 1.

Mode for Carrying Out the Invention

[0012] The present invention relates to a method for evaluating diet support functional components (hereinafter also simply referred to as functional components) using an iPS cell-derived adipocyte evaluation system based on the results of obesity-related gene testing, the method comprising: (1) identifying a genotype diet type by obesity-related gene testing; (2) establishing iPS cells of the identified genotype diet type and constructing an iPS cell-derived adipocyte evaluation system; and (3) identifying the diet support functional component using the constructed iPS cell-derived adipocyte evaluation system. Furthermore, the present invention provides supplements and foods containing the functional component found through the method for evaluating diet support functional components. The present invention will be explained in detail below, but it is not limited to the following specific embodiments and can be modified as appropriate within the scope of the technical idea.

[0013] [Obesity-related genes] The three obesity-related genes that Japanese people are more likely to have mutations in and that affect basal metabolic rate are the β3AR gene, the UCP1 gene, and the β2AR gene.

[0014] (β3AR gene) The β3AR gene is the β3 adrenergic receptor gene. By binding to adrenaline and noradrenaline, it activates adenylyl cyclase via GTP-binding protein, increasing the intracellular concentration of the secondary signaling molecule cAMP. cAMP activates PKA (protein kinase A), which in turn phosphorylates and activates hormone-sensitive lipase (a triglyceride-degrading enzyme). In this way, triglycerides accumulated in adipocytes are broken down into fatty acids and monoglycerides. The fatty acids broken down in white adipocytes travel through the bloodstream to brown adipocytes, where they bind to UCP1 (uncoupling protein 1) in mitochondria, promoting thermal energy production. Mutations in the β3AR gene reduce basal metabolic rate by 200 kcal, leading to visceral fat obesity. Individuals with this mutation are known to have a lower carbohydrate metabolism.

[0015] (UCP1 gene) The UCP1 gene is an uncoupling protein gene. Uncoupling proteins inhibit ATP production by ATP synthase via the proton gradient from the electron transport chain, and the energy that would have been used for ATP synthesis is instead produced as heat. The UCP1 gene is thought to be expressed only in brown adipose tissue, which is responsible for heat production, and is used as a marker for brown adipose tissue. A mutation in this UCP1 gene reduces basal metabolic rate by 100 kcal. Genetic testing to check for this mutation has shown that people with the mutation have low lipid metabolism.

[0016] (β2AR gene) The β2AR gene is the β2 adrenergic receptor gene, and like the β3AR gene, it is activated by binding to adrenaline and noradrenaline. It is known that mutations in the β2AR gene increase basal metabolic rate by 200 kcal, but because protein metabolism improves, it is difficult to build muscle, and once weight is gained, it is difficult to lose it.

[0017] By analyzing mutations in the three genes mentioned above, individuals are classified into genotypes (homozygous, heterozygous, wild) based on the presence or absence of mutations. These are categorized into three genotype diet types: the apple type, characterized by a tendency to accumulate visceral fat and a plump, belly-like body shape due to a mutation in the β3AR gene; the pear type, characterized by a tendency to accumulate subcutaneous fat around the hips and a plump, lower body shape due to a mutation in the UCP1 gene; and the banana type, characterized by a tendency to accumulate fat easily and a lean physique, but who finds it difficult to lose weight once they gain it, due to a mutation in the β2AR gene. The Adam and Eve type is characterized by no mutations in any of the β3AR, UCP1, or β2AR genes. This type is thought to be influenced more by lifestyle factors than by genetic factors in terms of obesity.

[0018] [iPS cell-derived adipocyte evaluation system] As mentioned above, since iPS cells are pluripotent stem cells similar to ES cells, an evaluation system will be constructed by further differentiating iPS cells created from characteristic somatic cells into target cells. Identifying functional components that exhibit specific effects in improving an individual's constitution or preventing disease using this constructed evaluation system is important from the perspective of integrated medicine, which is linked to pharmaceutical-centered medical care. This invention involves inducing differentiation into target cells, specifically white adipocytes, to construct an iPS cell-derived adipocyte evaluation system. Subsequently, the addition of the following diet support functional components to be evaluated induces the conversion (beigeing) of white adipocyte precursor cells into beige adipocytes in the iPS cell-derived adipocyte evaluation system.

[0019] [Dietary support functional ingredients] Numerous functional ingredients for diet support are commercially available and are generally classified into categories such as sugar and fat absorption and metabolism, and cholesterol level improvement. For example, ingredients involved in fat metabolism and breakdown include forskolin, alpha-lipoic acid, carnitine, and garcinia. Ingredients that slow the rise in blood sugar levels, suppress insulin secretion, and prevent the accumulation of body fat include gymnema, bitter melon, mulberry leaves, and white kidney bean extract. Beige adipose tissue is similar to brown adipose tissue and is rich in mitochondria that express UCP1. Gamma-tocopherol, a natural vitamin E, has the effect of enhancing mitochondrial function by eliminating reactive oxygen species generated in mitochondria.

[0020] [Ingredients that promote beigeing] In this invention, the effect of adding a diet support functional ingredient to an iPS cell-derived adipocyte evaluation system is used to evaluate the differentiation-promoting effect from white adipose progenitor cells to beige adipocytes. Among the many diet support functional ingredients, those that have the function of promoting the differentiation from white adipose progenitor cells to beige adipocytes are named "beige-promoting ingredients" in this invention. The beige-promoting components identified from the diet support functional ingredients include at least one selected from the group consisting of forskolin, γ-tocopherol, and α-lipoic acid. Preferably, the beige-promoting components include at least two selected from the group consisting of forskolin, γ-tocopherol, and α-lipoic acid. More preferably, the beige-promoting components include forskolin and γ-tocopherol.

[0021] [Step (1): Methods for testing obesity-related genes and identification of genotype diet type] Various methods can be used to analyze single nucleotide polymorphisms (SNPs) in obesity-related genes, namely the β3AR, UCP1, and β2AR genes. These methods include direct sequencing, PCR-RFLP, Invader, TaqMan Genotyping, and microarray. This invention uses the "DHC Gene Testing Diet Kit" manufactured by DHC Corporation and employs the TaqMan Genotyping method. For each of the three obesity-related genes mentioned above, three variants (wild, heterozygous, and homozygous) are determined, and the results are classified into 54 patterns based on the combination of variants for each gene and the combination of male and female, as described in "The Fastest Way to Lose Weight! Gene Type-Based Diet: Know Your Genotype and You Can Lose Weight" (Author: Takuji Shirasawa, DHC). Furthermore, the subjects' genotype diet types can be divided into four categories: apple type, pear type, banana type, and Adam and Eve type.

[0022] [Step (2): Establishment of iPS cells and construction of an iPS cell-derived adipocyte evaluation system] Mononuclear cells are collected from subjects whose genotype diet type has been identified. Four reprogramming factors (Oct3 / 4, Klf4, Sox2, c-Myc) are introduced into the obtained mononuclear cells by electroporation to establish iPS cells of each genotype diet type. Subsequently, the iPS cells are differentiated into white adipocytes to construct an iPS cell-derived adipocyte evaluation system. When establishing iPS cells, obtaining mononuclear cells by blood sampling is the most common method as it places less burden on the subject. However, if necessary, it is also possible to establish iPS cells by collecting somatic cells such as skin cells.

[0023] (Blood collection and mononuclear cell preparation in step (2)) Blood collection is performed using specialized blood collection tubes for mononuclear cell isolation, virus analysis, and gene analysis. After collecting 24 mL of blood, centrifugation is immediately performed using a swing rotor (18°C, 1,500-1,800 g, 20 minutes) to remove plasma components, red blood cells, platelets, and granulocytes from the blood to prepare mononuclear cells (a population of blood cells with a spherical nucleus, including T cells, B cells, monocytes, NK cells, and dendritic cells). Mononuclear cell isolation is performed according to standard methods, and viruses (HTL) are analyzed. We use mononuclear cells that are negative for both HIV (V, HBV, HCV, HIV) and mycoplasma.

[0024] The procedure to isolate mononuclear cells from the subjects' blood and establish human iPS cells requires approval from the ethics committee. Each blood sample subject will first receive a thorough written and verbal explanation from a physician, and written consent will be obtained from all (informed consent). Next, the physician performing the blood collection will confirm the subject's condition and ensure there are no obstacles to blood collection, based on a medical history (medical history, current medications, allergies, past blood collection problems, etc.), body temperature, pulse, blood pressure, and other information deemed necessary.

[0025] (Establishment of iPS cells in step (2)) Reprogramming factors (Oct3 / 4, Klf4, Sox2, c-Myc) are introduced into prepared mononuclear cells by electroporation. After culturing the gene-transfected cells in an on-feeder, colony picking is performed, and the cells are cultured from the on-feeder to a feeder-free state to establish iPS cells. Figure 1 shows the process of establishing human iPS cells from mononuclear cells prepared according to this invention. In addition, iPS cells can be established using the "Method for Establishing Human iPS Cells Using Episomal Vectors" published on the website of the Kyoto University Center for iPS Cell Research and Application (https: / / www.cira.kyoto-u.ac.jp / j / research / protocol.html). The established iPS cells undergo epizomal vector retention testing, karyotype analysis, and virus and mycoplasma testing. iPS cells that are free of epizomal vectors, karyotype abnormalities, virus-negative, and mycoplasma-negative are cryopreserved.

[0026] (Construction of an iPS cell-derived adipocyte evaluation system in step (2)) The established iPS cells can be induced to differentiate into white adipocytes. By adding well-known adipocyte differentiation-inducing factors, such as insulin, dexamethasone, and IBMX, to the culture medium, the differentiation of iPS cells into white adipocytes is induced. Figure 2 shows the differentiation process from iPS cells to white adipocytes performed according to this invention.

[0027] The iPS cell-derived adipocyte evaluation system of the present invention allows for differentiation confirmation at the morphological level through microscopic observation of cell structure, at the gene level through decreased expression of reprogramming genes expressed in iPS cells and increased relative expression levels of genes specifically expressed in white adipocytes, or at the protein level through immunostaining of protein expression of genes expressed in white adipocytes.

[0028] While the relative expression levels of target genes such as PPARγ (an early adipocyte differentiation marker), FABP4 (a mid-stage adipocyte differentiation marker), HOXA5 (a white adipocyte marker), and UCP1 (a brown adipocyte marker) can be confirmed by RT-PCR, the study is not limited to these markers.

[0029] Figure 3 shows the cell morphology at each time point during differentiation induction from iPS cells to white adipocytes in the present invention. Embryoid bodies (EBs) are formed by day 10 of culture, and differentiation into white adipocytes occurs via white adipocyte precursor cells on day 20 of culture. Differentiation into white adipocytes can be confirmed by methods such as cell morphology observation (Figure 3), oil red O staining (Figure 4), and expression analysis of various adipocyte differentiation markers by RT-PCR (Figure 5), as shown in Figures 3 to 5. The iPS cell-derived adipocyte evaluation system of the present invention includes white adipocytes.

[0030] [Step (3): Evaluation of diet support functional ingredients using an iPS cell-derived adipocyte evaluation system] The above functional components are added to the iPS cell-derived adipocyte evaluation system constructed in step (2). Furthermore, it induces beige coloration during the differentiation process from white adipose progenitor cells to white adipocytes. Specifically, first, prepare the iPS cell-derived adipocyte evaluation system constructed in step (2) (white adipocyte progenitor cells cultured on day 20 in Figures 2 and 6 in this invention). Next, by adding the above functional components along with transferring the cells to the adipocyte differentiation medium, differences in differentiation will appear during culture. In this invention, a functional component is added to white adipose-derived progenitor cells cultured for 20 days. In fact, the differentiation state of iPS cells can be confirmed by cell morphology observation, expression analysis of various adipocyte differentiation markers by RT-PCR, and analysis of mesenchymal cell-specific cell surface markers by flow cytometry, so it is possible to add the diet support functional component at the stage when white adipose-derived progenitor cells can be identified.

[0031] The addition of functional components is carried out using the same methods as adding nutrients or drugs to adipocyte differentiation induction medium. For example, a 1M solution of functional components is prepared and added to the adipocyte differentiation induction medium until the final concentration of the functional components reaches 100 μM.

[0032] Furthermore, differences in adipocyte differentiation can be confirmed from the lipid droplet accumulation level, gene level, protein level, etc., as described above. As shown in Figures 7 to 9, differences in adipocyte differentiation can be confirmed by observing adipocytes stained with oil red O under a microscope and by analyzing the expression of various adipocyte differentiation markers by RT-PCR.

[0033] The brown adipocyte markers UCP1 and PGC1α, and the beige adipocyte marker CD137 are used as evaluation indicators for beige coloration. After differentiation induction is complete, the gene expression level in each cell without the functional component is set to 1, and the relative expression level with the functional component added is calculated. An increase in the relative expression level of any one of these markers indicates that beige coloration of white adipocytes is induced. In other words, a functional component that increases the relative expression level of any one of these markers has a diet support function. This induction demonstrates that beige adipocytes are induced from the same white adipocyte precursor cells, as shown in Figure 6.

[0034] [Supplements containing functional ingredients that support weight loss] The supplement of the present invention contains functional ingredients whose weight loss support function can be confirmed by an iPS cell-derived adipocyte evaluation system. Furthermore, the supplement of the present invention contains at least one beige-promoting component. Among the beige-promoting components, it contains at least one selected from the group consisting of forskolin, γ-tocopherol, and α-lipoic acid. Furthermore, other nutrients may be added as needed, in addition to the functional components, as long as they do not hinder the effects of the present invention.

[0035] Regarding the amounts of forskolin, γ-tocopherol, and α-lipoic acid in the supplement of the present invention, each tablet of the supplement should contain 7-10 mg of forskolin, 10-30 mg of γ-tocopherol, and 25-33 mg of α-lipoic acid. The acceptable daily intake for adults of forskolin, γ-tocopherol, and α-lipoic acid is 100 mg, 300 mg, and 400 mg, respectively. As long as the daily intake does not exceed these acceptable amounts, there is no particular limit to the number of doses, but it is preferable to take it no more than three times a day. The supplement may take the form of granules, powder, capsules, or tablets, but is not limited to these forms.

[0036] [Foods containing functional ingredients that support weight loss as active ingredients] Foods containing the diet support functional ingredient of the present invention as an active ingredient include: Examples include foods containing the EtSupport functional ingredient or foods into which it is incorporated, and it can usually take the form of food. There are no particular restrictions on the type of food into which it is provided or incorporated; for example, it can be incorporated into liquid foods such as jelly drinks, fruit juices, soft drinks, soups, and teas, as well as solid foods such as rice, bread, and noodles, and into staple foods, side dishes, confectionery, and seasonings. Depending on the application, it can be molded into, but is not limited to, beverages, dairy products, fermented milk, bars, granules, powders, capsules, and tablets. Furthermore, to the extent that it does not impair the effects of the present invention, it may be appropriately mixed with excipients, bulking agents, binders, thickeners, emulsifiers, colorants, flavorings, food additives, seasonings, etc., as needed. In this case, the amount of additive should be such that, when the diet support functional ingredients for the above-mentioned food are forskolin, γ-tocopherol, and α-lipoic acid, the acceptable daily intake for adults is 100 mg, 300 mg, and 400 mg, respectively, and the daily intake should not exceed these acceptable amounts. Examples of these foods include health foods and functional foods. They can also be consumed in combination with other foods. [Examples]

[0037] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0038] [Reagents and Equipment] (1) Blood collection tubes used for preparing mononuclear cells BD Vacutainer (Registered Trademark) CPT TM Mononuclear cell isolation tube (8mL) (Becton Dickinson Japan Co., Ltd.), Venoject® II vacuum blood collection tube (6mL) (Terumo Corporation), vacuum-sealed blood collection tube NeoTube (EDTA-2N, 2mL) (Nipro Corporation).

[0039] (2) Establishment and stock creation of iPS cells Reagents, solvents, etc.: L-glutamine, penicillin / streptomycin, FBS (all from Thermo Fisher Scientific K.K.), Y-27632, Orange G (both from Fujifilm Wako Pure Chemical Corporation) Serum-free culture medium: StemPro®-34 SFM (Thermo Fisher Scientific K.K.) Enzyme: TrypLE TM Select (Thermo Fisher Scientific Co., Ltd.) Cytokines, growth factors, etc.: IL-6, SCF, TPO, Flt-3L, IL-3, G-CSF (all from Fujifilm Wako Pure Chemical Corporation) Culture medium: DMEM (Nacalai Tesque Co., Ltd.) Epizomal vectors: pCE-hOCT3 / 4, pCE-mp53DD, pCE-hSK, pCE-hUL, pCXWB-EBNA1 (all from the US non-profit organization Addgene) Culture media: Primate ES cell medium (Reprocell Co., Ltd.), iMatrix (Nippi Corporation), StemFitAK02N (Ajinomoto Healthy Supply Co., Ltd.) Cell cryopreservation solution: STEM-CELLBANKER® (registered trademark) (Xenoac Resources Co., Ltd.) Kit: DNeasy mini Kit (Qiagen Co., Ltd.), MycoAlert TM Mycoplasma detection kit (Lonza Corporation) Cells: SNL cells (Cosmo Bio Co., Ltd.)

[0040] (3) Differentiation into adipocytes (other than (2)) Reagents, solvents, etc.: b-FGF, SB431542 (both from Fujifilm Wako Pure Chemical Industries, Ltd.), D-PBS, Accutase, 4% Paraformaldehyde Phosphate Buffer, 2-Propanol, Sterile Water, Chloroform, Ethanol (both from Nacalai Tesque Co., Ltd.), Gelatin, IBMX, Dexamethasone, T3 (3,3',5-Triiodo-L-Chyrone Sodium), Rosiglitazone, Insulin, Dye Oil Red O (Sigma-Aldrich Japan LLC), Hoechst® 33342 (Thermo Fisher Scientific K.K.) Culture medium: DMEM / F-12, GlutaMAX supplement, KnockOut TM Serum replacement (KSR) (Thermo Fisher Scientific Co., Ltd.) Markers: HOXA5, FABP4, PPARγ, UCP1, TBP (all from Thermo Fisher Scientific K.K.) Kits: TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific K.K.), RNeasy® Lipid Tissue Mini Kit (Qiagen Co., Ltd.), ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd.)

[0041] (4) Evaluation of functional ingredients that support dieting (other than (2) and (3)) Alpha-lipoic acid, forskolin (both from Nacalai Tesque Co., Ltd.), gamma-tocopherol (Sigma-Aldrich Japan LLC), PGC1α, CD137 (both from Thermo Fisher Scientific Co., Ltd.)

[0042] [Identifying your genotype diet type] Six participants (women, aged 35-56) underwent obesity-related gene testing using the "DHC Genetic Testing Diet Support Kit" (product name). The results showed that two participants each had a pear-shaped, apple-shaped, and Adam & Eve-shaped genetic diet type.

[0043] [Blood sampling] Approval was obtained from the DHC Ethics Committee for the blood collection (Approval Number DHC-Rin 201801). The six individuals whose blood was collected received thorough written and verbal explanations from a physician, and all provided written consent (informed consent). Blood collection was performed at the medical clinic. Immediately after collection, centrifugation was performed to prepare mononuclear cells from the blood. Mononuclear cells that tested negative for both viruses (HTLV, HBV, HCV, HIV) and mycoplasma were used.

[0044] [iPS cell establishment] As shown in Figure 1, iPS cells were established from the prepared pear-shaped, apple-shaped, and Adam & Eve-shaped mononuclear cells. Specifically, in this invention, mononuclear cells cultured for 7 days were introduced into five types of episomatic vectors (pCE-hOCT3 / 4, pCE-mp53DD, pCE-hSK, pCE-hUL, pCXWB-EBNA1) containing reprogramming factors (Oct3 / 4, Klf4, Sox2, c-Myc) using Nucleofector2b (Lonza Corporation)'s CD34-positive cell program (U-008). The number of cells introduced was 3.0 × 10⁶. 6They were cells. After electroporation, feeder cells (SNL cells) were seeded onto plates. Primate ES cell medium was added on the 2nd, 4th, and 6th days, and medium changes were performed daily from the 8th day. Colony picking was performed between the 17th and 21st days after gene introduction. After culturing by the on-feeder method, culturing by the feeder-free method was performed from the 3rd passage. For feeder-free, StemFi with 1 / 1000 volume of 10 mM Y-27632 added to a plate coated with iMatrix (0.5 μg / μL) was replaced with tAK02N medium. 0.5×TripLE TM Select was added to detach the iPS cells. After that, cell counting was performed using Vi-CELLXR (Beckman Coulter, Inc.), and the number of cells considering the growth rate was seeded. The next day, it was replaced with StemFit AK02N medium, and after 4 days of culture, it was replaced with StemFit AK02N every other day, and passage was performed according to confluence. The frozen stock of iPS cells was 0.5×TripLE TM After Select treatment, StemFit AK02N medium with 1 / 1000 volume of 10 mM Y-27632 added was used to detach the iPS cells. After centrifugation, they were suspended in STEM-CELLBANKER (registered trademark) (2.4×10 6 cells / mL). They were dispensed into each frozen vial to reach 2.0×10 5 cells, left standing at -80°C for 4 hours to overnight, and then stored in liquid nitrogen (vapor phase). Before freezing, mycoplasma testing was performed using a MycoAlert TM mycoplasma detection kit (Lonza). For the episomal vector residual test, PCR was performed for OriP, EBNA1, and OCT3 / 4, and the presence or absence of bands of each PCR product was used to confirm the residual by electrophoresis. Karyotype testing was performed by the G-Band method.

[0045] [Induction of differentiation from iPS cells into white adipocytes (construction of an iPS cell-derived adipocyte evaluation system) and evaluation of differentiated cells] As shown in Figure 2, the established pear-shaped, apple-shaped, and Adam & Eve-shaped iPS cells were each induced to differentiate into white adipocytes. The iPS cell line 1231A3 established by Professor Shinya Yamanaka was derived from a healthy individual and was used as a comparative example, undergoing differentiation into white adipocytes using the same procedure.

[0046] Differentiation induction in this induction system was initiated by seeding undifferentiated iPS cells into a 6-well plate to form embryoid bodies (EBs), which were then cultured in suspension in DMEM / F12 medium containing 20% ​​KSR. SB431542 was added from day 3 to day 5 of the EB culture process. Subsequently, the EBs formed on day 10 were reseeded, and adherent culture was initiated. From day 20 onward, differentiation into adipocytes was promoted using DMEM / F12 medium containing 10% KSR with 0.5 mM IBMX, 0.25 μM dexamethasone, 0.2 nM T3, 1 μg / ml insulin, and 1 μM rosiglitazone. Differentiation induction from iPS cells to white adipocytes was completed on day 30 of culture.

[0047] Figure 3 shows photographs of cell morphology at 0, 10, 20, and 30 days during differentiation induction of iPS cells (1231A3 strain, pear-shaped, apple-shaped, and Adam & Eve-shaped human iPS cells) into white adipocytes. White adipocyte precursor cells were observed at 20 days after the start of culture, and at 30 days, many hypertrophied cells and cells accumulating lipid droplets, which are characteristic of adipocytes, were observed (Figure 4).

[0048] Figure 5 is a graph showing the expression analysis of various adipocyte differentiation markers by RT-PCR in human iPS cells of strain 1231A3, pear-shaped, apple-shaped, and Adam & Eve type (day 0 of culture) and after induction into white adipocytes (day 30). The relative expression level at day 30 was calculated by setting the gene expression level at day 0 of each cell type to 1. For HOXA5, since no gene expression was detected in any of the iPS cells at day 0, the relative expression level was calculated by setting the expression level at day 30 of strain 1231A3 to 1. As shown in the graph in Figure 5, in each cell line, an increase in the relative expression levels of PPARγ (an early adipocyte differentiation marker), FABP4 (a mid-stage adipocyte differentiation marker), and HOXA5 (a white adipocyte marker) was observed at day 30 compared to day 0. In comparisons between cell lines, HOXA5 expression tended to be higher in the pear-shaped and apple-shaped cells. No significant changes were observed in the expression levels of UCP1 (a brown adipocyte marker) in the pear-shaped, apple-shaped, and Adam & Eve-shaped cells.

[0049] Figures 3-5 show that even human iPS cells of the established diet-type gene were derived from healthy individuals. Similar to the iPS cell line 1231A3, we were able to induce white adipocytes.

[0050] [Evaluation of beigeing of iPS cell-derived adipocytes by adding functional ingredients] For white adipose-derived progenitor cells (Pear-shaped and Apple-shaped iPS cells) 20 days after differentiation induction into white adipocytes, the differentiation (beigeing) from white adipose-derived progenitor cells to beige adipocytes was evaluated by adding each functional component to be evaluated, namely forskolin (100 μM), γ-tocopherol (500 μM), and α-lipoic acid (100 μM) in this example. For Adam-Eve-shaped iPS cell-derived white adipose-derived progenitor cells, forskolin (100 μM) and γ-tocopherol (500 μM) were evaluated. However, the functional components that can be evaluated are not limited to forskolin, γ-tocopherol, and α-lipoic acid. Each functional component was added for 10 days, from day 20 of culture to day 30 of differentiation induction. White adipocytes derived from the same iPS cells without the functional components were used as a comparison group.

[0051] In the embodiments of the present invention, the effects of these functional components on genes related to lipid droplet accumulation and beige staining were evaluated. As evaluation methods, relative expression analysis of brown adipocyte markers (UCP1), (PGC1α), and beige adipocyte marker (CD137) was performed by RT-PCR, and microscopic observation and comparison of lipid droplet formation amounts in adipocytes were performed by oil red O staining (Figures 7, 8, and 9).

[0052] (Pear-shaped fat cells) White adipocytes derived from pear-shaped iPS cells were differentiated under conditions of supplementation with 100 μM forskolin or 500 μM γ-tocopherol, and the expression levels of genes specific to brown / beige adipocytes were quantified. As shown in Figure 7, at 30 days after the start of culture, the expression levels of the brown adipocyte markers UCP1 and PGC1α significantly increased in the forskolin-supplemented group. An increasing trend in the expression levels of UCP1 and PGC1α was also observed in the γ-tocopherol-supplemented group. The expression level of the beige adipocyte marker CD137 was higher in both treatments than in untreated white adipocytes. Furthermore, in the groups treated with forskolin and γ-tocopherol, a reduction in lipid droplet size, characteristic of beige adipocytes, was observed.

[0053] (Apple-shaped fat cells) White adipocytes derived from apple-type iPS cells were differentiated under conditions of supplementation with 100 μM forskolin or 500 μM γ-tocopherol, and the expression levels of genes specific to brown / beige adipocytes were quantified. As shown in Figure 8, at 30 days after the start of culture, the expression levels of the brown adipocyte markers UCP1 and PGC1α significantly increased in the forskolin-supplemented group. The expression level of the beige adipocyte marker CD137 showed a suppressive trend. In the γ-tocopherol-supplemented group, the expression levels of the brown adipocyte markers UCP1 and CD137 increased. The expression level of the brown adipocyte marker PGC1α showed a suppressive trend. Furthermore, in the groups treated with forskolin and γ-tocopherol, a reduction in lipid droplet size, characteristic of beige adipocytes, was observed.

[0054] (Adam and Eve type fat cells) White adipose-derived progenitor cells from Adam-Eve type iPS cells were differentiated under conditions of supplementation with 100 μM forskolin, and the expression levels of genes specific to brown / beige adipocytes were quantified. As shown in Figure 9, at 30 days after the start of culture, the expression level of the brown adipocyte marker UCP1 significantly increased in the group supplemented with forskolin. An increasing trend was observed in the expression level of the brown adipocyte marker PGC1α. Furthermore, in the group treated with forskolin, a reduction in lipid droplet size, characteristic of beige adipocytes, was observed.

[0055] In the evaluation system for functional components, the results from Oil Red O showed that all functional components used in this invention suppress differentiation into white adipocytes. Furthermore, the relative expression levels of UCP1 and PGC1α suggested that forskolin and γ-tocopherol, which were found to have more multilocular lipid droplets than white adipocytes, promote the beigeing of adipocytes. In particular, forskolin was suggested to promote thermogenesis mediated by UCP1 in mitochondria. As shown in Figure 10, in the comparison of gene expression levels by genotype diet type, forskolin increased the expression of the brown adipocyte marker UCP1 to a certain level in all three genotype diet types compared to the control group. In particular, the expression of the brown adipocyte marker PGC1α showed a significant increase in both the pear-type and apple-type diet types. These results clearly indicate that forskolin is a diet support functional component suitable for all three genotype diet types, and it can be said that it is the diet support functional component best suited to the genetic constitution of the pear-type and apple-type diet types. Based on these findings, consuming the evaluated functional ingredients is expected to have a weight-loss effect by burning fat and expending excess calories as heat.

[0056] In this way, by understanding one's genetic predisposition through genetic testing and providing personalized supplements incorporating verified functional ingredients as active ingredients, as well as foods containing these functional ingredients, it is possible to change from a body type prone to weight gain to one prone to weight loss, resulting in unprecedented and superior weight loss effects.

Claims

[Claim 1] A method for evaluating diet support functional components using an iPS cell-derived adipocyte evaluation system based on obesity-related genes, wherein in this method, (1) A step to identify the type of gene mutation associated with obesity-related genes through genetic testing, (2) The steps of establishing iPS cells of the identified gene mutation-related type and constructing an iPS cell-derived adipocyte evaluation system, (3) The step of identifying diet support functional components using the constructed iPS cell-derived adipocyte evaluation system, The aforementioned gene mutation-associated type is UCP1 mutation, β3AR mutation, or no mutation. The aforementioned diet support functional ingredient contains at least a beige-promoting ingredient, The aforementioned beige-promoting component is one or more selected from the group consisting of forskolin, γ-tocopherol, and α-lipoic acid, and contains at least forskolin. A method for evaluating a diet support functional component, wherein in step (3) above, the diet support functional component is added to the iPS cell-derived adipocyte evaluation system to increase the relative expression level of at least one selected from the group consisting of brown adipocyte markers UCP1, PGC1α, and beige adipocyte marker CD137.

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