Application of GDI2 as weight-losing target and method for screening obesity treatment drugs

Abstract

The invention relates to the technical field of biological medicine, in particular to application of GDI2 as a weight-losing target and a method for screening obesity treatment drugs. On one hand, GDI2 can be used as a weight-losing target for preparing obesity treatment drugs. On the second aspect, the method for screening the obesity treatment medicine comprises the following steps: S1, measuring the expression level of the GDI2 gene in adipose tissue of an obese individual; s2, applying candidate drugs to the obese individuals in the step S1; s3, measuring the expression level of the GDI2 gene in the adipose tissue of the obese individual after the candidate drug is applied; and S4, if the expression level of the GDI2 gene is significantly lower than the initial expression level after the candidate drug is applied, determining that the candidate drug is an obesity treatment drug. The invention proves that the knockout of GDI2 is practical and effective for treating obesity and ingestion increase induced by high fat diet, and provides an important basis and direction for developing a novel therapeutic drug targeting GDI2 to relieve obesity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of GDI2 as a weight loss target and a method for screening obesity therapeutic drugs. Background Art

[0002] Obesity, a chronic metabolic disease, has become a global public health concern. It not only affects personal appearance but is also closely linked to a variety of health issues, including diabetes, hypertension, hyperuricemia, dyslipidemia, fatty liver disease, chronic kidney disease, gallstones, and carotid artery plaques. Currently, common treatments for obesity include medication, surgery, and lifestyle interventions, but these traditional approaches all have limitations.

[0003] Lifestyle intervention mainly controls diet and increases exercise so that the body's energy consumption is greater than its intake, in order to achieve the goal of weight loss. However, this method requires patients to maintain regular living habits for a long time, which is extremely challenging for many people. Some patients find it difficult to give up high-calorie foods or cannot stick to regular exercise, resulting in repeated fluctuations in weight. Moreover, excessive dieting may lead to nutritional imbalance, and excessive exercise may cause problems such as muscle strain and joint injury. In addition, changes in lifestyle may also bring psychological pressure to patients, especially when no obvious results are seen in the short term, they are prone to anxiety. Surgical treatments for obesity primarily involve reducing stomach volume or inserting a gastric balloon to enhance satiety and reduce energy intake. However, surgical treatment carries significant risks, including potential complications such as infection, bleeding, and anesthesia reactions, which can be life-threatening in severe cases. Furthermore, surgery is traumatic and requires a long recovery time. For example, while removing part of the stomach or small intestine can reduce food intake, it can also affect nutrient absorption, leading to vitamin and mineral deficiencies and other nutritional issues.

[0004] Currently, the main medications used to treat obesity are drugs like orlistat, which can reduce fat absorption by inhibiting intestinal lipase activity, but may also cause adverse reactions such as incontinence. Furthermore, some weight-loss medications can cause discomfort such as dizziness, nausea, diarrhea, and insomnia, and long-term use can adversely affect the cardiovascular system and liver and kidney function. Furthermore, drug-based weight loss can be addictive, and if patients don't change their lifestyle habits once the medication is discontinued, weight can easily rebound.

[0005] With the advancement of gene therapy technology and in-depth research on genes related to glucose and lipid metabolism, treating obesity at the genetic level is becoming increasingly possible. Chinese invention patent CN 115518160 B proposes that the Tks4 gene could serve as an effective target for gene therapy. Intervention of the Tks4 gene could regulate the body's fat metabolism and energy balance, thereby achieving the goal of treating obesity. However, gene therapy for obesity is still in the research stage, with only a few effective gene targets discovered. Uncovering more gene therapy targets is crucial.

[0006] Rab proteins are small GTPases that act as molecular switches. By dynamically cycling between GTP-bound (active) and GDP-bound (inactive) states, they are widely involved in regulating various cellular processes, such as vesicle trafficking, organelle interactions, and signal transduction. Guanine nucleotide dissociation inhibitor 2 (GDI2), an inhibitor of the Rab GTPase family, binds to the GDP form of Rab proteins, preventing their dissociation and maintaining their inactive state. It also regulates the intracellular distribution of Rab proteins. Currently, little research has been conducted on GDI2, and its functional role and regulatory mechanisms in metabolic diseases remain unclear. Summary of the Invention

[0007] To address the current lack of research on GDI2 in gene therapy for obesity, the present invention provides the use of GDI2 as a weight loss target and a method for screening obesity therapeutics. This invention demonstrates that GDI2 knockout is effective in treating obesity and increased food intake induced by a high-fat diet. GDI2 is expected to become a potential therapeutic target for inhibiting the development of obesity, providing important evidence and direction for the development of novel therapeutic drugs targeting GDI2 to alleviate obesity.

[0008] The technical solutions of the present invention are as follows: In a first aspect, the present invention provides a use of GDI2 as a weight loss target in the preparation of a drug for treating obesity.

[0009] Furthermore, obesity treatment drugs are GDI2 inhibitors, such as: The GDI2 inhibitor can be a drug that inhibits the expression of the GDI2 gene, a drug that knocks down the GDI2 gene, or a drug that knocks out the GDI2 gene; Among them, the drug for knocking down the GDI2 gene can be any drug that can prevent the expression of the gene encoding the GDI2 protein. Specific methods of knocking down the gene include removing all or part of the coding gene sequence, introducing a frameshift mutation so that no functional protein is produced, removing or changing regulatory components (such as promoter editing) so that the coding gene sequence is not transcribed, and preventing translation by binding to mRNA. The drug for knocking out the GDI2 gene may be any drug that can cause the GDI2 gene to mutate and thereby lose its activity. The mutation form herein may be a deletion mutation, an insertion mutation and / or a base substitution.

[0010] The GDI2 inhibitor may also be a drug that inhibits the synthesis of the GDI2 protein, a drug that inhibits the activity of the GDI2 protein, or a drug that promotes the degradation of the GDI2 protein.

[0011] Furthermore, the GDI2 inhibitor is selected from nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, polypeptides, proteins or viruses.

[0012] Furthermore, the GDI2 inhibitor further comprises one or more pharmaceutically acceptable excipients.

[0013] Furthermore, the GDI2 inhibitor further includes one or more pharmaceutically acceptable carriers.

[0014] Furthermore, the dosage form of the GDI2 inhibitor is selected from tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal formulations or suppositories.

[0015] In a second aspect, the present invention provides a method for screening obesity therapeutic drugs, comprising the following steps: S1. Measuring the initial expression level of the GDI2 gene in adipose tissue of obese individuals other than humans; S2. administering the candidate drug to the obese individual of step S1; S3. measuring the expression level of the GDI2 gene in adipose tissue of the obese individual after administration of the candidate drug; S4. If, after administration of the candidate drug, the expression level of the GDI2 gene in the adipose tissue of the obese individual is significantly lower than the initial expression level, with a statistical difference of P < 0.05, the candidate drug is determined to be an obesity treatment drug.

[0016] Furthermore, the obese individual other than humans may be obese mice induced by a high-fat diet.

[0017] The beneficial effects of the present invention are: The present invention constructs mice with GDI2 adipose tissue-specific knockout and feeds them a high-fat diet. It is found that the loss of GDI2 in adipose tissue can alleviate the metabolic syndrome phenotype caused by overnutrition, including improving the weight and fat size of obese individuals, reducing the size of subcutaneous and reproductive fat droplets, inhibiting the increase of blood lipid and blood sugar levels, improving glucose intolerance, and enhancing individual energy expenditure. This proves that inhibiting GDI2 expression can effectively suppress the obesity phenotype and glucose and lipid metabolism, achieving the effect of weight loss. The present invention can be used to prepare drugs for the prevention and / or treatment of obesity.

[0018] In summary, GDI2 can be used as a biological marker for predicting obesity and a research target for preventing and / or treating obesity, and can be used to prepare and screen drugs for treating obesity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0020] Figure 1 This is the test result of the DIO group mice in Example 1. Figure 1 Figure 2 (A) shows the body size comparison between Ctrl and DIO mice; (B) shows the Western blot analysis of GDI2 protein expression in iWAT and BAT between Ctrl and DIO mice; (C) shows the statistical analysis of GDI2 expression in iWAT and BAT between Ctrl and DIO mice (n=6, t-test, *P<0.05, ***P<0.001); (D) shows the Western blot analysis of GDI2 protein expression over time after C3H10T1 / 2 induction; (E) shows the correlation analysis of GDI2 with different BMIs (n=21, nonparametric Spearman test); (F) shows the relative expression of GDI2 in non-obese (Lean) and obese (Obesity) subjects (**P<0.01).

[0021] Figure 2 It is constructed in Example 2 Gdi2 AKO Test results in mice. Figure 2 In the Gdi2 flox / + Mouse construction strategy; B is Gdi2 AKO Mating strategy of mice; C is the agarose gel electrophoresis result of mouse genotype identification; D is 8-week-old Gdi2 AKO Mice and Gdi2 f / f Body size comparison of mice; E is 8 weeks old Gdi2 AKO Mice and Gdi2 f / f Comparison of the body weight of mice (ns indicates no significant difference); F is Gdi2 f / f Mice and Gdi2 AKO Western blot bands of GDI2 protein expression levels in iWAT and BAT of mice; G is Gdi2 f / f Mice and Gdi2 AKO Western blot bands showing the expression levels of GDI2 protein in primary adipocytes of mice.

[0022] Figure 3 It is constructed in Example 2 Gdi2 AKO -Test results of DIO mice; Figure 3 A is the 16-week high-fat diet feeding period Gdi2 f / f -DIO mice and Gdi2 AKO -Line graph of dynamic changes in body weight of DIO mice (*P<0.05, **P<0.01); B is a graph showing the dynamic changes in body weight of DIO mice after 16 weeks of high-fat diet feeding. Gdi2 f / f -DIO mice and Gdi2 AKO -DIO mice body size, iWAT, eWAT, and BAT morphology comparison; C is after 16 weeks of high-fat diet feeding Gdi2 f / f -DIO mice and Gdi2 AKO -Statistical analysis of iWAT, eWAT, and BAT weights in DIO mice (*P<0.05, **P<0.01, ***P<0.001); D represents the weights of iWAT, eWAT, and BAT in DIO mice after 16 weeks of high-fat diet feeding. Gdi2 f / f -DIO mice and Gdi2 AKO -DIO mice glucose tolerance test results (ns indicates no significant difference, *P<0.05, **P<0.01); E is after 16 weeks of high-fat diet feeding Gdi2 f / f -DIO mice and Gdi2 AKO -Line graph of oxygen consumption dynamic changes over time in DIO mice; F represents the oxygen consumption after 16 weeks of high-fat diet feeding. Gdi2 f / f -DIO mice and Gdi2AKO Statistical analysis of oxygen consumption in DIO mice (n=6, t-test, *P<0.05); G represents oxygen consumption after 16 weeks of high-fat diet feeding. Gdi2 f / f -DIO mice and Gdi2 AKO -Line graph of the dynamic changes of heat production in DIO mice over time; H represents the temperature after 16 weeks of high-fat diet feeding Gdi2 f / f -DIO mice and Gdi2 AKO Statistical analysis of thermogenesis in DIO mice (n=6, t-test, *P<0.05); I represents the thermogenic rate after 16 weeks of high-fat diet feeding. Gdi2 f / f -DIO mice and Gdi2 AKO -Statistical analysis of triglyceride levels in serum of DIO mice (**P<0.01); J represents the difference between the two groups after 16 weeks of high-fat diet feeding. Gdi2 f / f -DIO mice and Gdi2 AKO -Statistical analysis of cholesterol in the eye serum of DIO mice **P<0.01; K represents the difference between the two groups after 16 weeks of high-fat diet feeding. Gdi2 f / f -DIO mice and Gdi2 AKO -Statistical analysis of glucose in the eye serum of DIO mice (*P<0.05); L is the difference between the two groups after 16 weeks of high-fat diet feeding. Gdi2 f / f -DIO mice and Gdi2 AKO Statistical analysis of free fatty acids in the ocular serum of -DIO mice (ns indicates no significant difference, **P<0.01); M represents the free fatty acids in the ocular serum of -DIO mice after 16 weeks of high-fat diet feeding. Gdi2 f / f -DIO mice and Gdi2 AKO -HE staining results of iWAT, eWAT, and BAT in DIO mice. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0024] The experimental methods in the following examples are conventional methods unless otherwise specified. The reagents, consumables, etc. involved in the following examples are common commercial products and can be purchased through conventional commercial channels unless otherwise specified.

[0025] In the following examples, SPF male C57BL / 6J mice (6-8 weeks old) were purchased from Beijing Weitonglihua Co., Ltd. (Animal License No.: SCXK (Beijing) 2021-0006). Adipoq-Cre mice were purchased from Saiye Biotechnology Co., Ltd. (No. C001186). This strain originated from the Jackson Laboratory, and its Adipoq gene is specifically expressed in adipose tissue.

[0026] Example 1 (1) Establishment of the DIO mouse model on a high-fat diet Male C57BL / 6J mice (6-8 weeks old) were divided into a normal diet group (Ctrl group) and a high-fat diet group (DIO group). The mice in the high-fat diet group were fed a high-fat diet containing 60% lipids to establish a DIO (diet-induced obesity) model for 16 weeks. During the feeding period, the mice's food intake, body weight, and blood sugar levels were monitored weekly. After the modeling was completed, the body weight and adipose tissue weight were used to determine whether the modeling was successful. Figure 1 As shown in middle A, after 16 weeks of feeding, the body size of mice in the DIO group was significantly larger than that of mice in the Ctrl group.

[0027] (2) Expression of GDI2 in adipose tissue of mice in the DIO group Western Blot analysis was performed to analyze the expression of GDI2 in the adipose tissue of mice in the DIO group. The specific experimental steps are as follows: A transverse incision was made in the neck of DIO mice, and the skin was cut diagonally upward to expose the white adipose tissue covering the dorsal scapula. Two patches of brown adipose tissue (BAT) were observed after the white adipose tissue was lifted. Subsequently, the dorsal skin was cut downward to the tail and torn apart to expose the inguinal white adipose tissue (iWAT).

[0028] RIPA lysis buffer (Shanghai Biyuntian, P0013B) supplemented with PMSF (Beijing Solebao, P0100) and phosphatase inhibitors (Shanghai Biyuntian, P1081) was added to the harvested adipose tissue and ground using an electric grinder until a homogenate was obtained. The mixture was centrifuged at 12,000 rpm and 4°C for 15 minutes, removing the upper oil layer and lower precipitate. The intermediate layer was aspirated into a new EP tube and centrifuged again to obtain the extracted protein. The extracted protein was quantitatively analyzed using a Bradford assay (Bio-rad, 5000205). The protein loading system was then prepared by mixing the protein with 5× SDS-PAGE protein loading buffer (Shanghai Biyuntian Biotechnology Co., Ltd., P0015L) based on the quantitative results. Next, proteins were separated by electrophoresis on a 10% SDS-PAGE gel (Shanghai Yazyme, PG112). After loading the proteins, the upper gel was run at 80 V, and the lower gel was run at 120 V. The membrane was then transferred to a NC membrane (PALL, USA, 66485). The membrane was transferred in a transfer tank placed on ice and transferred at 100 V for 120 minutes. After blocking with 5% skim milk powder (BD Biosciences, USA, 232100, diluted in 1× TBST) at room temperature for 1 hour, the NC membrane was incubated with primary antibodies overnight at 4°C. The primary antibodies used included GDI2 (1:4000, Wuhan Tri-Tech Co., Ltd., 10116-1-AP) and HSP90 (1:2000, Wuhan Tri-Tech Co., Ltd., 13171-1-AP).

[0029] The next day, the primary antibody was recovered and washed three times with 1× TBST on a shaker. After washing, the NC membrane was incubated with a secondary antibody corresponding to the species of the primary antibody for 1 hour at room temperature. Finally, the immunoblot was detected using an ultrasensitive ECL chemiluminescence detection kit (New Saimei Biotechnology Co., Ltd., P10300). Protein bands were visualized using an automated chemiluminescence imaging system (Bio-Rad, Chemi Doc, USA).

[0030] The results are as follows Figure 1 As shown in Figures B and C, it can be seen that the expression of GDI2 in iWAT and BAT of DIO mice was increased, which suggests that GDI2 may be involved in the occurrence of obesity.

[0031] (3) Induction of adipocyte differentiation and expression of GDI2 Mouse mesenchymal stem cells C3H10T1 / 2 (purchased from the Chinese Academy of Sciences Cell Bank, SCSP-506) were used for induction differentiation as follows: After passage, cells were transferred to 6-well plates or 10 cm culture dishes and cultured in complete medium for 16-18 hours. This time was designated as D0 of differentiation induction. Cells were then cultured in differentiation induction medium containing 100x penicillin-streptomycin solution (Shanghai Biotech Co., Ltd., C0222) and 0.25% trypsin-EDTA (Thermo Fisher Scientific, USA, 25200-072). The specific steps for inducing differentiation are as follows: Day 0: DMEM medium (Gibco, USA, 11966025) + 10% FBS (fetal bovine serum, Gibco, USA, A3161001C) + dexamethasone (Sigma, D-1756, 2 μg / mL) + 3,3',5-triiodo-L-thyronine (3,3',5-Triiodo-L-thyronine, T3, Sigma, T-2877, 1 nM) + insulin (Biyuntian, P3376, 5 μg / mL) + rosiglitazone (Sigma, R-2408, 0.5 μM) + 3-Isobutyl-1-methylxanthine (3-Isobutyl-1-methylxanthine, IBMX, Sigma, I-5879, 0.5 mM) + indomethacin (Sigma, I-7378, 125 μM); Day 2: DMEM + 10% FBS + dexamethasone (2 μg / mL) + T3 (1 nM) + insulin (5 μg / mL) + rosiglitazone (0.5 μM); Day 4: DMEM + 10% FBS + dexamethasone (2 μg / mL) + T3 (1 nM) + insulin (5 μg / mL) + rosiglitazone (0.5 μM); On the 6th to 7th day of culture, the effect of inducing differentiation into mature adipocytes was determined by observing the ratio of induced differentiated lipid droplets.

[0032] Following a method similar to that described in (2) the expression of GDI2 in adipose tissue of mice in the DIO group, the cell clumps were analyzed by Western Blot for the expression of GDI2 during the differentiation of mesenchymal stem cells C3H10T1 / 2 into mature adipocytes. The results showed that GDI2 was gradually highly expressed during the differentiation process (e.g. Figure 1 (D) indicating that GDI2 is an important target for lipid metabolism regulation.

[0033] (4) Correlation analysis between GDI2 expression level in adipose tissue and BMI On this basis, real-time fluorescence quantitative PCR (qPCR) detection was performed on subcutaneous adipose tissue of people with different body mass index (BMI) collected in clinical studies (patients were informed and consented), and it was found that GDI2 (GDP dissociation inhibitor 2, NCBI ID 14569, Ensembl number ENSMUSG00000021218) showed high expression characteristics in the adipose tissue of obese people. The correlation analysis of GDI2 expression level and BMI was further carried out, such as Figure 1 As shown in Figures E and F, GDI2 expression in adipose tissue is significantly positively correlated with BMI. This indicates that GDI2 expression is closely related to obesity. This finding provides important experimental evidence for GDI2 as an obesity-related biomarker and potential therapeutic target.

[0034] Example 2 In order to further explore the effect of GDI2 on obesity, the Crisper Cas9 technology was used to construct Gdi2 flox / + Mice, such as Figure 2 As shown in A, a gRNA plasmid was designed for the intron region before the second exon and after the third exon of the GDI2 gene to achieve site-directed cutting of the target gene. At the same time, a donor plasmid was designed to insert a loxP sequence and an endonuclease sequence into the two intronic cutting sites of GDI2 for PCR-RELP experimental identification of the positive mouse genotype. Subsequently, sgRNAs were transcribed in vitro, and after the donor plasmid was linearized with restriction endonucleases, the two sgRNAs, the donor plasmid, and the Cas9 plasmid were microinjected into the mouse in vitro fertilized eggs. By genotyping the born mice, F0 generation positive chimeric mice were obtained, and the positive mice were mated with wild-type mice to obtain F1 generation. Gdi2 flox / + mouse.

[0035] The F1 generation Gdi2 flox / + Mice mate with each other and reproduce Gdi2 flox / flox Mouse (abbreviated as Gdi2 f / f mice), Adipoq-Cre mice were crossed with Gdi2 flox / + Mice were mated to obtain GDI2 conditional knockout in adipose tissue. Gdi2 flox / flox Adipoq Cre Mouse, referred to as Gdi2 AKO Mice. For daily mouse breeding, use Gdi2 AKO Mice and Gdi2 f / f Mice are mated for breeding (e.g. Figure 2 The genotype of the mice was confirmed by tail genotyping (as shown in Figure B). Figure 2 Mouse genotype identification primers are as follows: GDI2 flox Forward (sequence 1): 5'-CAGCATTACACACTTAGACAAGGAGG-3' GDI2 flox Reverse (sequence 2): 5'-TTAGGTGCGGGAAATGAGGAAAC-3' Adipoq Cre Forward (Sequence 3): 5'-GGATGTGCCATGTGAGTCTG-3' Adipoq Cre Reverse (sequence 4): 5′-ACGGACAGAAGCATTTTCCA-3′.

[0036] right Gdi2 AKO Mice and Gdi2 f / f Mice were fed with normal feed and observed to be 8 weeks old. Gdi2 AKO Mice and Gdi2 f / f There were no significant differences in the body size and weight of the mice (e.g. Figure 2 (as shown in D and E in the figure).

[0037] To determine the knockout efficiency, Western Blot was used to detect Gdi2 AKO Mice and Gdi2 f / f The expression of GDI2 in mouse iWAT and BAT, such as Figure 2 As shown in F, Gdi2 AKO GDI2 is almost not expressed in mouse adipose tissue, and the small amount of residual expression comes from other cells in the adipose tissue, such as fibroblasts, endothelial cells, and macrophages.

[0038] from Gdi2 AKO Mice and Gdi2 f / f Primary adipocytes were extracted from iWAT and BAT of mice and induced to differentiate into mature adipocytes by stromal vascular fraction (SVF). The protein level of GDI2 in primary adipocytes was detected by Western Blot. Figure 2 As shown in G, after knocking out GDI2 Gdi2 AKO GDI2 is completely absent in primary mouse adipocytes.

[0039] To clarify Gdi2 AKO The sensitivity of mice to high-fat diet-induced obesity was compared with Gdi2 f / f The mice were fed with a high-fat diet containing 60% lipid for 16 weeks to establish the DIO mouse model. Gdi2 AKO -DIO mice and Gdi2 f / f -DIO mice. During the feeding period, the mice were weighed weekly and found Gdi2 AKO -DIO mice had significantly lower body weight Gdi2 f / f -DIO mice, and significant statistical differences appeared from week 7 (e.g. Figure 3 (as shown in A in the figure).

[0040] The growth of mice was observed and found that Gdi2 AKO iWAT, BAT, and epididymal white adipose tissue (eWAT) of DIO mice Gdi2 f / f significantly smaller than mice (e.g. Figure 3 B), and Gdi2 AKO -DIO mice had a significant reduction in adipose tissue weight (e.g. Figure 3 (as shown in C in the figure).

[0041] right Gdi2 AKO -DIO mice and Gdi2 f / f -DIO mice were subjected to oral glucose tolerance test. One week before the experiment, the mice were trained to adapt to the tail pinching to avoid stress-induced hyperglycemia. After the experimental mice were fasted and deprived of water for 12 hours, each mouse in each group was weighed, and blood was collected from the tail tip to remove the first drop to measure the basal blood glucose level. The glucose injection volume was calculated according to 2.0 g / kg mouse body weight, and the glucose dilution was injected into the abdominal cavity respectively. The blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes later using a portable blood glucose meter, and a line graph was drawn to observe the trend of curve changes between different groups, as shown in Figure 2. Figure 3 As shown in D, Gdi2 AKO -DIO mice have a higher energy tolerance to glucose Gdi2 f / f -DIO mice.

[0042] To understand the energy metabolism of mice, Gdi2 AKO -DIO mice and Gdi2 f / f -DIO mice were placed in a metabolic cage animal monitoring system and the mouse metabolic cage experiment was conducted at 22°C with an airflow rate of 380 mL / min. The data were monitored using the PhenoMaster high-throughput intelligent metabolic analysis system. After weight standardization, the changes in the mice's oxygen consumption and heat production were calculated. Figure 3 As shown in E, F, G and H, Gdi2 AKO -DIO mice had significantly higher oxygen consumption and heat production than Gdi2 f / f -DIO mice.

[0043] The eyeballs of mice were removed and blood was collected. After the blood was collected, the blood was centrifuged at 3000 rpm for 3 min and the supernatant was collected as the mouse eye serum. The test kit of Nanjing Jiancheng Bioengineering was used according to the instructions. Gdi2 AKO -DIO mice and Gdi2 f / f -DIO mouse eye serum triglyceride (TG), cholesterol (CHO), blood glucose (Glucose) and free fatty acids (NEFA). Figure 3 As shown in I, J, and K, it was found Gdi2 AKO -DIO mouse ocular serum TG, glucose and CHO levels were reduced, indicating that GDI2 deficiency inhibited the increase of blood lipids and blood glucose caused by obesity; Figure 3 As shown in L, Gdi2 AKO NEFA levels in the ocular serum of -DIO mice were elevated, indicating that GDI2 deficiency promoted the hydrolysis of lipid droplets.

[0044] Figure 3 Middle M shows the HE staining results of iWAT, eWAT and BAT of two groups of mice. The morphological changes of adipocytes were observed. Gdi2 AKO -DIO mice had significantly smaller fat cells.

[0045] In summary, adipose tissue-specific GDI2 knockout can alleviate high-fat diet-induced obesity and improve changes in metabolic indicators caused by obesity.

[0046] Example 3 Based on Examples 1 and 2, this Example 3 provides an application of GDI2 as a weight loss target, specifically its use in preparing a drug for treating obesity, especially its use in preparing a drug for treating physiological obesity caused by an overnutrition diet.

[0047] Obesity treatment drugs are GDI2 inhibitors. Inhibiting GDI2 can: 1) significantly improve the weight and fat cell size of obese individuals, reduce the fat droplets in subcutaneous and reproductive fat, directly reduce fat accumulation, and thus alleviate obesity symptoms; lactose effectively inhibits the increase in blood lipids (such as triglycerides and cholesterol) and blood sugar levels, and improves glucose intolerance; 3) enhance the individual's energy consumption, promote the body's energy metabolism balance, increase energy expenditure, and thereby reduce the storage of excess energy in the form of fat.

[0048] As a preferred embodiment, the GDI2 inhibitor can inhibit GDI2 through various pathways, including inhibiting the expression of the GDI2 gene, knocking down or knocking out the GDI2 gene, or inhibiting the synthesis and activity of the GDI2 protein or promoting its degradation.

[0049] Knocking down the GDI2 gene involves removing all or part of the coding gene sequence, introducing a frameshift mutation that prevents it from producing functional protein, removing or changing regulatory components such as the promoter to prevent transcription of the coding gene sequence, or preventing translation by binding to mRNA; knocking out the GDI2 gene involves causing the gene to undergo deletion mutations, insertion mutations, or base substitutions, resulting in its loss of activity.

[0050] In terms of substance type, GDI2 inhibitors can be selected from nucleic acid molecules, carbohydrates, lipids, small molecule chemical drugs, antibody drugs, peptides, proteins or viruses, etc.

[0051] At the same time, to meet the needs of pharmaceutical applications, GDI2 inhibitors may also contain one or more pharmaceutically acceptable excipients and carriers, and can be made into various dosage forms such as tablets, capsules, aerosols, pills, powders, solutions, etc. to meet different administration needs.

[0052] Wherein, the excipient can be selected from glucose, sucrose, sorbitol, mannose, starch, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose and / or water; The vector can be selected from a DNA plasmid vector, a lentiviral vector, a retroviral vector, a poxvirus vector, a herpes simplex virus vector, an adenovirus vector, an adeno-associated virus vector, a liposome bound to a DNA plasmid, a molecular couple bound to a DNA plasmid and / or a polymer bound to a DNA plasmid, etc.

[0053] GDI2 inhibitors can effectively improve the weight and fat mass of obese individuals, lower blood lipid and blood sugar levels, improve glucose intolerance, enhance energy consumption, etc., thereby inhibiting the obese phenotype and abnormal glucose and lipid metabolism, achieving the effect of weight loss, and can be used to prepare drugs for the prevention and / or treatment of obesity.

[0054] Example 4 Based on Examples 1 and 2, this Example 4 provides a method for determining the therapeutic potential of a candidate drug by detecting its inhibitory effect on GDI2 gene expression, that is, a method for screening obesity therapeutic drugs using GDI2, which specifically includes the following steps: S1. Select obese individuals other than humans as experimental models and measure the initial expression level of the GDI2 gene in their adipose tissue. The obese individuals other than humans herein may be high-fat diet-induced obese mice, rats, rabbits, or non-human primates such as rhesus monkeys; S2. administering the candidate drug to the obese individual of step S1; S3. After the drug administration is completed, the expression level of the GDI2 gene in the adipose tissue of the obese individual is measured again; S4. If, after administration of the candidate drug, the expression level of the GDI2 gene in the adipose tissue of obese individuals is significantly lower than that before administration of the candidate drug, with a statistical difference of P < 0.05, it indicates that the candidate drug can effectively inhibit the expression of the GDI2 gene, meets the screening criteria for obesity treatment drugs, and the candidate drug is determined to be an obesity treatment drug.

[0055] In order to more scientifically evaluate the regulatory effect of candidate drugs on GDI2 gene expression, in the method of screening obesity therapeutic drugs, in addition to the experimental group (candidate drug group) administered with the candidate drug, a blank control group and a model control group can also be set up.

[0056] Blank control group: Select individuals of the same strain and age as the obese individuals who are fed a normal diet, do not apply any candidate drugs, and only give an equal amount of solvent, such as normal saline.

[0057] Model control group: Obese individuals are also used, but no candidate drug is administered, and only an equal amount of solvent, such as normal saline, is given.

[0058] Before the experiment began, the initial expression levels of the GDI2 gene in the adipose tissue of the three groups of animals were recorded. During the administration process, the feeding conditions of the groups remained consistent. The candidate drug group was administered with the candidate drug, while the blank control group and the model control group were administered with an equal amount of solvent instead of the candidate drug. After the administration, the expression levels of the GDI2 gene in the adipose tissue of the three groups of animals were measured again, and the data were statistically analyzed.

[0059] During the statistical analysis, the GDI2 gene expression levels of the blank control group and the model control group were first compared to verify the high expression characteristics of the GDI2 gene in obese individuals; then, the candidate drug group and the model control group were compared. If the GDI2 gene expression level of the candidate drug group was significantly lower than that of the model control group (P<0.05) and close to or reached the level of the blank control group, the candidate drug could be determined to be a potential obesity treatment drug.

[0060] Although the present invention has been described in detail with reference to the accompanying drawings and in conjunction with preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be easily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. An application of GDI2 as a weight loss target in the preparation of obesity treatment drugs.

2. The use according to claim 1, characterized in that Obesity treatment drugs are GDI2 inhibitors.

3. The use according to claim 2, characterized in that A GDI2 inhibitor is a drug that inhibits the expression of the GDI2 gene, a drug that knocks down the GDI2 gene, or a drug that knocks out the GDI2 gene.

4. The use according to claim 3, characterized in that The drug for knocking down the GDI2 gene is any drug that can prevent the expression of the gene encoding the GDI2 protein, including but not limited to removing all or part of the coding gene sequence, introducing a frameshift mutation so that no functional protein is produced, removing or changing the regulatory components so that the coding gene sequence is not transcribed, and preventing translation by binding to mRNA.

5. The use according to claim 3, characterized in that The drug for knocking out the GDI2 gene is any drug that can cause the GDI2 gene to mutate and thereby lose its activity, and the mutation form is selected from deletion mutation, insertion mutation and / or base substitution.

6. The use according to claim 2, characterized in that GDI2 inhibitors are drugs that inhibit the synthesis of GDI2 protein, drugs that inhibit the activity of GDI2 protein, or drugs that promote the degradation of GDI2 protein.

7. The use according to any one of claims 2 to 6, characterized in that The GDI2 inhibitor may also include one or more pharmaceutically acceptable excipients.

8. The use according to any one of claims 2 to 6, characterized in that The GDI2 inhibitor further includes one or more pharmaceutically acceptable carriers.

9. A method for screening drugs for treating obesity, characterized in that: The steps include: S1. Measuring the initial expression level of the GDI2 gene in adipose tissue of obese individuals other than humans; S2. administering the candidate drug to the obese individual of step S1; S3. measuring the expression level of the GDI2 gene in adipose tissue of the obese individual after administration of the candidate drug; S4. If, after administration of the candidate drug, the expression level of the GDI2 gene in the adipose tissue of the obese individual is significantly lower than the initial expression level, with a statistical difference of P < 0.05, the candidate drug is determined to be an obesity treatment drug.

10. The method for screening a drug for treating obesity according to claim 9, wherein: The obese individuals other than humans were high-fat diet-induced obese mice.

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