Application of imidazole-4-acetic acid in preparation of drugs or health care products for inhibiting appetite, reducing blood sugar, losing weight or losing weight

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of imidazole-4-acetic acid in preparation of drugs or health care products for inhibiting appetite, reducing blood sugar, losing weight or losing weight. The invention discloses application of imidazole-4-acetic acid and / or pharmaceutically acceptable salts, solvates and precursors of imidazole-4-acetic acid in preparation of drugs or health care products for inhibiting appetite. The imidazole-4-acetic acid is a natural metabolite, so that toxic and side effects can be reduced, and the imidazole-4-acetic acid is relatively safe. The invention provides application of imidazole-4-acetic acid in preparation of drugs or health care products for inhibiting appetite, reducing blood sugar, losing weight or losing weight, and is expected to provide a new strategy for appetite treatment of obesity and reducing blood sugar and losing weight.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of imidazole-4-acetic acid in preparing medicines or health products for suppressing appetite, lowering blood sugar, reducing weight or losing weight. Background Art

[0002] Obesity caused by appetite disorders is a predisposing factor for many metabolic diseases such as diabetes, fatty liver, cancer, etc. Therefore, it is necessary for obese people to lose weight.

[0003] Suppressing appetite is an effective and relatively safe method for weight loss. Many appetite suppressants have been used clinically. In recent years, appetite suppressants have developed rapidly. The main drugs developed are: (1) Catecholamines such as fenfluramine and amphetamine can reduce appetite by increasing oxygen consumption and promoting energy release by acting on β receptors; common adverse reactions include insomnia, dry mouth, and constipation. (2) Opioid receptor blockers such as naltrexone can suppress appetite by acting on the central nervous system; common adverse reactions include nausea, vomiting, and headache. (3) Serotonin receptor agonists such as lorcaserin and fenfluramine can reduce food intake and promote satiety by selectively stimulating the hypothalamic satiety center; common adverse reactions include headache, dizziness, dry mouth, and fatigue. 4) Other drugs that can reduce appetite, energy intake, inhibit fat synthesis, increase metabolic rate, and increase energy consumption through central and peripheral effects, thereby reducing fat accumulation. The appetite suppressants currently on the market often cause many side effects. Therefore, the development of new and safe appetite suppressant health products or drugs has important clinical significance.

[0004] Imidazole-4-acetic acid (4-IAA) is one of the most important imidazole derivatives and a natural metabolite present in the brain. It is reported that 4-IAA has many uses, including analgesia, sedation, hypnosis, treatment of retinal diseases, and as a drug intermediate.

[0005] There are no reports on the use of 4-IAA in suppressing appetite, lowering blood sugar, reducing weight, or losing weight, or in the preparation of medicines or health products for suppressing appetite, lowering blood sugar, reducing weight, or losing weight. Summary of the invention

[0006] The technical problem to be solved by the present disclosure is that the health products or drugs for suppressing appetite, lowering blood sugar, reducing weight, and losing weight on the market often cause more side effects, and a new and safe health product or drug for suppressing appetite, lowering blood sugar, reducing weight, and losing weight is developed, and the use of imidazole-4-acetic acid in the preparation of drugs or health products for suppressing appetite, lowering blood sugar, reducing weight, and losing weight is provided.

[0007] In order to achieve the above technical objectives, the technical solution adopted by the present disclosure is:

[0008] In one aspect, the present disclosure provides a compound that binds to positions D114, Y115, C118, Y374, F398 and L401 of a histamine H3 receptor (HRH3) and / or a pharmaceutically acceptable salt, a solvate, or a precursor thereof for use in preparing a drug or health product, wherein the histamine H3 receptor sequence is: SEQ ID NO: 2, and the drug or health product is any one or more of the following:

[0009] (1) Appetite suppressing drugs or health products;

[0010] (2) Drugs or health products that lower blood sugar;

[0011] (3) drugs or health products for the treatment of diabetes;

[0012] (4) drugs or health products for weight loss;

[0013] (5) Weight loss drugs or health products;

[0014] (6) Fat-reducing drugs or health products;

[0015] (7) Drugs or health products for the treatment of fatty liver.

[0016] In some preferred embodiments of the present disclosure, Y115 and Y374 are specific for 4-IAA.

[0017] In some preferred embodiments of the present disclosure, W402 is specific for histamine binding.

[0018] In some preferred embodiments of the present disclosure, D114, Y115, C118, Y374, F398 and L401 are residues necessary for HRH3 to bind to 4-IAA.

[0019] In some preferred embodiments of the present disclosure, D114, C118, F398 and L401 are common sites in HRH3 required for binding to 4-IAA and histamine.

[0020] In the present disclosure, D114 is aspartic acid at position 114. Y115 is tyrosine at position 115. C118 is cysteine ​​at position 118. Y374 is tyrosine at position 374. F398 is phenylalanine at position 398. L401 is leucine at position 401. W402 is tryptophan at position 402.

[0021] Although the present disclosure provides some specific amino acid sequences or nucleotide sequences, such as the amino acid sequences or nucleotide sequences shown in the sequence listing, it should be understood that a specific amino acid sequence or nucleotide sequence includes variants with conservative sequence modifications thereof, such as sequences having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homology thereto, as long as the biological function or activity of the specific amino acid sequence or nucleotide sequence is not lost.

[0022] As used herein, the term "homology" has a meaning recognized in the art and is a central concept in comparative biology. The basic meaning of homology is that the two samples being compared (e.g., an amino acid sequence or a nucleotide sequence) have a common ancestor. Generally speaking, if two traits (states) in two species meet any of the following two conditions, the two traits can be called a pair of homologous traits: 1. They are the same as a trait found in the ancestral group of these species; 2. They are different traits with an ancestor-descendant relationship. Amino acid sequence homology can be determined by methods known per se. For example, amino acid sequence homology (%) can be determined using programs commonly used in the field (e.g., BLAST, FASTA, etc.) according to initial settings. On the other hand, homology (%) can be determined using any algorithm known in the field, such as the algorithm of Needleman et al. (1970) (J. Mol. Biol. 48: 444-453), Myers and Miller (CABIOS, 1988, 4: 11-17), etc. The algorithm of Needleman et al. is integrated into the GAP program of the GCG software package (available at www.gcg.com), and homology (%) can be determined, for example, by using a BLOSUM 62 matrix or a PAM250 matrix, and gap weights (weights): 16, 14, 12, 10, 8, 6 or 4 and any one of length weights: 1, 2, 3, 4, 5 or 6. In addition, the algorithm of Myers and Miller is integrated into the ALIGN program as part of the GCG sequence alignment software package. In the case of utilizing the ALIGN program for comparison of amino acid sequences, for example, a PAM120 weight residue table, gap length penalty, gap penalty can be used.

[0023] On the other hand, the present disclosure provides a compound that binds to the D114, Y115, C118, Y374, F398 and L401 positions of the histamine H3 receptor and / or its pharmaceutically acceptable salt, solvate, or precursor for use in preparing a drug or health product for suppressing appetite, lowering blood sugar, treating diabetes, reducing weight, losing weight, reducing fat, and / or treating fatty liver mediated by hypothalamic AgRP neurons, wherein the histamine H3 receptor sequence is: SEQ ID NO: 2.

[0024] In some preferred embodiments of the present disclosure, the compound is imidazole-4-acetic acid and / or a pharmaceutically acceptable salt, a solvate, or a precursor thereof, and the imidazole-4-acetic acid has a structure as shown in formula (I):

[0025]

[0026] In some preferred embodiments of the present disclosure, the medicine or health product is used for one or more purposes selected from the following: reducing body fat percentage, increasing muscle proportion, improving glucose tolerance, improving insulin sensitivity, inhibiting or reducing the weight of adipose tissue, reducing white fat or inhibiting the weight of white adipose tissue, reducing the size of white fat cells or reducing the area of ​​white fat cells, maintaining the browning of brown fat, lowering liver triglyceride levels, preventing and treating obesity, and / or for regulating obesity, for obese, overweight or obesity-prone subjects.

[0027] In some preferred embodiments of the present disclosure, the medicine or health product is used to reduce body fat percentage, increase muscle proportion, improve glucose tolerance, and improve insulin sensitivity.

[0028] In some preferred embodiments of the present disclosure, the medicine or health product is used for one or more purposes selected from the following: inhibiting or reducing the weight of adipose tissue, reducing white fat or inhibiting the weight of white adipose tissue, reducing the size of white fat cells or reducing the area of ​​white fat cells, maintaining the browning of brown fat, and reducing liver triglyceride levels.

[0029] In some preferred embodiments of the present disclosure, the medicine or health product is used for one or more purposes selected from the following: inhibiting or reducing the weight of adipose tissue, reducing white fat or inhibiting the weight of white adipose tissue, reducing the size of white fat cells or reducing the area of ​​white fat cells, and / or maintaining the browning of brown fat.

[0030] In some preferred embodiments of the present disclosure, the medicine or health product is used to reduce liver triglyceride levels.

[0031] In some preferred embodiments of the present disclosure, the medicine or health product is used for preventing and treating obesity, and / or for regulating obesity.

[0032] In some preferred embodiments of the present disclosure, the medicine or health product is used to improve glucose tolerance.

[0033] In some preferred embodiments of the present disclosure, the medicine or health product is used to improve insulin sensitivity.

[0034] In some preferred embodiments of the present disclosure, the medicine or health product is used to reduce body fat percentage and increase muscle proportion.

[0035] In some preferred embodiments of the present disclosure, the medicament or health product is for use in obese, overweight or obesity prone subjects.

[0036] In some preferred embodiments of the present disclosure, the medicine or health product is used to inhibit fatty liver.

[0037] In some preferred embodiments of the present disclosure, the medicine or health product is used to alleviate the pathological changes of fatty liver.

[0038] In some preferred embodiments of the present disclosure, the medicine or health product is used to inhibit or reduce the weight of adipose tissue.

[0039] In some preferred embodiments of the present disclosure, the medicine or health product is used to reduce white fat or inhibit the weight of white adipose tissue.

[0040] In some preferred embodiments of the present disclosure, the medicine or health product reduces the size of white fat cells or reduces the area of ​​white fat cells.

[0041] In some preferred embodiments of the present disclosure, the medicine or health product is used to maintain the browning of brown fat.

[0042] In some preferred embodiments of the present disclosure, the medicine or health product has at least one effect selected from the following:

[0043] Does not affect the metabolic phenotype of a normal healthy body;

[0044] Does not affect exercise;

[0045] Does not cause nausea;

[0046] It does not affect preferences;

[0047] Does not affect learning and cognition; and / or

[0048] It will not cause anxiety or depression.

[0049] In some preferred embodiments of the present disclosure, the medicine or health product has an effect of not affecting the metabolic phenotype of a normal healthy body.

[0050] In some preferred embodiments of the present disclosure, the medicine or health product has the effect of not affecting exercise.

[0051] In some preferred embodiments of the present disclosure, the medicine or health product has the effect of not affecting the total activity.

[0052] In some preferred embodiments of the present disclosure, the medicine or health product has the effect of not causing nausea.

[0053] In some preferred embodiments of the present disclosure, the medicine or health product has an effect that does not affect preference.

[0054] In some preferred embodiments of the present disclosure, the medicine or health product has the effect of not affecting learning and cognition.

[0055] In some preferred embodiments of the present disclosure, the medicine or health product has the effect of not causing anxiety or depression.

[0056] In some preferred embodiments of the present disclosure, the medicine or health product has the effect of not causing nausea and not affecting preference.

[0057] In some preferred embodiments of the present disclosure, the medicine or health product has the effect of not affecting learning and cognition and not causing anxiety and depression.

[0058] In some preferred embodiments of the present disclosure, the medicine or health product has the effects of not causing nausea, not affecting preferences, not affecting learning and cognition, and not causing anxiety and depression.

[0059] In some preferred embodiments of the present disclosure, the medicine or health product forms an appetite suppressant preparation; the preparation contains the imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate, or precursor as the sole active ingredient.

[0060] In the present disclosure, the dosage form of the preparation is not limited, and can be tablets, granules, capsules, pills, oral liquids, injections, etc.

[0061] In some preferred embodiments of the present disclosure, the medicine or health product forms an appetite suppressant preparation; the preparation comprises the imidazole-4-acetic acid and / or a pharmaceutically acceptable salt, a solvate, or a precursor thereof, and further comprises a pharmaceutically acceptable carrier. The type of the pharmaceutically acceptable carrier is not limited.

[0062] In some preferred embodiments of the present disclosure, the pharmaceutically acceptable carrier includes one or more of a solvent, a solubilizer, a cosolvent, an emulsifier, a flavoring agent, an olfactory agent, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a pH regulator, a stabilizer, a surfactant, and a preservative. Fillers are also called diluents, such as wheat starch, tapioca starch, corn starch, potato starch, dextrin, microcrystalline cellulose, lactose, etc. Examples of flavoring agents include, but are not limited to, stevioside, glycyrrhizin, mogroside, acesulfame potassium, aspartame, sucralose, isomaltulose, etc. Examples of lubricants include, but are not limited to, magnesium stearate, talc, micropowdered silica gel, magnesium lauryl sulfate, etc.

[0063] In some preferred embodiments of the present disclosure, the medicine or health product forms an appetite suppressant preparation; the preparation comprises imidazole-4-acetic acid and / or a pharmaceutically acceptable salt, a solvate, a precursor thereof, and one or more other active ingredients that suppress appetite.

[0064] In some preferred embodiments of the present disclosure, the medicine or health product forms an appetite suppressing preparation; the amount of imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate, or precursor thereof in a unit preparation for suppressing appetite is 5 to 100 mg, preferably 10 to 60 mg.

[0065] In some preferred embodiments of the present disclosure, the amount of imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, or precursors thereof in a unit preparation for resisting diet-induced obesity is 20 to 300 mg, preferably 30 to 200 mg.

[0066] In some preferred embodiments of the present disclosure, the amount of imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, or precursors thereof in a unit preparation for weight loss is 50 to 1000 mg, preferably 100 to 500 mg.

[0067] In some preferred embodiments of the present disclosure, the drug forms an appetite suppressant preparation; the dosage of imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, or precursors thereof in a unit preparation for suppressing appetite in mice is 75 mg / kg for a single intraperitoneal injection or 120 mg / kg for a single oral gavage.

[0068] In some preferred embodiments of the present disclosure, the drug forms an appetite suppressant preparation; the dosage of imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, and precursors thereof in the unit preparation for suppressing appetite in mice is a single intraperitoneal injection of 75 mg / kg.

[0069] In some preferred embodiments of the present disclosure, the drug forms an appetite suppressant preparation; the dosage of imidazole-4-acetic acid and / or its pharmaceutically acceptable salts, solvates, or precursors thereof in the unit preparation for suppressing appetite in mice is 120 mg / kg by single oral gavage.

[0070] In some preferred embodiments of the present disclosure, the drug forms an appetite suppressant preparation; the dosage of imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate, or precursor thereof in a unit preparation for mice to resist diet-induced obesity is to prepare the drug into a 3 mg / ml aqueous solution, and allow the mice to drink water freely. Combined with the daily water intake of the mice, the dosage of the mouse is about 3 to 25 mg, preferably 5 to 20 mg, and preferably 8 to 15 mg.

[0071] In some preferred embodiments of the present disclosure, the drug forms an appetite suppressant preparation; the dosage of imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate, or precursor thereof in a unit preparation for weight loss in obese mice is to prepare the drug into a 12 mg / ml aqueous solution, and allow the obese mice to drink water freely. Combined with the daily water intake of mice, the dosage for mice is about 10 to 80 mg, preferably 30 to 60 mg, and preferably 40 to 55 mg.

[0072] A unit preparation refers to a preparation of a unit for preparation and use, such as a tablet, a bag of granules, a capsule, a bottle of oral solution, etc. When the content of imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate, or precursor thereof in the unit preparation is within the above range, it is convenient to take and also convenient to exert the appetite suppressing effect.

[0073] In some preferred embodiments of the present disclosure, the compounds act by inhibiting the activity of hypothalamic AgRP neurons.

[0074] In some preferred embodiments of the present disclosure, the drug or health product is administered by one or more of oral, intravenous, intraperitoneal, intramuscular, rectal or subcutaneous administration.

[0075] In some preferred embodiments of the present disclosure, the drug is administered by one or more of oral, intravenous, and intraperitoneal administration.

[0076] In some preferred embodiments of the present disclosure, the preparation is a solid preparation, a semisolid preparation or a liquid preparation; preferably, the solid preparation is a tablet, a capsule, a granule or a pill; the semisolid preparation is a gel, a suppository or a paste; the liquid preparation is an emulsion, a mixture, a suspension or a solution.

[0077] In some preferred embodiments of the present disclosure, the medicine or health product is a tablet, capsule, injection, powder, pill, granule, syrup, chewable tablet and patch.

[0078] In some preferred embodiments of the present disclosure, the medicine or health product is a tablet, a capsule, an injection, a powder, a pill, a granule, a syrup and a chewable tablet.

[0079] The present disclosure has the following advantages:

[0080] (1) The present invention provides the use of imidazole-4-acetic acid in the preparation of a drug or health product for suppressing appetite, and a drug or health product composition containing the same. Since imidazole-4-acetic acid is a natural metabolite, the present invention can reduce toxic side effects and is relatively safe.

[0081] (2) The present disclosure provides the use of imidazole-4-acetic acid in the preparation of appetite suppressing drugs or health products, which can effectively alleviate metabolic symptoms such as weight gain, increased blood sugar, and insulin resistance caused by a high-fat diet by suppressing appetite, and is expected to provide a new strategy for appetite treatment of obesity and blood sugar reduction and weight loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the prior art description. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can also be obtained based on these drawings without creative work. The drawings herein are incorporated into the specification and constitute a part of the specification, showing embodiments that conform to the specification, and are used together with the specification to explain the principles of the specification.

[0083] Figure 1 Identification and functional validation of the metabolite 4-IAA are shown; values ​​are presented as mean ± standard error, and statistical significance was analyzed using Student's t-test. Figure 1 A is the daily food intake of mice trained to have a restricted diet; Figure 1 B is the food intake of mice trained with restricted diet at different time points; Figure 1 C shows the identification of 4-IAA using non-targeted metabolomics, and the structural formula of 4-IAA is shown below the figure; Figure 1 D is the plasma level of 4-IAA in mice at different time points after feeding; Figure 1 E (left) shows the food intake of mice at different time points after intraperitoneal injection of 4-IAA; Figure 1 E (middle) shows the food intake of mice at different time points after intragastric administration of 4-IAA; Figure 1E (right) shows the food intake of mice at different time points after 4-IAA was mixed into the drinking water of mice; Figure 1 F is the food intake of mice at different time points after intraperitoneal injection of 4-IAA or 1-IAA. The left side of the figure is the structural formula of 1-IAA. Figure 1 G is the metabolomics analysis of 4-IAA concentration in the blood of Chow (normal), HFD-fed, and ob / ob obese mice in the hungry and fed states; Figure 1 H is the change of 4-IAA in human blood before and after eating; Figure 1 I is the correlation between the BMI index of a person and the 4-IAA in his blood, where BMI refers to body mass index, also known as body mass index; R 2 The coefficient of determination is an important concept in statistics, used to measure the predictive ability of a statistical model. It is mainly used in regression analysis to explain the variation of the response variable and the proportion of the variation explained by the free variable. 2 The values ​​range from 0 to 1, where higher values ​​indicate that the model better explains the coefficient of variation; P values ​​were calculated by two-tailed unpaired t-test without adjustment for multiple comparisons. Figure 1 J is the change of 4-IAA in blood before and after food restriction weight loss. Values ​​represent mean ± standard error, *p≤0.05, **p≤0.01, ***p≤0.001.

[0084] Figure 2 It was shown that 4-IAA improved abnormal metabolic indicators in HFD obese mice; Figure 2 A is the food intake of HFD obese mice under 4-IAA, 1-IAA, and water conditions; Figure 2 B shows the weight gain of HFD obese mice under 4-IAA, 1-IAA, and water conditions; Figure 2 C shows the glucose tolerance of HFD obese mice under 4-IAA, 1-IAA, and water conditions; Figure 2 D is the insulin sensitivity of HFD obese mice under 4-IAA, 1-IAA, and water conditions; Figure 2 E is the weight of liver and adipose tissue of HFD obese mice under 4-IAA, 1-IAA, and water conditions, respectively, where iWAT refers to inguinal white adipose tissue, gWAT refers to gonadal white adipose tissue also known as visceral white adipose tissue, and BAT refers to brown fat; Figure 2 F shows the effects of 4-IAA, 1-IAA, and water on liver triglyceride content in HFD obese mice; Figure 2 G is a flow chart of the model of treating obese mice with 12 mg / ml 4-IAA in drinking water; Figure 2 H is the percentage change in body weight of mice after intervention; Figure 2I is the glucose tolerance of HFD obese mice under 4-IAA and water conditions; Figure 2 J is the insulin sensitivity of HFD obese mice under 4-IAA and water conditions; Figure 2 K is the liver weight of HFD obese mice under 4-IAA and water conditions, respectively; Figure 2 L is the liver triglyceride content of HFD obese mice under 4-IAA and water conditions; liver TAG refers to liver triglyceride content. Values ​​are expressed as mean ± standard error, and Student's t-test was used for statistical significance analysis; *p≤0.05, **p≤0.01, ***p≤0.001.

[0085] Figure 3 The metabolic cage analysis of the effects of 4-IAA on metabolic parameters in HFD obese mice is shown; Figure 3 A is the food intake (Cumulative food intake), O2 consumption, CO2 exhalation, respiratory quotient, heat production (Heat), and total activity (Total-Activity) of HFD obese mice; Figure 3 B is the fat ratio and muscle ratio of HFD obese mice under 4-IAA, 1-IAA, and water conditions, where the fat ratio refers to the percentage of fat / body weight, and the muscle ratio refers to the percentage of muscle / body weight; Figure 3 C shows the morphology of adipose tissue and liver in HFD obese mice under 4-IAA, 1-IAA, and water conditions, respectively, where iWAT refers to inguinal white adipose tissue, gWAT refers to gonadal white adipose tissue also known as visceral white adipose tissue, and BAT refers to brown fat; Figure 3 D is the effect of HE pathology in liver and adipose tissue of HFD obese mice; Figure 3 E is the quantitative analysis of white adipocyte area in HFD obese mice; Figure 3 F is the liver HE pathology of HFD obese mice treated with 12 mg / ml 4-IAA in drinking water. The values ​​are expressed as mean ± standard error, and the Student's t-test was used for statistical significance analysis, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

[0086] Figure 4 It was shown that 4-IAA improved abnormal metabolic indicators in ob / ob obese mice; Figure 4 A is the effect of 4-IAA on feeding in the ob / ob obese mouse model; Figure 4 B is the effect of 4-IAA on body weight gain in the ob / ob obese mouse model; Figure 4 C is the effect of 4-IAA on glucose tolerance in the ob / ob obese mouse model; Figure 4 D is the effect of 4-IAA on insulin sensitivity in the ob / ob obese mouse model; Figure 4 E is the effect of 4-IAA on the liver and adipose tissue weights of the ob / ob obese mouse model, where iWAT refers to inguinal white adipose tissue, gWAT refers to gonadal white adipose tissue also known as visceral white adipose tissue, and BAT refers to brown fat; Figure 4 F is the effect of 4-IAA on HE pathology in liver and adipose tissue of ob / ob obese mouse model; Figure 4 G is the area of ​​white fat cells; Figure 4 H is the effect of 4-IAA on liver triglyceride content in ob / ob obese mouse model. Values ​​are expressed as mean ± standard error, and statistical significance was analyzed using Student's t-test, *p≤0.05, **p≤0.01, ***p≤0.001.

[0087] Figure 5 It was shown that 4-IAA slightly improved the metabolic phenotype of mice in normal physiological state; Figure 5 A is the effect of 4-IAA on the feeding of normal healthy mice; Figure 5 B is the effect of 4-IAA on the weight gain of normal healthy mice; Figure 5 C is the effect of 4-IAA on glucose tolerance in normal healthy mice; Figure 5 D is the effect of 4-IAA on insulin sensitivity of normal healthy mice; Figure 5 E is the effect of 4-IAA on the body fat percentage of normal healthy mice; Figure 5 F is the effect of 4-IAA on the weight of adipose tissue and liver of normal healthy mice; Figure 5 G is the effect of 4-IAA on the liver and adipose tissue morphology of normal healthy mice; Figure 5 H is the effect of 4-IAA on HE pathology in normal healthy mice; Figure 5 I is the effect of 4-IAA on the white adipocyte area of ​​normal healthy mice; Figure 5 J is the effect of 4-IAA on the triglyceride content in the liver of normal healthy mice. The values ​​are expressed as mean ± standard error, and the statistical significance was analyzed by Student's t-test; *p≤0.05, **p≤0.01, ***p≤0.001.

[0088] Figure 6 The effects of 4-IAA on mouse behavior are shown; Figure 6 A is the metabolic cage analysis of the oxygen intake (VO2), exhaled carbon dioxide (VCO2), respiratory quotient (RER), heat production (Heat), and total exercise volume of mice after administration of 4-IAA; Figure 6 B shows the effect of 4-IAA on mice fed with normal diet (left) and kaolin (right). Figure 6 C is a schematic diagram of the preference experiment design (left), and the effect of 4-IAA on the preference of mice under normal diet conditions (right); Figure 6 D is the water maze test of the effect of 4-IAA on the learning ability of mice under normal diet conditions; Figure 6 E is the anxiety-depression forced swimming test of 4-IAA on mice fed a normal diet, where immobility means no movement at all; immobility low means low-frequency movement; Figure 6 F is the strong tail suspension test of anxiety and depression induced by 4-IAA on mice fed with normal diet; Figure 6 G is the open field test of 4-IAA on the anxiety level of mice under normal diet conditions; 6H is the movement trajectory of mice, and there is no difference between the groups. ***P<0.001. Values ​​are expressed as mean ± standard error, and Student's t-test was used for statistical significance analysis; *p≤0.05, **p≤0.01, ***p≤0.001.

[0089] Figure 7 The metabolic pathway of 4-IAA and the main metabolic enzyme AOC1 are shown; Figure 7 A is the metabolic pathway diagram for the synthesis of 4-IAA from histidine; Figure 7 B. Me-IAA was administered to test mice for food intake; Figure 7 C is the transcriptional expression profile of HDC, AOC1 and HNMT; Figure 7 D is the expression diagram of AOC1 enriched in the small intestine analyzed using the Mouse Cell Atlas [MCA] database, where Vil1 is a marker gene for the small intestine, representing the small intestine; Figure 7 E is the transcriptional expression of AOC1 in the small intestine under the TRF model, where calnexin is an internal reference protein for control correction; Figure 7 F is the protein expression in the small intestine under the TRF model; Figure 7 G is the plasma 4-IAA content after administration of histamine and histidine to mice; Figure 7 H means that giving mice histidine does not affect their food intake; Figure 7 I is the design diagram of AOC1 knockout mice; Figure 7 J is the DNA verification of AOC1 knockout; Figure 7 K is the verification of AOC1 transcription knockout efficiency in the small intestine of knockout mice; Figure 7 L is the verification of the knockout efficiency of AOC1 protein in the duodenum of knockout mice; Figure 7 M is the plasma 4-IAA content of AOC1+ / + and AOC1- / - mice before and after feeding; Figure 7N is the plasma 4-IAA content of AOC1+ / + and AOC1- / - mice after oral administration of histamine. The values ​​are expressed as mean ± standard error, and the Student's t-test was used for statistical significance analysis; *p≤0.05, **p≤0.01, ***p≤0.001.

[0090] Figure 8 It was shown that 4-IAA inhibits the activity of AgRP neurons in the ARC region of the hypothalamus. Figure 8 A is the brain area that changes after 4-IAA administration found by c-Fos staining of whole brain clearing. The upper part is the FOS staining process of whole brain clearing. The left side below is the brain area loop for appetite regulation. The right side is the screening of the significant decrease of c-Fos in the ARC area after 4-IAA injection. Among them, BNST represents the bed nucleus of the stria terminalis, PVT represents the paraventricular nucleus of the thalamus, PVH represents the paraventricular nucleus of the hypothalamus, ARC represents the arcuate nucleus of the hypothalamus, and PBN represents the parabrachial nucleus. 8B is the staining result of c-Fos in AgRP neurons after 4-IAA and I-IAA were given to mice. Among them, DAPI represents the nuclear dye, which is used to dye the cell nucleus; merged represents merging to indicate the co-localization; Enlarged represents amplification. Figure 8 C is the statistical result of c-Fos in AgRP neurons after administration of 4-IAA and I-IAA to mice. Figure 8 D is the experimental process of injecting DIO-GCaMP-AAV into the ARC region of the hypothalamus in AgRP-Cre mice, where adenovirus: AAV-DIO-GCaMP. Figure 8 E: In vivo calcium imaging recorded by optical fiber in mice injected with 4-IAA. Figure 8 F shows that the ARC of CAG-tdTomato / AgRP mice was cut and then treated with 50 μM 4-IAA. The action potentials of AgRP neurons in the brain slices recorded by patch clamp were significantly inhibited by 4-IAA. Among them, ACSF refers to artificial cerebrospinal fluid and represents the blank control group. Figure 8 G is the statistical result of action potentials emitted by AgRP neurons. Figure 8 H is a schematic diagram of using CNO to activate AgRP neurons in hM3Dq / AgRP-Cre mice. Figure 8 I: Injection of 4-IAA after activation of AgRP can significantly inhibit the feeding of mice. Values ​​are expressed as mean ± standard error, and statistical significance was analyzed using Student's t-test; *p≤0.05, **p≤0.01, ***p≤0.001.

[0091] Fig. 9The whole brain clearing was performed to find the target brain regions of 4-IAA and to verify the results. The values ​​are expressed as mean ± standard error, and the Student's t-test was used for statistical significance analysis. Fig. 9 A represents the food intake of mice after injection of 4-IAA (12 μg) or 1-IAA (12 μg) into the third ventricle of mice; BNST represents the bed nucleus of the stria terminalis, PVT represents the paraventricular nucleus of the thalamus, PVH represents the paraventricular nucleus of the hypothalamus, ARC represents the arcuate nucleus of the hypothalamus, and PBN represents the parabrachial nucleus. Fig. 9 B is the verification of the changes in non-ARC brain areas caused by 4-IAA after whole-brain clearing c-Fos staining; DAPI represents the nuclear dye used to stain the cell nucleus; merged represents merging to indicate co-localization; Enlarged represents magnification. Fig. 9 C is the effect of 4-IAA on the activity of POMC neurons in the ARC region; DAPI represents a nuclear dye used to stain the cell nucleus; merged represents merging to indicate co-localization; Enlarged represents amplification. Fig. 9 D is the result of c-Fos staining in AgRP neurons in the ARC brain region after 4-IAA treatment. Fig. 9 E is the statistical result of verifying the c-Fos expression of AgRP neurons in the ARC brain region after treatment with 4-IAA.

[0092] Fig.10 Receptor screening for the action of the metabolite 4-IAA is shown. Fig.10 A shows the measurement of cAMP in isolated wild-type mouse hypothalamus treated with Forskolin (10 μM) for 15 minutes in the presence or absence of 1 μM 4-IAA; wherein, Hypothalamus represents hypothalamus, Forskolin represents forskolin; Forskolin is an inducer of intracellular cAMP formation and is used here to induce cAMP formation; Homogenate represents homogenate. Fig.10 B shows the effect of 1 μM 4-IAA stimulation on Forskolin-induced mouse hypothalamic cAMP levels; the 15 GPCRs with the highest expression levels in the mouse ARC transcriptome database (GSE96627) were selected for verification; Fig.10 B is the flowchart of candidate screening of 4-IAA receptors and GloSensor cAMP analysis. Fig.10 C is a detailed concentration curve of 4-IAA inhibiting cAMP generation through HRH3; EC50 is the half-effective concentration, which indicates the binding concentration of HRH3 and 4-IAA, representing the mutual binding ability. Fig.10D: HEK293T cells were transfected with HRH3, Gβγ and Gαi1, Gαi2, Gαi3 or Gαo. The cells were treated with different concentrations of 4-IAA and then subjected to BRET analysis. Each value represents the mean ± sem of 4 samples. Fig.10 E is a schematic diagram of the structural determination of the 4-IAA-HRH3-mG complex; wherein, 4-IAA-HRH3-mG. 4-IAA-HRH3 coupled mG. pathway complex. (4-IAA binds to the HRH3 receptor and activates the downstream mG. signaling pathway). Fig.10 F is a schematic diagram of the two-dimensional structure, wherein 4-IAA-HRH3-mG. 4-IAA-HRH3 coupled mG. pathway complex. (4-IAA binds to the HRH3 receptor and activates the downstream mG. signaling pathway). Fig.10 G is the cryo-electron microscopy density map and ribbon diagram of the 4-IAA-HRH3-mGo complex. The 3D structure shows in detail the interaction between 4-IAA and HRH3 in the ligand binding pocket; W402 is the tryptophan at position 402, F398 is the phenylalanine at position 398, L401 is the leucine at position 401, Y374 is the tyrosine at position 374, Y115 is the tyrosine at position 115, C118 is the cysteine ​​at position 118, and L401 is the leucine at position 401. The number in the upper right corner is the ballesteros–weinstein number (used to determine the TMH region of GPCRs proteins). Fig.10 H is a comparison of the binding sites of 4-IAA and histamine to HRH3. The top of the figure shows the residue positions with Ballesteros-Weinstein numbers related to 5-HTRs. Purple circles indicate HRH3 residues that interact with both 4-IAA and histamine, and orange circles indicate residues that interact with 4-IAA or histamine through hydrogen bonds. Among them, TM (transmembrane domain) refers to the membrane expansion region. Fig.10 I is the BRET assay of the binding activity of different mutants to 4-IAA; each value represents the mean ± SEM of 4 samples; BRET (bioluminescence resonance energy transfer) refers to bioluminescence resonance energy transfer; NR means no response to the ligand; aND means active but not determined due to low signal. Fig.10 J is the cAMP level of different mutants binding activity with 4-IAA; each value represents the mean ± SEM of 4 samples; RLU (relative light unit) refers to relative light unit; NR means no response to ligand; aND means active but not determined due to low signal.

[0093] Fig.11The structural screening of the interaction between 4-IAA and HRH3 is shown; Fig.11 A and 11B are the expressions of Hrh1-4 in AgRP neurons (GSE68177) or ARC regions (GSE96627) of hungry and fed mice; TPM refers to Transcript per Kilobase per Million mapped reads; RPKM refers to Reads per kilobase per million mapped reads; in RNA-Seq analysis, TPM and RPKM are used as quantitative expression methods for transcriptome data. Fig.11 C describes the binding of Gαo of HRH1, HRH2, and HRH3 to 4-IAA. Dissociation is the dissociation curve. This figure shows the BRET analysis of HEK293T cells expressing G protein probes and HRH1, HRH2, or HRH3 after treatment with different concentrations of 4-IAA to evaluate the dissociation of Gαo-Gβγ. The values ​​represent the mean ± sem of 3 independent experiments. Fig.11 D shows the separation and purification of 4-IAA-HRH3-mGo complex. Fig.11 E is the high-resolution analysis of the 4-IAA-HRH3-mGo complex. Fig.11 F is a single transmembrane domain of the 4-IAA-HRH3-mGo complex; wherein α5 refers to the α5 helix of Go in the G protein. Fig.11 G represents the interaction between histamine and HRH3, which comes from the histamine-HRH3-Gi complex structure, namely the histamine-HRH3 complex (PDB: 8YUU).

[0094] Fig.12 It was shown that 4-IAA acts on HRH3 receptors on AgRP neurons in the ARC region to suppress appetite. Fig.12 A shows the ARC brain slices of AgRP-Cre; CAG-LSL-tdTomato mice, which were treated with 4-IAA and HRH3 inhibitors Ciproxifan (37.5 μM) and GSK189254 (20 μM), and the action potentials of AgRP neurons in the brain slices were recorded by patch clamp. GSK189254 and Ciproxifan are two HRH3 inhibitors. The spontaneous frequency of action potentials is indicated in the figure, which represents the activity of neurons. Fig.12 B is the statistical result of action potentials emitted by AgRP neurons. Fig.12C shows the food intake of mice after injection of HRH3 inhibitor Ciproxifan (3 μg) and 4-IAA (12 μg) into the third ventricle. Fig.12 D is the statistical result of c-Fos staining of AgRP neurons after unilateral injection of DIO-shHRH3-AAV into the hypothalamic ARC region of AgRP-Cre mice to knock down HRH3. Fig.12 E: The food intake of AgRP-Cre mice was measured after HRH3 was knocked down by bilateral injection of DIO-shHRH3-AAV into the ARC region of the hypothalamus. Fig.12 F shows the changes in food intake of mice after HRH3 knockdown and high-fat induction. Fig.12 G shows the changes in body weight gain in mice after HRH3 knockdown and high-fat induction. Fig.12 H shows the changes in glucose tolerance in mice after HRH3 knockdown and high-fat induction. Fig.12 I shows the changes in insulin sensitivity of mice after HRH3 knockdown and high-fat induction. Fig.12 J is the change in liver and adipose tissue weight of mice after HRH3 knockdown and high-fat induction. The values ​​are expressed as mean ± standard error, and the Student's t-test was used for statistical significance analysis.

[0095] Fig.13 It was shown that HRH3 receptor knockout increased appetite and induced obesity. Fig.13 A is the verification of the knockdown efficiency of HRH3 shRNA; after overexpressing Flag-hrh3 and hrh3 shRNA, the knockdown efficiency of shRNA was detected, and it was found that hrh3-shRNA3 had the best knockdown efficiency, and hrh3-shRNA3 was selected for subsequent experiments. Fig.13 B: The effect of 4-IAA on the activity of AgRP neurons in the ARC region of the hypothalamus after bilateral injection of DIO-shHRH3-AAV into the ARC region of AgRP-Cre mice to knock down HRH3. Fig.13 C shows the changes in body fat percentage after HRH3 was knocked down by bilateral injection of DIO-shHRH3-AAV into the ARC region of the hypothalamus in AgRP-Cre mice. Fig.13 D shows the morphological changes of liver and adipose tissue after HRH3 was knocked down by bilateral injection of DIO-shHRH3-AAV into the ARC region of the hypothalamus in AgRP-Cre mice. iWAT refers to inguinal white adipose tissue, gWAT refers to gonadal white adipose tissue also known as visceral white adipose tissue, and BAT refers to brown fat. Fig.13 E: Pathological HE changes in liver and adipose tissue after HRH3 was knocked down by bilateral injection of DIO-shHRH3-AAV into the hypothalamic ARC region of AgRP-Cre mice. Fig.13F shows the changes in white adipocyte size after HRH3 was knocked down by bilateral injection of DIO-shHRH3-AAV into the ARC region of the hypothalamus in AgRP-Cre mice. Fig.13 G is the measurement of liver TAG (liver triglyceride) content after HRH3 was knocked down by bilateral injection of DIO-shHRH3-AAV into the hypothalamic ARC region of AgRP-Cre mice. The values ​​are expressed as mean ± standard error, and the Student's t-test was used for statistical significance analysis.

[0096] Fig.14 A schematic diagram of the synthesis and action of 4-IAA is shown; wherein HRH3-mG complex refers to HRH3-mG HRH3 coupled mG pathway complex (mG signaling pathway downstream of HRH3 receptor). DETAILED DESCRIPTION

[0097] Definition and Description

[0098] In order to make it easier to understand the present disclosure, some technical and scientific terms are specifically defined below. In the present disclosure, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell and tissue culture, microbiology-related terms and laboratory operation steps used herein are terms and routine steps widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definition and explanation of the relevant terms are provided below. It should be understood that the present disclosure is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course the above can be changed. It should also be understood that the terms used in the present application are only for describing specific embodiments and are not intended to be limited.

[0099] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0100] As used herein, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, device, product or apparatus comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or apparatuses.

[0101] In the description of this document, reference is made to “some embodiments”, “some implementation schemes” or “some implementation plans”, which describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0102] In the present disclosure, imidazole-4-acetic acid (4-IAA) is an important imidazole derivative. The molecular formula of imidazole-4-acetic acid is C5H6N2O2, and the molecular weight is 126.1. Imidazole-4-acetic acid is a known compound and can be prepared by chemical synthesis, for example, α-hydroxy-β-imidazolyl-4-propionic acid oxidation method and 4-nitrilomethylimidazole hydrolysis method. Imidazole-4-acetic acid can also be obtained by biosynthesis, for example, it can be prepared according to the method disclosed in patent number CN107177642B.

[0103] In the present disclosure, pharmaceutically acceptable salts of imidazole-4-acetic acid refer to those salts that are non-toxic in dosage administration. Because the imidazole-4-acetic acid in the present disclosure contains a carboxyl group, base addition salts may be present. Pharmaceutically acceptable base addition salts include metal salts and organic salts. More preferred metal salts include (but are not limited to) suitable alkali metal (Ia group) salts, alkaline earth metal (IIa group) salts and other physiologically acceptable salts. Such salts can be prepared from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc. Preferred organic salts can be prepared from tertiary amines and quaternary ammonium salts including (partial) tromethamine, diethylamine, N, N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.

[0104] In the present disclosure, examples of the solvate of imidazole-4-acetic acid include, for example, hydrates, alcoholates, and the like.

[0105] In the present disclosure, the precursor of imidazole-4-acetic acid refers to a compound that is inactive or less active in vitro and releases imidazole-4-acetic acid through enzymatic or non-enzymatic conversion in vivo to exert its pharmaceutical effect.

[0106] Disorders in energy intake and expenditure lead to obesity, which is the main cause of many chronic diseases such as diabetes, fatty liver, and cancer. The central nervous system regulates systemic metabolic activities such as food acquisition, energy intake, and energy expenditure, and therefore plays an important regulatory role in the occurrence and development of obesity.

[0107] The brain-gut axis plays an important role in the regulation of energy metabolism. The Gut-Brain Axis refers to the interaction between the intestine and the brain, involving complex interactions between the intestine, nervous system, and immune system, and plays an important role in human physical and mental health. The components of the gut-brain axis include: ① Gut flora: microorganisms present in the intestine, including bacteria, fungi, and viruses, which affect human psychological and emotional states through interactions with the immune and nervous systems. ② Immune system: The intestine is an important part of the immune system and works closely with the intestinal flora to maintain intestinal immune balance. The immune system's inflammatory response and immunomodulatory substances can affect brain function and emotional state. ③ Nervous system: There is a wealth of neurotransmission and chemical signals between the intestine and the brain. The intestine communicates with the brain through neurotransmitters and neuropeptides, affecting functions such as sleep, emotion, cognition, and behavior.

[0108] Nutritional perception and energy homeostasis signals from peripheral tissues are projected to the CNS through the nucleus of the solitary tract (NTS) in the brainstem via hormones (such as leptin, insulin, etc.), cytokines (such as inflammatory factors), and nerve impulses (such as mechanical tension), and are further processed, integrated, and released in the hypothalamic neuronal circuits. Among them, the main neurons that regulate food acquisition and intake are located in the arcuate nucleus (ARC) of the hypothalamus. There are two types of neurons in the ARC that are closely related to appetite: orexigenic neurons that express neuropeptide Y (NPY) and agouti-related protein (AgRP); and anorexigenic neurons that express proopiomelanocortin (POMC) and cocaine and amphetamine-regulated transcriptpeptide (CART).

[0109] Secretory factors derived from gastrointestinal tissues, such as gherlin and glucagon-like peptide-1 (GLP-1), can be projected to the paraventricular nucleus (PVN) of the hypothalamus through the vagus nerve or directly through the blood-brain barrier via AgRP / NPY and POMC / CART neurons in the ARC, respectively, to regulate food intake and energy metabolism. Spinal afferents and vagal afferents in gastrointestinal tissues can directly sense changes in signaling molecules such as neurotransmitters and cytokines and mechanical tension in the tissues and further regulate the body's energy metabolism through the central nervous system. Intestinal microorganisms can also regulate appetite through the brain-gut axis. For example, a variety of metabolites produced by intestinal microorganisms regulate food intake: the short-chain fatty acids (SCFAs) produced regulate appetite by promoting the secretion of GLP-1 and gastrointestinal hormone peptide (PYY); the production of neurotransmitters such as γ-aminobutyric acid (GABA) directly regulates appetite; the production of tryptophan as a synthetic precursor of the neurotransmitter 5-hydroxytryptamine (5-HT) regulates appetite.

[0110] The ARC blood-brain barrier is relatively weak, so neurons in the ARC can not only perceive peripheral metabolic information through neural pathways, but also perceive peripheral metabolic signals through humoral pathways, and regulate the body's energy intake through specific neural projection pathways. For example, recent studies have shown that metabolites can also directly regulate appetite through the ARC blood-brain barrier. For example, bile acid reduces the secretion of AgRP / NPY through the TGR5 receptor on the surface of AgRP / NPY neurons, thereby suppressing appetite.

[0111] AgRP (Agouti-related protein) is a neuropeptide produced in the brain by AgRP / NPY neurons. AgRP is synthesized only in the cell bodies containing neuropeptide Y (NPY) located in the ventral part of the arcuate nucleus of the hypothalamus. AgRP is co-expressed with NPY and acts to increase appetite, reduce metabolism and reduce energy expenditure. AgRP is one of the most potent and long-lasting appetite stimulants. In humans, AgRP is encoded by the AgRP gene. The relevant sequence of human AgRP can be found in the Gene Sequence Information section.

[0112] The principle of c-fos staining is mainly based on antigen-antibody reaction, through the specific antibody binding to c-fos protein, and then dyeing the conjugate with a dye, so as to achieve the localization and quantification of c-fos protein. Commonly used staining methods include immunohistochemical staining, Western Blot, etc.

[0113] BMI (Body Mass Index) refers to the body mass index, also known as the body mass index, which is a commonly used standard internationally to measure the degree of fatness and health of the human body. The calculation formula is: BMI = weight ÷ height squared; the unit of weight is kilograms; the unit of height is meters.

[0114] Histamine, as a biogenic amine, plays an important role in a variety of pathophysiological conditions. In peripheral tissues, histamine is mainly stored in mast cells and basophils. In allergic diseases, histamine is released from these cells and causes several common symptoms in skin and respiratory allergic diseases. Histamine exerts its effects through at least four different receptor subtypes (histamine H1 receptor, histamine H2 receptor, histamine H3 receptor, histamine H4 receptor). Molecular biological methods have shown that all histamine receptors belong to the large family of G protein-coupled receptors.

[0115] Histamine H3 receptor, or HRH3, also known as histamine receptor H3, is a member of the G protein-coupled receptor (GPCR) family. It is mainly expressed on histamine neurons in the central nervous system and also plays a role in peripheral tissues such as immune cells. The mechanism of action of HRH3 mainly involves regulating the synthesis and release of neurotransmitters. It inhibits the synthesis and secretion of histamine as an autoreceptor in the presynaptic region and regulates the activity of other neurotransmitters as a heteroreceptor in the postsynaptic region. The relevant sequence of human histamine H3 receptor can be found in the gene sequence information section.

[0116] White adipose tissue is a type of adipose tissue in humans or animals. Its main function is to store excess fat in the body.

[0117] Brown adipose tissue refers to brown adipose tissue in humans or animals.

[0118] Body fat percentage refers to the proportion of body fat weight in the total body weight, also known as body fat percentage, which reflects the amount of fat in the human body.

[0119] Muscle ratio, also known as muscle rate, is the percentage of muscle tissue in the body's total body weight. Generally speaking, the normal range of muscle ratio varies from person to person.

[0120] Glucose tolerance refers to the body's ability to absorb and utilize ingested glucose. When the body's tolerance to glucose decreases, insulin secretion is insufficient or cells become less sensitive to insulin, leading to increased blood sugar levels. To assess glucose tolerance, doctors usually recommend an oral glucose tolerance test (OGTT). In addition, fasting blood sugar testing is also one of the routine screening methods.

[0121] D114 is aspartic acid at position 114.

[0122] Y115 is the tyrosine at position 115.

[0123] F398 is phenylalanine at position 398.

[0124] L401 is the leucine at position 401.

[0125] W402 is the tryptophan at position 402.

[0126] C118 is cysteine ​​at position 118.

[0127] Y374 is the tyrosine at position 374.

[0128] The Ballesteros–Weinstein number is a universal numbering scheme used to identify the transmembrane helix (TMH) region and binding site (BS) of G protein-coupled receptors (GPCRs) proteins; the scheme was developed by Ballesteros and Weinstein to correspond the position of each amino acid in the GPCR sequence to a specific generalized number, thereby deriving the relative position of each amino acid and the specific amino acid information at that position.

[0129] Detailed description of the specific implementation method

[0130] Example

[0131] In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all the embodiments. The following is only a further description of the present disclosure, and the protection scope of the present disclosure is not limited thereto.

[0132] The experimental conditions and methods involved in the following Examples 1-9 are as follows:

[0133] (1) Population sample

[0134] To study the changes in plasma 4-IAA before and after meals, we recruited 17 healthy male student volunteers at Fudan University. The volunteers did not eat within 14 hours (8 pm to 10 am) and did not drink water within 12 hours (10 pm to 10 am) before the experiment. At 10 am the next day, the volunteers began to eat. Blood samples were collected before and 2 hours after the meal. The study was approved by the Human Research Ethics Committee of Fudan University (B2022-553R).

[0135] To investigate the relationship between plasma 4-IAA levels and body mass index (BMI), we collected plasma samples from 42 healthy volunteers. The informed consent form for blood collection from healthy volunteers was reviewed and approved by the Ethics Committee of Shanghai Xuhui Central Hospital (2011-37).

[0136] Zhongshan Hospital Affiliated to Fudan University conducted a randomized clinical trial (NCT04283942) to compare the efficacy and safety of intermittent caloric restriction (ICR) with continuous caloric restriction (CCR). All 10 participants were required to consume a prescribed daily caloric intake (25 kcal / kg x [height (cm) -100] kg) and eat regular food without time restrictions. During the 12-week intervention, the average daily calorie consumption in the CCR group decreased by 673.7 kcal (95% CI, -884.9 to -462.5 kcal) compared with baseline, and the average weight loss was 7.6%. Serum samples were collected for measurement of 4-IAA after fasting for 10 hours before and after the 12-week dietary intervention. Written informed consent was obtained from all patients and approved by the Ethics Committee of Zhongshan Hospital Affiliated to Fudan University (B2019-256).

[0137] (2) Mice

[0138] All mice were housed in group cages at 22°C with a 12-h light / 12-h dark cycle. The dark cycle started at 7:00 PM. All animal studies were approved by the Animal Care and Research Advisory Committee of Fudan University and Xiamen University. Mice were euthanized by isoflurane at appropriate times during the study, and tissue samples were collected for further experiments.

[0139] Normal diet (Xietong Organism, Nanjing, China) and high-fat diet (HFD, Research Diet, D12492) contained 12% and 60% of calories from fat, respectively. C57BL / 6, db / db, Aoc1- / - (T027589), and CAG-LSL-tdTomato (T006163) mice were purchased from GemPharmatech Co., Ltd. AgRP-Cre (JAX: 012899) mice were purchased from Jackson laboratory. R26-LSL-hM3Dq-DREADD (JAX: 026220) mice were generously provided by Dr. Ying Chen, Xiamen University, China. AgRP-Cre and CAG-LSL-tdTomato mice were crossed to generate AgRP-Cre; CAG-LSL-tdTomato mice for specific expression of tdTomato in AgRP neurons. AgRP-Cre; R26-LSL-hM3Dq mice generated by hybridization of AgRP-Cre and R26-LSL-hM3Dq-DREADD mice can specifically express hM3Dq in AgRP neurons. All transgenic mice used in this study were on a C57BL / 6 background. Primer sequences are shown in Table 1.

[0140] Table 1. Primers used in the study (related to the STAR method)

[0141]

[0142] *, Primer sequences are available at http: / / pga.mgh.harvard.edu / primerbank.

[0143] (3) Compounds

[0144] 4-Imidazoleacetic acid hydrochloride (4-IAA, S64493), 2-(1-imidazolyl)acetic acid (1-IAA, S63507), 1-methyl-4-imidazoleacetic acid hydrochloride (1-Me-4-IAA, Y35050), histamine (S20188) and L-histidine hydrochloride (S20127) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Ciproxifan maleate (HY-15289), GSK189254 (HY-14111) and clozapine-N-oxide (CNO, clozapine N-oxide) (HY-17366) were purchased from MCE (MedChemExpress).

[0145] (4) Plasmid and adenovirus (Adeno-associated virus, AAV)

[0146] Mouse Hrh3 was cloned into the pcDNA3.1-Flag vector. AAV2 / 9-hsyn-FLEX-GCaMP6s-WPRE-pA (S0226-9-H20) was purchased from Taitool. We modified 2543C to 2543T to create a cleavage site for AgeI in PT-2788 (rAAV-CMV-DIO-(mCherry-U6)-shRNA-WPRE-hGH, BrainVTA), which we called PT-2788*. The shRNA for HRH3 was cloned into PT-2788*. AAV particles were prepared and isolated as described in the reference "Wang, X. et al. Receptor-Mediated ER Export of Lipoproteins Controls Lipid Homeostasis in Mice and Humans. Cell Metab 33, 350-366e357 (2021)" and stored at -80°C before use. Primer sequences are shown in Table 1.

[0147] (5) Diet training of mice

[0148] Single-housed mice were switched from ad libitum feeding to time-restricted feeding (TRF), in which mice could eat between 1 and 5 pm. After 6 days of training, mice completed food intake measurements within 4 h. Plasma samples were collected at 0, 0.5, 1, 2, and 4 h after feeding on days 0 and 6 of TRF and subjected to nontargeted metabolomics studies.

[0149] (6) Quantification of plasma 4-IAA

[0150] To extract and quantify 4-IAA, 10 μL of plasma was mixed with 40 μL of MeOH:ACN (v / v, 1 / 1), 62.5 nM L-phenyl-d5-alanine (BePure, MD-4580D5) was added as an internal standard, and vortexed for 30 seconds. The sample was sonicated in a water bath at 4°C for 10 minutes. After incubation at -20°C for 1 hour, the sample was centrifuged at 20,000 g for 15 minutes at 4°C. The supernatant was transferred to a new tube and blown dry using a nitrogen blower at 4°C. The sample was reconstituted with 20 μL of MeOH:ACN:H2O (v / v / v, 2 / 2 / 1) and sonicated in a water bath at 4°C for 10 minutes. After centrifugation at 20,000 g for 10 minutes, the supernatant was collected and analyzed by AB SCIEX QTRAP 6500+LC-MS / MS.

[0151] (7) Measurement of food intake in mice

[0152] Mice were housed individually for 3-5 days. Mice were fasted for 20 hours (from 5 pm to 1 pm the next day), treated with the compounds indicated in the figure legends, and then re-fed. Food intake was recorded at the indicated times.

[0153] (8) Mouse metabolic cage analysis

[0154] Metabolic cage analysis was performed in a system (Columbus, CLAMS-16M) at 22°C with a 12-hour light / 12-hour dark cycle. Mice were housed individually for one week and allowed to acclimate to the metabolic cages for 2 days before the experiment. Food intake, body weight, oxygen consumption, carbon dioxide production, and locomotor activity of the mice were monitored.

[0155] (9) c-Fos signal staining in the whole brain of mice

[0156] Briefly, mice were perfused with 4% paraformaldehyde (PFA), and the dissected brains were immersed in 4% PFA overnight at 4°C for further fixation. The brains were immersed in a 10% hot gelatin solution and then placed in ice-cold water for gelatin solidification. The embedded samples were returned to 4% PFA for further fixation for 24 hours. The brains were cut into 300 μm thick brain slices using a Leica VT1200S Vibratome slicer. All parts of each brain were collected sequentially. The slices were washed with 0.5% TritonX-100 in PBS solution at 37°C with gentle shaking for 6 hours. The slices were immunostained with anti-Fos (#2250, CST, 1:1500) and anti-rabbit secondary antibodies (#711-165-152, Jackson ImmunoResearch, 1:500). For ease of imaging, slices of each brain were mounted on custom slides (100 mm x 100 mm). The sample slides were transferred to refractive index matching solution (RIMS) overnight. The slides were imaged with the VISoR imaging system as described previously. Images were reconstructed using Imaris9.5.

[0157] (10) Histology and immunofluorescence analysis

[0158] For histological analysis, hematoxylin and eosin (H&E) staining was performed. Immunofluorescence was performed. Briefly, after the mice were perfused with 4% PFA and fixed for 5 hours, the brains were transferred to a 30% sucrose solution in PBS for 24 hours and then embedded in OCT embedding medium (Sakura, 4583) at -80°C. 16 μm coronal sections were made continuously with a freezing microtome. After drying at room temperature, the brain slices were stored at -80°C. For immunostaining, the brain slices were returned to room temperature (RT), placed in an oven at 55°C for 30 minutes, and then transferred to a boiling antigen retrieval solution (0.3782 g / L citric acid and 2.4116 g / L sodium citrate in MQ) at low temperature for 10 minutes. Afterwards, the brain slices were washed 3 times with PBS and permeabilized with a blocking solution (0.3% Triton X-100, 3% BSA in PBS) for 0.5 hours at room temperature. The sections were then incubated with anti-Fos (sc-271243, Santa Cruz, 1:500) and anti-AgRP (A00272, BOSTER, 1:1200) or anti-POMC (BM5411, BOSDER, 1:250) at room temperature for 24 hours. After washing with PBS, the sections were incubated with Alexa Fluor 488 goat anti-rabbit or Alexa Fluor 594 goat anti-mouse IgG secondary antibodies (CST, 1:50) at room temperature for 6 hours. The sections were then stained with DAPI and then mounted. The slides were imaged under a Leica LSM880 confocal microscope.

[0159] (11) Gene expression analysis

[0160] Total RNA was isolated and quantitative real-time PCR (qRT-PCR) was performed. Primers are listed in Table 1. All reactions were performed in triplicate. qRT-PCR was performed on ABIQuantStudio 7Flex using Hieff qPCR SYBR Green Master Mix (High ROX Plus) (YEASEN, 11201ES08). The relative amount of each mRNA was calculated by using the comparative threshold cycle (CT) method. Cyclophilin or 36B4 mRNA was used as a control.

[0161] (12) Oral glucose tolerance test and insulin sensitivity test

[0162] Mice were fasted for 16 h (from 5 pm to 9 am) and orally administered D-glucose at a dose of 2 mg / g body weight for mice fed with normal diet, 1 mg / g body weight for mice fed with HFD, and 0.1 mg / g body weight for db / db mice. Blood was collected from the tail vein at 0, 15, 30, 60, 90, and 120 min, and blood glucose was measured using a Bayer glucometer.

[0163] For the insulin sensitivity test, mice were fasted for 6 h (from 8 am to 14 pm), and insulin was injected IP at 0.5 U / kg body weight for mice fed with normal diet and 1 U / kg body weight for mice fed with HFD and db / db mice. Blood was collected from the tail vein at 0, 15, 30, 60, 90 and 120 min, and blood glucose was measured using a Bayer glucometer.

[0164] (13) CNO chemical genetic activation experiment

[0165] After fasting for 20 hours, AgRP-Cre; R26-LSL-hM3Dq mice were IP injected with CNO (0.5 mg / kg), given saline or 4-IAA (75 mg / kg), and given food. Food intake was recorded at 0, 0.5, 1, and 2 hours.

[0166] (14) 3D brain surgery

[0167] Mice were anesthetized with tribromoethanol (200 mg / kg). The skull was exposed and cleaned with a cotton swab soaked in 5% hydrogen peroxide. For third ventricular cannulation, a guide cannula was implanted in the ventral third ventricle at the midline coordinates of 1.8 mm posterior to bregma and 5.45 mm below the skull surface. Drug delivery was delivered using a microinjection pump (R462, RWD Instruments).

[0168] For AAV delivery, 300-350 nL AAV was injected stereotaxically (bregma: AP: -1.55 mm, ML: ±0.2 mm, DV: 5.95 mm) at a rate of 50 nL / min by a microinjector (R-480 Nanoliter Microinjection Pump, RWD Instruments). For fiber optic analysis experiments, a fiber optic cannula (400 μm, NA = 0.50) was implanted 0.1 mm above the virus injection site and fixed with dental cement. The experiment was performed at least 2 weeks after surgery.

[0169] (15) Fiber Optic Recording and Analysis of AgRP Neurons

[0170] AAV2 / 9-hsyn-FLEX-GCaMP6-WPRE-pA was delivered to the target brain region of AgRP-Cre mice by stereotaxic injection, followed by implantation of fiber optic cannulas. Fluorescence in AgRP neurons was recorded using an Inper multichannel fiber photometer device, with the excitation channels set to wavelengths of 410 nm and 470 nm. The excitation light power at the top of the fiber was adjusted to 20-30 mW, delivered at a frequency of 30 Hz and 10 ms pulses. To minimize the autofluorescence of the fiber, the recording fiber was pre-photobleached using a high-power light source.

[0171] Photometric data were analyzed using Inper data analysis software with the remove interval function (retention duration set to 3 s, no baseline correction) to eliminate redundancy in both channels. Background autofluorescence and baseline correction were processed using default settings (Polyflt, Poly Order = 2). F0 was defined as the average fluorescence within the first 30 min of saline or 4-IAA recording, and ΔF / F0 was calculated based on this. 11 .

[0172] (16) Brain slice preparation and whole-cell patch clamp recording

[0173] Mice were anesthetized with 2.5% tribromoethanol and decapitated. The brain was quickly dissected and cut into 250 μm thick coronal slices in 95% O2 / 5% CO2 saturated ice-cold N-methyl-D-glucosamine (NMDG, SigmaAldrich M2004) cutting solution using a Leica VT1200S Vibratome slicer (Leica, Germany). The solution contained 92mM NMDG, 1.2mM KCl, 30mM NaHCO3, 1.2mM KH2PO4, 25mM D-glucose, 20mM HEPES, 5mM L-ascorbic acid, 3mM sodium pyruvate, 2mM thiourea, 10mM MgSO4 and 0.5mM CaCl2 (310 ± 5mOsmol / L during pH 7.2 ± 0.1). Brain slices were incubated at 37°C in artificial cerebrospinal fluid (ACSF) saturated with 95% O2 / 5% CO2 for at least 45 minutes and allowed to recover for approximately 1 hour at room temperature before recording. ACSF contained 125 mM NaCl, 25 mM NaHCO3, 1.25 mM KH2PO4, 25 mM D-glucose, 0.4 mM L-ascorbic acid, 2 mM sodium pyruvate, 2 mM CaCl2, and 1 mM MgCl2. After recovery, slices were placed in a recording chamber and continuously perfused with ACSF at a rate of 1-2 mL / min.

[0174] Brain slices were visualized under an Olympus BX51WI microscope, infrared (IR) differential interference contrast (DIC), and a charge-coupled device (CCD) camera (IR-2000; DAGE-MTI). Electrodes were pulled from borosilicate glass capillaries (OD = 1.5 mm, ID = 0.86, Sutter Instrument) using a Model P-97 puller (Sutter Instrument) to a final tip resistance of 4-7 MΩ. For recording action potentials (current clamp), pipettes were filled with a solution containing 26 mM potassium gluconate, 10 mM HEPE, 4 mM KCl, 4 mM adenosine 5′-triphosphate magnesium salt (ATP-Mg), 0.3 mM guanosine 5′-triphosphate sodium salt hydrate (5′-GTP-Na), and 10 mM creatine phosphate. All recordings were performed using a MultiClamp 700B amplifier (Axon Instruments), and electrophysiological data were filtered at 2.9 kHz and sampled at 10 kHz using Clampex 11.1, MultiClamp700B software8.

[0175] (17) Conditioned flavor test

[0176] To test the effect of 4-IAA on flavor preference, a conditioned flavor test was designed. The protocol involved an 11-day protocol in which mice were introduced to a test arena with two water bottles for 90 minutes each day from 9 am to 10:30 am. From day 0, mice had free access to food but could only drink water in the test arena from 9 am to 10:30 am. From day 0 to day 6 (habituation day), mice were provided with 2 bottles of water during this period. On day 7 (conditioning day), mice were provided with a cherry-flavored solution (0.36 g / L Kool-Aid sweet cherry) containing 0.2% saccharin and an IP injection of saline at 10:30 am. On day 8 (recovery day), mice only drank water. On day 9, mice were divided equally into 3 groups and given a grape-flavored solution (0.36 g / L Kool-Aid sweet grape) and an IP injection of saline, LiCl (200 mg / kg), or 4-IAA (75 mg / kg). Mice were then given water on day 8. On day 11 (test day), cherry and grape flavored solutions were provided to 3 groups of mice on random sides between 9:00 and 10:30 AM, and the consumption of each water bottle was recorded.

[0177] (18) Kaolin ingestion test

[0178] To test the effect of 4-IAA on nausea, we performed a kaolin ingestion test. Five days before the experiment, mice were housed individually and allowed free access to kaolin pellets (Research Diets, K50001), food, and water. On the day of the experiment, mice were fasted for 20 h, IP injected with saline or 4-IAA (75 mg / kg), and re-fed with food and kaolin. The intake of food or kaolin was recorded.

[0179] (19) Behavioral test

[0180] After Morris water maze test training, mice received IP injection of 4-IAA (75 mg / kg) and were tested. CleverSys TopScanLite system was used for result analysis.

[0181] For the forced swimming test, mice received an IP injection of 4-IAA (75 mg / kg) or saline before the test. The duration of cumulative immobility was recorded in the last 4 minutes of the 6-minute test period. The immobility was defined as the mouse floating motionlessly in the water without struggling.

[0182] For the immobility time in the tail suspension test, mice were IP injected with 4-IAA (75 mg / kg) after 30 minutes of tail suspension test and then subjected to a 6-minute tail suspension test.

[0183] Open field test (OFT) to assess locomotor activity and anxiety-like behavior. In the test, mice were injected IP with 4-IAA (75 mg / kg).

[0184] (20) cAMP assay

[0185] To measure cAMP levels in the arcuate nucleus (ARC) of the hypothalamus, freshly isolated hypothalamus was treated with 10 μM forskolin for 15 minutes with or without 1 μM 4-IAA. Tissues were homogenized in cold 0.1 M HCl and centrifuged at 10,000 g for 10 minutes. The supernatant was neutralized with 1 M NaOH and cAMP levels were quantified using a commercial ELISA kit (R&D Systems, KGE012B).

[0186] (21) GloSensor cAMP analysis

[0187] To screen for 4-IAA receptors, the GloSensor cAMP assay was performed. On day 0, HEK293 cells were transiently co-transfected with the GloSensor cAMP probe, and each of the first 15 cells abundantly expressed the GPCR in the arcuate nucleus. On day 2, the transfected cells were plated at 5 × 10 per well. 4Cells were seeded into 96-well plates at a concentration of 10 cells / mL. On day 3, cells were incubated with serum-free DMEM containing 2% (v / v) GloSensor cAMP substrate stock solution (Progema) at 37°C for 2 h. Different concentrations of 4-IAA or 1-IAA were added, and the ligand-induced luminescence intensity changes were recorded using an EnVision Multilabel microplate reader (Perkin-Elmer).

[0188] (22) Bioluminescence resonance energy transfer (BRET)

[0189] To characterize G protein signaling, bioluminescence resonance energy transfer (BRET) analysis was performed. On day 0, HEK293 cells were transfected with GPCR and G protein probes (Gαi1-3 or Gαo fused to Nluc, Gβ, Gγ fused to GFP2). On day 2, cells were plated at 5x10 4 On day 3, cells were treated with different concentrations of 4-IAA in the presence of the luciferase substrate coelenterazine 400a (5 μM, Cayman), and the BRET signal between Nluc (440-480 nm) and GFP2 (510 nm) emission was measured using a Mithras LB 940 multimode microplate reader (Berthold Technologies).

[0190] (23) Purification of HRH3 and G protein complex

[0191] Spodopterafrugiperda (Sf9) cells were cultured in insect ESF921 cell culture medium (Sino Biological). To express HRH3, miniGαo1, Gβ1, Gγ2, and scFv16, high titer recombinant baculovirus was generated using the Bac-To-Bac baculovirus system (Invitrogen) according to the previously described protocol. 19 Sf9 cells were transfected with recombinant baculoviruses carrying HRH3, miniGαo1, Gβ1, Gγ2, and scFv16 using FuGENE HD transfection reagent (Promega) to produce high titers of recombinant viruses. Sf9 cells were infected with a mixture of these baculoviruses at a ratio of 1 / 1 / 1 / 1. After shaking at 27°C for 48 h, cells were collected by centrifugation at 1000 g for 20 min, and the pellets were frozen in liquid nitrogen and stored at -80°C.

[0192] To prepare 4-IAA-HRH3-mGo complexes, frozen cell pellets were thawed and resuspended in lysis buffer (100 μM 4-IAA, 20 mM HEPES pH 7.5, 100 mM NaCl, 3 mM MgCl2, 5 mM CaCl2, 2.5 mg / mL leupeptin, and 0.2 mg / mL benzamidine) at room temperature with end-to-end rotation at 50 rpm for 1.5 h. The supernatant containing 4-IAA-HRH3-mGo complex was incubated with M1 anti-FLAG affinity resin at 4°C for 2 h by adding 0.5% (w / v) dodecylmaltose neopentyl glycol (LMNG, Anatrace) and 0.1% (w / v). The mixture was then centrifuged at 100000 g for 20 min, and the supernatant containing 4-IAA-HRH3-mGo complex was incubated with M1 anti-FLAG affinity resin at 4°C for 2 h. Flag-M1 beads were washed three times with a buffer containing 100 μM 4-IAA, 0.01% (w / v) LMNG, 0.002% (w / v) CHS, 0.2 mg / mL benzamidine and 2.5 mg / mL leupeptide. The beads were washed three times with a buffer containing 100 μM 4-IAA, 20 mM HEPES pH 7.5, 100 mM NaCl, 0.01% (w / v) LMNG, 0.002% (w / v) CHS, 5 mM EGTA and 0.2 mg / mL 4-IAA-HRH3-mGo complexes were eluted from M1 beads using buffer containing FLAG peptide.

[0193] The eluted complex was concentrated using an Amicon ultracentrifugal filter with a molecular weight cutoff of 100 kDa and then injected onto a Superose 6 Increase 10 / 300 GL column pre-equilibrated with a buffer containing 100 μM 4-IAA, 20 mM HEPES pH 7.5, 100 mM NaCl, 0.00075% (w / v) LMNG and 0.00025% (w / v) CHS for complex separation. The peak fractions containing the 4-IAA-HRH3-mGo complex were pooled and concentrated to approximately 8 mg / mL for cryogrid preparation.

[0194] (24) Cryogrid preparation and EM data acquisition

[0195] To prepare cryo-EM grids, 3 μL of purified 4-IAA-HRH3-mGo complex was applied to glow-discharged holey carbon grids. After rapid freezing using a FEI Vitrobot Mark IV (Thermo Fisher Scientific), the samples were placed in a storage box and stored in liquid nitrogen. The sample grids were loaded into a Titan Krios electron microscope (300 kV) equipped with a spherical aberration (Cs) corrector for data collection. Images of the 4-IAA-HRH3-mGo complex were taken using a Falcon 4 camera with a nominal magnification of 120,000x and a pixel size of SerialEM software equipped with custom scripts was used to facilitate automated low-dose image acquisition. The defocus range of the image stack was set between -1.0 and -2.0 μm. The cumulative dose was set to electrons, and each micrograph contains 32 frames.

[0196] (25) Data processing and 3D reconstruction

[0197] All cryo-EM film stacks of 4-IAA-HRH3-mGo were motion corrected and dose weighted using MotionCor2. Patch CTF estimation was subsequently used to estimate the contrast transfer function (CTF) parameters. The cryo-EM data were then processed using CryoSPARCv4.4.1. A total of 8580 imaging records were manually inspected to exclude low-quality images containing crystalline ice or other contaminants. To enhance particle picking, we used the traditional neural network-based Topaz method in CryoSPARC. Afterwards, the dataset underwent two rounds of reference-free two-dimensional (2D) classification. The selected good 1367298 particle projections were used for ab initio reconstruction to generate an initial reference map, followed by three rounds of non-uniform refinement in CryoSPARC. A high-quality subset of 565614 particle projections was re-extracted and subjected to 3D classification in Relion 4.4.1 to further exclude particles belonging to poorly defined classes. This subset was further refined using uniform and non-uniform refinement to obtain a 2D image with a resolution of 1.33 million. The final map was determined by the gold standard FSC criteria. The overall density was refined by automatic local sharpening using an EM-ready method.

[0198] (26) Model construction and optimization

[0199] The histamine-HRH3-Gi complex model (PDB: 8YUU) was used as the initial model for the structure determination of HRH3 in the 4-IAA-HRH3-Go complex. The Go heterotrimer (mGo, Gβ1, and Gγ2) and scFv16 were generated using the beclomethasone-GPR97-mGo complex model (7D76) as the initial model. These initial models were docked into the EM density map using UCSF Chimera. Manual adjustments and model reconstruction based on the EM density map were subsequently performed in the COOT software and then refined using the Phenix tool. Residues or side chains that could not be accurately fitted into the explicit electron microscopy density were excluded from the final model. The goodness of fit of the refined model was assessed using the model-map Fourier shell correlation (FSC). The structural map was created using UCSF Chimera and UCSF ChimiraX. 4-IAA was generated using the eLBOW program in Phenix, and the model was validated using the MolProbity tool.

[0200] Molprobity is an online model analysis tool, the address is: http: / / molprobity.biochem.duke.edu / index.php; Molprobity has now become a relatively comprehensive protein structure detection tool in structural biology.

[0201] (27) Quantification and statistical analysis

[0202] All statistical analyses were performed using Student's two-tailed paired t-test. The values ​​represent mean ± SEM. Statistical details for all experiments can be found in the figure legends, including the exact number of cell samples or mice. Ns represents no difference, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. No data were excluded from all experiments.

[0203] Example 1

[0204] Identification of 4-IAA, a substance that regulates feeding:

[0205] In order to find metabolites related to appetite regulation, we established a time-restricted diet model. Male mice (n=8) aged 8 to 10 weeks and weighing 22 to 25 g were allowed to eat freely from 1 to 5 pm every day, and no food was given to the mice at other times. The training lasted for 7 days. The mice that had not been trained were defined as the non-training group (Day 0), and the mice that had been trained for 7 days were defined as the experimental group (Day 6). The total daily diet of normal mice is about 4 g. We found that the mice in the Day 0 group could only reach about 1 g during the restricted feeding time. For the experimental group mice, each day of training, the experimental group mice would eat more than the previous day. By the 7th day of training, the mice in the Day 6 group were able to eat an amount equivalent to the total daily diet of normal mice during the restricted feeding time ( Figure 1 A). We found that the food intake of mice in the Day 0 group and the Day 6 group was twice as much after 1 hour of eating, and the food intake of mice in the Day 0 group stopped increasing after 1 hour, while the mice in the Day 6 group continued to eat ( Figure 1 B). Therefore, we trained 8-10 week old male mice and divided them into two groups: Day 0 and Day 6. We performed non-targeted metabolomics analysis on the plasma of mice before (0h) and 1h after eating (n=3) to find substances that control the eating of mice ( Figure 1 C), and the metabolite 4-IAA ( Figure 1 C, 1D), the metabolite 4-IAA plays the function of suppressing appetite.

[0206] To prove that 4-IAA can inhibit food intake, we injected 4-IAA into male mice aged 8 to 10 weeks (single dose of 75 mg / kg, n=6), orally administered 4-IAA to the mice (single dose of 120 mg / kg, n=6), and supplemented 4-IAA with drinking water (the drug was made into a 3 mg / ml aqueous solution and the mice were allowed to drink water freely, n=6). All of these results showed that the food intake of mice could be significantly inhibited ( Figure 1 E) These results suggest that the metabolite 4-IAA exerts a significant appetite suppressant effect.

[0207] We also found that 1-IAA, the isomer of 4-IAA, did not suppress appetite, indicating that the appetite suppressing effect of 4-IAA was structurally specific. Male mice aged 8 to 10 weeks (n = 6) ( Figure 1 F).

[0208] In the correlation analysis, we found that the levels of the metabolite 4-IAA in the blood of three types of mice were significantly upregulated after eating normal diet (Chow, 12-14 weeks old mice, weighing about 26-28g), high-fat diet-induced obese mice (HFD, high-fat induction for 14 weeks, weighing 45g), and genetic defect-induced obese model mice (ob / ob, about 10 weeks old mice, weighing about 55g) ( Figure 1 G).

[0209] 17 healthy male student volunteers were recruited. The volunteers were required to fast for 14 hours (8 pm to 10 am) and abstain from water for 12 hours (10 pm to 10 am). The volunteers began to eat at 10 am the next day. Blood samples were collected from the volunteers before and 2 hours after the meal. The changes in 4-IAA in their blood before and after the meal were measured. We found that the level of 4-IAA in the blood was significantly increased after people ate ( Figure 1 H).

[0210] In order to study the relationship between 4-IAA levels in plasma and body mass index (BMI), the researchers collected plasma samples from 42 healthy volunteers and calculated the correlation between the BMI index and 4-IAA in their blood. The plasma 4-IAA content in the population showed a high positive correlation with its BMI ( Figure 1 I).

[0211] Ten adults were randomly selected from the continuous caloric restriction (CCR) group, and all 10 participants were asked to consume a prescribed amount of calories per day (25 kcal / kg x [height (cm) -100] kg) and eat regular food at any time. During the 12-week intervention, the average daily calorie consumption in the CCR group decreased by 673.7 kcal (95% CI, -884.9 to -462.5 kcal) compared with baseline, and the average weight loss was 7.6%. Before and after the 12-week dietary intervention, patients fasted for 10 hours overnight, and serum samples were collected the next morning to determine changes in 4-IAA ( Figure 1 J). The 4-IAA content in the blood of obese patients decreased significantly after weight loss. These results indicate that 4-IAA can inhibit food intake and regulate obesity. The values ​​are expressed as mean ± standard error, and the Student's t-test was used for statistical significance analysis.

[0212] Example 2

[0213] 4-IAA can resist high-fat diet-induced obesity and can treat obesity:

[0214] Since appetite regulation plays an important role in maintaining metabolic homeostasis, excessive eating can lead to metabolic homeostasis imbalance, thereby inducing metabolic diseases such as obesity. Our study found that the metabolite 4-IAA has an inhibitory effect on appetite, indicating that 4-IAA may inhibit the progression of obesity. Using the HFD high-fat diet-induced obese mouse model, we evaluated the role of 4-IAA in the occurrence of obesity. Mice were induced with HFD at 4 weeks of age (starting at 20g body weight, n=9), and drinking water experiments were performed until 12 weeks; appetite was measured in the 3rd week of treatment; glucose tolerance tests were performed in the 10th week of treatment, insulin sensitivity tests were performed in the 11th week, and body composition analysis and anatomical processing were performed in the 12th week.

[0215] We found that 4-IAA can specifically inhibit the feeding of HFD obese mice compared with 1-IAA ( Figure 2 A) and significantly reduced the body weight of mice ( Figure 2 B), improve their glucose and insulin sensitivity ( Figure 2 C, Figure 2 D), reduce the body fat rate of mice and increase the muscle proportion of mice ( Figure 3 B).

[0216] 4-IAA can significantly inhibit the weight of fatty liver and adipose tissue in mice ( Figure 2 E), alleviated fatty liver lesions in HFD mice, reduced white fat, and maintained browning of brown fat ( Figure 3 C, 3D), reduced the area of ​​white fat cells ( Figure 3 E), significantly reduced the level of triglycerides in the liver of mice ( Figure 2 F).

[0217] At the same time, metabolic cage analysis revealed that 4-IAA could significantly reduce the food intake of mice induced by a high-fat HFD diet, increase the oxygen consumption and carbon dioxide exhalation of mice, and increase the heat production level of mice without changing the total amount of exercise of mice ( Figure 3 A). These results indicate that 4-IAA can improve multiple metabolic abnormalities in obese mice induced by a high-fat diet. We found that 4-IAA can resist obesity and improve metabolic abnormalities. Therefore, we used a high-fat diet to induce obesity in mice, and then gave the obese mice daily drinking water containing 12 mg / ml of 4-IAA to study whether 4-IAA can treat obese mice ( Figure 2 G). First, we found that drinking 4-IAA water significantly reduced the body weight of obese mice ( Figure 2 H), significantly improved glucose tolerance and insulin sensitivity in obese mice ( Figure 2 I-2J), administration of 4-IAA can significantly reduce the liver weight of fatty lesions in obese mice ( Figure 2K), significantly reduced the severity of fatty liver lesions in obese mice ( Figure 3 F). At the same time, 4-IAA can significantly reduce the content of triglycerides in the liver of obese mice ( Figure 2 These results indicate that 4-IAA can significantly treat metabolic abnormalities in obese mice, helping them to lose weight and improve abnormal metabolic indicators.

[0218] Example 3

[0219] 4-IAA improves various metabolic abnormalities in obese mice with gene defects: ob / ob mice were treated with drinking water at 4 weeks of age (starting at 25g body weight, n=5) for 8 weeks. Appetite was measured in the 3rd week of treatment. Glucose tolerance test was performed in the 6th week of treatment, insulin sensitivity test was performed in the 7th week, and body composition analysis and autopsy were performed in the 8th week.

[0220] To further prove that 4-IAA can inhibit the occurrence and development of obesity, we used a gene-deficient obese mouse model (ob / ob) to evaluate the role of 4-IAA in ob / ob obese mice. We found that 4-IAA can specifically inhibit the feeding of ob / ob obese mice ( Figure 4 A) and significantly reduced the body weight of mice ( Figure 4 B), 4-IAA did not significantly alter glucose tolerance in ob / ob obese mice ( Figure 4 C), but significantly improved the insulin sensitivity of ob / ob mice ( Figure 4 D). 4-IAA can significantly inhibit the weight of fatty liver and white adipose tissue in ob / ob obese mice ( Figure 4 E) and reduce the degree of fatty liver lesions in ob / ob obese mice ( Figure 4 F), reduced the size of white fat cells ( Figure 4 G). At the same time, 4-IAA can significantly reduce the content of triglycerides in the liver of ob / ob obese mice ( Figure 4 H), these results indicate that 4-IAA can significantly improve multiple metabolic abnormalities in gene-deficient obese mice.

[0221] Example 4

[0222] 4-IAA does not affect the metabolic phenotype of mice in normal physiological state: Mice were treated with drinking water experiment at 4 weeks of age (starting with 20g body weight, n=7) until 14 weeks. Appetite was measured in the 3rd week of treatment. Glucose tolerance test was performed in the 11th week of treatment, insulin sensitivity test was performed in the 12th week, and body composition analysis and autopsy were performed in the 14th week.

[0223] We found that 4-IAA can significantly improve metabolic abnormalities in mice with high-fat diet and genetically defective obesity, as well as in diabetic mice with genetic defects, indicating that 4-IAA may be able to significantly affect mice under pathological conditions. However, whether 4-IAA will have an effect on mice in a healthy and normal physiological state, we used a normal physiological state mouse model fed a normal diet (Chow Diet) to evaluate the effects of 4-IAA on normal healthy mice. We found that long-term drinking water treatment with 4-IAA did not change the feeding of normal mice ( Figure 5 A), nor does it change the body weight of mice ( Figure 5 B) and body fat percentage ( Figure 5 E), administration of 4-IAA did not alter glucose tolerance in normal healthy mice ( Figure 5 C), but can improve insulin sensitivity in mice ( Figure 5 D). 4-IAA does not change the weight, morphology and pathology of liver and adipose tissue in normal healthy mice ( Figure 5 F, 5H), without changing the size of white adipocytes in normal healthy mice ( Figure 5 I) without changing the triglyceride content in the liver of normal healthy mice ( Figure 5 These results suggest that 4-IAA does not affect the metabolic phenotype of normal healthy mice.

[0224] Example 5

[0225] 4-IAA does not affect the movement of mice; 4-IAA does not cause nausea, preference, learning cognition, anxiety and depression:

[0226] Male mice aged 8 to 10 weeks were intraperitoneally injected (single dose 75 mg / kg, n = 8). We used metabolic cages to analyze the metabolic phenotype of mice before and after administration of 4-IAA. We found that administration of 4-IAA inhibited feeding and naturally significantly reduced the oxygen intake (VO2), carbon dioxide exhalation (VCO2), respiratory quotient (RER) and heat production (Heat) of mice, but administration of 4-IAA did not affect the total activity (Total-Activity) of mice ( Figure 6 A) It also proved that 4-IAA did not produce anesthetic or hypnotic effects on mice to inhibit their eating.

[0227] In order to prove that 4-IAA can suppress appetite and improve abnormal metabolic indicators without affecting behavior and cognition, and to evaluate its influence and drugability, we conducted a feeding test on male mice aged 8 to 10 weeks after starvation for 20 hours by intraperitoneal injection of kaolin (single 75 mg / kg, n=5), and proved that 4-IAA does not cause nausea in mice ( Figure 6B) The flavor preference experiment was conducted using male mice aged 8 to 10 weeks, with a single intraperitoneal injection of 75 mg / kg (n=6) to test whether 4-IAA would cause mice to develop a preference for flavor ( Figure 6 C), 4-IAA did not change the learning and cognitive ability of mice by intraperitoneal injection of 75 mg / kg (n=7) in male mice aged 8 to 10 weeks in a water maze test. Figure 6 D), male mice aged 8 to 10 weeks old were forced to swim and injected intraperitoneally with a single dose of 75 mg / kg (n = 13) Figure 6 E), tail suspension test: male mice aged 8 to 10 weeks were intraperitoneally injected with a single dose of 75 mg / kg (n = 13) Figure 6 F) and open field test: male mice aged 8 to 10 weeks were intraperitoneally injected with a single dose of 75 mg / kg (n = 13) Figure 6 G) 4-IAA does not cause anxiety and depression in mice. Through the above behavioral experiments, we have proved that 4-IAA does not cause nausea and changes in preferences in mice, nor does it affect the learning and cognitive abilities of mice, and does not cause anxiety and depression in mice.

[0228] Example 6

[0229] Analyze the synthesis and production pathway of 4-IAA in the body. 4-IAA is synthesized through the intestinal pathway:

[0230] The mice used in this example were all 8 to 10 weeks old.

[0231] We further analyzed the synthesis pathway of 4-IAA and found that 4-IAA is produced by a series of metabolism: histidine (L-Histidine)-histamine (Histamine)-tetraimidazole acetaldehyde-tetraimidazole acetic acid (4-IAA) ( Figure 7 A), the main enzyme that catalyzes L-histidine to produce histamine is HDC, which is less expressed in the intestine. The enzyme that catalyzes tetraimidazole acetaldehyde to produce 4-IAA is mainly AOC1, which is highly expressed in the intestine, while another histidine catalytic enzyme HNMT is also almost not expressed in the intestine ( Figure 7 C) Since 4-IAA is a metabolite of histamine in the intestine, we gave mice another metabolite of histamine, methylimidazole acetic acid (Me-IAA), and found that only 4-IAA could specifically suppress the appetite of mice ( Figure 7 B), and thus speculated that the physiological significance of 4-IAA synthesis is that histidine in food is metabolized into histamine, and then metabolized into 4-IAA in the intestine to regulate appetite and metabolism. Using the mouse cell atlas [MCA], it was found that AOC1 is enriched in intestinal cells ( Figure 7D), and we also found that the expression level of AOC1 in the duodenum and jejunum in the upper and middle part of the small intestine was significantly reduced after TRF ( Figure 7 EF), indicating that the body needs to synthesize less 4-IAA to promote appetite. In order to prove the direct source of 4-IAA synthesis, we detected the plasma 4-IAA content of mice after giving histidine and histamine, and found that only histamine could significantly increase the 4-IAA content, proving that histamine is the direct substrate for the synthesis of 4-IAA ( Figure 7 G). Giving mice histidine did not suppress their appetite ( Figure 7 H), these results indicate that 4-IAA is synthesized from histamine. To further prove how histamine synthesizes 4-IAA, we constructed a mouse model with systemic knockout of AOC1 ( Figure 7 I) to verify that AOC1 was knocked out in the AOC1- / - intestine ( Figure 7 J, 7K, 7L), we detected the 4-IAA levels in the blood of control mice AOC1+ / + and knockout mice AOC1- / - before and after feeding, and found that the 4-IAA levels in the blood of knockout mice AOC1- / - after starvation were significantly lower than those in control mice. After refeeding (refed), the increase in 4-IAA levels in the blood of knockout mice AOC1- / - was also significantly lower than that in control mice ( Figure 7 M), proving that AOC1 is the main catalytic enzyme of 4-IAA. Furthermore, we found that the upregulation of 4-IAA content in the blood of AOC1- / - mice after oral administration of histamine was significantly lower than that of the control mice ( Figure 7 N), the above experiments prove that AOC1 catalyzes histamine to synthesize tetraimidazoleacetic acid (4-IAA) to regulate the body's appetite and metabolism.

[0232] Example 7

[0233] 4-IAA suppresses appetite by inhibiting the activity of hypothalamic AgRP neurons:

[0234] The hypothalamic nuclei of the brain play an important role in appetite regulation. We hypothesized that the metabolite 4-IAA acts in the brain and can affect the activity of certain appetite-regulating neurons. We first supplemented the brains of 8-10 week-old mice with 4-IAA (12 μg) by third cerebroventricular injection to observe its effects in the brain. We found that supplementation of 4-IAA in the brain can significantly and specifically inhibit the eating of mice ( Fig. 9 A). To understand where in the brain 4-IAA acts, we performed whole-brain clearing staining analysis ( Figure 8 A) It was found that the administration of 4-IAA caused changes in c-Fos signals in some brain regions ( Figure 8A, 9B), and we mainly focused on the significant decrease of c-Fos signal in the ARC region of the hypothalamus ( Figure 8 A). Since AgRP neurons in the ARC region have been reported to play an important role in promoting appetite in appetite regulation, we further analyzed the effect of 4-IAA on the activity of AgRP neurons. By c-Fos staining, we found that 4-IAA can specifically and significantly inhibit the activity of AgRP neurons ( Figure 8 B, 8C, Fig. 9 D, 9E). Moreover, 4-IAA did not affect the activity of POMC neurons in the ARC region (which have been reported to play an important role in suppressing appetite in appetite regulation) ( Fig. 9 C), the above results indicate that 4-IAA may be able to specifically act on AgRP neurons in the ARC region of the hypothalamus to regulate appetite. To further prove that 4-IAA can specifically act on AgRP neurons in the ARC region of the hypothalamus, we injected DIO-GCaMP AAV into the ARC region of AgRP-Cre mice and buried optical fibers for in vivo calcium imaging experiments. We found that after injection of 4-IAA, the fluorescent calcium activity of mice was significantly reduced (n=8) ( Figure 8 D, 8E), proving that 4-IAA can inhibit the activity of AgRP neurons. Further, we cut brain slices from the ARC region of AgRP-Cre; CAG-LSL-tdTomato mice (green fluorescence labeled AgRP), used patch clamp to record the electrophysiology of AgRP neurons in the ARC region, and found that 4-IAA dose-dependently inhibited the firing of action potentials of AgRP neurons ( Figure 8 F, 8G), then we used CNO (0.5 mg / kg) to activate the AgRP neuron activity of AgRP-Cre; R26-LSL-hM3Dq mice and found that 4-IAA could significantly inhibit the feeding of mice with activated AgRP neurons (n=9) ( Figure 8 H, 8I). These experiments together indicate that 4-IAA suppresses appetite by inhibiting the activity of AgRP neurons in the ARC region of the hypothalamus.

[0235] Example 8

[0236] Receptor screening identified HRH3 as the receptor for 4-IAA:

[0237] We next investigated the mechanism by which the metabolite 4-IAA inhibits the activity of AgRP neurons. We first demonstrated that 4-IAA was able to significantly reduce Forskolin-induced cAMP levels in the wild-type mouse hypothalamus. Using the effect of 4-IAA stimulation on Forskolin-induced cAMP levels in the wild-type mouse hypothalamus, we measured cAMP ( Fig.10 A). Candidate screening of 4-IAA receptors: On day 0, the 15 GPCRs with the highest expression in ARC (derived from GSE96627) were transfected into HEK293T cells expressing GloSensor; on day 2, cells were treated with Forskolin (10 μM) in the presence of 100 pM, 1 nM, or 100 nM 4-IAA or 1-IAA, and cAMP levels were monitored by GloSensor analysis. Histamine receptor H3 (HRH3) was identified as a potential receptor for 4-IAA with an EC50 of 10.7 ± 2.01 nM ( Fig.10 B, 10C). Furthermore, we found that the 4-IAA-HRH3 complex triggers inhibitory signals through Gαo ( Fig.10 D). Since histamine receptors have four members, we detected the binding of 4-IAA to other receptors and found that 4-IAA can also trigger Gαo signaling through HRH2, but its EC50 (315±42nM) is 12 times that of HRH3 (25.1±6.5nM) ( Fig.11 C). Considering that the expression level of Hrh3 is much higher than that of other HRH family members ( Fig.11 A, 11B), and it has been reported that knocking out Hrh3 would increase the appetite of mice and lead to obesity. We speculated that HRH3 might be the functional receptor of 4-IAA to inhibit appetite.

[0238] To further confirm this result, we expressed HRH3, miniGαo1, Gβ1, Gγ2, and scFv16 in insect cells, purified the 4-IAA-HRH3-mGo complex, and resolved its structure by cryo-electron microscopy ( Fig.10 E-10G; Fig.11 D-11F). Resolution is ( Fig.10 E). According to the structure, the binding pocket of 4-IAA is located in transmembrane 3 (TM3) and TM6-TM7 ( Fig.10 G-10H). Through structural analysis and point mutation experiments, we found that D114, Y115, C118, Y374, F398 and L401 are essential residues for HRH3 to bind to 4-IAA ( Fig.10IJ). The imidazole group and carboxyl group of 4-IAA are located in the same plane. D114, C118, F398 and L401 are the common sites in HRH3 that need to bind to 4-IAA and histamine ( Fig.10 H). Y115 and Y374 are specific for 4-IAA, and W402 is specific for histamine binding ( Fig.10 H).

[0239] Example 9

[0240] 4-IAA acts on HRH3 receptors, thereby inhibiting the activity of AgRP neurons to regulate appetite:

[0241] We further studied the mechanism by which 4-IAA binds to the above GPCRs and inhibits the activity of AgRP neurons. We cut brain slices from the ARC brain region of AgRP-Cre; CAG-LSL-tdTomato mice and used patch clamps to record the electrophysiology of AgRP neurons in the ARC brain region. We found that when the brain slices were treated with HRH3 inhibitors Ciproxifan (37.5 μM) and GSK189254 (20 μM), 4-IAA could not inhibit the firing of action potentials of AgRP neurons ( Fig.12 A, 12B). Furthermore, we also found that 4-IAA lost its appetite-suppressing effect after injecting Ciproxifan (3 μg) into the third ventricle of mice. n=5 ( Fig.12 C). We further knocked down HRH3 by unilaterally injecting DIO-shHRH3 AAV virus into the ARC region of AgRP-Cre mice and found that 4-IAA lost its function of inhibiting the activity of mouse AgRP neurons in HRH3 knockdown mice ( Fig.12 D, 13B), these results together prove that 4-IAA acts on the HRH3 receptor on AgRP neurons in the ARC region of the hypothalamus, thereby inhibiting the activity of AgRP neurons to exert its function of suppressing appetite and regulating metabolism.

[0242] To further prove that 4-IAA acts on HRH3 receptors on AgRP neurons in the ARC region of the hypothalamus, thereby inhibiting the activity of AgRP neurons and exerting the function of suppressing appetite and regulating metabolism. We knocked down HRH3 by unilaterally injecting DIO-shHRH3 AAV virus into the ARC region of male AgRP-Cre mice aged 8 to 10 weeks. We found that 4-IAA could not inhibit the feeding of HRH3 knockdown mice. Fig.12 E), and then induced mice to eat a high-fat diet for 9 weeks (n=6) to observe whether the loss of HRH3 could affect the metabolic phenotype of mice. We found that when HRH3 was knocked down, the mice's food intake was significantly increased ( Fig.12F), and the weight gain was significantly higher than that of the control group mice ( Fig.12 G), the knockdown group mice showed worse glucose tolerance at 7 weeks ( Fig.12 H) and worse insulin sensitivity at week 8 ( Fig.12 I). At the 9th week, the knockdown group mice were killed and the weights of the liver and three types of adipose tissues were significantly higher than those of the control group mice ( Fig.12 J), higher body fat percentage ( Fig.13 C). The morphology of the liver and three types of adipose tissue in the knockdown group mice were larger than those in the control group mice ( Fig.13 D), HE showed that the liver lipid droplet deposition in the knockdown group mice was significantly more than that in the control group mice (13E), and the white adipocytes were also significantly larger than those in the control group mice ( Fig.13 E, 13F), and the triglyceride content in the liver of the knockdown group mice was significantly higher than that of the control group mice ( Fig.13 G), these results indicate that when the HRH3 receptor is missing, 4-IAA cannot play its significant role in treating metabolic abnormalities in obese mice, loses its effect in improving abnormal metabolic indicators, and leads to obesity and abnormal metabolism in mice.

[0243] The present disclosure proposes a new appetite suppressant metabolite 4-IAA, explores its synthesis source, and clarifies the specific mechanism of action of 4-IAA, which is to suppress appetite and regulate metabolism by acting on the HRH3 receptor on the AgRP neurons in the ARC region. Its regulatory mode is summarized as follows: Fig.14 We discovered for the first time that 4-IAA regulates appetite and reduces blood sugar and weight through neuro-metabolic regulation, and also provided a very promising preclinical target receptor HRH3 for metabolic diseases such as obesity, which has very important theoretical and application value for the research and development of new drugs for suppressing appetite and reducing blood sugar and weight.

[0244] Gene sequence information:

[0245] Human HRH3 sequence: (from

[0246] https: / / www.ncbi.nlm.nih.gov / CCDS / CcdsBrowse.cgi? REQUEST=GENEID&DATA=11255&BUIL DS=CURRENTBUILDS)HRH3 histamine receptor H3[Homo sapiens(human)]

[0247] Gene ID: 11255

[0248] SEQ ID NO: 1: Nucleotide sequence of human HRH3 (1338 nt):

[0249]

[0250] SEQ ID NO: 2: Translation of human HRH3 (445 amino acids):

[0251] MERAPPDGPLNASGALAGEAAAAGGARGFSAAWTAVLAALMALLIVATVLGNALVMLAFVADSSLRTQNNFFLLNLAISDFLVGAFCIPLYVPYVLTGRWTFGRGLCKLWL VVDYLLCTSSAFNIVLISYDRFLSVTRAVSYRAQQGDTRRAVRKMLLVWVLAFLLYGPAILSWEYLSGGSSIPEGHCYAEFFYNWYFLITASTLEFFTPFLSVTFFNLSIY LNIQRRTLRLDGAREAAGPEPPPPEAQPSPPPPPGCWGCWQKGHGEAMPLHRYGVGEAAVGAEAGEATLGGGGGGGSVASPTSSSGSSSRGTERPRSLKRGSKPSASSASL EKRMKMVSQSFTQRFRLSRDRKVAKSLAVIVSIFGLCWAPYTLLMIIRAACHGHCVPDYWYETSFWLLWANSAVNPVLYPLCHHSFRRAFTKLLCPQKLKIQPHSSLEHCWK

[0252] Human AgRP sequence: (from

[0253] https: / / www.ncbi.nlm.nih.gov / CCDS / CcdsBrowse.cgi? REQUEST=CCDS&GO=MainBrowse&DAT A=CCDS10839.1)AGRP agouti related neuropeptide[Homo sapiens(human)]

[0254] Gene ID: 181

[0255] SEQ ID NO: 3: Nucleotide sequence of human AgRP (399 nt):

[0256] ATGCTGACCGCAGCGGTGCTGAGCTGTGCCCTGCTGCTGGCACTGCCTGCCACGCGAGGAGCCCAGATGGGCTTGGCCCCCATGGAGGGCATCAGAAGGCCTGACCAGGCCCTGCTCCCAGAGCTCCCAGGCCTGGGCCTGCGGGCCCCACTGAAGAAGACAACTGCAGAACAGGCAGAAGAGGATCTGTTGCAGGAGGCTCAGGCCTTGGCAGAGGTACTAGACCTGCAGGACCGCGAGCCCCGCTCCTCACGTCGCTGCGTAAGGCTGCATGAGTCCTGCCTGGGACAGCAGGTGCCTTGCTGTGACCCATGTGCCACGTGCTACTGCCGCTTCTTCAATGCCTTCTGCTACTGCCGCAAGCTGGGTACTGCCATGAATCCCTGCAGCCGCACCTAG

[0257] SEQ ID NO: 4: Translation of human AgRP (132 amino acids):

[0258] MLTAAVLSCALLLALPATRGAQMGLAPMEGIRRPDQALLPELPGLGLRAPLKKTTAEQAEEDLLQEAQALAEVLDLQDREPRSSRRCVRLHESCLGQQVPCCDPCATCYCRFFNAFCYCRKLGTAMNPCSRT

[0259] Mouse HRH3 sequence: (from

[0260] https: / / www.ncbi.nlm.nih.gov / CCDS / CcdsBrowse.cgi?REQUEST=GV&DATA=603534&BUILDS=CURRENTBUILDS) Hrh3 histamine receptor H3 [Mus musculus (house mouse)]

[0261] Gene ID: 99296.

[0262] SEQ ID NO: 5: Nucleotide sequence of mouse HRH3 (1338 nt):

[0263]

[0264] SEQ ID NO: 6: Translation of mouse HRH3 (445 amino acids):

[0265] MERAPPDGLMNASGALAGEAAAAGGARGFSAAWTAVLAALMALLIVATVLGNALVMLAFVADSSLRTQNNFFLLNLAISDFLVGAFCIPLYVPYVLTGRWTFGRGLCKLWL VVDYLLCASSVFNIVLISYDRFLSVTRAVSYRAQQGDTRRAVRKMALVWVLAFLLYGPAILSWEYLSGGSSIPEGHCYAEFFYNWYFLITASTLEFFTPFLSVTFFNLSIY LNIQRRTLRLDGGREAGPEPPPDAQPSPPPAPPSCWGCWPKGHGEAMPLHRYGVGEAGPGVETGEAGLGGGSGGGAAASPTSSSGSSSRGTERPRSLKRGSKPSASSASL EKRMKMVSQSITQRFRLSRDKKVAKSLAIIVSIFGLCWAPYTLLMIIRAACHGHCVPDYWYETSFWLLWANSAVNPVLYPLCHYSFRRAFTKLLCPQKLKVQPHGSLEQCWK

[0266] Mouse AgRP sequence: (from

[0267] https: / / www.ncbi.nlm.nih.gov / CCDS / CcdsBrowse.cgi? REQUEST=GV&DATA=613553&BUILDS=CURRENTBUILDS)Agrp agouti related neuropeptide[Mus musculus(house mouse)]

[0268] Gene ID: 11604.

[0269] SEQ ID NO: 7: Nucleotide sequence of mouse AgRP (396 nt):

[0270] ATGCTGACTGCAATGTTGCTGAGTTGTGTTCTGCTGTTGGCACTGCCTCCCACACTGGGGGTCCAGATGGGCGTGGCTCCACTGAAGGGCATCAGAAGGCCTGACCAGGCTCTGTTCCCAGAGTTCCCAGGTCTAAGTCTGAATGGCCTCAAGAAGACAACTGCAGACCGAGCAGAAGAAGTTCTGCTGCAGAAGGCA GAAGCTTTGGCGGAGGTGCTAGATCCACAGAACCGCGAGTCTCGTTCTCCGCGTCGCTGTGTAAGGCTGCACGAGTCCTGCTTGGGACAGCAGGTACCTTGCTGCGACCCGTGCGCTACGTGCTACTGCCGCTTCTTCAATGCCTTTTGCTACTGCCGCAAGCTGGGTACGGGCCACGAACCTCTGTAGTCGCACCTAG

[0271] SEQ ID NO: 8: Translation of mouse AgRP (131 amino acids):

[0272] MLTAMLLSCVLLLALPPTLGVQMGVAPLKGIRRPDQALFPEFPGLSLNGLKKTTADRAEEVLLQKAEALAEVLDPQNRESRSPRRCVRLHESCLGQQVPCCDPCATCYCRFFNAFCYCRKLGTATNLCSRT

[0273] The foregoing description of specific exemplary embodiments of the present disclosure is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present disclosure to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present disclosure and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present disclosure and various different selections and changes. The scope of the present disclosure is intended to be defined by the claims and their equivalents.

Claims

1. Use of a compound that binds to positions D114, Y115, C118, Y374, F398 and L401 of a histamine H3 receptor (HRH3) and / or a pharmaceutically acceptable salt, solvate or precursor thereof in the preparation of a drug or health product, wherein the histamine H3 receptor sequence is: SEQ ID NO: 2, and the drug or health product is any one or more of the following: (1) Appetite suppressing drugs or health products; (2) Drugs or health products that lower blood sugar; (3) drugs or health products for the treatment of diabetes; (4) drugs or health products for weight loss; (5) Weight loss drugs or health products; (6) Fat-reducing drugs or health products; (7) Drugs or health products for the treatment of fatty liver.

2. Use of a compound that binds to positions D114, Y115, C118, Y374, F398 and L401 of a histamine H3 receptor and / or its pharmaceutically acceptable salt, solvate or precursor in the preparation of a drug or health product for suppressing appetite, lowering blood sugar, treating diabetes, reducing weight, losing weight, reducing fat and / or treating fatty liver mediated by hypothalamic AgRP neurons, wherein the histamine H3 receptor sequence is: SEQ ID NO:

2.

3. The use according to claim 1 or 2, characterized in that The compound is imidazole-4-acetic acid and / or a pharmaceutically acceptable salt, a solvate, or a precursor thereof. The imidazole-4-acetic acid has a structure as shown in formula (I):

4. The use according to any one of claims 1 to 3, characterized in that The medicine or health product is used for one or more purposes selected from the following: reducing body fat percentage, increasing muscle proportion, improving glucose tolerance, improving insulin sensitivity, inhibiting or reducing the weight of adipose tissue, reducing white fat or inhibiting the weight of white adipose tissue, reducing the size of white fat cells or reducing the area of ​​white fat cells, maintaining the browning of brown fat, reducing liver triglyceride levels, preventing and treating obesity, and / or for regulating obesity, for obese, overweight or obesity-prone subjects; Preferably, the medicine or health product is used to reduce body fat percentage, increase muscle proportion, improve glucose tolerance, and improve insulin sensitivity; Preferably, the medicine or health product is used for one or more purposes selected from the following: inhibiting or reducing the weight of adipose tissue, reducing white fat or inhibiting the weight of white adipose tissue, reducing the size of white fat cells or reducing the area of ​​white fat cells, maintaining the browning of brown fat, and reducing liver triglyceride levels; Preferably, the medicine or health product is used for one or more purposes selected from the following: inhibiting or reducing the weight of adipose tissue, reducing white fat or inhibiting the weight of white adipose tissue, reducing the size of white fat cells or reducing the area of ​​white fat cells, and / or maintaining the browning of brown fat; Preferably, the medicine or health product is used to reduce liver triglyceride levels; Preferably, the medicine or health product is used to improve insulin sensitivity; Preferably, the medicine or health product is used to reduce body fat percentage and increase muscle proportion; Preferably, the medicament or nutraceutical is for use in obese, overweight or obesity prone subjects.

5. The use according to any one of claims 1 to 4, characterized in that The medicine or health product has at least one effect selected from the following: Does not affect the metabolic phenotype of a normal healthy body; Does not affect exercise; Does not cause nausea; It does not affect preferences; Does not affect learning and cognition; and / or It will not cause anxiety or depression.

6. The use according to any one of claims 1 to 5, characterized in that The medicine or health product forms an appetite suppressing preparation; the preparation uses the imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate or precursor as the sole active ingredient.

7. The use according to any one of claims 1 to 6, characterized in that The medicine or health product forms an appetite suppressant preparation; the preparation comprises the imidazole-4-acetic acid and / or a pharmaceutically acceptable salt, a solvate, or a precursor thereof, and further comprises a pharmaceutically acceptable carrier; Preferably, the pharmaceutically acceptable carrier includes one or more of a solvent, a solubilizer, a cosolvent, an emulsifier, a flavoring agent, an olfactory agent, a colorant, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a pH regulator, a stabilizer, a surfactant, and a preservative.

8. The use according to any one of claims 1 to 7, characterized in that The medicine or health product forms an appetite suppressing preparation; the preparation comprises imidazole-4-acetic acid and / or a pharmaceutically acceptable salt, a solvate, a precursor thereof, and one or more other active ingredients for suppressing appetite.

9. The use according to any one of claims 1 to 8, characterized in that The medicine or health product forms an appetite suppressing preparation; the amount of imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate, or precursor thereof used to suppress appetite in a unit preparation is 5 to 100 mg, preferably 10 to 60 mg; The amount of imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate, or precursor thereof in a unit preparation for resisting diet-induced obesity is 20 to 300 mg, preferably 30 to 200 mg; The amount of imidazole-4-acetic acid and / or its pharmaceutically acceptable salt, solvate, or precursor thereof in a unit preparation for weight loss is 50 to 1000 mg, preferably 100 to 500 mg.

10. The use according to any one of claims 1 to 9, characterized in that The compounds work by inhibiting the activity of hypothalamic AgRP neurons; Preferably, the administration of the drug or health product is one or more of oral, intravenous, intraperitoneal, intramuscular, rectal or subcutaneous administration; Preferably, the administration of the drug is one or more of oral, intravenous, intraperitoneal administration; Preferably, the preparation is a solid preparation, a semisolid preparation or a liquid preparation; preferably, the solid preparation is a tablet, a capsule, a granule or a pill; the semisolid preparation is a gel, a suppository or an ointment; the liquid preparation is an emulsion, a mixture, a suspension or a solution; Preferably, the medicine or health product is in the form of tablets, capsules, injections, powders, pills, granules, syrups, chewable tablets and patches.

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