Method and composition for regulating GLP-1

Abstract

The compositions and methods for improving or regulating GLP-1 levels, stimulating GLP-1 secretion, ameliorating metabolism including losing weight, managing glucose or insulin tolerance including stimulating insulin secretion, inhibiting glucagon secretion and reducing fasting glucose levels. The compositions comprise β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative, or mixture thereof.

Description

Method and composition for regulating GLP-1BACKGROUND

[0001] Glucagon like peptide-1 (GLP-1), a peptide hormone from the intestinal tract, plays a central role in the coordination of postprandial glucose homeostasis through actions on insulin secretion, food intake and gut motility. Exogenous GLP-1 acutely stimulates insulin secretion, inhibits glucagon secretion, and lowers plasma glucose concentrations in people. In addition, GLP-1 inhibits gastric emptying, and reduces appetite in these patients. In rats, GLP-1 reverses age-dependent glucose decline in beta-cell function and stimulates beta-cell proliferation and neogenesis. In one report, after six weeks of GLP-1 treatment, 20 patients with type 2 diabetes in the study lost an average of 1.9 kg. Thus, GLP-1 could be effective on glycaemic control, body weight, insulin resistance, glucose tolerance management and beta-cell function.

[0002] The effect of β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid, or tributyrin on GLP-1 has not been reported yet. In this invention, β-aminoisobutyric acid, tributyrin and 3-hydroxy-3-methylbutanoic acid have been found to regulate GLP-1 levels.SUMMARY

[0003] In a first aspect, the present invention provides a method for improving or regulating GLP-1 level, or stimulating GLP-1 secretion in a subject, comprising administrating to the subject in need thereof a composition comprising: an effective amount of β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, tributyrin or tributyrin derivative, or mixture thereof.

[0004] In some embodiments, the method is used for ameliorating metabolism including losing weight, managing glucose or insulin tolerance, the said managing glucose or insulin tolerance include stimulating insulin secretion, inhibiting glucagon secretion and reducing fasting glucose levels.

[0005] In some embodiments, β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, tributyrin or tributyrin derivative, or mixture thereof is administrating in an effective amount.

[0006] In some embodiments, a proportion of the β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid or tributyrin is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0007] In some embodiments, the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or mixture thereof is administrated in an amount of 1-3000 mg. In some embodiments, the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or mixture thereof is administrated in an amount of 1-2500 mg, 1-2000 mg,1-1500 mg, 10-2000 mg,10-1500 mg, 20-1000 mg, 10-800 mg, 20-600 mg, 30-300 mg, 40-200 mg, 40-100 mg. In some embodiments, the tributyrin or tributyrin derivative thereof is administrated in an amount of 0.05 g-50 g. In some embodiments, the tributyrin or tributyrin derivative thereof is administrated in an amount of 0.05 g-50 g, 0.1 g-50 g, 0.5 g-50 g, 1 g-50 g, 5 g-50 g, 8 g-50 g, 10 g-50 g, 15 g-50 g, 20 g-50 g, 10 g-45 g, 15 g-45 g. In some embodiments, the daily dose is administered in divided doses or a single dose. In some embodiments, the administration is at least once a day or more times a day. In some embodiments, the administration is at least 7 days and above in one period.

[0008] In some embodiments, the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative, or mixture thereof is administrated in an amount of 0.1 μM to 1 M or 0.01 to 50.0wt% (w / w). In some embodiments, the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative, or mixture thereof may be administrated in an amount of 0.1 μM to 500 μM, 1 μM to 500 μM, 1 μM to 5 mM, 1 μM to 500 mM, 5 μM to 500 μM, 5 μM to 5 mM, 5 μM to 100 mM, 5 μM to 500 mM, 50 μM to 500 μM, 50 μM to 5 mM. In some embodiments,the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative, or mixture thereof may be administrated in an amount of 0.05% to 45%, 0.05% to 40%, 0.05% to 30%, 0.05% to 25%, 0.1% to 40%, 0.1% to 30%, 0.1% to 25%, 0.1% to 15%, 0. 5% to 30%, 0.5% to 20%, 0.5% to 10%, 1% to 35%, 1% to 25%, 1% to 10%, 2% to 25%, 2% to 10%, 5% to 9%, 1% to 7%, 2% to 7%, 0.05% to 7%, 0.05% to 9wt% (w / w).

[0009] In some embodiments, the subject is a mammal.

[0010] In some embodiments, the subject is human or animal. In some embodiments, the subject is human.

[0011] In some embodiments, the composition is prepared as a food, a drink, a supplement, a biochemical composition, or animal’s food, or a nutraceutical composition.

[0012] In some embodiments, the administration is through various routes selected from oral, intravenous injection, intramuscular injection, intraperitoneal injection, external use, or sublingual application.

[0013] In some embodiments, the composition is formulated in solutions, aqueous suspensions, liquid suspensions, parenteral solutions, injections, microemulsion, (micro)capsules, drops, granules, liquids, powders, aerosols, tonics, syrups, tablets, pills, film, functionalized foods, beverages, toothpaste, nourishments, snacks, gums, bars, sugars, and sublingual articles.

[0014] In a second aspect, the present invention provides a composition comprising an effective amount of β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative, or mixture thereof, for improving or regulating GLP-1 level, or stimulating GLP-1 secretion in a subject.

[0015] In some embodiments, the composition is used for ameliorating metabolism including losing weight, managing glucose or insulin tolerance, the said managing glucose or insulin tolerance include stimulating insulin secretion, inhibiting glucagon secretion and reducing fasting glucose levels.

[0016] In some embodiments, a proportion of the β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid or tributyrin is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0017] In some embodiments, the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or mixture thereof is administrated at a daily dose of 1-3000 mg. In some embodiments, the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or mixture thereof is administrated at a daily dose of 1-2500 mg, 1-2000 mg,1-1500 mg, 10-2000 mg, 10-1500 mg, 20-1000 mg, 10-800 mg, 20-600 mg, 30-300 mg, 40-200 mg, 40-100 mg. In some embodiments, the tributyrin or tributyrin derivative thereof is administrated in an amount of 0.05 g-50 g. In some embodiments, the tributyrin or tributyrin derivative thereof is administrated in an amount of 0.05 g-50 g, 0.1 g-50 g, 0.5 g-50 g, 1 g-50 g, 5 g-50 g, 8 g-50 g, 10 g-50 g, 15 g-50 g, 20 g-50 g, 10 g-45 g, 15 g-45 g. In some embodiments, the daily dose is administered in divided doses or a single dose. In some embodiments, the administration is at least once a day or more times a day. In some embodiments, the administration is at least 7 days and above in one period.

[0018] In some embodiments, the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative, or mixture thereof is administrated in an amount of 0.1 μM to 1 M or 0.01 to 50.0wt% (w / w). In some embodiments, the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative, or mixture thereof may be administrated in an amount of 0.1 μM to 500 μM, 1 μM to 500 μM, 1 μM to 5 mM, 1 μM to 500 mM, 5 μM to 500 μM, 5 μM to 5 mM, 5 μM to 100 mM, 5 μM to 500 mM, 50 μM to 500 μM, 50 μM to 5 mM. In some embodiments, the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative, or mixture thereof may be administrated in an amount of 0.05% to 45%, 0.05% to 40%, 0.05% to 30%, 0.05% to 25%, 0.1% to 40%, 0.1% to 30%, 0.1% to 25%, 0.1% to 15%, 0. 5% to 30%, 0.5% to 20%, 0.5% to 10%, 1% to 35%, 1% to 25%, 1% to 10%, 2% to 25%, 2% to 10%, 5% to 9%, 1% to 7%, 2% to 7%, 0.05% to 7%, 0.05% to 9wt% (w / w).

[0019] In some embodiments, the subject is a mammal.

[0020] In some embodiments, the subject is human or animal. In some embodiments, the subject is human.

[0021] In some embodiments, the composition is prepared as a food, a drink, a supplement, a biochemical composition, or animal’s food, or a nutraceutical composition.

[0022] In some embodiments, the composition is formulated in solutions, aqueous suspensions, liquid suspensions, parenteral solutions, injections, microemulsion, (micro)capsules, drops, granules, liquids, powders, aerosols, tonics, syrups, tablets, pills, film, functionalized foods, beverages, toothpaste, nourishments, snacks, gums, bars, sugars, and sublingual articles.

[0023] In a third aspect, the present invention provides use of a β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative, or mixture thereof in a preparation composition for improving or regulating GLP-1 level, or stimulating GLP-1 secretion in a subject.

[0024] In some embodiments, the composition is used for ameliorating metabolism including losing weight, managing glucose or insulin tolerance, the said managing glucose or insulin tolerance include stimulating insulin secretion, inhibiting glucagon secretion and reducing fasting glucose levels.

[0025] In some embodiments, a proportion of the β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid or tributyrin is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0026] In some embodiments, the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or mixture thereof is administrated at a daily dose of 1-3000 mg. In some embodiments, the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or mixture thereof is administrated at a daily dose of 1-2500 mg, 1-2000 mg, 1-1500 mg, 10-2000 mg, 10-1500 mg, 20-1000 mg, 10-800 mg, 20-600 mg, 30-300 mg, 40-200 mg, 40-100 mg. In some embodiments, the tributyrin or tributyrin derivative thereof is administrated in an amount of 0.05 g-50 g. In some embodiments, the tributyrin or tributyrin derivative thereof is administrated in an amount of 0.05 g-50 g, 0.1 g-50 g, 0.5 g-50 g, 1 g-50 g, 5 g-50 g, 8 g-50 g, 10 g-50 g, 15 g-50 g, 20 g-50 g, 10 g-45 g, 15 g-45 g. In some embodiments, the daily dose is administered in divided doses or a single dose. In some embodiments, the administration is at least once a day or more times a day. In some embodiments, the administration is at least 7 days and above in one period.

[0027] In some embodiments, the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative, or mixture thereof is administrated in an amount of 0.1 μM to 1 M or 0.01 to 50.0wt% (w / w). In some embodiments, the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative, or mixture thereof may be administrated in an amount of 0.1 μM to 500 μM, 1 μM to 500 μM, 1 μM to 5 mM, 1 μM to 500 mM, 5 μM to 500 μM, 5 μM to 5 mM, 5 μM to 100 mM, 5 μM to 500 mM, 50 μM to 500 μM, 50 μM to 5 mM. In some embodiments, the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative, or mixture thereof may be administrated in an amount of 0.05% to 45%, 0.05% to 40%, 0.05% to 30%, 0.05% to 25%, 0.1% to 40%, 0.1% to 30%, 0.1% to 25%, 0.1% to 15%, 0. 5% to 30%, 0.5% to 20%, 0.5% to 10%, 1% to 35%, 1% to 25%, 1% to 10%, 2% to 25%, 2% to 10%, 5% to 9%, 1% to 7%, 2% to 7%, 0.05% to 7%, 0.05% to 9wt% (w / w).

[0028] In some embodiments, the subject is a mammal.

[0029] In some embodiments, the subject is human or animal. In some embodiments, the subject is human.

[0030] In some embodiments, the administration is through various routes selected from oral, intravenous injection, intramuscular injection, intraperitoneal injection, external use, or sublingual application.

[0031] In some embodiments, the composition is formulated in solutions, aqueous suspensions, liquid suspensions, parenteral solutions, injections, microemulsion, (micro)capsules, drops, granules, liquids, powders, aerosols, tonics, syrups, tablets, pills, film, functionalized foods, beverages, toothpaste, nourishments, snacks, gums, bars, sugars, and sublingual articles.

[0032] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1a is percentage change curve of body weight of groups 1-3 and 6. Figure 1b is percentage change curve of body weight of groups 1-2, 4-5 and 7-8.

[0034] Figure 2a is changes in the proportion of fat content of groups 1-3 and 6. Figure 2b is changes in the proportion of fat content of groups 1-2, 4-5 and 7-8.

[0035] Figure 3a is area under the curve (AUC) of oral glucose tolerance test (OGTT) of groups 1-3 and 6. Figure 3b is area under the curve (AUC) of oral glucose tolerance test (OGTT) of groups 1-2, 4-5 and 7-8.

[0036] Figure 4a is area under the curve (AUC) of insulin intraperitoneal tolerance test (IPTT) of groups 1-3 and 6. Figure 4b is area under the curve (AUC) of insulin intraperitoneal tolerance test (IPTT) of groups 1-2, 4-5 and 7-8.

[0037] Figure 5a is the pararenal fat weight of groups 1-3 and 6. Figure 5b is the pararenal fat weight of groups 1-2, 4-5 and 7-8.

[0038] Figure 6a is the insulin levels in serum of groups 1-3 and 6. Figure 6b is the insulin levels in serum of groups 1-2, 4-5 and 7-8.

[0039] Figure 7a is the TC levels in serum of groups 1-3 and 6. Figure 7b is the TC levels in serum of groups 1-2, 4-5 and 7-8.

[0040] Figure 8a is the TG levels in serum of groups 1-3 and 6. Figure 8b is the TG levels in serum of groups 1-2, 4-5 and 7-8.

[0041] Figure 9a is the LDL levels in serum of groups 1-3 and 6. Figure 9b is the LDL levels in serum of groups 1-2, 4-5 and 7-8.

[0042] Figure 10a is the HDL levels in serum of groups 1-3 and 6. Figure 10b is the HDL levels in serum of groups 1-2, 4-5 and 7-8.

[0043] Figure 11a is the GLP-1 levels in serum of groups 1-3 and 6. Figure 11b is the GLP-1 levels in serum of groups 1-2, 4-5 and 7-8.DETAILED DESCRIPTION

[0044] As used herein, the term “or” is meant to include both “and” and “or.” In other words, the term “or” may also be replaced with “and / or.”

[0045] As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0046] As used herein, the term “comprise” or “include” and their conjugations, refer to a situation wherein said terms are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It also encompasses the more limiting verb ‘to consist essentially of’ and ‘to consist of’.

[0047] As used herein, the term "effective amount" refers to the amount required to achieve the effect as taught herein. The specific effective dose level for any particular subject will depend upon a variety of factors including the conditions being treated and the severity of the conditions; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid or tributyrin or tributyrin derivative employed; the duration of the treatment; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired effect and to gradually increase the dosage until the desired effect is achieved.

[0048] One of skill in the art recognizes that an amount may be considered “effective” even if the condition is not totally eradicated or prevented, but it or its symptoms and / or effects are improved or alleviated partially in the subject.

[0049] As used herein, the term “physiologically acceptable” means pharmaceutically, physiologically, alimentarily, and / or nutritionally acceptable, and refers to those compositions or combinations of agents, materials, or compositions, and / or their dosage forms, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0050] As used herein, the term “mammal” or “subject” may be used interchangeably to refer to any animal to which the presently disclosed methods and compositions may be applied or administered. The animal may have an illness or other disease, but the animal does not need to be sick to benefit from the presently disclosed methods and compositions. As such any animal may apply the disclosed combinations, compositions or kits, or be a recipient of the disclosed methods. “Mammal” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, and, in particular, humans. Although the animal subject is preferably a human, the methods and compositions of the invention have application in veterinary medicine.

[0051] The dosage of β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid or tributyrin thereof and / or composition comprising the same may range broadly, depending upon the desired effects and the indication. The dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the subject. In some embodiments, the compounds are administered for a period of time, for example for a week or more, or for months or years.

[0052] As used herein, the term "administration" refers to the process of delivering a disclosed composition or active ingredient to a subject. The compositions of the invention can be administered in a variety of ways, including orally, intragastrically, and parenterally (e.g., intravenous and intraarterial as well as other suitable parenteral routes), in external use, and the like.

[0053] As used herein, a “parenteral solution” refers to a solution that can be administered elsewhere in the body than the mouth and alimentary canal. It is not delivered via the intestinal tract. For example, parenteral solution can be delivered intravenously.

[0054] As used herein, a “tonic” refers to a medicinal substance taken to give a feeling of vigor or well-being.

[0055] As used herein, a “syrup” refers to a thick sticky liquid derived from a sugar-rich plant, for example, sugar cane, corn, and maple.

[0056] Multiple techniques of administering a composition exist in the art including, but not limited to, oral, rectal, topical, aerosol, injection and parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal and intraocular injections.

[0057] “Intraperitoneal” as used here means within or administered through the peritoneum. The peritoneum is a thin, transparent membrane that lines the walls of the abdominal (peritoneal) cavity and contains / encloses the abdominal organs such as the stomach and intestines.

[0058] As used herein, “sublingual” refers to situated or applied under the tongue.

[0059] A “functionalized food composition” includes a food composition that has a potentially positive effect on health beyond basic nutrition.

[0060] Various embodiments of the present invention provide for methods for administrating β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid or tributyrin thereof to a subject for effectively improving or regulating GLP-1 level, or stimulating GLP-1 secretion in a subject. For instance, the method is used for losing weight, managing glucose tolerance, or reducing fasting glucose levels. For example, an amount of approximately 10 mg to approximately 1000 mg of β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid or tributyrin may be administered to a human. β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid or tributyrin may be administrated in a variety of forms, such as solutions, aqueous suspensions, liquid suspensions, parenteral solutions, injections, microemulsion, (micro)capsules, drops, granules, liquids, powders, aerosols, tonics, syrups, tablets, pills, film, functionalized foods, beverages, toothpaste, nourishments, snacks, gums, bars, sugars, and sublingual articles.

[0061] In some implementations, one or more additives may be included in the composition, such as flavorings (e.g., natural and / or artificial), vitamins, minerals, binders, and / or any other appropriate additive. The additives may alter flavor, color, and / or texture. The additives may increase palatability and / or facilitate inclusion in a delivery vehicle (e.g., tablet, food product, beverage product such as a drink mix, etc.). In some implementations, the β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid or tributyrin may be processed to increase bioavailability, solubility, palatability, and / or combination with other compounds.

[0062] Any titles or subheadings used herein are for organization purposes and should not be used to limit the scope of embodiments disclosed herein.

[0063] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

[0064] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.Example 1

[0065] Human NCI-H716 cells (colorectal adenocarcinoma cells) were grown in suspension at 37℃, 5% CO2. The culture medium was RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 100 IU / mL penicillin and 100 μg / mL streptomycin. Endocrine differentiation was induced by seeding cells in dishes coated with Matrigel, in high-glucose DMEM, 10% FBS, 2 mM L-glutamine, 100 IU / mL penicillin, and 100 μg / mL streptomycin. Two days before the experiments, 1.5 x 106cells were seeded in 12-well culture plates coated with Matrigel and containing highglucose DMEM, 10% FBS, 2 mM L-glutamine, 100 IU / mL penicillin and 100 μg / mL streptomycin. On the day of the experiment, medium was replaced by Krebs-Ringer bicarbonate buffer (KRB) buffer (128.8 mmol / L NaCl, 4.8 mmol / L KCl, 1.2 mmol / L KH2PO4, 1.2 mmol / L MgSO4, 2.5 mmol / L CaCl2, 5 mmol / L NaHCO3, and 10 mmol / L HEPES, pH 7.4) containing 0.2% BSA and different concentrations of β-aminoisobutyric acid (BAIBA) or 3-hydroxy-3-methylbutanoic acid (HMB) (0 μM, 3 μM, 10 μM or 30 μM), or tributyrin (0 mM, 2 mM, 5 mM or 10 mM). Following incubating at 37℃ for 2 h, the supernatants were collected with the addition of 50 μg / mL phenylmethylsulfonyl fluoride and stored at -80℃ for analysis. The cells were scraped off and sonicated in a homogenization buffer (1 M HCl containing 5% formic acid, 1% trifluoroacetic acid and 1% NaCl). GLP-1 in supernatant and cells was measured by a GLP-1 active ELISA kit, respectively, according to the manufacturer's protocol. Protein content of the cells was determined using the Bradford protein assay. The GLP-1 content was normalized for the total protein of the cells. A total of 3 x 106cells were seeded in 6-well culture plates coated with Matrigel and incubated for 24 h. Medium was replaced by serum-free medium containing 0.2% BSA without or with 30 μM β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid, or 10 mM tributyrin. The cells were re-incubated for 24 h and were washed with cold-PBS and stored at -80℃ for RT-PCR analysis. Total RNA was isolated from each well with Trizol reagent (Invitrogen Co., USA). RT-PCR was performed according to the protocol provided with the TwoStep RT-PCR kit (BestBio, Shanghai, China). The sequences of the forward and reverse primers were: 5'-GTAATGCTGGTACAAGGCAG-3' and 5'-TTATAAAGTCCCTGGCGGCA-3' for the proglucagon gene, 5'-CGCTGACCTGCACAATGACT-3' and 5'-CAGACAACCAGGTGCTGCAT-3' for prohormone convertase 3 gene, 5'-GCTGAGAACGGGAAGCTTGT-3' and 5'-TCTCCATGGTGGTGAAGACG-3' for internal control GAPDH gene. After denaturing at 94℃ for 5 min, the amplification was obtained by 33 cycles of 94℃ for 30 s, 59℃ for 30 s and 72℃ for 30 s each. A final extension step at 72℃ for 5 min was performed. 27 cycles were performed for the amplification for GAPDH gene. PCR products were subjected to electrophoresis on 2.5% agarose gel, and visualized by means of ethidium bromide staining. Densitometric quantification was recorded using Jeda image analysis system 3.3 (Jiangsu Jeda ScienceTechnology Co. Ltd., Nanjing, China). Genes implicated in GLP-1 synthesis and secretion (i.e. Gcg, Pcsk1, and Slc5a1) were also detected by q-PCR.

[0066] Measurement of GLP-1: After incubation for 2 hours, GLP-1 content in supernatant and cell lysate was detected by ELISA.

[0067] mRNA expression of proglucagon and prohormone convertase 3: After incubation for 24 hours, mRNA of cells was extracted, cDNA was obtained by inversion, and the amplified fragments were amplified by PCR, and horizontal electrophoresis was performed to evaluate the relative expression levels of proglucagon mRNA and prohormone convertase 3 mRNA.

[0068] β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid or Tributyrin treatment may increase GLP-1 secretion (medium) in a concentration-dependent manner. β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid also or Tributyrin increased cellular GLP-1 level, significant increases were found in cells treated without β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid or Tributyrin.Example 2

[0069] At the beginning of the experiment, the mice were divided randomly into control group (NC, Group 1, n=8), HFD group (HED, Group 2, n=8), HFD + tributyrin group (Group 3, n=8), HFD + BAIBA group (Group 4, n=8) and HFD + HMB group (Group 5, n=8), NC + tributyrin group (Group 6, n=8), NC + BAIBA group (Group 7, n=8) and NC+ HMB group (Group 8, n=8). The dosage of tributyrin supplementation is 100 mg / kg. The dosage of BAIBA or HMB supplementation is 100 mg / kg. The group 1 is the control group and is given the normal chow; the groups 2, 3 and 4, 5 are the high-fat-diet induced obese mice and continue high fat diet for 8 weeks; the groups 3 and 4, 5 also continued to eat a high-fat diet and received daily intragastric administration of sample for 8 weeks, the groups 6-8 are control groups and received daily intragastric administration of sample for 8 weeks. Record the body fat mass and weight of each group of mice every week. Complete oral glucose tolerance test (OGTT) / insulin intraperitoneal tolerance test (IPTT) on the last day of week 8, then anesthesia the mice and take serum -80℃ for storage.

[0070] Measurement of Blood Glucose: Oral glucose tolerance test (OGTT): At week 16th of the experiment, after fasting for 15h, glucose was given intravenously (2 g / kg), and blood glucose was measured at 0, 15, 30, 60, 90, 120 mins. Insulin intraperitoneal tolerance test (IPTT): At week 16th of the experiment, after fasting for 15h, insulin (1 U / kg) was injected intraperitoneally and blood glucose was measured at 0, 15, 30, 60, 90, 120 mins.

[0071] Body composition analysis: During the experiment, the fat mass and lean mass was measured by Nuclear Magnetic Resonance Body Composition Analyzer once a week.

[0072] Serum detection by ELISA: At week 8th of the experiment, the whole blood is extracted from heart. The serum was acquired after centrifugation (3000 rpm 10 mins), stored at -80℃ for later use. Determinate the concentration of insulin (INS), cholesterol (TC), triglycerides (TG), LDL (low density lipoprotein), HDL (high density lipoprotein), GLP-1 in serum by ELISA kits.

[0073] Weight of the fat pad: At the 17th week, the mice were sacrificed, and the fat pads (subcutaneous, epididymis and perirenal) were dissected and weighed.

[0074] Change of body weight: Weigh once a week at a fixed time from beginning of the experiment.

[0075] Figure 1a is percentage change curve of body weight of groups 1-3 and 6. In Figure 1a, after taking Tributyrin for 8 weeks, body weight in HFD + Tributyrin group decreased by 5.8% compared with HFD group, NC + Tributyrin group decreased by 4.5% at the 8th week compared with NC group. Figure 1b is percentage change curve of body weight of groups 1-2, 4-5 and 7-8. After taking BAIBA and HMB for 8 weeks, respectively, body weight in HFD + BAIBA group decreased by 6.8% compared with HFD group, NC + BAIBA group decreased by 5.1% at the 8th week compared with NC group, while HFD + HMB showed a 3.2% reduction, NC + HMB group decreased by 2.2% at the 8th week compared with NC group. Thus, the body weight curve showed that Tributyrin or BAIBA or HMB administration is helpful in inhibiting high fat induced obesity. Compared with the untreated control group, the body weight of Tributyrin or BAIBA or HMB treated group was decreased approximately 2%, preferably decreased 5%, 7%.

[0076] Figure 2a displays fat content percentage changes of groups 1-3 and 6. HFD + Tributyrin reduced fat accumulation by 12.7%, and the fat content in NC + Tributyrin group was decreased by 22.1% as compared to control group. Figure 2b displays fat content percentage changes of groups 1-2, 4-5 and 7-8. HFD + BAIBA reduced fat accumulation by 17.5% and the fat content in NC + BAIBA group was decreased approximately by 32.8% as compared to control group, and HFD + HMB by 8.0% and the fat content in NC + HMB group was decreased approximately by 14.6% as compared to control group.

[0077] Figure 3a is area under the curve (AUC) of oral glucose tolerance test (OGTT) of groups 1-3 and 6, which reveals HFD + Tributyrin improved glucose tolerance by 9.6% as compared to HFD group, and NC + Tributyrin improved glucose tolerance by 4.6% as compared to NC group. Figure 3b is area under the curve (AUC) of oral glucose tolerance test (OGTT) of groups 1-2, 4-5 and 7-8, which reveals HFD + BAIBA improved glucose tolerance by 16.2% as compared to HFD group, and NC + BAIBA improved glucose tolerance by 6.9% as compared to NC group, and HFD + HMB by 3.8% as compared to HFD group, and NC + HMB improved glucose tolerance by 1.4% as compared to NC group.

[0078] Figure 4a is area under the curve (AUC) of insulin intraperitoneal tolerance test (IPTT) of groups 1-3 and 6, which shows HFD + Tributyrin improved insulin sensitivity by 7.1% as compared to HFD group, and NC + Tributyrin improved glucose tolerance by 7.2% as compared to NC group. Figure 4b is area under the curve (AUC) of insulin intraperitoneal tolerance test (IPTT) of groups 1-2, 4-5 and 7-8, which shows HFD + BAIBA enhanced insulin sensitivity by 11.4% as compared to HFD group, and NC + BAIBA improved glucose tolerance by 10.5% as compared to NC group, with HFD + HMB improving 5.2% as compared to HFD group, and NC + HMB improved glucose tolerance by 2.0% as compared to NC group.

[0079] Figure 5a is the pararenal fat weight of groups 1-3 and 6, which shows HFD + Tributyrin lowered pararenal fat weight by 11.7% as compared to HFD group, and NC + Tributyrin by 16.3% as compared to NC group. Figure 5b is the pararenal fat weight of groups 1-2, 4-5 and 7-8, which shows HFD + BAIBA lowered pararenal fat weight by 19.6% as compared to HFD group, and NC + BAIBA by 21.6% as compared to NC group, and HFD + HMB lowered pararenal fat weight by 8.0% as compared to HFD group, and NC + HMB by 6.1% as compared to NC group.

[0080] From Figure 1 to Figure 5, the results indicated that supplementing Tributyrin or BAIBA or HMB can achieve beneficial effects on metabolic health compared to the group without supplementation. This includes inhibition of weight gain, reduction in fat accumulation, amelioration of glucose intolerance, and enhancement of insulin sensitivity. In particular, compared with the untreated control group, the body weight of Tributyrin or BAIBA or HMB treated group was decreased approximately 2%, preferably decreased 5%, 7%; the fat content of Tributyrin or BAIBA or HMB treated group reduced by approximately 8%, preferably decreased 12%, 14%, 17%, 20%, 32%.

[0081] Figures 6-11 are serum lipid metabolism indexes including insulin (INS), cholesterol (TC), triglycerides (TG), LDL (low density lipoprotein), HDL (high density lipoprotein), GLP-1 levels in groups 1-4. Insulin regulates glucose levels in the bloodstream and induces glucose storage in the liver, muscles, and adipose tissue. HDL have long been considered as "good cholesterol," beneficial to the whole body and, in particular, to cardio-vascular health. TG is a very important component of blood fat, and elevated levels may lead to heart disease. LDL, the "bad cholesterol" has a strong effect on atherosclerosis. Therefore, LDL is also known as atherogenic factor. When LDL, especially oxidized low density lipoprotein (OX-LDL) is excessive, the cholesterol it carries accumulates in the artery wall, which is easy to cause arteriosclerosis for a long time. The importance of GLP-1 was described above. GLP-1 (Glucagon-Like Peptide-1) is an incretin hormone that enhances glucose-dependent insulin secretion, inhibits glucagon release, slows gastric emptying, and promotes satiety, playing a crucial role in glucose homeostasis and energy balance.

[0082] Figure 6a is insulin (INS) levels in serum showing HFD + Tributyrin decreased INS by 23.3% relative to HFD and a 9.2% reduction in NC + Tributyrin group compared with NC group. Figure 6b is insulin (INS) levels in serum showing HFD + BAIBA decreased INS by 30.8% relative to HFD and a 17.4% reduction in NC + BAIBA group compared with NC group, and HFD + HMB decreased INS by 15.9% relative to HFD and a 7.3% reduction in NC + HMB group compared with NC group. ​

[0083] Figure 7a is cholesterol (TC) levels revealing HFD + Tributyrin reduced TC by 20.4% relative to HFD and a 17.2% reduction in NC + Tributyrin group compared with NC group. Figure 7b is cholesterol (TC) levels revealing HFD + BAIBA reduced TC by 34.6% relative to HFD and a 36.2% reduction in NC + BAIBA group compared with NC group, and HFD + HMB reduced TC by 3.8% relative to HFD relative to HFD and a 2.6% reduction in NC + HMB group compared with NC group.

[0084] Figure 8a is triglycerides (TG) levels demonstrating HFD + Tributyrin lowered TG by 17.3% relative to HFD and a 23.0% reduction in NC + Tributyrin group compared with NC group. Figure 8b is triglycerides (TG) levels demonstrating HFD + BAIBA lowered TG by 23.8% relative to HFD and a 33.6% reduction in NC + BAIBA group compared with NC group, and HFD + HMB lowered TG by 5.4% relative to HFD and a 4.1% reduction in NC + HMB group compared with NC group.

[0085] Figure 9a is LDL levels where HFD + Tributyrin reduced LDL by 14.2% relative to HFD and a 20.9% reduction in NC + Tributyrin group compared with NC group. Figure 9b is LDL levels where HFD + BAIBA reduced LDL by 33.3% relative to HFD and a 32.4% reduction in NC + BAIBA group compared with NC group and HFD + HMB reduced LDL by 5.7% relative to HFD relative to HFD and a 6.4% reduction in NC + HMB group compared with NC group.

[0086] Figure 10a is HDL levels indicating HFD + Tributyrin increased HDL by 11.3% compared with HFD group and an 10.6% increase in NC + Tributyrin group compared with NC group. Figure 10b is HDL levels indicating HFD + BAIBA increased HDL by 19.1% compared with HFD group and an 24.5% increase in NC + BAIBA group compared with NC group, and HFD + HMB increased HDL by 1.8% relative to HFD and an 1.6% increase in NC + HMB group compared with NC group. In addition, as for the ratio of HDL / LDL, compared with the HFD group, HFD + Tributyrin group showed a 29.8% increase, and the NC + Tributyrin group showed a 39.7% increase compared with NC group. Compared with the HFD group, HFD + BAIBA group showed a 78.7% increase, and the NC + BAIBA group showed an 84.1% increase compared with NC group. Compared with the HFD group, HFD + HMB group showed a 4.1% increase, and the NC + HMB group showed an 8.6% increase compared with NC group.

[0087] Figure 11a is GLP-1 levels showing HFD + Tributyrin increased GLP-1 by 75.4% compared with HFD group and a 55.3% increase in NC + Tributyrin group compared with NC group. Figure 11b is GLP-1 levels showing HFD +BAIBA increased GLP-1 by 50.3% compared with HFD group and a 36.7% increase in NC + BAIBA group compared with NC group, and HFD + HMB increased GLP-1 by 11.0% relative to HFD and a 4.2% increase in NC + HMB group compared with NC group.

[0088] Compared with the untreated control group, the TC of Tributyrin or BAIBA or HMB treated group was reduced by 2%, preferably reduced by 15%, 20%, 35%; the TG of Tributyrin or BAIBA or HMB treated group was reduced by 4%, preferably reduced by 15%, 20%, 30%, 35%; the LDL of Tributyrin or BAIBA or HMB treated group was reduced by 5%, preferably reduced by 15%, 20%, 30%, 35%; the HDL of Tributyrin or BAIBA or HMB treated group was increased by 2%, preferably increased by 10%, 20%, 25%; especially the ratio of HDL / LDL of Tributyrin or BAIBA treated group was increased by 30%, preferably increased by 40%, 70%, 80%, 85%; the GLP-1 of Tributyrin or BAIBA treated group was increased by 30%, preferably increased by 40%, 50%, 55%, 70%, 80%. From the point of view of serum indexes (Figures 6-11), supplementing Tributyrin, BAIBA or HMB could significantly increase HDL and GLP-1, and decrease INS, TC, TG, and LDL relative to the HFD group without supplementation, indicating that Tributyrin, BAIBA and HMB all have the effect of improving obesity and ameliorating metabolism. This effect persists for as long as the test protocol is continued.

[0089] The results indicated that supplementing Tributyrin or BAIBA or HMB can achieve the beneficial effects on metabolic health than without Tributyrin or BAIBA or HMB supplementation.

[0090] Although specific embodiments and examples of this invention have been illustrated herein, it will be appreciated by those skilled in the art that any modifications and variations can be made without departing from the spirit of the invention. The examples and illustrations above are not intended to limit the scope of this invention. Any combination of embodiments of this invention, along with any obvious their extension or analogs, are within the scope of this invention. Further, it is intended that this invention encompass any arrangement, which is calculated to achieve that same purpose, and all such variations and modifications as fall within the scope of the appended claims.

Claims

1. A method for improving or regulating GLP-1 level, or stimulating GLP-1 secretion in a subject, comprising administrating to the subject in need thereof a composition comprising: an effective amount of β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative, or mixture thereof.

2. The method of claim 1, wherein the method is used for ameliorating metabolism including losing weight, managing glucose or insulin tolerance, the said managing glucose or insulin tolerance include stimulating insulin secretion, inhibiting glucagon secretion and reducing fasting glucose levels.

3. The method of claim 1 or 2, wherein a proportion of the β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid or tributyrin is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

4. The method of any of claims 1-3, wherein the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or mixture thereof is administrated in an amount of 1-3000 mg, the tributyrin or tributyrin derivative is administrated in an amount of 0.05 g-50 g.

5. The method of any of claims 1-4, wherein the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or the tributyrin or tributyrin derivative or mixture thereof is administrated in an amount of 0.1 μM to 1 M or 0.01 to 50.0wt% (w / w).

6. The method of any of claims 1-5, wherein the subject is a mammal.

7. The method of any of claims 1-6, wherein the subject is human or animal.

8. The method of any of claims 1-7, wherein the composition is prepared as a food, a drink, a supplement, a biochemical composition, animal’s food, or a nutraceutical composition.

9. The method of any of claims 1-8, wherein the administration is through various routes selected from oral, intravenous injection, intramuscular injection, intraperitoneal injection, external use, or sublingual application.

10. The method of any of claims 1-9, wherein the composition is formulated in solutions, aqueous suspensions, liquid suspensions, parenteral solutions, injections, microemulsion, (micro)capsules, drops, granules, liquids, powders, aerosols, tonics, syrups, tablets, pills, film, functionalized foods, beverages, toothpaste, nourishments, snacks, gums, bars, sugars, and sublingual articles.

11. A composition comprising an effective amount of β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, the tributyrin or tributyrin derivative, or mixture thereof, for improving or regulating GLP-1 level, or stimulating GLP-1 secretion in a subject.

12. The composition of claim 11, wherein the composition is used for ameliorating metabolism including losing weight, managing glucose or insulin tolerance, the said managing glucose or insulin tolerance include stimulating insulin secretion, inhibiting glucagon secretion and reducing fasting glucose levels.

13. The composition of claim 11 or 12, wherein a proportion of the β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid or the tributyrin is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

14. The composition of any of claims 11-13, wherein the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or mixture thereof is administrated at a daily dose of 1-3000 mg, the tributyrin or tributyrin derivative is administrated at a daily dose of 0.05 g-50 g.

15. Use of β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative or mixture thereof in a preparation of composition for improving or regulating GLP-1 level, or stimulating GLP-1 secretion in a subject.

16. The use of claim 15, wherein the composition is used for ameliorating metabolism including losing weight, managing glucose or insulin tolerance, the said managing glucose or insulin tolerance include stimulating insulin secretion, inhibiting glucagon secretion and reducing fasting glucose levels.

17. The use of claim 15 or 16, wherein the subject is a mammal.

18. The use of any of claims 15-17, wherein a proportion of the β-aminoisobutyric acid or 3-hydroxy-3-methylbutanoic acid or tributyrin is at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

19. The use of any of claims 15-18, wherein the β-aminoisobutyric acid, a physiologically acceptable salt, polymer, ester, or 3-hydroxy-3-methylbutanoic acid, a physiologically acceptable salt, polymer, ester, or tributyrin or tributyrin derivative or mixture thereof is administrated at a daily dose of 1-3000 mg or in an amount of 0.1 μM to 1 M or 0.01 to 50.0wt% (w / w).

20. The use of any of claims 15-19, wherein the composition is formulated in solutions, aqueous suspensions, liquid suspensions, parenteral solutions, injections, microemulsion, (micro)capsules, drops, granules, liquids, powders, aerosols, tonics, syrups, tablets, pills, film, functionalized foods, beverages, toothpaste, nourishments, snacks, gums, bars, sugars, and sublingual articles.

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