Application of DEC2 gene as target in obesity research

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of DEC2 gene as a target spot in obesity research. It is found for the first time that in a mouse obesity model, DEC2 expression in hypothalamic arcuate nucleus (ARC) is remarkably increased, and leptin resistance and obesity can be remarkably induced through DEC2 overexpression in ARC and paraventricular nucleus (PVH); the ARCPOMC DEC2 plays a key role in mediating obesity, and the effect of the ARCPOMC DEC2 is located at the downstream of a leptin receptor; the overall behavior, morphology and nerve electrophysiological function prove that the conditional overexpression DEC2 of the neurons of the ARCPOMC significantly induces the POMC neuronal leptin resistance, but the influence on the insulin reactivity is small. The invention proves that the knockout of DEC2 is practical and effective for treating obesity and ingestion increase induced by high fat diet, and is expected to be developed into a novel therapeutic drug to make up for the deficiency of current drug choices.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of DEC2 gene as a target in obesity research. Background Art

[0002] Obesity is a chronic systemic disease caused by excessive fat accumulation (Rubino F, Cummings DE, Eckel RH, et al. Definition and diagnostic criteria of clinical obesity. Lancet Diabetes Endocrinol. 2025 Jan 9: S2213-8587 (24) 00316-4.), and has become the most prominent and severe health challenge in the world. According to the latest data from the World Obesity Report (2024 Edition), in 2020, there were about 2.2 billion obese and overweight people in the world (accounting for 42% of the world's total population), and this number is expected to grow rapidly to 3.3 billion (about 54%) in 2035 (World Obesity Federation. World Obesity Atlas 2024. London: World Obesity Federation, 2024.). The obesity problem in my country is also not optimistic. By 2018, the proportion of overweight and obese people in China had reached 50.7%, and is expected to reach 70.5% (about 610 million) by 2030. China is one of the countries with the fastest growing proportion of obese population in the world (National Health Commission. Obesity Diagnosis and Treatment Guidelines (2024 Edition). 2024.10.12.).Against the backdrop of an aging global population, obesity and the metabolic disorders it causes have become a major public health problem that increases the global medical and economic burden (Global Nutrition Target Collaborators. Global, regional, and national progress toward the 2030 global nutrition targets and forecasts to 2050: a systematic analysis for the Global Burden of Disease Study 2021. Lancet. 2025 Dec 21; 404(10471): 2543-2583; GBD 2021 Risk Factors Collaborators. Global burden and strength of evidence for 88 risk factors in 204 countries and 811 subnational locations, 1990-2021: a systematic analysis for the Global Burden of Disease Study 2021. Lancet. 2024 May 18; 403(10440): 2162-2203; World Obesity Federation. The economic impact of overweight & obesity in 2020and 2060.Second edition with estimates for 161 countries.2022.). Therefore, it is of great significance to conduct research on the pathogenic mechanism of obesity and weight loss strategies.

[0003] The arcuate nucleus of the hypothalamus (ARC) is one of the most sensitive brain regions for sensing metabolic changes in the body (Lei Y, Liang X, Sun Y, et al. Region-specific transcriptomic responses to obesity and diabetes in macaque hypothalamus. Cell Metab. 2024 Feb 6; 36 (2): 438-453. e6.), but the specific mechanism of how ARC mediates metabolic disorders during the development of obesity is still not completely clear. The arcuate nucleus of the hypothalamus (ARC) is the brain region most significantly affected by obesity and is extremely sensitive to metabolic disorders. Different types of neurons in ARC (such as POMC and AgRP neurons) can sense nutrition-related signals in blood and cerebrospinal fluid through humoral pathways. The two antagonize each other and play a key role in regulating feeding behavior and maintaining systemic energy metabolism homeostasis (Jin K, Yao Z, van Velthoven CTJ, et al. Brain-wide cell-type-specific transcriptomic signatures of healthy ageing in mice. Nature. 2025 Jan 1.). Among them, AgRP neurons are mainly responsible for sensing hunger signals (such as growth hormone releasing peptide), mediating food intake and reducing energy expenditure, while POMC neurons sense satiety signals (such as leptin, insulin and glucagon-like peptide), reducing food intake and increasing energy expenditure (Chen J, Cai M, Zhan C. Neuronal Regulation of Feeding and Energy Metabolism: A Focus on the Hypothalamus and Brainstem. Neurosci Bull. 2024 Dec 20.).

[0004] As a key satiety signal, leptin is an important regulatory factor secreted by adipose tissue and regulates eating behavior and energy metabolism. It inhibits eating behavior, increases energy consumption, and promotes fat utilization by acting on multiple neuronal subpopulations expressing leptin receptors in the brain (such as POMC neurons) (Tilg H, Ianiro G, Gasbarrini A, et al. Adipokines: masterminds of metabolic inflammation. Nat Rev Immunol. 2024 Nov 7.). However, when leptin resistance occurs, this negative feedback mechanism of energy perception and regulation is blocked, inducing the occurrence and development of obesity (Tadross JA, Steuernagel L, Dowsett GKC, et al. A comprehensive spatio-cellular map of the human hypothalamus. Nature. 2025 Feb 5.). Although significant progress has been made in the analysis of leptin receptors and their downstream signaling in the past 30 years (Münzberg H, Heymsfield SB, Berthoud HR, et al. History and future of leptin: Discovery, regulation and signaling. Metabolism. 2024 Dec; 161: 156026.), the specific molecular mechanism of leptin resistance is still not completely clear, so there has been no major breakthrough in the development of drugs targeting leptin resistance (Perakakis N, Mantzoros CS. Evidence from clinical studies of leptin: current and future clinical applications in humans. Metabolism. 2024 Dec; 161: 156053.). Leptin resistance can occur at various levels such as signal perception, receptor binding, and signal transduction. The most widely studied is the mechanism of leptin resistance-induced obesity caused by defects and mutations at the leptin receptor level.The team of Dr. Guan Dongxian and Professor Umut Ozcan discovered that the leptin receptor has dynamic acetylation modification, and histone deacetylase 6 (HDAC6) can deacetylate the leptin receptor and promote its activity decline, participating in leptin resistance (Guan D, Men Y, Bartlett A, et al. Central inhibition of HDAC6 re-sensitizes leptin signaling during obesity to induce profound weight loss. Cell Metab. 2024 Apr 2; 36 (4): 857-876. e10.). Professor Kazuhiro Nakamura found that the cilia of neurons expressing melanocortin-4 receptor (MC4R) gradually shorten with age, weakening the activation of the leptin-mediated melanocortin signaling pathway in POMC neurons, leading to leptin resistance and age-related obesity (Oya M, Miyasaka Y, Nakamura Y, et al. Age-related ciliopathy: Obesogenic shortening of melanocortin-4 receptor-bearing neuronal primary cilia. Cell Metab. 2024 May 7; 36 (5): 1044-1058. e10.). These two works are important breakthroughs in recent years in analyzing the mechanism of leptin resistance at the level of leptin signal perception, but the specific mechanism of abnormal leptin signal transduction during leptin resistance is still not fully understood. Summary of the invention

[0005] The present invention found that in the mouse obesity model, the expression of DEC2 in the arcuate nucleus (ARC) of the hypothalamus increased significantly, and the POMC neurons in the ARC were the key neuron type for DEC2-induced leptin resistance. POMC Neuronal conditional knockout of DEC2 significantly improves obesity and increased food intake induced by a high-fat diet. Based on this, the present invention was completed.

[0006] In a first aspect, the present invention provides a biomarker for diagnosing obesity, wherein the biomarker is DEC2; when the expression of DEC2 in a biological sample of a patient increases significantly, it indicates that the patient is at risk of obesity.

[0007] Furthermore, the patient biological sample is hypothalamus.

[0008] Furthermore, when DEC2 expression in the arcuate nucleus or paraventricular nucleus of the hypothalamus of a patient is significantly increased, it indicates that the patient is at risk of obesity.

[0009] Furthermore, when the expression of DEC2 in the arcuate nucleus of the patient's hypothalamus is significantly increased, it indicates that the patient is at risk of obesity.

[0010] Preferably, when the expression of DEC2 in POMC neurons in the arcuate nucleus of the hypothalamus of a patient is significantly increased, it indicates that the patient is at risk of obesity.

[0011] In a second aspect, the present invention provides the use of DEC2 in the preparation of a reagent for diagnosing obesity, wherein the reagent contains a reagent for detecting the content of DEC2 in a biological sample of a patient; when the expression of DEC2 in the biological sample of a patient increases significantly, it indicates that the patient is at risk of obesity.

[0012] Furthermore, the patient biological sample is hypothalamus.

[0013] Furthermore, when DEC2 expression in the arcuate nucleus or paraventricular nucleus of the hypothalamus of a patient is significantly increased, it indicates that the patient is at risk of obesity.

[0014] Furthermore, when the expression of DEC2 in the arcuate nucleus of the patient's hypothalamus is significantly increased, it indicates that the patient is at risk of obesity.

[0015] Preferably, when the expression of DEC2 in POMC neurons in the arcuate nucleus of the hypothalamus of a patient is significantly increased, it indicates that the patient is at risk of obesity.

[0016] In a third aspect, the present invention provides a kit for diagnosing obesity, the kit comprising a reagent for detecting the content of DEC2 in a biological sample of a patient; when the expression of DEC2 in the biological sample of a patient increases significantly, it indicates that the patient is at risk of obesity.

[0017] Furthermore, the patient biological sample is hypothalamus.

[0018] Furthermore, when DEC2 expression in the arcuate nucleus or paraventricular nucleus of the hypothalamus of a patient is significantly increased, it indicates that the patient is at risk of obesity.

[0019] Furthermore, when the expression of DEC2 in the arcuate nucleus of the patient's hypothalamus is significantly increased, it indicates that the patient is at risk of obesity.

[0020] Preferably, when the expression of DEC2 in POMC neurons in the arcuate nucleus of the hypothalamus of a patient is significantly increased, it indicates that the patient is at risk of obesity.

[0021] Furthermore, the kit may be one or more of an ELISA detection kit, a colloidal gold detection kit, an immunohistochemistry kit, an immunofluorescence kit and / or an in situ hybridization staining kit.

[0022] Furthermore, the diagnostic method of the kit includes one or more of a direct method, an indirect method, a double antibody sandwich method and / or a competitive method.

[0023] In a fourth aspect, the present invention further provides a method for screening drugs for treating obesity, wherein the method is to screen drugs that inhibit the activity of DEC2 gene or reduce the expression of DEC2 gene, and the method comprises the following steps:

[0024] S01: administering the screened drug to the model animal;

[0025] S02: Detect the expression level of DEC2 gene in model animals;

[0026] S03: If the expression level of DEC2 is significantly reduced, it is considered that the screened drug can be used as a drug for treating obesity.

[0027] Furthermore, the animal model refers to an animal with high expression of DEC2.

[0028] Furthermore, the drug includes a small molecule compound or a gene drug that specifically inhibits the function of the DEC2 protein.

[0029] Furthermore, the small molecule compound that specifically inhibits the function of DEC2 protein is obtained by screening a compound library.

[0030] Furthermore, the compound library includes a diversity compound library, a target compound library, a known active compound library, a marketed drug library, a natural product compound library, a fragment library, a building block or a hotspot.

[0031] In a fifth aspect, the present invention provides an obese animal model, wherein the animal model refers to an animal with high expression of DEC2.

[0032] In a sixth aspect, the present invention provides use of a preparation for inhibiting DEC2 in the preparation of a drug for treating obesity, wherein the drug targets DEC2 and inhibits DEC2 expression.

[0033] Furthermore, the drug exerts a weight loss effect by improving leptin resistance.

[0034] Furthermore, the agent for inhibiting DEC2 may be a protein, a polypeptide or a small molecule compound that inhibits the function of DEC2 protein.

[0035] Furthermore, the DEC2-inhibiting agent may be a substance that inhibits DEC2 protein synthesis or promotes DEC2 protein degradation or knocks down or knocks out the DEC2 gene.

[0036] Furthermore, the DEC2 knockdown agent may be any substance that can render the gene encoding the DEC2 protein unexpressable.

[0037] Furthermore, the DEC2 knockout preparation can be a substance that achieves the goal of preventing the hypothalamic neurons of obese patients from producing the functional protein product of the DEC2 gene in any manner, such as 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, preventing translation by binding to mRNA, etc.

[0038] Furthermore, the DEC2 knockout agent may be any substance that can cause the DEC2 gene to mutate (the mutation may be a deletion mutation and / or an insertion mutation and / or a base substitution) and thereby lose its activity.

[0039] Furthermore, one or more pharmaceutically acceptable carriers may be added to the drug.

[0040] Furthermore, the drug can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories.

[0041] Furthermore, the preparation may be one or more of a conventional preparation, a sustained-release preparation and / or a controlled-release preparation.

[0042] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0043] Furthermore, the drug can be administered via injection, cavity administration or respiratory tract administration.

[0044] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration includes nasal administration.

[0045] Beneficial Effects

[0046] 1. The present invention first discovered that in a mouse obesity model induced by a high-fat diet, the expression of DEC2 in the ARC was significantly increased; overexpression of DEC2 in the ventromedial nucleus (VMH) and dorsomedial nucleus (DMH) of the hypothalamus did not affect food intake and body weight, but overexpression of DEC2 in the ARC and paraventricular nucleus (PVH) could significantly induce leptin resistance and obesity;

[0047] 2. The present invention found that only ARC POMCConditional overexpression of DEC2 significantly induced obesity in mice, which was manifested by increased food intake, weight gain, fat accumulation, and decreased metabolism; however, conditional overexpression of DEC2 in AgRP neurons (AgRP+), microglia (CX3CR1+), and astrocytes (ALDH1L1+) had no significant effect on the food intake and body weight of mice; this proved that ARC POMC It is the key neuron for DEC2-mediated obesity;

[0048] 3. The present invention discovers ARC POMC Neuronal conditional knockout of DEC2 significantly improved obesity and increased food intake induced by a high-fat diet, but had no effect on the body weight and food intake of leptin receptor mutant (db / db) mice; POMC DEC2 plays a key role in mediating obesity, and its action is downstream of the leptin receptor;

[0049] 4. The present invention discovers ARC POMC Conditional overexpression of DEC2 in neurons had no significant effect on insulin, OGG, and ITT in mice, but significantly induced an increase in leptin levels in mice and significantly reduced leptin-mediated food intake, weight loss, and p-STAT3 expression; the results of this part confirmed ARC in terms of overall behavior and morphology POMC Conditional overexpression of DEC2 in neurons significantly induced leptin resistance in POMC neurons, but had little effect on insulin responsiveness;

[0050] 5. The present invention discovers ARC POMC Conditional overexpression of DEC2 in neurons significantly inhibited the leptin-mediated increase in firing frequency and depolarization in POMC neurons. This part of the results further confirmed the role of ARC in neurophysiological function. POMC Neuronal conditional overexpression of DEC2 significantly induced leptin resistance in POMC neurons.

[0051] The above results prove that the key target of leptin resistance in the arcuate nucleus (ARC) is DEC2, and gene regulation targeting DEC2 can significantly improve central leptin resistance and play an effective role in weight loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Analysis of hypothalamic transcriptome in mice with high-fat diet-induced obesity. A. Volcano plot of differentially expressed genes; B. Heat map of differentially expressed genes; C. Functional enrichment of differentially expressed genes.

[0053] Figure 2The effect of ARC overexpression of DEC2 on mouse obesity. A. Schematic diagram of stereotaxic positioning; B. Example of stereotaxic positioning of ARC overexpression of DEC2 and statistical analysis of its effect on mouse body weight; C. Example of stereotaxic positioning of VMH overexpression of DEC2 and statistical analysis of its effect on mouse body weight; D. Example of stereotaxic positioning of DMH overexpression of DEC2 and statistical analysis of its effect on mouse body weight; E. Example of stereotaxic positioning of PVH overexpression of DEC2 and statistical analysis of its effect on mouse body weight. ***P<0.001.

[0054] Figure 3 For ARC POMC Effects of conditional overexpression of DEC2 on obesity in mice. A. Experimental flow chart; B. Example of two groups of mice; ARC POMC Effects of conditional overexpression of DEC2 on mice: C. Statistical analysis of body weight gain; D. Statistical analysis of cumulative food intake; E. Statistical analysis of the effect of daily cumulative food intake; F. Statistical analysis of the number of daily cumulative food intake; G. Statistical analysis of daily cumulative food intake time; H. Statistical analysis of the effect of daily energy expenditure; I. Statistical analysis of daily respiratory exchange ratio; J. Statistical analysis of daily activity; K. Examples of magnetic resonance fat imaging and white fat HE staining; L. Statistical analysis of visceral fat and subcutaneous fat fraction (PDFF); M. Statistical analysis of white fat mass and volume. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0055] Figure 4 The effect of conditional overexpression of DEC2 in other types of ARC cells on food intake and body weight. A. Experimental flow chart; B. Example of two groups of mice; ARC AgRP Effects of conditional overexpression of DEC2 in neurons on mice: C. Statistical analysis of cumulative body weight gain; D. Statistical analysis of cumulative food intake; E. Experimental flow chart; F. Example of two groups of mice; Effects of conditional overexpression of DEC2 in ARC microglia on mice: G. Statistical analysis of cumulative body weight gain; H. Statistical analysis of cumulative food intake; I. Experimental flow chart; J. Example of two groups of mice; Effects of conditional overexpression of DEC2 in ARC astrocytes on mice: K. Statistical analysis of cumulative body weight gain; L. Statistical analysis of cumulative food intake.

[0056] Figure 5 Conditional knockout of ARC POMC Effects of DEC2 on body weight and food intake in mice. A. Experimental flow chart; conditional knockout of ARC POMC DEC2 knockdown in mice: B. Statistical analysis of cumulative body weight gain; C. Statistical analysis of cumulative food intake; D. Experimental flow chart; ARC DEC2 knockdown in db / db mice: E. Statistical analysis of cumulative body weight gain; F. Statistical analysis of cumulative food intake. ****P<0.0001.

[0057] Figure 6 Single-cell omics sequencing and biological function analysis of ARC overexpressing DEC2. A. Dimensionality reduction distribution of single cells in the arcuate nucleus of the hypothalamus; B. Expression profiles of marker molecules of different subpopulations of neurons; C. Differential enrichment of transcription factors and their activities in POMC neurons; D. Effects of DEC2 overexpression in POMC neurons on adipogenesis, promelanocortin signaling pathway, and insulin signaling pathway.

[0058] Figure 7 For ARC POMC Effects of conditional overexpression of DEC2 in neurons on glucose and insulin tolerance and leptin sensitivity in mice. POMC Effects of conditional overexpression of DEC2 in neurons on mice: A. Statistical analysis of the effects on random blood glucose; B. Statistical analysis of the effects on fasting blood glucose; C. Statistical analysis of the effects on plasma insulin; D. Statistical analysis of the effects on ITT; E. Statistical analysis of the effects on GTT; F. Statistical analysis of the effects on plasma leptin; G. Statistical analysis of the effects on ARC Lepr gene expression; H. Statistical analysis of leptin-mediated reduction in food intake; I. Statistical analysis of leptin-mediated weight loss; J. Statistical analysis of leptin-mediated ARC POMC Statistical analysis of p-STAT3 expression; K. Statistical analysis of leptin-induced ARC p-STAT3 expression; L. Statistical analysis of leptin-induced LV-Bhlhe41 stably transfected GT1-7 cells p-STAT3 expression.

[0059] Figure 8 For ARC POMC Effects of conditional overexpression of DEC2 on the electrophysiological responses of POMC neurons in mice to leptin. A. Discharge pattern of POMC neurons in the arcuate nucleus; B. Discharge pattern of ARC neurons in two groups of mice POMC Examples of spontaneous discharges before and after leptin perfusion; C. ARC of two groups of mice POMC The number of cells recorded in different firing patterns; ARC of two groups of mice POMC Before and after leptin perfusion: D. Statistical analysis of action potential frequency changes; E. Statistical analysis of action potential amplitude changes; F. Statistical analysis of cell membrane depolarization; G. Statistical analysis of spontaneous discharge frequency ratio changes. **P<0.01; ****P<0.0001. DETAILED DESCRIPTION

[0060] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0061] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0062] DEC2 (UniProtNo.Q99PV5): It is an important rhythm regulation gene (coding gene Bhlhe41). It is known as the famous "short sleep gene" because the P384R mutation of this gene affects the circadian rhythm and sleep time. In addition, as a transcriptional regulatory factor, the role of DEC2 in the occurrence and development of tumors has also attracted much attention.

[0063] The complete amino acid sequence of DEC2 is shown in SEQ ID NO.1:

[0064] MDEGIPHLQERQLLEHRDFIGLDYSSLYMCPKRSLKRDDTKDTYKLPHRLIEKKRRDRI

[0065] NECIAQLKDLLPEHLKLTTLGHLEKAVVLELTLKHLKALTALTEQQHQKIIALQNGERSLK

[0066] SPVQADLDAFHSGFQTCAKEVLQYLARFESWTPREPRCAQLVSHLHAVATQLLTPQVPSG

[0067] RGSGRAPCSAGAAAASGPERVARCVPVIQRTQPGTEPEHDTDTDSGYGGEAEQGRAAVK

[0068] QEPPGDSSPAPKRPKLEARGALLGPEPALLGSLVALGGGAPFAQPAAAPFCLPFYLLSPSAA

[0069] AYVQPWLDKSGLDKYLYPAAAAPFPLLYPGIPAAAAAAAAAAFPCLSSVLSPPPEKAGAT

[0070] AGAPFLAHEVAPPGPLRPQHAHSRTHLPRAVNPESSQEDATQPAKDAP

[0071] AAV-CMV: As a control virus for AAV-CMV-Bhlhe41, it has the same vector and promoter structure as AAV-CMV-Bhlhe41 and does not carry the gene sequence for overexpressing Bhlhe41.

[0072] Example 1 Main research techniques and methods

[0073] 1. Obese mouse model

[0074] A. High-fat diet-induced obesity (DIO) mouse model: 8-10 week-old C57BL / 6J mice were fed a high-fat diet (Research Diets, D12492) for 8 weeks, with an average body weight gain of 10.8±5.1 g.

[0075] B.ARC POMC DEC2 overexpression obese mouse model: 8-10 week old POCM-Cre mice (C57BL / 6J) were used, and AAV-Bhlhe41-dio was stereotactically injected into bilateral ARC to conditionally overexpress DEC2. Regular diet conditions induced obesity in mice, and the average body weight increased by 28.2±6.6 g after 8 weeks.

[0076] C. Lepr spontaneous mutation db / db mouse model: db / db mice and control db / m mice were purchased from Sibeifu (Suzhou) Biotechnology Co., Ltd., with C57BLKS / Niu mice as the background, Dock7m was introduced to maintain the breeding of Lepr mutant mice, and Lepr gene mutant mice were constructed; sgRNA sites were designed in exon19 and both sides of the Lepr gene, and CRISPR / Cas9 technology and fertilized egg injection technology were used to cut exon19 and delete 479bp, resulting in gene function destruction. This study used 8-10 week old db / db and db / m mice to study the upstream and downstream relationship between DEC2 and Lepr.

[0077] 2. Construction of transgenic mice

[0078] A. Taking the conditional knockout of DEC2 in POMC neurons as an example: select the POMC neuron cell-specific promoter POMC and cross the POMC-Cre mice with Bhlhe41 fl / fl Mating of mice to produce POMC-Bhlhe41 - / - In addition, stereotaxic injection and the Cre / loxP system can also be used to achieve conditional knockout of the Bhlhe41 gene in POMC neurons within the ARC.

[0079] B. Take POMC-tdTomato fluorescently labeled transgenic mice as an example: POMC-Cre mice were mated with Rosa26-LSL-tdTomato mice to produce POMC-tdTomato mice, and the Cre / 1oxP system was used to achieve tdTomato fluorescent labeling of POMC neurons in the brain.

[0080] C. Taking conditional overexpression of DEC2 in microglia as an example: select the microglia-specific promoter CX3CR1 (CX3CR1-Cre-ERT2 mice), and use stereotaxic injection, Cre / loxP system, and tamoxifen induction to achieve conditional overexpression of DEC2 in microglia in ARC.

[0081] 3. Sugar, insulin tolerance and leptin sensitivity test

[0082] A. Glucose tolerance test (GTT): After overnight fasting, mice were intraperitoneally injected with 2 g / kg 20% ​​glucose, and the blood glucose level in the tail vein of mice was measured at 0, 15, 30, 60, 90 and 120 min after glucose injection.

[0083] B. Insulin tolerance test (ITT): After fasting, mice were intraperitoneally injected with 0.75 U / kg insulin, and the blood glucose level in the tail vein of the mice was measured at 0, 15, 30, 60, 90 and 120 min after insulin injection.

[0084] C. Leptin sensitivity experiment: Mice were adapted to single cage housing 1 week in advance. Mice received intraperitoneal injection of normal saline twice a day (10am and 6pm) for 3 days, followed by intraperitoneal injection of 1 mg / kg recombinant leptin for 3 days. Body weight and food intake were measured once a day.

[0085] 4. Metabolic monitoring

[0086] Metabolic monitoring was performed using a comprehensive laboratory animal monitoring system (Columbus Instruments, CLAMS). After the mice adapted to the metabolic monitoring system for 3 days, their food intake, water intake, and metabolic parameters were continuously monitored for 3 days, including: oxygen consumption, carbon dioxide production, respiratory exchange rate, heat production, horizontal and vertical movement, food intake, water intake, etc.

[0087] 5. Stereotaxic injection

[0088] After anesthesia with 2% isoflurane, the mice were fixed in a supine position on a stereotaxic apparatus after skin preparation. After disinfection, the skin on the top of the skull was incised and the skull was fully exposed. The plane of the skull was corrected according to Bregma and Lamda points, and the coordinates were determined with reference to the standardized mouse brain atlas. The skull was drilled with a dental drill and the dura mater was removed. The AAV virus was stereotaxically injected into the bilateral ARC (Bregma-1.70mmAP, ±0.20mmML,-5.80mmDV; 200nL, 40nL / min). After the needle was retained for 10min, it was disinfected and the skin on the top of the skull was sutured layer by layer. 0.5ml of normal saline was injected subcutaneously in the neck of the mice, and the mice were kept warm and housed in a single cage until they woke up.

[0089] 6. Single-cell transcriptomics

[0090] The bilateral ARC was frozen in liquid nitrogen, total RNA was extracted to construct a library, and the data was obtained by sequencing. The quality of the original data was controlled and compared with the reference sequence. After exon quantification, gene expression was quantified. According to the differential gene expression of different cells, dimensionality reduction was performed to cluster, and gene differences of various types of cells and cell subgroups were analyzed (including: GO enrichment analysis, Pathway enrichment analysis, cluster analysis, specific expression and common expression analysis, protein network interaction analysis, transcription factor analysis, etc.).

[0091] 7. Electrophysiology

[0092] A. Preparation of ex vivo brain slices: After anesthesia, mice were killed by cervical dislocation, and the whole brain tissue was quickly peeled off and rinsed with pre-cooled oxygenated solution. A vibrating slicer was used to cut brain slices with a thickness of about 400 μm along the coronal plane and then infiltrated with 95% O 2 and 5% CO 2 The cells were incubated in artificial cerebrospinal fluid (ACSF) saturated with the gas mixture.

[0093] B. Spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs): Place a brain slice at the bottom of a full immersion recording bath (0.5-1.0 mL), fix it with a U-shaped metal frame nylon mesh, and continuously perfuse ACSF at a rate of 3 mL / min. Record neuronal discharge activity in voltage clamp mode. Clamp the voltage at -70 mV and find tdTomato + ARC POMC Patch clamp recording was performed on neurons to record sEPSCs; 0.5 mM TTX was added to the perfusion fluid, and mEPSCs were recorded 10 minutes later.

[0094] C. Spontaneous discharge (AP): Find tdTomato + ARC POMC The discharge activity of neurons was recorded. 100 nM leptin was added to the perfusion fluid, and the changes in AP of POMC neurons were recorded 5 minutes before and after leptin perfusion.

[0095] Example 2 Analysis of hypothalamic transcriptome in high-fat diet-induced obese mice

[0096] After 8 weeks of high-fat diet-induced obesity (DIO) in WT mice, the hypothalamus was removed for transcriptomic sequencing, and bioinformatics was used to analyze differential gene expression and related biological function changes.

[0097] The results showed that the expression of Bhlhe41 in the hypothalamus of high-fat diet-induced obese mice was significantly increased, and its related differential functions were mostly enriched in biological functions such as synapse formation, vesicle release and intersynaptic transmission ( Figure 1 ). The results of this part preliminarily found that hypothalamic DEC2 may play an important role in the occurrence and development of obesity.

[0098] Example 3 Stereotaxic injection of AAV-CMV-Bhlhe41 into different nuclei of the hypothalamus of WT mice induces obesity in mice

[0099] To further study the role of Bhlhe41 gene-encoded protein DEC2 in the hypothalamus, the present invention used WT mice to stereotactically inject AAV-CMV-Bhlhe41 or control virus (AAV-CMV) into different hypothalamic nuclei ARC, VMH, DMH, and PVH, and observed the weight changes of the two groups of mice.

[0100] The results showed that only ARC overexpression of DEC2 significantly induced obesity in mice, while overexpression of other hypothalamic nuclei such as VMH, DMH, and PVH did not show a significant obesity phenotype ( Figure 2 ). The results of this part prove that hypothalamic ARC is the key nucleus of DEC2-mediated obesity.

[0101] Example 4 Stereotaxic injection of AAV-CMV-Bhlhe41-dio into the ARC of POMC-Cre mice to conditionally overexpress DEC2 and significantly induce obesity in mice

[0102] POMC-Cre mice were stereotactically injected with AAV-CMV-Bhlhe41-dio or control virus (AAV-CMV-dio) at the hypothalamic ARC, and the changes in food intake, body weight and metabolism of the two groups of mice were observed; AgRP-Cre, CX3CR1-Cre-ERT2, and ALDH1L1-Cre-ERT2 mice were stereotactically injected with AAV-CMV-Bhlhe41-dio or control virus (AAV-CMV-dio) at the hypothalamic ARC, and the changes in body weight and food intake of the two groups of mice were observed.

[0103] The results show that only ARC POMC Conditional overexpression of DEC2 significantly induced obesity in mice, manifested by increased food intake, weight gain, fat accumulation, and decreased metabolism ( Figure 3 ); while AgRP neurons (AgRP + ), microglia (CX3CR1 + ) and astrocytes (ALDH1L1 + )Conditional overexpression of DEC2 had no significant effect on food intake and body weight in mice ( Figure 4 ). This part of the results proves that ARC POMC It is a key neuron in DEC2-mediated obesity.

[0104] Example 5 Bhlhe41 fl / fl Conditional knockout of DEC2 by stereotaxic injection of AAV-POMC-Cre into ARC of obese mice effectively improves obesity in mice

[0105] Bhlhe41 fl / fl After the mice had been fed a high-fat diet for 8 weeks, AAV-POMC-Cre or control virus (AAV-POMC) was stereotaxically injected into the ARC, and the changes in body weight and food intake of the two groups of mice were observed. AAV-CMV-Bhlhe41-shRNAi was stereotaxically injected into the ARC of db / db mice and db / m mice to knock down DEC2, and the changes in body weight and food intake of the two groups of mice were observed.

[0106] Results Tip: ARC POMC Neuronal conditional knockout of DEC2 significantly improved obesity and increased food intake induced by a high-fat diet, but had no effect on the body weight and food intake of leptin receptor mutant (db / db) mice ( Figure 5 ). This result further proves that ARC POMC DEC2 plays a key role in mediating obesity and its action is located downstream of the leptin receptor.

[0107] Example 6 Single-cell omics sequencing and biological function analysis of ARC overexpressing DEC2

[0108] POMC-Cre mice were stereotaxically injected with AAV-CMV-Bhlhe41-dio or control virus (AAV-CMV-dio) at the ARC. Eight weeks later, the hypothalamus was removed for single-cell omics sequencing, and bioinformatics was used to analyze differential gene expression and related biological function changes.

[0109] The results showed that overexpression of DEC2 in POMC neurons mainly affected the transcriptional activity of STAT3, a key molecule in the leptin signaling pathway, and had little effect on the insulin signaling pathway. Figure 6 ). The results of this part suggest that the biological function of DEC2 in affecting POMC neurons is mainly focused on leptin resistance.

[0110] Example 7 ARC POMC Effects of conditional overexpression of DEC2 in neurons on glucose and insulin tolerance and leptin sensitivity in mice

[0111] POMC-Cre mice were stereotaxically injected with AAV-CMV-Bhlhe41-dio or control virus (AAV-CMV-dio) at the ARC, and tail vein blood was collected 4 weeks later to detect plasma insulin and leptin levels. GTT and ITT experiments were performed by intraperitoneal injection of glucose or insulin, and tail vein blood was collected at 0, 15, 30, 60, 90, and 120 min after injection to detect changes in blood glucose. Normal saline (0.2 ml) and leptin were continuously injected intraperitoneally for 3 days and 3 days, and changes in food intake and body weight were observed. ARC was collected 30 minutes after intraperitoneal injection of leptin or normal saline for immunofluorescence staining to detect ARC POMC Expression of p-STAT3.

[0112] The results show that: ARC POMC Conditional overexpression of DEC2 in neurons had no significant effect on insulin, OGG, and ITT in mice, but significantly induced an increase in leptin levels and significantly reduced leptin-mediated food intake, weight loss, and p-STAT3 expression in mice. Figure 7 ). The results of this part confirm that ARC POMC Neuronal conditional overexpression of DEC2 significantly induced leptin resistance in POMC neurons but had little effect on insulin responsiveness.

[0113] Example 8 ARC POMC Effects of conditional overexpression of DEC2 on the electrophysiological responses of leptin in mouse POMC neurons

[0114] POMC-tdTomato mice were stereotaxically injected with AAV-CMV-Bhlhe41-dio or control virus (AAV-CMV-dio) at the ARC, and brain slices containing the ARC were removed 4 weeks later for electrophysiological testing.

[0115] The results show that: ARC POMC Conditional overexpression of DEC2 in neurons significantly inhibited the leptin-mediated increase in firing frequency and depolarization in POMC neurons ( Figure 8 ). The results of this part further confirmed that ARC POMC Neuronal conditional overexpression of DEC2 significantly induced leptin resistance in POMC neurons.

Claims

1. A biomarker for diagnosing obesity, wherein the biomarker is DEC2; when the expression of DEC2 in a patient's biological sample increases significantly, it indicates that the patient is at risk of obesity.

2. The biomarker as claimed in claim 1, when the expression of DEC2 in the arcuate nucleus and paraventricular nucleus of the patient's hypothalamus is significantly increased, it indicates that the patient is at risk of obesity; when the expression of DEC2 in the arcuate nucleus of the patient's hypothalamus is significantly increased, it indicates that the patient is at risk of obesity; when the expression of DEC2 in POMC neurons in the arcuate nucleus of the patient's hypothalamus is significantly increased, it indicates that the patient is at risk of obesity.

3. The use of DEC2 in the preparation of a reagent for diagnosing obesity, wherein the reagent contains a reagent that can detect the content of DEC2 in a patient's biological sample; when the expression of DEC2 in the patient's biological sample increases significantly, it indicates that the patient is at risk of obesity.

4. A kit for diagnosing obesity, the kit comprising a reagent for detecting the content of DEC2 in a patient's biological sample; when the expression of DEC2 in the patient's biological sample is significantly increased, it indicates that the patient is at risk of obesity.

5. The kit according to claim 4, wherein the kit can be one or more of an ELISA detection kit, a colloidal gold detection kit, an immunohistochemistry kit, an immunofluorescence kit and / or an in situ hybridization staining kit.

6. A method for screening drugs for treating obesity, wherein the method is to screen drugs that inhibit DEC2 gene activity or reduce DEC2 gene expression, and the method comprises the following steps: S01: administer the screened drugs to model animals; S02: Detect the expression level of DEC2 gene in model animals; S03: If the expression level of DEC2 is significantly reduced, it is considered that the screened drug can be used as a drug for treating obesity.

7. An obese animal model, wherein the animal model has high expression of DEC2.

8. Use of an agent for inhibiting DEC2 in the preparation of a drug for treating obesity, wherein the drug targets DEC2 and inhibits DEC2 expression.

9. The use according to claim 8, wherein the drug exerts a weight loss effect by improving leptin resistance.

10. The use as claimed in claim 8, wherein the drug can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories.

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