Application of MEN1 gene and encoded protein thereof in preparation of medicine for preventing or treating obesity and metabolic complications thereof

Abstract

The invention discloses an application of an MEN1 gene and an encoded protein thereof in preparation of a medicine for preventing or treating obesity and metabolic complications thereof, and belongs to the technical field of biology. In order to overcome the defect that in the prior art, technical gaps exist in the action of an MEN1 gene and Menin protein coded by the MEN1 gene in small intestine lipid metabolism, the invention provides application of the MEN1 gene and protein coded by the MEN1 gene in preparation of drugs for preventing or treating obesity and metabolic complications thereof, and the MEN1 gene and the coded protein Menin serve as drug targets; by down-regulating MEN1 gene expression or inhibiting Menin protein functions, fat entering blood is reduced, and obesity and metabolic disorder are improved. Therefore, the invention provides the medicine for reducing intestinal lipid entering blood, blocking excessive fat accumulation and improving insulin resistance, the HFD-induced obesity and metabolic disorder can be effectively relieved, and a novel therapeutic molecule is provided for preventing and treating obesity and complications thereof.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to the use of the MEN1 gene and its encoded protein in the preparation of drugs for preventing or treating obesity and its metabolic complications. Background Art

[0002] Obesity and its complications (e.g., type 2 diabetes, atherosclerosis, etc.) are major global public health issues. Their primary pathological mechanism is attributed to a metabolic imbalance in which long-term energy intake exceeds energy expenditure. The small intestine is a key organ for lipid absorption, and the uptake and metabolism of lipids in intestinal epithelial cells play an important role in the development of obesity. After dietary lipids are absorbed, they are primarily transported into the bloodstream via chylomicrons and distributed to the liver, adipose tissue, and other sites, promoting fat storage and the development of obesity.

[0003] Studies have shown that inhibiting chylomicron production can effectively reduce the body's lipid burden. For example, overexpression of APOC3 can reduce plasma chylomicron levels, significantly reducing obesity in mice. Knockout of DGAT1, the rate-limiting enzyme for triglyceride synthesis, in the small intestine, without affecting food intake and energy expenditure, exhibits a pronounced anti-obesity phenotype. Notably, targeted inhibition of small intestinal lipid absorption does not significantly affect systemic energy metabolism; therefore, regulating small intestinal lipid metabolism is a highly safe and effective anti-obesity strategy.

[0004] Prior art has identified the Menin protein, encoded by the MEN1 (Multiple Endocrine Neoplasia 1) gene, as a tumor suppressor. Research has primarily focused on its role in neuroendocrine tumors and leukemia. However, recent studies have revealed that Menin also plays an important role in cell proliferation, differentiation, and metabolic regulation. For example, it can regulate genes involved in lipogenesis and glucose metabolism, influencing metabolic homeostasis. While the functions of Menin in other metabolic organs have been extensively studied, its role in small intestinal lipid metabolism remains unclear. Summary of the Invention

[0005] The present invention addresses the technical gap in the prior art regarding the role of the MEN1 gene and the Menin protein it encodes in small intestinal lipid metabolism, and provides the use of the MEN1 gene and the protein it encodes in the preparation of drugs for preventing or treating obesity and its metabolic complications.

[0006] One of the objectives of the present invention is to provide the use of the MEN1 gene in the preparation of a drug for preventing or treating obesity and its metabolic complications.

[0007] In a preferred embodiment of the present invention, the nucleotide sequence of the MEN1 gene is shown as SEQ ID NO.1.

[0008] In a preferred embodiment of the present invention, the application refers to improving obesity and metabolic disorders by inhibiting the expression of the MEN1 gene and thereby inhibiting lipid transport in the small intestinal epithelium.

[0009] A second object of the present invention is to provide a small molecule inhibitor of Menin, which is a molecule designed to inhibit MEN1 gene expression or block Menin protein activity by targeting the MEN1 gene. The Menin is a protein encoded by the MEN1 gene.

[0010] In a preferred embodiment of the present invention, the amino acid sequence of Menin is shown as SEQ ID NO.2.

[0011] The third object of the present invention is to provide the use of the above-mentioned Menin small molecule inhibitor in the preparation of a method for preventing or treating obesity and its metabolic complications.

[0012] Beneficial effects of the present invention: The present invention proposes the use of the MEN1 gene and its encoded protein in the preparation of a drug for preventing or treating obesity and its metabolic complications, taking the MEN1 gene and its encoded protein Menin as drug targets, and reducing the entry of lipids from the small intestinal epithelium into the blood to improve obesity and metabolic disorders; the present invention also provides a Menin small molecule inhibitor, which includes any of the following: (1) double-stranded siRNA of the MEN1 gene; (2) a mutant of the MEN1 gene; (3) other small molecule inhibitors that can inhibit the expression of the MEN1 gene or the function of the Menin protein; wherein the double-stranded siRNA uses RNA interference to silence the MEN1 gene, thereby intervening in the occurrence process of obesity; the mutant of the MEN1 gene, after being injected into cells, competes for the substrate of the MEN1 gene prototype, thereby inhibiting the function of the MEN1 gene, thereby achieving the purpose of reducing lipid accumulation and improving obesity.

[0013] Effect experiments have shown that knocking out Men1 to reduce Menin expression, or using a small molecule inhibitor of Menin, can significantly reduce mouse body weight and subcutaneous fat, decrease blood glucose levels in the small intestinal epithelial cells, significantly reduce TAG content in the small intestinal epithelial cells, effectively reduce lipid accumulation in the intestine, significantly reduce the size of chylomicrons in the small intestinal epithelial cells, and reduce the expression of genes related to the fatty acid oxidation pathway in the intestinal epithelial cells. This suggests that knocking out or inhibiting Men1 expression can reduce intestinal lipid transport, block excessive fat accumulation, improve insulin resistance, and effectively alleviate HFD-induced obesity and metabolic disorders, providing a new therapeutic molecule for the prevention and treatment of obesity and its complications.

[0014] The present invention found that the MEN1 gene has a promoting effect on small intestinal lipid metabolism and the occurrence and development of obesity in the body. Knocking out or inhibiting the expression of the MEN1 gene can effectively block the transport of lipids in the intestine, thereby significantly reducing the occurrence of obesity and improving systemic metabolic disorders.

[0015] The present invention proposes that MEN1 and the Menin protein it encodes can serve as potential targets for the development of therapeutic molecules or intervention strategies for the prevention, alleviation and / or treatment of obesity and related complications, and provides new breakthroughs and prospects for the clinical treatment of obesity using Menin small molecule inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 For Men1 ΔIEC Mouse detection results; A is the Men1 expression detection diagram, B is the small intestinal epithelial cell staining results;

[0017] Figure 2 This is a feeding flowchart for the high-fat diet (HFD)-induced obesity mouse model;

[0018] Figure 3 Figure 1 shows the effect of Men1 knockout on the obesity phenotype of mice; A shows the detection of Men1 expression; B shows the white adipose tissue; C shows the comparison of the final body weight of mice; D and E show the HE staining of subcutaneous and retroperitoneal adipose tissue of mice; F shows the detection of TAG content in liver tissue;

[0019] Figure 4 Figure 1 is a graph showing the results of a glucose tolerance test (GTT); A is a graph showing changes in blood glucose at different time points; B is a statistical graph showing the area under the curve (AUC);

[0020] Figure 5 For Men1 f / f With Men1 ΔIEC Comparison of triglyceride TG content in small intestinal epithelial cells of mice in the two groups; A is the TG content change curve at different time points; B is the area under the curve AUC statistical graph;

[0021] Figure 6 Figure 2 is a diagram of lipid accumulation in the mouse intestine detected by Nile red staining; A is an image of lipid droplets under a fluorescence microscope; B is a quantitative statistical graph of fluorescence intensity grayscale value;

[0022] Figure 7 Figure 2 is a graph showing the size of chylomicrons (CMs) in small intestinal epithelial cells; A is a transmission electron microscope image of subcellular structures; B is a graph showing the statistical analysis of CM diameters;

[0023] Figure 8 This is the expression map of fatty acid oxidation-related genes in mouse small intestinal epithelial cells;

[0024] Figure 9 A is a graph showing the detection of APOB48 levels in serum; A is a graph showing the detection of Western blot; B is a graph showing the statistical grayscale intensity of APOB48;

[0025] Figure 10 Figure 1 is a diagram showing the lipid transport function assay in CACO-2 cells; A is a diagram showing protein validation in a MEN1 and CES1 double knockout cell line; B is a schematic diagram showing the structure of the Transwell system; C is a comparison of TG content in cells and the lower chamber; D is a diagram showing the Bodipy fluorescence intensity assay;

[0026] Figure 11 Detection graphs of key lipid catabolism genes expression in CACO-2 or MODE-K cells after MEN1 knockdown; A is the detection graph of Ces1g expression in MODE-K cells, B is the detection graph of Cpt1a expression in MODE-K cells, C is the detection graph of CES1 expression in CACO-2 cells, and D is the detection graph of CPT1A expression in CACO-2 cells;

[0027] Figure 12 Figure 1 shows the weight change of mice treated with Menin inhibitor MI-463; A is the treatment flow chart; B is the comparison of weight gain curves;

[0028] Figure 13 The following is a graph showing the postprandial serum TG levels in mice treated with the Menin inhibitor MI-463; A is the TG level change curve; B is the quantitative comparison of TG levels;

[0029] Figure 14 Figure 2: Detection of intestinal lipid accumulation after treatment with the Menin inhibitor MI-463. A is a Nile red staining image of the mouse intestine after MI-463 administration; B is a fluorescence grayscale quantitative image.

[0030] Figure 15The expression level of APOB48 in serum was detected after treatment with Menin inhibitor MI-463. A is a gel image of protein expression; B is a grayscale quantitative statistical graph;

[0031] Figure 16 These are graphs showing the improvement of glucose metabolism function after treatment with the Menin inhibitor MI-463; A is the glucose tolerance GTT test graph, B is the glucose tolerance GTT test AUC statistical graph, C is the insulin sensitivity test graph, and D is the insulin sensitivity test UC statistical graph. DETAILED DESCRIPTION

[0032] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0034] The sequences involved in the following examples are:

[0035] The nucleotide sequence of MEN1 in the human genome is shown in SEQ ID NO.1, and the amino acid sequence of the Menin protein encoded by MEN1 in the human genome is shown in SEQ ID NO.2;

[0036] Example 1: Use of the MEN1 gene and its encoded protein in the preparation of drugs for preventing or treating obesity and its metabolic complications

[0037] 1. Experimental Animals and Rearing Conditions

[0038] This experiment was performed by changing the genotype to Men1 f / f However, mice of the same littermate that do not carry Cre recombinase (purchased from Shanghai Nanmo Biological Company) were crossed with transgenic mice that specifically express Cre recombinase in intestinal epithelial cells (Villin-Cre) to obtain mice with Men1 knockout in small intestinal epithelial cells (Men1 ΔIEC ).

[0039] The results are as follows Figure 1 Compared to Men1f / f Control mice, Men1 ΔIEC Mice in this group lacked Menin protein expression in the small intestinal epithelial cells. All mice were housed in the animal laboratory of Harbin Medical University (SPF) at room temperature of 22-24°C, relative humidity of 50%-60%, and a 12-hour / 12-hour light cycle. Mice had free access to food and water. All animal experiments were conducted in compliance with the Animal Ethics Committee and relevant regulations.

[0040] 2. High-fat diet-induced obesity model and related index detection

[0041] 1. High-fat diet induction: Feeding flow chart of high-fat diet (HFD)-induced obesity model in mice, such as Figure 2 The following table shows the treatment cycles and key points of experimental animals under normal diet and high-fat diet conditions; 6-8 week old Men1 f / f Control mice, and Men1 ΔIEC Male mice were randomly divided into two groups and then fed a standard diet (10% of calories from fat) or a high-fat diet (approximately 60% of calories from fat) for 6-12 weeks to induce obesity. During this period, body weight and status were regularly measured. At week 18, the mice were tested for:

[0042] (1) Weigh the mice weekly and record their growth curves;

[0043] (2) At the end of the experiment, body fat content was measured using a metabolic cage. At the same time, the main adipose tissues (such as subcutaneous fat, mesenteric fat, brown fat, and retroperitoneal white adipose tissue) were dissected and weighed after sacrifice, and the proportion of adipose tissue weight to body weight was calculated. The subcutaneous fat and retroperitoneal white adipose tissue were stained with HE to show the size and distribution of fat droplets in the tissue.

[0044] The results are as follows Figure 3 As shown, Men1 ΔIEC The weight gain of mice in group A was significantly reduced ( Figure 3 A);Men1 ΔIEC The subcutaneous fat of mice was significantly reduced, and the proportion of subcutaneous fat, mesenteric fat, brown fat and retroperitoneal white fat tissue to body weight was significantly lower than that of Men1 f / f Control group mice ( Figure 3 BC). Men1 ΔIEC Lipid accumulation in the liver of mice was significantly reduced ( Figure 3 DE), the accumulation of TAG was also significantly lower than that of Men1 f / f Control group mice ( Figure 3 F).

[0045] 2. Blood glucose and glucose tolerance test: Glucose tolerance test (GTT) was performed on mice fed HFD for 12 weeks, and blood glucose was recorded at different time points after glucose administration. Figure 4 As shown, compared with Men1 f / f Control mice, Men1 ΔIEC The changes in blood glucose in the small intestinal epithelial cells of the mice in the control group showed a downward trend.

[0046] 3. Serum lipid analysis: A lipid tolerance test (TTT) was performed on mice fed with HFD for 12 weeks. After fasting, olive oil was gavaged and a lipase inhibitor was injected into the tail vein. Blood was collected from the orbit at different time points to measure triglyceride (TG) indicators. Figure 5 Compared to Men1 f / f Control mice, Men1 ΔIEC The TAG content in the small intestinal epithelial cells of the mice in the 44th group was significantly decreased.

[0047] 3. Analysis of lipid accumulation and chylomicron formation in intestinal epithelial cells

[0048] 1. Histological analysis of intestinal epithelial lipid droplet size: After the experiment, approximately 1 cm of tissue was obtained from the proximal small intestine or jejunum of the mouse. The tissue was fixed with 4% paraformaldehyde for 24 hours, dehydrated with gradient ethanol, embedded in paraffin, and sectioned (approximately 5 μm). Nile red staining was performed to observe and evaluate the accumulation of lipid droplets in the intestinal epithelium. Nile red staining can be performed directly on frozen sections or after dewaxing the sections to visualize the distribution of neutral lipids. The fluorescence area or fluorescence intensity of lipid droplets in the cells was statistically analyzed to determine the effect of Men1 knockout on intracellular lipid accumulation.

[0049] Liposomes typically appear as red fluorescence in images taken by fluorescence microscopy after staining with Nile red, e.g. Figure 6 As shown, compared with Men1 f / f Control mice, Men1 ΔIEC The accumulation of lipids in the intestine of the mice in the Men1 group was significantly reduced; it can be seen that the knockout of Men1 can effectively reduce the accumulation of lipids in the intestine.

[0050] 2. Transmission electron microscopy for chylomicron size detection: The small intestine of mice after the FTT experiment was fixed with 2.5% glutaraldehyde at 4°C for 12 hours, then post-fixed with 1% osmium acid to enhance contrast. The sample was then dehydrated with graded ethanol (30%, 50%, 70%, 90%, 100%) or acetone, and embedded in epoxy resin for solidification. After the embedded block solidified, it was cut into ultrathin sections with a thickness of 70-90 nm using an ultramicrotome, attached to a nickel grid, and then stained with uranyl acetate and lead citrate for heavy metals to enhance contrast. Finally, observation and photography were performed under a transmission electron microscope, during which intracellular subcellular structures such as CM, lipid droplets, mitochondria, and endoplasmic reticulum were visualized. To ensure statistical accuracy, all fields of view were randomly selected and photographed under the same conditions, and the microscope magnification was recorded or the built-in ruler was used. The images were then imported into ImageJ, and the CM area was segmented by setting a threshold or manually circling the area. The software then automatically calculated the CM diameter. The CM diameter measurement data for all fields of view were summarized and the diameter distribution range was calculated using Origin software.

[0051] The results are as follows Figure 7 As shown, with Men1 f / f Compared with the CM volume in the mouse intestine (284.48±12.9), Men1 ΔIEC The volume of mouse CM (203.02±5.19) was significantly reduced. ΔIEC The size of chylomicrons in the small intestinal epithelial cells of mice in the treatment group was significantly lower than that in the control group.

[0052] 4. Detection and Analysis of Key Genes and Pathways

[0053] 1. Extract Men1 f / f Control mice and Men1 ΔIEC Total RNA from small intestinal epithelial cells or CACO-2 cells of mice in the two groups was reverse transcribed and synthesized into cDNA (KR118-02, TIANGEN). The mRNA levels of Cpt1a, Acox1, Acadm, Acadl, and Ehhadh genes were detected using SYBR Green real-time fluorescence quantitative PCR kit (AQ131-02, Quanshijin Company). β-actin was used as an internal reference and 2 -ΔΔCT Relative quantitative analysis was performed using the method.

[0054] The results are as follows Figure 8 As shown, Men1 ΔIEC The expression levels of genes related to fatty acid oxidation pathway in intestinal epithelial cells of mice in group A were significantly higher than those in group B. f / f Control mice; specifically, Cpt1a, Acox1, Acadl, and Ehhadh genes were expressed in Men1 ΔIEC The expression of β-actin was upregulated in group mice.

[0055] 2. The level of APOB48 in the serum of mice after FTT was detected by Western blot. SDS-PAGE electrophoresis was performed, and the membrane was transferred and incubated with the primary antibody against APOB48. ECL color development was performed, and grayscale analysis was performed using albumin as an internal control.

[0056] The results are as follows Figure 9 As shown, compared with Men1 f / f Control mice, Men1 ΔIEC The level of APOB48 in the serum of group A mice was significantly decreased.

[0057] 5. CACO-2 Cell Lipid Absorption and Transport Model

[0058] 1. Cell culture and Transwell model establishment: Schematic diagram of the Transwell culture system, as shown in Figure 10 As shown in Part B, the protein verification results of MEN1 and CES1 double knockout cell lines are as follows Figure 10 As shown in A; human colon cancer cells CACO-2 were seeded on the upper layer of the Transwell chamber. After the cells grew, merged, and polarized to form a barrier similar to the small intestinal epithelium (21 days), the cell monolayer permeability was tested by transepithelial electrical resistance or sodium-fluorescein leakage test and met the standards; CRISPR-Cas9 was used to knock out MEN1, CES1, or double knock out MEN1 and CES1 genes in CACO-2 cells. After cell fusion, CACO-2 epithelial cells were cultured in the Transwell chamber and the cells were allowed to grow on the membrane surface for 21 days. 0.5 mL of the upper chamber was used. The culture medium was supplemented with a lipid mix (0.6 mM oleic acid, 2 mM sodium bile acid, 0.2 mM monoglyceride, 0.05 mM cholesterol, and 0.2 mM lysophosphatidylcholic acid) with or without 5 μM BODIPY oleic acid, along with 1.5 mL of serum-free DMEM medium in the lower chamber. After incubating the cells with the lipid mix for 10 minutes, the mix was discarded and replaced with serum-free DMEM. After 4 hours, 50 μL of serum-free DMEM medium from the lower chamber was collected and fluorescence intensity was measured using excitation at 480 nm and emission at 510 nm. After CACO-2 cells were cultured in a Transwell chamber and the lipid mix was added, TG levels in the cells and the lower chamber were measured, along with 12C Bodipy fluorescence in the lower chamber.

[0059] The results are as follows Figure 10 As shown in Figures C and D, the lipid transport capacity of MEN1-knockdown CACO-2 cells was significantly reduced compared with Vector cells; however, the lipid transport capacity was significantly restored after further knockout of CES1 in MEN1-knockdown CACO-2 cells.

[0060] To further verify the Menin-mediated regulatory role in lipid metabolism, this study used RT-qPCR to detect the mRNA expression levels of Ces1g, CES1, and Cpt1a in Men1 knockout MODE-K cells and MEN1 knockout CACO2 cells incubated with or without oleic acid, respectively.

[0061] The results are as follows Figure 11 As shown, downregulation of MEN1 significantly promoted the expression of lipid catabolism genes CES1 and CPT1A.

[0062] 6. MI-463 Pharmacological Intervention Experiment

[0063] Obese mice induced by a high-fat diet (HFD) for 6 weeks were treated with the menin inhibitor MI-463 (#S7816, Selleck) or a solvent by gavage. MI-463 was dissolved in 25% DMSO, 25% PEG400, and 50% PBS. Mice were gavaged once daily at a dose of 30 mg / kg for 6 consecutive weeks. Body weight changes and serum TAG and APOB48 levels were measured in the MI-463 and solvent-treated groups. Fasting blood glucose and insulin levels, glucose tolerance, and insulin sensitivity were also measured.

[0064] The results are as follows Figure 12-16 As shown, compared with the control solvent-treated group, the weight gain of mice in the Menin inhibitor MI-463-treated group was significantly slowed down ( Figure 12 ); serum TAG levels decreased after meals ( Figure 13 ), the accumulation of lipids in the intestine was significantly reduced, and the lipid load in the small intestine was reduced ( Figure 14 ); serum APOB48 levels decreased ( Figure 15 ); and glucose tolerance of mice in the MI-463 group ( Figure 16 AB) and insulin sensitivity was significantly improved ( Figure 16 Therefore, targeting Menin inhibitors can effectively alleviate HFD-induced obesity and metabolic disorders, providing new ideas for clinical intervention.

[0065] Any matters not described in detail in this specification are well known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. Use of the MEN1 gene in the preparation of drugs for preventing or treating obesity and its metabolic complications.

2. The use according to claim 1, characterized in that The nucleotide sequence of the MEN1 gene is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that The application refers to improving obesity and metabolic disorders by inhibiting the expression of the MEN1 gene or the function of the Menin protein, thereby reducing the entry of small intestinal epithelial lipids into the blood.

4. A small molecule inhibitor of Menin, characterized in that The Menin small molecule inhibitor is a molecule designed with the MEN1 gene and its encoded protein Menin as targets and capable of inhibiting MEN1 gene expression or blocking Menin protein activity. The Menin is a protein encoded by the MEN1 gene.

5. The Menin small molecule inhibitor according to claim 4, characterized in that The amino acid sequence of Menin is shown in SEQ ID NO.

2.

6. Use of the small molecule Menin inhibitor according to any one of claims 4 to 5 in the preparation of a drug for preventing or treating obesity and its metabolic complications.

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